93.4... v A.” i. . 23.5.»: x. .. (Ix. . 93:3,, 5. “9..“ .... . -1 .. , .2 .. ., if; 1: t , .Hfiifiéa‘ y... 04.: 0'. J2. 3 A4PUA .. ... a a ...a....m.ia.au. 3. .24331? 004 81¢ . n1... . art! «$.51. elrnflh..}‘ , li‘t’ s «13".» I» :. .v u. . . . z. A . ‘ ‘ 9.3.3. . 4 “a. . 5:51: .. 3. 25“ ... ‘ I J .. .5 ,1 , (:54! 1.»... ‘5. .nil[ .7} 13:34.7 THESOS 2004 6t; (32?. 6 ”i LIBRARY Michigan State This is to certify "Tat the U n We [Sity dissertation entitled MOLECULAR MECHANISMS OF SPACE RELATED BONE LOSS presented by Christopher Scott Ontiveros has been accepted towards fulfillment of the requirements for the PhD degree in Microbiology and Molecular Genetics fl/JWM A Major Professor’s Signature Jew/05 Date MSU is an Ati‘innative Action/Equal Opportunity Institution -.--.-.- ....--...-.--..--.O o o n u a a . u . e . . . . . - a -a PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/ClRC/DateDue.p65-p. 15 MOLECULAR MECHANISMS OF SPACE RELATED BONE LOSS By Christopher Scott Ontiveros A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology and Molecular Genetics 2003 ABSTRACT MOLECULAR MECHANISMS OF SPACE RELATED BONE LOSS BY Christopher Scott Ontiveros Mechanical loading is a major player in the maintenance of a normal bone phenotype. Increasing load or force on bone promotes bone formation while decreasing load promotes bone loss. The molecular events mediating responses to alterations in skeletal loading are largely unknown. My thesis focuses on determining the molecular effects of altered mechanical load on bone forming osteoblasts using a system of decreased load. The NASA designed rotating wall vessel (RWV) allows for the culture of cells in solid phase rotation around a horizontal axis such that gravity vectors are randomized to near zero thereby modeling microgravity. Osteoblasts exposed to 24 hours of RWV conditions exhibit suppressed expression of markers of osteoblast differentiation, namely alkaline phosphatase and osteocalcin. Furthermore, transcription factors that regulate the expression of these genes and progression to a fully differentiated osteoblast are also suppressed in the RWV. Specifically, c-fos and c-jun expression, members of the AP-1 family, are suppressed after 24 hours of RWV culture. Consistent with this finding AP-1 DNA binding and transactivation are suppressed. The osteoblast transcription factor runx2 displays similar effects to AP-1 in modeled microgravity conditions. After 24 hours of RWV culture, runx2 mRNA levels are decreased along with runx2 DNA binding and runx2 promoter activation. This demonstrates that conditions of decreased loading may be involved in suppressed osteoblast phenotype through alterations in transcription factor activities. While the RWV models microgravity, there is also a hypoxic component associated with the culture of differentiating osteoblasts. To uncouple the effects of hypoxia from modeled microgravity, external oxygen was supplied to RWV cultured cells to normoxia. Under normoxia, the suppression of c-fos, c-jun, and runx2 expression does not occur suggesting that the hypoxic component of the RWV and not modeled microgravity mediates the initial suppression of these genes we observed. Interestingly, skeletal unloading induces a hypoxic microenvironment to which bone cells are subjected. My findings suggest that bone loss associated with unloading, as seen during space flight, may be directly due to hypoxia. ...dedicated to my parents for the many years of neverending, unwavering support and encouragement that allowed me to get as far as I have. . .. ACKNOWLEDGEMENTS I would like to thank all the members of the labs l have been a part of during my graduate years for contributing to my personal and professional growth. In particular, I would like to thank my current lab mates for the dynamic relationship we shared along with the informal, congenial atmosphere we maintained in the lab and good times we shared outside the lab. Thank you Laura McCabe, Sergiu Botolin, Brian Coates, Moushumi Hossaln, Regina “The Hills Have Eyes” Inivin, Andrew Reinink and Jianwei Xie. These people have become my Michigan family and will be sorely missed and never forgotten. In particular, I would like to thank my good friend, colleague, and mentor Laura McCabe for coming along at the right time in my graduate career. Besides serving as the foundation of the lab, her support, guidance, one on one attention over latte and chai, and continuous, infectious enthusiasm over the past years have had the greatest impact on my professional growth. I would have been lost without her and am truly indebted. TABLE OF CONTENTS LIST OF TABLES ................................................................................. viii LIST OF FIGURES ................................................................................ ix ABBREVIATIONS ................................................................................. xii INTRODUCTION ................................................................................... 1 CHAPTER I. LITERATURE REVIEW ...................................................... 2 I. What is bone? ................................................................................... 2 A. Bone Structure/Function ............................................................. 2 B. Osteoblast Biology/Differentiation ................................................ 8 1 , Proliferation ................................................................. 1 1 2. Extracellular Matrix Maturation ........................................ 13 3. Matrix Mineralization ..................................................... 15 C. Osteoporosis ......................................................................... 16 ll. Molecular Regulation of Runx2 and AP-1 in Osteoblast Differentiation .............................................................................. 19 A. Runx2 .................................................................................. 20 1 . Structure/Function ........................................................ 20 2. Osteoblast Differentiation ............................................... 21 B. AP-1 .................................................................................... 28 1 . Structure/ Function ........................................................ 28 2. Osteoblast Differentiation ............................................... 31 Ill. Osteoblast Response to Alterations in Force ......................................... 35 A. Increased Force ..................................................................... 35 1 . Physiology .................................................................. 39 2. Mechanosensors .......................................................... 4O 3. Model Systems ............................................................ 42 a. Centrifugation ...................................................... 42 b. Fluid Flow ............................................................ 44 c. Hydrostatic Compression ........................................ 44 B. Decreased Force .................................................................... 45 1 . Spaceflight .................................................................. 46 2. Hindlimb unloading ....................................................... 50 3. Clinostat ..................................................................... 52 IV. Hypoxia ........................................................................................ 55 A. Molecular Cell Response ......................................................... 56 B. Bone and the Role of Hypoxia ................................................... 57 Vi C. Relationship to Decreased Load ................................................ 58 REFERENCES .................................................................................... 61 Chapter II. SIMULATED MICROGRAVITY SUPPRESSES OSTEOBLAST PHENOTYPE, RUNX2 LEVELS, AND AP-1 ........................................................................ 91 Abstract .................................................................................... 92 Introduction ................................................................................ 93 Methods .................................................................................... 98 Results .................................................................................... 101 Discussion ............................................................................... 104 Table ...................................................................................... 109 References .............................................................................. 1 10 Figure Legends ......................................................................... 118 Figures .................................................................................... 121 Chapter III. HYPOXIA SUPPRESSES RUNX2 INDEPENDENT OF MODELED MICROGRAVITY ............................................. 127 Abstract ................................................................................... 128 Introduction .............................................................................. 130 Methods .................................................................................. 133 Results .................................................................................... 138 Discussion ............................................................................... 141 References .............................................................................. 145 Figure Legends ......................................................................... 150 Figures .................................................................................... 154 Chapter IV. RWV ASSOCIATED HYPOXIA SUPPRESSES AP-1 IN OSTEOBLASTS ......................................................... 162 Abstract ................................................................................... 163 Introduction .............................................................................. 1 64 Methods .................................................................................. 167 Results .................................................................................... 175 Discussion ............................................................................... 178 References .............................................................................. 1 85 Table ...................................................................................... 195 Figure Legends ......................................................................... 196 Figures .................................................................................... 200 Chapter V. DISCUSSION .................................................................. 209 References ...................................................................... 220 Figure Legend .................................................................. 224 Figure ............................................................................ 225 vii LIST OF TABLES Table 1. PCR Primers ......................................................................... 109 Table 2. PCR Primers ......................................................................... 195 viii Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. LIST OF FIGURES Schematic of a longitudinal section through a long bone ............... 3 Cortical and Cancellous Bone ................................................ 5 Cross sectional top view through cortical bone ........................... 6 Bone Cells and Remodeling ................................................... 9 Osteoblast differentiation and changes in cell morphology .......... 10 Preosteoblast differentiation to a mature osteoblast involves three discrete stages marked by changes in gene expression......12 Functional Organization of Runx2 .......................................... 25 Functional Organization of AP-1 Family Members ..................... 29 Bone is subjected to three stress components .......................... 36 Experimental Design ......................................................... 121 Acute RWV exposure decreases alkaline phosphatase and osteocalcin expression ...................................................... 122 Acute RWV exposure does not alter RNA integrity or total cellular transcription .......................................................... 123 Acute RWV exposure results in decreased runx2 expression ..... 124 Acute RWV exposure results in modest increase in collagen l expression ...................................................................... 125 Acute RWV exposure results in decreased AP-1 transactivation but does not alter general cellular transcription ....................... 126 Three RWV experimental approaches are used to uncouple Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25 Figure 26. Figure 27 Figure 28. Figure 29. Modeled microgravity from hypoxic conditions ........................ 154 GAPDH and VEGF mRNA levels are increased in osteoblasts grown in horizontal or vertical rotating RWV ........................... 155 Runx2 mRNA levels are decreased in osteoblasts grown in horizontal or vertical rotating RWV .................................... 156 OSE2 DNA binding is decreased in osteoblasts grown in horizontal or vertical rotating RWV .................................... 157 Horizontal or vertical RWV rotation suppresses runx2 promoter activity ............................................................... 158 Normoxic conditions in the RWV prevent GAPDH and VEGF mRNA induction ...................................................... 159 Normoxic conditions in the RWV prevent runx2 mRNA suppression ..................................................................... 160 Hypoxia alters GAPDH, VEGF and Runx2 mRNA levels in osteoblasts .................................................................. 161 c-Fos and c-Jun mRNA levels decrease 24 hours following 24 hours following culture ................................................... 200 c-jun protein levels decrease 24 hours following RWV culture...202 AP-1 DNA binding is decreased in the RWV ........................... 203 c-fos and c-jun DNA binding is decreased in the RWV .............. 204 AP-1 transactivation is decreased and reversible up to 48 hours in the RWV ......................................................... 205 Oxygenation of the RWV to normoxic conditions prevents Figure 30. Figure 31. Figure 32. c-Fos and c-Jun mRNA suppression .................................... 206 RWV conditions do not alter osteoblast proliferation ................. 207 RWV conditions do not induce osteoblast apoptosis ................ 208 Normoxic RWV conditions do not prevent suppression of alkaline phosphatase and osteocalcin expression ................... 224 xi ABBREVIATIONS 3H d-MEM AEF1 uCi U9 pl AD AML1 AML2 AML3 AP AP-1 ATF2 ATP BMP-2 BrdU cAMP CAT cbf beB CBFA1 CBFA2 CBFA3 CBP ccd cDNA C/EBP c-fos c-myc C02 Col I cox-2 CRE c-src CTX DNA DPyr EDTA EGF EMSA ERK ETS tritium a minimal essential media delta ets related factor 1 microCurie microgram microliter activation domain acute myeloid leukemia factor 1 acute myeloid leukemia factor 2 acute myeloid leukemia factor 3 alkaline phosphatase activator protein 1 activating transcription factor 2 adenosine triphosphate bone morphogenic protein bromodeoxyuridine cyclic adenosine monophosphate chloramphenicol acetyltransferase core binding factor core binding factor beta core binding factor A1 core binding factor A2 core binding factor A3 CREB binding protein cleidocranial dysplasia complementary deoxynucleic acid CAAT/enhancer binding protein FBJ osteosarcoma oncogene myelocytomatosis oncogene carbon dioxide collagen I cyclooxygenase 2 cAMP response element Rous sarcoma oncogene C-telopeptide deoxyribonucleic acid deoxypyridinoline ethylenediaminetetra acetate trihydrate epidermal growth factor electrophoretic mobility shift assay extracellular regulated kinase E26 avian leukemia protein xii FBJ FBS FHL2 9 G GAPDH HARV HCI HDAC6 HEPES HIF HOB HOS-TE85 HRE hRWV Hu09 IKB lL-6 lL-8 JAB1 JNK KCI kDa MEF2A M mg MgCIz mM mmHg MMP13 MMPIIA MORF MOZ mRNA MSX2 MYST NaCl NASA NFAT NF KB ng NLS NMP—2 NMTS NP40 Finkel Biskis Jinkins fetal bovine serum floating head 2 gravity unit gravity glyceraldehyde phosphate dehydrogenase horizontal aspect rotating vessel hydrochloric acid histone deacetylase 6 hydroxyethylpiperazine ethanesulfonate hypoxia inducible factor human osteoblast human osteosarcoma TE85 hypoxia response element horizontal rotating wall vessel human osteoblast-like 9 inhibitor of K3 interleukin 6 interleukin 8 jun activation domain binding protein 1 Jun N-terminal kinase potassium chloride kilodalton myocyte enhancer factor 2A modeled or simulated microgravity milligrams magnesium chloride millimolar millimeters mercury matrix metalloprotein 13 matrix metallothionein IIA mortality factor monocytic leukemia zinc finger protein messenger ribonucleic acid msh homeobox homolog 2 M02, YBF2/SA83, SASZ, Tip60 histone acetyltransferase sodium chloride National Aeronautics and Space Administration nuclear factor of activated transcription nuclear factor of K3 nanogram nuclear localization signal nuclear matrix protein 2 nuclear matrix targeting signal nonident P40 xiii OC OSE Pa PAS PBS PCR PEBPZB PEBPqA PEBPqB PEBPoC PDGFB PGE2 PGHSZ PKA PKC PMA pmol F302 PPARv PST PTH QA thMP-2 RIP140 ROS17/2.8 rpm RT—PCR runx1 runx2 runx3 RWV SDS SE shc SOX9 SP-1 SRO-1 STLV STS SV40 TAZ TBE TBP TCA TFllB TGFB osteocalcin osteoblast specific element Pascal Per Arnt Sim domain phosphate buffered saline polymerase chain reaction polyoma enhancer binding protein 2 beta polyoma enhancer binding protein alpha A poyoma enhancer binding protein alpha B polyoma enhancer binding protein alpha C platelet derived growth factor B prostaglandin E2 prostaglandin H synthase 2 protein kinase A protein kinase C phorbol myristate acetate picomole partial pressure of oxygen peroxisome proliferators activator receptor gamma proline/serine/threonine parathyroid hormone glutamine/alanine recombinant human morphogenic protein receptor interacting protein 140 rat osteosarcoma 17/2.8 revolutions per minute reverse transcription polymerase chain reaction runt related transcription factor 1 runt related transcription factor 2 runt related transcription factor 3 rotating wall vessel sodium dodecyl sulfate standard error src homology 2 domain-containing transforming protein sex determining region Y box 9 trans acting transcription factor 1 steroid receptor coactivator 1 slow turning lateral vessel space transportation system simian virus 40 transcriptional coactivator with PDZ binding motif tris borate EDTA TATA binding protein trichloroacetic acid transcription factor II B transforming growth factor beta xiv TLE2 TNFa TPA TRE US VDR/RAR VEGF VP16 vRWV VWRPY transducin-like enhancer of split 2 tumor necrosis factor a 12-O-tetradecan-oylphorbol 1 3-acetate TPA response element United States vitamin D receptor/retinoic acid receptor vascular endothelial growth factor viral protein 16 vertical rotating wall vessel valine/tryptophan/arganine/proline/tyrosine XV INTRODUCTION This thesis provides a background on the understanding of mediators of osteoblast differentiation and subsequent bone formation. In particular, the work here focuses on the role of mechanical loading on osteoblast differentiation. To address the molecular changes associated with unloading l utilized the NASA designed rotating wall vessel (RWV) to model microgravity conditions. Osteoblasts cultured in the RWV exhibit suppressed expression of markers of differentiation consistent with true unloading conditions. This effect correlates with suppression of transcription factors important for normal osteoblast differentiation and expression of these differentiation markers. Surprisingly, in addition to modeling microgravity, culture of differentiating osteoblasts in the RWV results in hypoxia. This was an important finding since the RWV is used by many labs and has not been reported to cause hypoxic stress. In addition, this finding suggests that our observed effects of the RWV might not be due to modeled microgravity, but rather hypoxia. This idea was tested by culturing osteoblasts in the RWV while maintaining normoxic conditions. Using this approach, I found that the initial suppression in expression of markers of differentiation was in fact due to hypoxia and not modeled microgravity of the RWV. This hypoxic effect also has implications with regards to disuse associated bone formation in vivo since skeletal unloading induces a hypoxic microenvironment in bone. Knowing this, it is attractive to speculate if unloading induced bone loss is due to hypoxia directly and not decreased load. CHAPTER I LITERATURE REVIEW I. What is bone? Because of a common evolutionary descendent, vertebrates have a common body plan. While bodies of fishes and mammals are very different in many respects, they share common elements that identify them as vertebrates. This is particularly evident in the skeletons of different groups of vertebrates, in which homologous bones are relatively easy to find. Along with the gross morphological similarities, the structure and function of bone is also similar among vertebrates. A. Bone Structure/Function Bone consists of two types of morphological organization: compact (cortical) and spongy (cancellous, trabecular) [1]. Compact bone provides both the mechanical and protective functions, while spongy bone provides the metabolic functions of bone. The outer portion of the cortical bone is called the periosteum and the inner portion contacting the bone marrow is called the endosteum (Figure 1) [2]. Compact bone is most abundant in the long bone shafts of the appendicular skeleton and can be visualized best under polarized light or by electron microscopy where the organization of collagen fibers that alternate from layer to layer is more evident (Fig 23). This layered or lamellar . .- } Epiphysis Growth plate Metaphysis Cortical bone Endosteum Diaphysis < Periosteum Fused growth plate Figure 1. Schematic of a longitudinal section through a long bone. (From: Jee WSS, The skeletal tissues. In: Weiss L, ed. Histology, cell and tissue biology. New York: Elsevier Biomedical, 1983: 200-255.) structure allows for the highest density of collagen per unit volume of bone thus contributing to the strong nature of compact bone. The strength of compact bone is further enhanced by perpendicular planes of fibrils in adjacent lamellae which lend to it a plywood characteristic [3]. Compact bone can also be deposited surrounding a blood vessel resulting in a concentric ring of bone through which the vessel runs. This is called a haversian system (Fig 3). The second type of organization of bone is spongy or cancellous bone (Fig 2b). Spongy bone is not as dense as compact bone and can be found predominately in the skull, vertebrae, ribs, pelvis, and ends of long bones. Its distinguishing morphological feature is its porous nature. If one considers that a real sponge is made of sponge and air, then spongy bone is made of bone or trabeculae and marrow. Though trabecular bone constitutes roughly 20% of bone mass, it composes about 80% of total bone surface area due to the networking trabeculae [4]. This type of architecture makes cancellous bone better suited to resist compressive forces as well as provide strength and flexibility to the skeleton. The structure of bone makes it well suited to perform three main functions - protection, storage, and support. Firstly, bone protects organs and bone marrow. The brain and spinal cord are protected by the skull and vertebrae, organs of the thoracic cavity are protected by the sternum and ribs, and the bladder and reproductive organs are protected by the pelvic girdle. The long bones including the femur and humerus also serve to protect bone marrow-a source for many progenitor blood cells. Secondly, bone serves as the largest reservoir for calcium in the body and participates in maintaining calcium Figure 2. Cortical and Cancellous Bone. A micrograph cross section of cortical bone (a) through a haversian system showing the lamellar organization of collagen in mature matrix.and a microCT scan of cancellous bone demonstrating its porous nature. Interstitial lamellae "first" (In between eons) I ., ‘- I .:1 I I ti.‘ I .1 .' 0818061088 Circumferential lamellae Figure 3. Cross sectional top view through cortical bone. The haversian system is a series of canals containing blood vessels traveling through the center of each osteon. Also note the lamellar or layering organization of bone and osteocytes surrounding the haversian system giving it a concentric ring appearance. homeostasis in the body [5, 6]. In the extracellular fluids and cytosol, the concentration of calcium is critical for the maintenance and control of a number of biochemical and physiological processes. In order to maintain the calcium balance, calcium intake and loss have to be matched. If, for example, serum calcium is low, signals including but not restricted to parathyroid hormone (PTH) promote mobilization of calcium from bones [7-9]. The most extreme case of this PTH mediated release of calcium can be seen in people who have hyperparathyroidism. In these patients, decreased bone density and increased fracture occur due to increased mobilization of calcium from the bones [10-12]. Conversely, the hormone calcitonin alters a natural balance between bone formation and bone resorption by preventing bone resorption [13-16]. This allows for a net increase in bone formation through removal of extra serum calcium. Thirdly, bone serves supportive and mechanical functions that allow us to stand upright as well as move by allowing attachment sites for tendons and muscles [17, 18]. The bones of the skeleton are used as levers and fulcrums to which muscle attaches and contracts to create a force. This force in turn resists or moves the load against which the force is exerted. Bone is a dynamic system which is constantly being remodeled to accommodate varying loading stresses [19-21]. When this occurs, old or damaged bone is removed and new bone is deposited. The balance between bone formation and bone resorption allows for maintenance of a normal skeletal homeostasis. When the balance between bone formation and resorption is not maintained, skeletal abnormalities result. Too much bone formation can result in a condition called osteopetrosis characterized by thickening of the cortical region [22]. Pathological bone thickening can then lead to reduced marrow space and sclerosis of the base of the skull and vertebral bodies. Too little bone formation can result in osteoporosis characterized by a net decrease in bone mineral density and thinning of the cortical and cancellous bone [23]. Under these conditions bone becomes fragile and more susceptible to increased fractures. Normal bone homeostasis is regulated in large part by three different types of bone cells: osteoblasts, osteoclasts, and osteocytes (Fig 4). Osteoblasts make new bone and are predominately found on the periosteal and endosteal surface of bone, osteoclasts are recruited from the blood stream and resorb bone at sites of remodeling, and osteocytes are found completely surrounded by mineralized bone matrix in both cortical and cancellous bone [24-31]. While the function of osteocytes is not well understood, it is thought that they mediate signals between the outer and inner portions of bone via a lacuno-canalicular network to regulate biomechanical regulation of bone mass and structure [32-34]. B. Osteoblast Biology/Differentiation Osteoblasts are responsible for the formation of new bone. They are derived from the mesenchymal stem cell lineage and go through discrete stages from an immature proliferative preosteoblast to a fully differentiated osteoblast that is able to secrete osteoid (Fig 5) [35]. Subsequent to maturation, these cells The Remodeling Cycle @ osteoclast I} & precursors CT) /' .________________-. '\ Activation FEEQ .7 'Cu—i‘escent I phase 0 I <3: 0 Q a: <1» W osteoblasts '1 . Q Resorption Figure 4. Bone Cells and Remodeling. (Taken from http://www.ifcc.org/ejifcc/vol13no4/130401004fig1.gif) The three primary cells involved in bone remodeling are osteoblasts, osteoclasts, and osteocytes. During remodeling, activated osteocytes are recruited to the area and resorb old bone then apoptose. Osteoblasts then secrete bone matrix to fill in resorbed area and become either quiescent bone lining cells, embed themselves in the matrix and become osteocytes, or undergo apoptosis. A. bone Ilnlng cell 0" © IO]: - to); _. mesenchymal osteoprogenltor preosteoblast mature osteoblast cell stem cell osteoblast apoptosis \ osteocyte Figure 5. Osteoblast differentiation and changes in cell morphology. Differentiation to a mature osteoblast involves a stepwise progression from a stem cell where it receives cues to begin proliferation and differentiation to discrete cell types along the osteoblast lineage. 10 apoptose, enter a quiescent state and become bone lining cells, or they completely embed themselves in the osteoid matrix they secrete and become osteocytes [36, 37]. These stages of differentiation are marked by the temporally regulated osteoblast gene expression associated with changes in cell phenotype [38-40]. One of the earliest progenitors of a mature osteoblast is the mesenchymally derived osteoprogenitor cell found predominately in the bone marrow stroma [41]. In addition to becoming osteoblasts, these pluripotent mesenchymal stem cells, under the correct cellular cues, have the ability to become other cell types including chondrocytes, myocytes, and adipocytes [42-45]. Markers of osteoblastic progression include alkaline phosphatase activity and formation of bone nodules. In the past several years, studies have better defined factors important for the regulated development of mesenchymal stem cells into osteoprogenitor cells, however the molecular mechanisms of this transition into committed stages of osteoblast differentiation are still limiting. If one considers the characterized linear progression of differentiation from a preosteoblast to a fully mature osteoblast, one could divide the stages into cell proliferation, matrix maturation, and mineralization (Fig 6). 1. Proliferation Proliferation characterizes the first stage of preosteoblast differentiation from an osteoprogenitor cell. Notice of these cells was first taken in 1967 by 11 Proliferation Matrix Maturation Mineralization - runx2 % Maximal expression Days In culture Figure 6. Preosteoblast differentiation to a mature osteoblast involves three discrete stages marked by changes in gene expression. Preosteoblasts first undergo proliferation followed by matrix maturation where matrix components are layed down. The final stage is potentiation of matrix mineralization to complete formation of new bone. AlkPhos = alkaline phosphatase, CC = osteocalcin 12 Scott et al. where they described preosteoblasts as an osteoblastic cell possessing some of the characteristics of the adult osteoblast but is still able to proliferate [46]. The predominate genes expressed during this stage are associated with DNA replication and cell cycle progression. These genes include histones and the protooncogenes c-src, c-myc, and c-fos among others [47]. In cell culture, it has been shown that while these cells are able to proliferate, they have limited proliferative potential before they begin differentiation. Using microscopic time lapse cinematography, preosteoblasts have been demonstrated to undergo roughly 8 rounds of proliferation before they begin to differentiate and form bone nodules in culture [48]. As cells stop proliferating, there is a concomitant increase in expression of genes associated with extracellular matrix maturation. 2. Extracellular Matrix Maturation The second stage of differentiation is characterized by high amounts of secreted collagen and expression of alkaline phosphatase. Because of the highly secretory nature of an active osteoblast, the cells are characterized as having a large nucleus, enlarged golgi, and highly extended endoplasmic reticulum. Type I collagen makes up roughly 90% of the total extracellular matrix components secreted during this stage of differentiation [49]. In addition, this collagen constituent contributes to the flexibility and elasticity of bone [50, 51]. A good example of this can be seen in patients with a nonfunctioning collagen l 13 allele where there is reduced collagen l content in bone [52]. Many of these patients have increased fractures due to brittle bones normally imparted by adequate amounts of collagen [53]. Minor components which contribute to the mature matrix formation include fibronectin and TGFB. Among the best genetic markers of the matrix maturation stage is alkaline phosphatase. Expression and activity of alkaline phosphatase gradually increase during early matrix maturation, peaks about midway, then decreases to levels seen at the earliest stages when matrix maturation is complete. Though its presence coincides with matrix maturation, alkaline phosphatase is also important for progression to a mature osteoblast phenotype [54]. The exact mechanistic role of alkaline phosphatase is not known, however it is believed to play a role in increasing the amount of phosphate to be using during the mineralized stage of bone consistent with alkaline phosphatase levels being positively correlated with bone formation [55]. When alkaline phosphatase activity is inhibited by levamisole, initiation of mineralization in fetal rat calvarial cells is also inhibited in vitro [56]. Similarly, primary preosteoblastic cultures from heterozygous alkaline phosphatase knockout mice demonstrate delayed mineralization of bone, while homozygous knockouts were completely unable to become mineralized [57]. In humans, a missense mutation in the bone alkaline phosphatase gene inactivating alkaline phosphatase can cause hypophosphatasia, a disease characterized by decreased bone mineralization in children (rickets) and adults (osteomalacia) [58]. These studies implicate alkaline 14 phosphatase as a marker of matrix maturation as well as a necessary player in the formation of a mature osteoblast phenotype in rodents and humans. 3. Matrix Mineralization Mineralization of the extracellular matrix is the third step to formation of bone. Through an incompletely understood mechanism, matrix mineralization occurs within the collagen matrix. Once crystallization, via calcium phosphate (hydroxyapatite) deposition into the collagenous matrix begins, a wave of branching hydroxyapatite spreads toward the bone surface. When mineralization surrounds the osteoblasts, they become embedded within the matrix while staying connected to other osteoblasts via gap junctions. Fully surrounded in mineral, these cells form the next layer of osteocytes. While osteocyte function is not completely understood, these cells are thought to have a role in maintaining bone structure by responding to mechanical stimuli experienced by the bone [59]. An alternative fate of mature osteoblasts is to become bone lining cells. This occurs if mineralization is localized to only the bone surface and the cell does not become embedded. It enters a quiescent state poised for matrix maturation and mineralization when necessary. It has also been proposed that bone lining cells remove collagen left over following demineralization by osteoclasts during bone resorption, though work on this is still limiting [60]. The removal of demineralized collagen in turn allows for a new round of preosteoblast proliferation, matrix maturation and then mineralization. Several genes characterize the stage of 15 matrix mineralization including bone sialoprotein, osteopontin, and osteocalcin, among others [61-63]. All of these proteins are noncollagenous proteins present in bone matrix. Because it is predominately expressed in osteoblasts, one of the most widely studied of these matrix proteins is osteocalcin [48, 64, 65]. Its expression is highest during mineralization both in vitro and in vivo and is highly associated with hydroxyapatite [62, 66]. In vitro studies demonstrate that while osteocalcin has a high affinity for calcium, it inhibits crystallization of hydroxyapatite [67, 68]. Similarly, osteocalcin deficient mice show increased bone formation [69]. In combination with in vitro data, these findings suggest a role for osteocalcin in activation of osteoclasts for subsequent resorption [70-72]. C. Osteoporosis Osteoporosis is marked by the loss of bone mass and deterioration of skeletal microarchitechture. It is often associated with old age and skeletal disuse, and can result in frailty and increase risk of fractures. The 2002 US Surgeon General’s Workshop on Osteoporosis estimated that more than 10 million Americans over age 50 have osteoporosis (7.8 million women and 2.3 million men). By 2020, the prevalence of osteoporosis in Americans is projected to reach almost 14 million. Aging assoicated osteoporosis predominantly results from the loss of sex hormones. While aging men and women both lose bone mass, women lose it much faster for the first 5-10 years following menopausal loss of estrogen [73-76]. 16 Decreased testosterone is also coupled with bone loss in men, however because the male sex hormone testosterone exhibits a slow progressive decline, bone loss in men occurs at a slower rate than it does in women. Bone loss is thought to result from increased bone remodeling [77, 78] possbily as a result of an extension of the working lifespan of the osteoclast and a shortening of the working lifespan of the osteoblast [79]. In addition to sex hormone related effects, there appears to be a role for senescence in age related apoptosis. As age increases in both men and women, the amount of bone formed during each remodeling cycle decreases-especially in trabecular bone [80, 81]. This is consistent with actual studies of human bone marrow that demonstrates as age increases, there is a concurrent decrease in osteoblastogenesis [82, 83]. These studies suggest that the decrease in osteoblast bone formation with age related osteoporosis is largely due to an inability to keep up with osteoclast resorption rate, a decrease in the lifespan of osteoblasts, as well as a decrease in osteoprogenitor cells able to function normally. Osteoporosis can also occur in response to decreased mechanical stress on bones. Conditions where disuse associated osteoporosis occurs include prolonged bed rest, localized immobilization as in using a cast to heal fractures, spinal cord injury resulting in limb disuse, and space related microgravity conditions [84-89]. In one study, 5 weeks of bed rest resulted in a roughly 1% decrease in vertebral bone mineral density [90]. It has also been reported that one month space flight results in a decrease of 1% total bone mineral density [91-93]. These conditions are characterized by an overall decrease in bone 17 mineral density, thinning of the cortical bone, and resorption of cancellous bone which consequently becomes fragile and more susceptible to increased fractures. While some studies have demonstrated that increased bone resorption by osteoclasts contributes to a net decrease in bone mineral density, one common effect of disuse osteoporosis is a decrease in osteoblast function. For this reason, most of the work done on understanding decreased bone formation addresses the effects of mechanical factors on osteoblast function. Under normal loading conditions, it has been demonstrated that flow of fluid, compression, and tension within bone contributes to maintenance of a normal bone homeostasis [94-97]. However, when loading conditions are decreased or absent, these mechanical signals are also decreased or absent and bone loss ensues. Several mechanisms have been proposed to account for these changes in bone formation. One proposed mechanism suggests that osteocytes within bone respond to normal fluid flow through extended osteocyte processes to promote bone formation [20, 32, 98]. In vitro evidence demonstrates that both osteoblasts and osteocytes exhibit significant responses to shear stress induced by continuous and pulsating fluid flow [99-101]. Under disuse conditions, osteocytes do not experience the same flow effects they experience under loading conditions and in turn do not facilitate promotion of bone formation. Another proposed regulator of bone formation is cell deformation. Though the amount of deformation a bone cell feels under loading conditions is roughly 0.1%, it is proposed that the signal is amplified within the cell [102]. An attractive alternative proposed mechanism for disuse associated 18 decreases in bone formation involves a role for hypoxia. Conditions of disuse or unloading are associated with decreased fluid flow within bone and formation of a hypoxic microenvironment [103]. Furthermore, this hypoxic environment has been shown to result in suppression of a mature osteoblast phenotype associated with decreased bone formation [104, 105]. Because of the highly metabolic needs of differentiating osteoblasts, it is conceivable that when local oxygen concentration is decreased, function of osteoblasts is decreased or prevented [106]. Though the exact mechanism of disuse osteoporosis is not understood, much work is being done to understand the effects and possible treatments for bone loss. Il. Molecular Regulation of Runx2 and AP-1 in Osteoblast Differentiation A precise regulated orchestration of gene regulation is important for the progression of a preosteoblast to a mature osteoblast. This temporally regulated alteration in gene expression is controlled in part by regulators of transcription, namely transcription factors. In addition, post transcriptional processes have also been implicated as regulators of osteoblast gene expression and differentiation. While the importance of many of these factors has been identified in human and animal genetic models, cell culture has also provided invaluable information. Furthermore, some factors appear to be important at different stages of development or for specific cells in bone. The focus here will be to review the importance of runx2 and AP-1 transcription factors in the development 19 of bone with a particular focus on their roles for differentiation to a mature osteoblast phenotype. A. Runx2 1. Structure/Function Runx2 belongs to the cbf family of transcription factors. The cbf transcription factors were originally identified as the gene runt, which regulates embryonic segmentation during Drosophila development [107]. In mammals, this family is composed of three known a subunit members and one known 8 member. The G subunits are composed of runx1 (PEBPoB, CBFA2, AML1), runx2 (PEBPqA, CBFA1, AML3), and runx3 (PEBPoC, CBFA3, AML2). The 0 subunits heterodimerize with the one beB (PEBPZB) subunit and activate transcription by binding to their response elements. The importance of these members has been demonstrated in both animal and human models and disease. It has been shown that runx1 and runx3 are important for definitive hematopoiesis and gastric cancer formation, respectively, while runx2 is understood to be involved in bone formation or more specifically in osteoblast differentiation [108-112]. Elucidation of the role of runx2 in bone development and its regulation of osteoblast differentiation has come about very quickly in the past few years and has contributed much to increased understanding of osteoblast biology. 20 2. Osteoblast Differentiation Several studies have demonstrated that differentiation of cells from different lineages is triggered by cell specific transcription factors acting as switches of gene expression which regulate cell phenotype. This is exemplified by cells of the mesenchymal lineage. In this lineage, cells differentiate from mesenchymal stem cells into connective tissue, blood, cartilage, bone, fat cells, and the outer layers of blood vessels. While many of the transcription factors are established for promoting differentiation into specific lineages, an osteoblast lineage specific transcription factor remained elusive until the late 19903 [113- 118]. Discovery of this factor came predominately from two different labs around the same time. Gary Stein at the University of Massachusetts demonstrated that a protein he called NMP-2 (nuclear matrix protein 2), is a cell type-specific, 38- kDa “promoter factor” localized in the nuclear matrix, important for expression of the osteoblast specific protein, osteocalcin [119]. Further analysis revealed that NMP-2 was osteoblast specific and recognized a cis acting site that resembled the CCAAT/enhancer binding protein (C/EBP) consensus sequence. Though the initial focus of this work was more directed at understanding the relationship between the nuclear matrix and NMP-2, this was the first identification of an osteoblast specific transcription factor. Developmental regulation of this factor and a more thorough study of its DNA binding site came from the lab of Karsenty. Karsenty et al. cloned the osteocalcin promoter, performed 5' deletion analysis 21 and reporter assays in tissue culture and found that a fragment of the mouse osteocalcin promoter was expressed only in preosteoblasts and increased as cells became mature osteoblasts [120]. Further analysis revealed the presence of two cis acting sequences in the osteocalcin promoter which he called the osteoblast specific element 1 and 2 (OSEI and OSE2). These sequences turn out to be the same elements Stein identified one year earlier that bound NMP-2. Soon thereafter, two additional independent studies appeared from these same labs [121, 122]. In both studies, it was demonstrated that the factor which bound the OSE 1 and 2 had immunological identity to PEBPd/AMLB/beaIIrunx2 transcription factors. The importance of runx2 for skeletal formation came from knockout studies and the human disease cleidocranial dysplasia. A radiation induced mutant mouse (ccd) created in 1978 by Selby and Selby resulted in a phenotype marked most prominently by absence of clavicles, patent fontanelles, supernumerary teeth, and short stature [123]. Roughly twenty years later, heterozygous runx2 mutant mice displayed this exact phenotype [124]. The ccd mutants were subsequently found to have mutations which mapped to one of the runx2 alleles proving that the cod mutant phenotypes were due to mutations in runx2. Patients diagnosed with cleidocranial dysplasia exhibit a similar phenotype and were found to carry mutations in one of the runx2 alleles resulting in inactivation of the corresponding protein proving that the human disease cleidocranial dysplasia was due to runx2 mutations [125]. The absolute necessity of runx2 for skeletal development came from a report by Komori et al. 22 [126]. In this study, homozygous runx2 mutant mice showed a complete lack of skeletal ossification and died immediately of asphyxiation since the lack of ossified ribs were not strong enough to provide the negative pressure needed for lung expansion. Osteoblasts from the runx2 homozygous mutants did not differentiate suggesting that the lack of ossification is due to maturational arrest of osteoblasts. Work by Karsenty et al. demonstrated that runx2 is expressed in osteoprogenitor cells during embryonic development where it binds to and activates several osteoblast specific genes [112]. Forced expression of runx2 in nonosteoblastic cells induces expression of osteoblast specific genes such as bone sialoprotein and osteocalcin. These studies indicate that runx2 is critical for commitment of precursor cells to the osteoblast lineage, which in turn result in normal embryonic skeletal development. The importance of runx2 also extends to postembryonic bone development [127]. Transgenic mice were created which were unable to express runx2 until shortly after birth. These mice had a progressively shorter stature and more lucent long bones with thinner cortices at two weeks of age. Furthermore, collagen and alkaline phosphatase as well as bone formation rate were decreased when runx2 expression was suppressed following birth. This work demonstrates that in addition to being important for embryonic skeletal development, postembryonic bone formation during bone remodeling also requires runx2. Cloning of runx2 cDNA gave clues to the functional organization of the protein (Fig 7) [112]. The 528 amino acid protein contains elements common to many transcription factors. An evolutionarily conserved region of 128 amino 23 acids known as the runt domain, homologous to the Drosophila pair-rule gene, runt, mediates DNA binding [107, 128]. Members of the runt domain gene family heterodimerize with the ubiquitously present be8 transcriptional coactivator which stabilize binding of the runx2 transcription factors to their PyGPyGGTPy consensus sites on DNA [129]. Runx2 also contains a 9 amino acid nuclear localization signal as well as a 38 amino acid subnuclear localization signal which targets runx2 to the nuclear matrix [130, 131]. It has been demonstrated that removal of this nuclear localization signal results in an inability to activate an OSE2 dependent reporter in cells expressing this exogenously transfected runx2 mutant. Subnuclear localization also appears to be very important for runx2 function. The 38 amino acid nuclear matrix targeting signal promotes colocalization of runx2 with hyperphosphorylated RNA polymerase II into discrete nuclear foci [132, 133]. Removal of the subnuclear targeting signal significantly reduces transactivation of the runx2 regulated osteocalcin and transforming growth factor [3 RI promoters further demonstrating the importance of subnuclear targeting for runx2 function. The transactivation potential of runx2 is conferred by three activation domains and one repression domain. The first 19 amino acids compose the first activation domain (AD1), a 47 amino acid glutamine/alanine (QA) rich domain makes up a second activation domain (AD2), and the third activation domain is located in the N-terminal half of the proline/serine/threonine (PST) rich domain [130]. Deletion of each of these domains result In similar decreases in transactivation of the protein suggesting that these domains are functionally 24 Functional Organization of Runx2 B u VWRPY . NLS I A01 A02 080 NMTS <— —> c t 1 01A PST 528 D Repression (VWRPY) st DNA binding domain (DBD) Actlvation domain (AD) _ QIA =polyglutamlnelpolyalanine Nuclear localization I " slgnal (NLS) repeats E Nuc|°ar matrix targeting PST = prollne, serine, threonine rich signal (NMTS) Figure 7. Functional Organization of Runx2. 25 dependent on each other. In addition, it was found that the last 154amino acids make up a large repression domain that confers transcriptional repression to the entire PST domain. Removal of this region derepressed transactivation potential of the A03 in transactivation studies. The last five amino acids, the VWRPY motif, act as potent inhibitors of transactivation in Gal4-VP16 transactivation assays independent of the context of the entire protein. A clue to a possible role for this domain in transcriptional repression of runx2 comes from studies in Drosophila where the transcriptional repressor Groucho has been shown to interact with this VWRPY motif to repress runt mediated transcription [134]. Cotransfection of TLE2, the mouse homologue of Groucho, with runx2 resulted in a significant decrease in runx2 transactivation. Cotransfection with a runx2 mutant lacking a portion of the repression domain resulted in a restoration of the transactivation potential of AD3. Additionally, TLE2, has been shown to interact with this repression region in coimmunoprecipitation studies suggesting a conserved role for runx2 mediated transcriptional repression [130, 135]. Others have also described the importance of this VWRPY motif in transcriptional repression. Javed et al. demonstrated that runx2 mediated suppression of the bone sialoprotein promoter is dependent on this C terminal VWRPY motif and not on the TLE proteins previously described to interact with it. This suggests that other protein interactions in this motif confer runx2 mediated transcriptional repression [136]. Another study found that a region of the runx2 protein N- terminal to the VWRPY motif was important for repression of transcription from the p21 promoter [137]. This repression appeared to be due to recruitment of 26 HDACG to chromatin through interaction with runx2. Thus, runx2 is able to repress transcription through interaction with corepressors. A direct interaction of runx2 with the PPARy transcription factor has been shown to result in downregulation of transcription from runx2 regulated promoters [138]. This suggests that runx2 repression occurs at least in part through its own interaction with corepressors. While there are demonstrations of the role of runx2 mediated transcriptional repression, the overwhelming amount of research has demonstrated that runx2 has a major role in the activation of transcription. In fact, the discovery of runx2 as a mediator in osteoblast differentiation was facilitated by its ability to activate transcription of the osteoblast specific osteocalcin promoter [119]. Since this time, runx2 has been shown to have a major role in expression of several genes important for the differentiation from some of the earliest osteoprogenitor cells to a mature osteoblast [117]. Genes important for osteoblast differentiation for which runx2 binding sites exist include type I collagen and osteopontin [139, 140]. Activation of transcription from these promoters is mediated in part by runx2 and its association with other transcription factors or coactivators. A detailed analysis of the osteocalcin promoter has demonstrated that regulation of osteocalcin transcription is mediated through runx2 and its interaction with several other transcription factors and coactivators including the vitamin D receptor, C/EBPB and 6, and p300 [141-143]. In addition, members of the MYST acetyltransferase family of transcription factors, M02 and MORF, have been demonstrated to potentiate transcriptional activation from a 27 OSE2 dependent promoter reporter [144]. Interestingly, this activation did not occur through acetylation of runx2 by either M02 or MORF. Most recently, Aukhil et al. demonstrated that the transcriptional coactivator, TAZ, interacted with runx2 and induced a dose dependent increase in a osteocalcin promoter reporter construct [145]. Further analysis suggested that the relationship between runx2 and TAZ may be important for nuclear import of the factors to the nucleus. A full understanding of the mechanism of activation is still being investigated. Of particular interest to our lab was the demonstration of a physical interaction between runx2 and AP-1 family members in some promoter contexts. Upon stimulation of osteoblasts with PTH, c-fos/c-jun heterodimers along with runx2 transcription factors bind their Individual consensus sites in the collagenase 3 promoter [146-148]. It was subsequently determined that there was a physical interaction between c-fos and c-jun with runx2 at this site and that this interaction was required for maximal activation of the collagenase 3 promoter in transient transfection assays [149, 150]. B. AP-1 1. Structure/Function The AP-1 family of transcription factors is composed of fos and jun members (Fig 8). They were first identified as transcriptional regulators by their 28 Functional Organization of AP-1 Members c-fos IE‘ .I , E" 1 1?, g. _m fosB FEEL] it It: I Ell - I I“ c-jun IIII _ llllllll- m IIJ E4 I junB (III—F IIIIIIII I11] I E21 junD III— "III" - IIII — I_§I [1111]] Activation domain E] Basic region E Leucine zipper [:1 Glutamine-proline rich Highly conserved, unknown function Figure 8. The AP-1 family of transcription factors is composed of fos and jun members. All AP-1 members have conserved basic regions which bind DNA. Fos-jun and jun-jun dimerization is conferred through leucine zipper interactions Along with some highly conserved regions in the fos family members with an unknown function, c-jun contains a glutamine-proline rich region of unknown function. 29 ability to regulate expression of the human metallothionein IIA (MMPIIA) promoter and SV40 enhancer [151]. Soon thereafter, AP-1 was found to respond to the TPA phorbol ester by increasing DNA binding of AP-1 to the thereafter called TRE (TPA response element) [152]. In addition, these TREs conferred AP-1 binding and inducibility to heterologous pieces of DNA upon TPA treatment. Since these initial studies, a better understanding of the structure and function of AP-1 and its members has come about. The AP-1 family of transcription factors is composed of seven known members divided into two separate groups. The first group is composed of the jun family members: c-jun, jun B, and jun D. The second group is composed of the fos members: c-fos, fos B, fra 1, and fra 2. While there are a number of conserved domains among the AP-1 members, the only homology between the fos and jun members is the DNA binding domain. Heterodimers of fos and jun members or homodimers of jun members form via a leucine zipper motif. Upon dimerization, AP-1 transcription factors bind the consensus TGA C/G TCA motif and modulate expression of AP-I regulated genes. While providing for a better understanding of how AP-1 functions, studies on AP-1 have also led to the understanding that the nature of AP-1 regulation is extremely complex and much work must be done to completely understand its function. While AP-1 regulates expression of many genes in many different cell and tissue types, this review will focus on AP-1 transcription factors and their role in bone formation with a particular emphasis on osteoblast differentiation. 30 2. Osteoblast Differentiation Regulation of AP-1 as a transcription factor occurs at several different levels. One level of regulation involves the abundance of individual fos and jun members available for dimerization [153-155]. Work from our lab has demonstrated that AP-1 family member expression and protein abundance of different AP-1 members changes from early to late stages of osteoblast differentiation [47, 153]. Specifically, late stages are marked by an abundance of fra 2 and jun D heterodimers which compose the AP-1 transcription factor present. In addition to protein abundance, AP-1 member composition is important for regulation of AP-1 activity. This is exemplified in studies which demonstrate preferential binding of fos/jun heterodimers to DNA compared to binding by jun homodimers [156, 157]. AP-1 can also be modulated by the consensus and surrounding elements to which they bind [158]. In addition to binding the AP-1 response element, AP-1 members are also able to bind other promoter elements including the cAMP response element (CRE) (5’- TGACGTCA—3’) [159]. Several reviews discuss these basic aspects of AP-1 in detail [160-163]. Another level of AP-1 regulation occurs at the level of protein modification. Posttranslation modification of AP-1 members is also a major regulator of AP-1 activity. Jun N-terminal kinase (JNK) is able to phosphorylate c-jun on serines 63 and 73, thus potentiating the activation of c-jun [164, 165]. Mutation of the phosphorylation site in c—jun prevented the PMA induced transcriptional 31 activation of Gal4-c—jun CAT reporters. Alternatively, phosphorylation of the DNA binding domains of different jun members has been shown to repress transactivation. Glycogen synthase kinase 3 and casein kinase II phosphorylate jun members in or near the DNA binding region, thus preventing their transactivation [166, 167]. Another posttranslational mechanism to regulate AP-1 is reduction/oxidation (redox) state of AP-1 as seen for c-fos and c-jun. These studies demonstrate that AP-1 binding to DNA is favored when a cysteine located in the DNA binding region of c-fos and c-jun is reduced and not oxidized [168]. Interestingly, S-glutathiolation of a cysteine in the DNA binding region of the c-jun protein has been shown to prevent c-jun DNA binding [169]. Lastly, the transactivating potential of AP-1 can be regulated by the multiprotein context in which AP-Iassociates. Because AP-1 is activated by so many different stimuli, a question arises as to what confers specificity of AP-1 to those responses. One explanation for this specificity comes about through interaction of AP-1 factors with other proteins in or near an AP-1 binding site. There are also coactivators and corepressors which interact with AP-1 and not the DNA thereby influencing the context in which AP-1 binds and regulates transcription. Some of the transcription factors known to interact and influence AP-1 activity through protein complex interactions include ATF2, VDR/RAR, glucocorticoid receptor, NFAT and ETS among others [170-174]. Jun protein family members have even been reported to directly interact with TBP and TFIIB members of the general transcription factors [175]. Moreover, AP-1 interacts with coactivators and 32 corepressors which influence the activity of AP-1. Some of these cofactors include CBP/p300, SRC-1, FHL2, RIP140, and JAB1 [176-180]. Genetically modified mice have provided much insight into the importance of different AP-1 members for bone formation. The first indication that the c-fos protein plays a role in bone development came from the observation that v-fos, the transforming oncogene related to c-fos and derived from the FBJ murine sarcoma virus, induces osteosarcomas in vivo [181]. This was consistent with transgenic mice which overexpress a c-fos transgene and develop bone lesions which ultimately become osteosarcomas [182]. An even higher frequency of bone osteosarcomas occur in double fos-jun transgenic mice implicating c-jun as the partner of c-fos in promoting osteosarcoma formation [183]. Since these initial observations in bone phenotype, osteoblasts have been found to be the target cells for transformation in these transgenic mice. These studies suggest that overexpression of c-fos disrupts normal growth control of osteoblastic cells which eventually lead to osteosarcomas in bone. Consistent with a role for c-fos in regulation of growth control, homozygous c-fos knockout mice are less than half the size of heterozygous Iittermates at 5 weeks [184]. Homozygous c-fos knockouts lack osteoclasts, which lead to an osteopetrotic phenotype. This phenotype is rescued by fra 1 overexpression demonstrating some functional redundancy among AP-1 members [185]. Fra 1 overexpressing mice display a progressive increase in bone mass which ultimately leads to osteosclerosis of the entire skeleton [186]. Histological and cytological analysis revealed that the increased bone formation is attributed to the ability of fra 1 to promote osteoblast 33 differentiation as seen by a marked Increase in the number of mature osteoblasts and higher levels of expression of collagen l, alkaline phosphatase, and osteocalcin. Transgenic mice expressing 3 splice variant of fos B, called Afos B also exhibit a progressive increase in bone mass and osteosclerosis [187]. Interestingly, adipogenesis is decreased in the Afos B transgenic mice at least in part by downregulating gene markers of adipocyte differentiation. Since osteoblasts and adipocytes share a common progenitor cell, it is believed that the increased osteoblastogenesis might occur at the expense of adipocyte differentiation. One interesting aspect of the fra 1 and Afos B transgenic mice is that they both lack transactivation domains suggesting that the effects they confer could be due to the AP-1 binding partners or cofactors with which they associate. In vitro studies also suggest a role for AP-1 in the regulation of osteoblast gene expression and phenotype. Work from our lab has demonstrated that selective suppression of Ira 2 during osteoblast differentiation prevents formation of bone nodules in culture [153]. In addition, when different AP-I members are overexpressed in ROSI7/2.8 cells containing an osteocalcin promoter-CAT reporter, fra 2 and jun D pairs increased osteocalcin promoter activation, while other AP-1 members suppressed or did not alter osteocalcin promoter activity. This is consistent with fra 2 and jun D being the principle AP-1 members present in differentiated osteoblasts. In addition, an AP-I binding site was discovered that mediates osteoblast specific activation of the runx2 promoter in ROSI7/2.8 cells [188]. Not surprisingly, the regulation of AP-1 member expression has 34 proven to be quite complex. Much more work needs to be performed to establish a firm understanding of the role of AP-1 in the process of regulation of bone formation Ill. Osteoblast Response to Alterations in Force Understanding how the skeletal system responds to alterations in mechanical load helps to appreciate the influence of mechanical properties on bone remodeling. This idea came about first by Wolff in the late 19th century where he posited that bone will remodel in response to the forces put upon it [189]. This adaptation of bone to mechanical force requires that bone cells detect the signal and integrate the signals to cells. The cells then convert this mechanically induced stimulus to a biochemical signal which ultimately is responsible for alterations in gene expression and cell phenotype. This process whereby molecular events induced by cell stresses ultimately lead to biological and physiological signals is called mechanotransduction. A. Increased Force During bone deformation, three major forces to which the cells within bone are subjected are tension, compression, and shear (Fig 9). Even the slightest deformations in bone can be broken down into these three components. 35 MECHANICAL LOAD Figure 9. Stress patters on bone can be resolved into three basic types: compression, tension, and shear. Compression results from two forces acting along the same line but directed toward each other, tension is produced when two forces are directed away from each other, and shear occurs when two loads act in parallel but in opposite directions from one another. 36 Bending, for example, produces a combination of tension on the convex side of bone and compression on the concave side. In addition, twisting or torsion produces shear stress along the entire length of the bone. The production of these forces by mechanical loading has been shown to promote bone formation both in vivo and in vitro. In fact, compressive force is even used to promote bone formation in some fracture pathologies [190]. In vivo studies demonstrate that a single bout of 36 loading cycles to the right tibia of rats was enough to increase osteoblast surface length and width in the periosteum, consistent with an increase in number of osteoblasts needed for de novo bone formation [191]. Mice subjected to 12 weeks of tibial loading using the four point bending model showed increased bone formation rate compared to nonloaded contralateral controls [192]. In humans, bone mineral density in the racquet arms (loaded limbs) of prepubescent female tennis players was 11-14% greater in the loaded arm than in the nonloaded arm [193]. Some studies suggest that the force applied to bone contributes more to bone formation when it is cyclic and not static. Roblin et al. demonstrated that 360 cycles of uninterrupted loading of rat ulnas for 16 weeks lead to significantly greater bone mineral density compared to the nonloaded contralateral control ulnas [194]. These parameters were increased further when the 360 cycles were divided into 4 separate bouts separated by three hour intervals. Consistently, vertebrae of rats subjected to a single bout of 360 loading cycles for 9 days showed a 4 fold increase in bone formation, while animals subjected to 36 daily 37 loading cycles showed a 30 fold increase in bone formation [195]. While loading promotes higher degrees of bone formation, these studies suggest that periodic loading contributes to an even higher amount of bone formation. Several models propose that regulation of bone formation in vivo involves interactions among different cell types, however when loading is analyzed on osteoblasts directly, effects from in vivo experiments are maintained. For example, tension studies on UMR106 osteoblastic cells showed that alkaline phosphatase activity increases with increasing tension levels [196]. Consistently, osteoblasts and osteoblast progenitor cells from fetal mouse calvaria subjected to intermittent hydrostatic compression upregulate expression of alkaline phosphatase [197]. To address the effects of shear stress, the flow of fluid over cells can be performed in culture. These studies are more extensive than effects of other stresses because there actually is a flow of fluid within bone. Fluid shear stress has been shown to activate several signaling molecules including cAMP, PKA, ERK, p38, IKB kinase, and calcium [198-201]. These pathways activate transcription factors including AP-1. Given the important role of AP-1 in regulating skeletal homeostasis, it is not surprising that alterations in mechanical load in bone also influence AP-1 activity [202-210]. In most studies of increased load, AP-1 function/expression is increased, while decreased load results in decreased AP-1 function/expression. Peake et al. demonstrated that primary human osteoblast-like (HOB) cells and the human MG63 osteosarcoma cell line upregulated expression of c-fos expression in response to stretch using the four point bending model [211]. 38 Furthermore, this mechanical effect was dependent on intracellular calcium and integrin binding. The c—fos promoter contains a consensus shear stress response element and serum response element which are necessary for the increased loading induced expression of c-fos, thus implicating c-fos as a target of mechanical loading [212]. Kletsas et al. exposed human periodontal osteoblastic cells to continuous stretch and found that upon stimulation, cells exhibited a very rapid and relatively sustained increase in c-fos and c—jun mRNA consistent with the just described reports [213]. Similarly, Hughes-Fulford et al. demonstrated that 15 minutes of hypergravity loading induced a 15 fold increase in expression of c-fos mRNA in MCST3 E1 osteoblasts [214]. These effects of mechanical load on AP-1 translate down to regulation of gene expression. For example, mechanical induced activation of cox-2 mRNA expression in MC3T3 E1 osteoblasts is in part due to increased AP-1 activation and binding to the cox-2 promoter [215]. Gene markers of osteoblast differentiation also respond to alterations in fluid flow. Several studies report that fluid flow induced shear stress promotes expression of both alkaline phosphatase and osteocalcin [216-219]. Moreover, marrow stromal osteoblasts cultured on three dimensional scaffolds under flow perfusion for extended periods of time resulted in significant enhancement of cell proliferation and mineralized matrix throughout the scaffold [220]. 1. Physiology 39 One of the aspects where increased loading regulates bone mass is seen in athletes. When the bone mineral density of elite athletes are compared to age matched, nonexercising control subjects, the athletes demonstrate significantly higher bone mineral density [221-226]. These effects are most demonstrable in high impact loading exercise. A low impact sport like swimming does not seem to increase bone mineral density and in some cases has shown to be less than in nonathletic controls [227, 228]. This is contrary to higher impact exercises. For example, when premenopausal women undenivent 50-8.5cm vertical jumps daily for 5 months, they obtained a 2-3% increase in bone mineral density of the femoral head [229]. Postmenopausal women showed no change in bone mineral density with this regime. Similarly, female gymnasts who experience up to 15-20 times body weight during routines, show some of the highest increases in bone mineral densities compared to controls [230]. These studies demonstrate a strong role for higher impact exercise in promotion of higher bone mineral densities. 2. Mechanosensors While bone is known to respond to alterations in load, the mechanosensor is not well understood. It is presumed that mechanical loads applied to bone are transduced through the skeleton and received by a network of interconnected bone cells which in turn respond to alterations in force. It is widely believed that mechanical signals are detected by certain receptor cells which either act directly 4O to remodel bone or indirectly by signaling to other cells to remodel bone. The most widely favored model of mechanosensors involve the osteoblast lineage derived osteocytes and the lacuno-canalicular network through which they extend [32, 59, 231]. In 1977, Piekarski et al. demonstrated that cyclic loading produces a flow of interstitial fluid through the canaliculi [232]. It is believed that this flow of interstitial fluid through the canaliculi transduce signals to the osteocytes which in turn signal bone remodeling cells to form new bone and/or resorb old bone. This lacuno-canalicular networking might also play a role in facilitating the transport of nutritional factors and waste. Studies have demonstrated that mechanical loading does indeed facilitate transport of molecules within bone [233]. Whether it is the flow of fluid and/or nutrients which contribute to osteocyte response to mechanical loading, movement of fluid and nutrients through this network appear to be Important. One interesting aspect of unloaded animals is that cells within bone are hypoxic [103, 234]. It is intriguing to speculate that normal loading conditions facilitate oxygen delivery to cells within bone via interstitial fluid. When normal loading does not occur, cells would in turn be under hypoxic conditions and in turn result in decreased bone formation. It still remains to be determined whether cells other than osteocytes experience hypoxia in response to loading conditions. Mechanosensing has also been postulated to occur through direct sensing of cell bone deformation in vitro. A report from Cowin et al. discusses the role of strain induced membrane deformation. He discussed how the mechanical induced small strains applied to a whole bone might be amplified at the 41 membrane of the osteocyte buried within the matrix [102, 235, 236]. Support for this theory comes from different studies. Weinbaum et al. calculated fluid induced shear stresses to be 0.8 to 3.0 Pa during peak physiologic loading regimes, while bone cells in vitro actually respond biochemically to fluid shear stresses of 0.2 to 6 Pa [34, 201, 237-240]. Since strain levels required to elicit a response in vitro may be higher than the actual strain applied during peak physiologic regimes, some amplification of the signal must occur. Exactly how this occurs is not known. An alternative study by You et al. suggests that strains applied to whole bone are much smaller than the strains that are necessary to cause bone signaling in deformed cell cultures [241]. Because the amount of data regarding this effect of mechanical induced deformation of bone cells in not very extensive, it is difficult to determine with sufficient confidence whether cell deformation actually regulates bone remodeling in vivo. 3. Model Systems To address the effects of loading on bone formation directly, models of increased loading on osteoblasts have been developed. While most loading conditions on bone can be broken down into the three components of tension,, shear, and compression, these model systems allow for determination of the contribution of the individual stresses on bone formation in osteoblasts. a. Centrifugation 42 One of the models used to address the effects of increased load is to directly subject cells in culture to centrifugation. By utilizing different speeds of centrifugation, it is possible to increase the gravitational force to which cells are exposed. Osteoblastic cells respond as early as 30 minutes following hypergravity conditions. When MCBT3 E1 preosteoblasts were subjected to hypergravity conditions which mimic the increased gravity felt during a space shuttle launch (39), significantly induced expression of the growth related c-fos gene occurs [242]. Interestingly, expression of the late differentiation marker gene, osteocalcin, was significantly decreased by 44% as early as three hours following centrifugation suggesting that prolonged force may in fact lead to decreased bone formation. This was supported by another study that demonstrated transient stimulation of MC3T3 E1 preosteoblasts at 509. These conditions resulted in PKC induced expression of c-fos mRNA [243]. Two separate studies by the same group determined that while lower 9 forces promote expression of genes seen during preosteoblast proliferation, higher g forces induced activation and expression of genes necessary for a more differentiated state. While proliferation of ROS17/2.8 cells subjected 1.5-2 9 force was significantly increased and expression of alkaline phosphatase and osteocalcin were decreased relative to 1 g controls, higher levels of hypergravity (40-80 g) suppressed proliferation and stimulated expression of alkaline phosphatase and osteocalcin [244, 245]. This is consistent with studies demonstrating higher impact exercise correlates with increased bone formation. 43 b. Fluid Flow Increased force has also been addressed by subjecting cells to fluid flow. Cytoskeletal rearrangement is apparent soon after subjecting cells to flow conditions suggesting cytoskeletal architecture maybe involved in mechanosensing of fluid flow [101, 246]. These alterations are involved In alterations in gene expression in response to flow conditions. Inhibiting flow induced cytoskeletal reorganization prevented c-fos and cox-2 expression in MC3T3 E1 osteoblasts [101]. Further studies determined that the flow induced cytoskeletal reorganization and increases in c-fos and cox-2 expression were also inhibited by preventing mobilization of intracellular calcium in osteoblasts. Other studies have also reported similar findings. Cox-2 mediates synthesis of PGE2, a chemical involved in osteoblast mediated bone formation. When chicken osteocytes were subjected to pulsed fluid flow, inhibitors of actin cytoskeletal rearrangement as well as inhibition of calcium activity resulted in suppression of flow induced synthesis of PGE2 [247]. This data supports a role for the cytoskeleton as a mechanosensor of flow induced increases in mechanical loading [246]. c. Hydrostatic Compression 44 A third model used to address increased loading is the hydrostatic compression model. Hydrostatic compression can be created by increasing the gas pressure above the medium of cells in a closed culture chamber. An important study that demonstrated the importance of hydrostatic compression on bone formation was performed by Klein-Nulend et al. [248]. In this study, both intermittent and continuous hydrostatic compression promoted bone formation and inhibited bone resorption in cultured fetal mouse calvaria. Cell culture studies also demonstrate that bone cells respond to varying degrees to hydrostatic compression. Embryonic chicken osteocytes, osteoblasts, and bone lining cells were all subjected to hydrostatic compression [34]. Osteocytes and osteoblasts but not bone lining cells responded to 6 hours hydrostatic compression with modest synthesis of PGE2. When gene expression and activity in response to hydrostatic compression was analyzed, it was found that alkaline phosphatase, actin, and collagen expression were increased in osteoblasts, but not in osteoprogenitors. This data all demonstrates that increasing mechanical load by hydrostatic compression promotes markers of bone formation In committed osteoblast lineage cells. B. Decreased Force Decreasing force on bone most often leads to a decrease in bone formation. These skeletal changes are evident in several pathological as well as nonpathological examples and are usually termed disuse or immobilization 45 induced osteoporosis. This disuse associated osteoporosis has been shown to occur in such circumstances as spinal cord injury patients where limb disuse is a result, chronic bed rest, and space related microgravity [89, 249-252]. All of these conditions are common in that force is decreased relative to normal physiological loading conditions. Understanding decreases in bone formation in one system may contribute to the understanding of the more generalized idea of disuse associated osteoporosis. In particular, extended periods of time in outer space are known to result in decreases in bone mineral density, much of which is thought to be due to changes in osteoblast function and subsequent bone formation. Since the accessibility of true spaceflight conditions are limited, ground based models of modeled microgravity have been developed. Two of the most popular model systems are mouse or hindlimb unloading and clinostat studies. 1. Spaceflight Effects of spaceflight on bone formation and bone resorption has been addressed by analysis of biochemical markers of bone turnover. These studies have however proven difficult to interpret because both bone formation and resorption markers show variation from increasing, decreasing, or not changing at all when comparing spaceflight and ground controls [253-256]. In one joint French and American space mission, two astronauts were analyzed for serum alkaline phosphatase and osteocalcin whose presence are indicative of bone 46 formation and found they were unchanged compared to preflight samples [257]. Alternatively, another study from astronauts flight samples of serum alkaline phosphate and osteocalcin were 27% and 38%, respectively compared to preflight samples [258]. In this same study, deoxypyridinoline (DPyr) and procollagen C-telopeptide (CTX), both markers of bone resorption, were increased 54% and 78% during the flight compared to preflight samples demonstrating increased bone resorption was also occurring in these individuals. Difficulty comes about in analyzing this data because some of the samples were taken during flight while others were taken following space conditions and may really reflect responses to reloading. Further clues to the idea that spaceflight conditions had a role in skeletal alterations came about through the observation that Gemini, Apollo, and Skylab mission astronauts exhibited a negative calcium balance and a calcium loss of at least 200 mg/day [259-263]. Because bones are the largest reservoir of calcium, it was only logical to first look at how bone changes following actual spaceflight missions. While human studies are limiting with regard to actual bone mass measurements, it has been documented that spaceflight results in loss of bone mostly from the weight bearing bones [264]. Because the calcaneus, or “heel,” bears most of the body weight, the effects of space conditions have often been addressed in humans by determining alterations of the calcaneus. Two crewman from the 14 day US Apollo 15 mission showed a loss of calcaneus mineral [265]. Similarly three of the six crewman aboard the 28 day Skylab 3 and 4 missions experienced a loss of 7.4%, 4.5%, and 7.9% mineral of the calcaneus [266]. One 47 month aboard the MIR space station caused a 2.27% and 7.74% decreases in the weight bearing trabecular component of the tibia and calcaneus, respectively, and no change in the nonweightbearing ulna [253]. After 6 months aboard the space station MIR, a decrease of 4.5% and 2.9% was evident in the trabecular and cortical compartments of the tibia, respectively. While human studies provide important information regarding space related bone loss, the information animal studies has provided for a more extensive analysis of bone loss. Consistent with human measurements, animals also experience bone loss in the weight bearing bones. While young rats subjected to 7-14 days of flight conditions resulted in a marked reduction in trabeculae of the tibia, there was no difference between flight and ground controls in thoracic vertebrae[267, 268]. 83 day old rats aboard the COSMOS 1129 biosatellite for 18.5 days induced a decreased mass of mineralized tissue in the proximal tibial and humeral metaphysis [269]. While these studies showed a consistent loss of bone following spaceflight conditions, they did not address whether the effects were due to a decrease in bone formation, increase in bone resorption, or a combination of both effects. To get a better idea of the effects of spaceflight on bone formation and bone resorption, biochemical markers of bone turnover have also been investigated. These studies have however proven difficult to interpret because both bone formation and resorption markers show variation from increasing, decreasing, or not changing at all when comparing spaceflight and ground controls [253-256]. 48 Specialized hardware has been developed for the analysis of the direct effect of microgravity on bone cells aboard NASA shuttle missions. Because there is a strong correlation in animal studies between decreased bone formation and microgravity conditions, most studies using cell culture in true microgravity use osteoblastic cells. Hughes-Fulford has reported several findings from MC3T3 E1 osteoblasts grown aboard shuttle flights. When cells were launched in a serum starved state then serum stimulated and allowed to grow for 4 days, cells proliferated roughly three time slower than ground controls [270]. This was accompanied by reduced stress fibers, abnormal morphology, and decreased PGE2 synthesis compared to controls. Contrary to the previous findings, Kumei et al. showed that culture for 5 days aboard a shuttle spacelab flight (STS-65) resulted In an increase in PGE2 synthesis with a consistent increase in PGHSZ and lL-6 mRNA [271]. While this discrepancy is more difficult to explain in relation to microgravity conditions and bone formation, other observations have proven consistent in relation to a decreased bone formation phenotype. Evidence suggests microgravity reduces osteoblast differentiation as can be seen by alterations In basal expression of genes associated with progression to a more differentiated phenotype. Culture of embryonic chick bone cells for 9 days aboard STS-59 showed a roughly 50% decrease in both collagen I and osteocalcin when compared to ground controls [272]. When alkaline phosphatase activity from human osteoblastic MG-63 cells flown for 9 days aboard the Foton 10 satellite were analyzed, no significant difference was observed [273, 274]. However, cells did display an attenuated upregulation in 49 activity and expression of alkaline phosphatase and osteocalcin by the normally stimulating TGF82 and vitamin D. A 5 day shuttle flight determined that vitamin D induced expression of PDGFB receptor, the shc adaptor protein, and c-fos mRNA were decreased by 62%, 55%, and 25%, respectively [275]. Furthermore, sounding rocket experiments which expose cells to short durations of true microgravity demonstrate that MC3T3 E1 preosteoblasts suppressed EGF induced upregulation of c-fos mRNA expression [276]. 2. Hindlimb Unloading One of the ground based in vivo models developed to address the role of unloading is the hindlimb suspension model. This hindlimb suspension, head down method was originally developed by Morey in 1979 to mimic microgravity through the suspension of rat hind limbs by a suspension harness [277]. It creates a condition that somewhat resembles conditions of astronauts in outer space-unloading of weight bearing limbs and cephalic fluid shifts. Adaptation of this model using tail suspension has also been used and shows similar effects. As with actual spaceflight and other conditions of decreased loading, hindlimb suspended rats have decreased bone formation [278-281]. Magnetic resonance imaging was used to analyze the tibial trabecular bone structure in mice hindlimb unloaded for 28 days [282]. This study determined that trabecular number and thickness were decreased and trabecular spacing was increased in unloaded animals consistent with decreased bone formation. This decrease in bone 50 formation has been shown to have an associated decrease in preosteoblast proliferation by other labs [283, 284]. In addition to decreases in proliferation, markers of differentiation in hindlimb unloaded animals also suggest suppression of differentiation. It has been reported that alkaline phosphatase activity decreases as early as two days following hindlimb suspension and can persist for extended periods of unloading [285-287]. In 5 day hindlimb unloaded animals, isolated osteogenic cells derived from bone marrow demonstrated a reduced number and size of alkaline phosphatase positive colonies compared to cells isolated from normally loaded animals [288]. This suggests that unloading conditions reduce the osteogenic potential of osteoprogenitor cells and osteoblasts. Consistent with decreased alkaline phosphatase activity, alkaline phosphatase mRNA expression is also decreased 8 days following hindlimb unloading [289]. This is accompanied by a decrease in serum osteocalcin and femur osteocalcin mRNA levels from unloaded rats [278, 290]. As these studies demonstrate, the decreased bone mass in unloaded animals correlates with decreases in markers of differentiation, namely alkaline phosphatase activity and expression, and serum osteocalcin and mRNA expression. There also is an apparent defect in responsiveness in hindlimb unloaded limbs which correlate with decreased bone formation. It has been reported that recombinant human BMP-2 promotes osteoblast differentiation [291-293]. Infusion of 14 day hindlimb unloaded rats with thMP-2 did not prevent decreased bone formation suggesting some defect in thMP-2 responsiveness 51 [294]. Like BMP-2, intermittent treatment of animals with PTH is also known to promote bone formation [295]. When hindlimb unloaded rats were intermittently treated with PTH, osteoprogenitor cells showed decreased proliferation, alkaline phosphatase activity, and mineralization compared to intermittently treated and normally loaded controls [296]. These studies are consistent with suppression of a mature osteoblast phenotype. 3. Clinostat The clinostat is an earth based model of microgravity. Culture of cells in a clinostat is based on growth of cells under solid phase rotation to maintain cells in suspension, with or without attachment to microcarrier beads as an attachment substrate. Under these conditions, cells experience randomized g vectors and minimal shear stress, thus modeling the decreased 9 force experienced under true microgravity conditions [297-299]. This method of cell culture allows for a more direct molecular analysis of cell biological alterations in response to decreased loading. Specifically, the system provides defined culture environment and allows the growth and analysis of a single cell type so effects of decreased loading can be determined independent of surrounding cells. When the effects of clinorotation on growth of osteoblastic cells were analyzed, most studies demonstrate a decrease in proliferation. One study by Hughes-Fulford et al. reported that serum treated MCBT3 E1 osteoblasts aboard the STS-56 shuttle flight grew to roughly 40% of ground controls [270]. This 52 result was similar to those reported by Al-Ajmi et al. where 48 hours of clinorotation resulted in a 35% and 39% decrease in cell numbers in ROS17.28 and rat calvarial cells, respectively [300]. Alternatively, work from our lab demonstrates no alteration in growth using thymidine incorporation and BrdU incorporation studies. Another study reports similar findings to ours. Kunisada et al. suggested that growth of human osteoblast-like cells in a horizontal clinostat had no effect on proliferation [301]. While the data is limiting with regards to osteoblasts, some labs attribute the decrease in cell number under clinorotation to increases in cell death [302, 303]. We found no difference in apoptosis as determined by no alterations in caspase 3 activation and DNA Iaddering up to 48 hours of clinorotation compared to stationary controls. Because of the lack of data, it is difficult to discern a reason for the difference between cell growth and death studies. With regards to proliferation, it is possible that clinostat conditions do in fact alter proliferation of osteoblastic cells, however since proliferation of a more mature osteoblast is extremely low, it is conceivable that only a very small, insignificant subpopulation of our cells might still be undergoing proliferation. When osteoblasts are cultured during the early stage of proliferation in the clinostat, because there are many cells undergoing proliferation, it is possible that effects on proliferation are more pronounced. In our studies, cells are first grown for 14 days prior to being subjected to RWV conditions. Usually by 14 days of growth in culture, most osteoblasts are no longer proliferating and are undergoing differentiation to a mature osteoblast phenotype. This could prevent one from being able to detect any really significant changes in proliferation. 53 Some studies utilize cells with a transformed phenotype which have an inherent altered growth/differentiation characteristic displayed by simultaneous expression of early and late gene markers, namely alkaline phosphatase and osteocalcin, while still undergoing proliferation. A last explanation for the discrepancy is that involves the stage of differentiation of the cell. The studies performed here were done at roughly the midstage of differentiation when cells are no longer undergoing proliferation. If we were to test the effects of the RWV on an earlier stage of differentiation when osteoblasts are proliferating, we might have observed a difference compared to unit gravity controls. Markers of osteoblast differentiation have also been analyzed following clinorotation. Kunisada et al. determined that alkaline phosphatase activity was significantly decreased following clinorotation of human osteoblast-like cells (Hu09) [301]. Stimulation of alkaline phosphatase activity by vitamin D treatment was also lower compared to vitamin D treated control cells. Consistently, osteoblasts grown for up to 21 days under normal culture conditions produced well defined calcified bone nodules in culture, while 21 days of growth in a clinostat failed to both calcify and produce bone nodules consistent with suppression of osteoblast differentiation [304]. Work from our lab has demonstrated that as early as 24 hours of clinorotation, alkaline phosphatase and osteocalcin expression are decreased by 80% and 50%, respectively [305]. Furthermore, expression of a key regulator of osteoblast specific gene expression was consistently reduced by more than 60%. Other transcription factors important for osteoblast function also show altered regulation under 54 clinorotation. TNFd functions in part promoting production of cytokines and cell adhesion molecules in osteoblasts. Kobayashi et al. demonstrated that clinorotation of human osteoblastic HOS-TE85 cells for 72 hours reduced suppressed TNFa induced transactivation of NFKB as determined by decreased DNA binding and activation of a NFKB dependent luciferase reporter [306]. The TNFd induced, NFKB dependent expression of lKBq and lL-8 was also significantly attenuated. AP-1 also shows altered activity. Granet et al. reported that 1 hour of clinorotation induces expression and translocation of all AP-1 members except fos B [307]. Longer durations seem to show different effects. We have demonstrated that 24 hours of clinorotation results in decreased expression of c- fos and c-jun, and not other AP-1 members. In addition, AP-1 DNA binding and transactivation were also decreased in our studies. These reports are consistent with a requirement for AP-1 in development of a mature osteoblast phenotype and AP-1 suppression in spaceflight and hindlimb studies [186, 187, 208, 275, 284, 308-310]. IV. Hypoxia Normal cell function requires that it receives an adequate supply of oxygen. When oxygen supply is decreased (hypoxia), pathologic conditions can result. These conditions include such things as heart attack, stroke, and shock [311-313]. Responses to hypoxia in turn result In several cellular alterations 55 including changes in respiration, apoptosis, and/or proliferation with consistent changes in gene expression. While the study of the effects of hypoxia have been studied for several different tissue types, the effects on bone is extremely limited A. Molecular Cell Response Mammalians cells require a constant supply of oxygen to ensure cell survival [314-318]. When there is not enough oxygen, metabolic alterations ensue and alterations in gene expression occur as demonstrated in different cell types include neural, cardiac, bone, lung, and kidney cells [103, 319-326]. Most changes in gene expression are directed toward promoting the facilitation of oxygen to the hypoxic area. When the kidney experiences hypoxic conditions, oxygen sensing cells release erythropoietin which stimulates production of red blood cells thereby increasing the oxygen carrying capacity of blood [327, 328]. While this response of the kidney to hypoxia is a specialized function of the kidney, hypoxia also induces responses intrinsic to all hypoxic cells suggesting that each cell has its own kind of oxygenating sensor. Under hypoxic conditions, cells are no longer able to derive most of its energy thorough oxidative phosphorylation. The cell must predominately rely on anaerobic respiration to derive most of its energy, that is glycolysis for ATP production. These metabolic changes are associated with an increase in expression of genes associated with glycolysis including glucose transporters, GAPDH, and enolase [329, 330]. In addition to glycolytic genes, other genes common to most cell types are also 56 expressed to promote increased oxygen to the local microenvironment including vascular endothelial growth factor (VEGF). VEGF promotes vascularization to areas which in turn increases blood flow and oxygen delivery to the hypoxic area [331-335]. Hypoxic induction of transcription of many genes occurs through hypoxia inducible factors (HIF). The discovery of HIFs originally came about through erythropoietin gene reporter studies [336, 337]. These studies resulted in elucidation of a 50 nucleotide enhancer region in the 3’ end of the gene that conferred hypoxic inducibility [338, 339]. Semenza et al. discovered the transcription factor that works through this DNA binding sequence and called it hypoxia inducible factor 1 (HIF-1) [340]. HIF-1 was found to function as an 08 heterodimer with each subunit containing a basic-helix-loop-helix motif and a PAS protein-protein interaction domain, motifs similar to many different transcription factors [341, 342]. Regulation of this transcription factor occurs predominately through inhibition of constitutive degradation then accumulation. Upon exposure to hypoxic conditions, HIF protein levels are no longer degraded, are stabilized, and able to regulate transcription from hypoxic response elements (HRE). As might be expected, many genes whose expression increases during hypoxic conditions also contain HREs in their promoters. These genes include GAPDH and VEGF, among others [343-348]. B. Bone and the Role of Hypoxia 57 Most studies that address hypoxia in the context of bone refer to the vascular interruption that occurs when bones fracture and normal blood supply to the bone is disrupted. When a fracture occurs, a hypoxic gradient forms where oxygen tension at the center of the wound is very low [349-351]. This hypoxic response in turn results in expression of genes leading to fracture repair including angiogenesis and growth factors to promote revascularization of the fracture and formation of new bone [352, 353]. Furthermore, studies demonstrate that osteoblasts may be critical regulators of angiogenesis during fracture healing. Reports have demonstrated that osteoblasts produce the angiogenic factor VEGF in response to hypoxic conditions [354-356]. Activation of these genes also correlate with increased levels of HIF transcription factors and activation of HREs [357, 358]. Understanding the regulation of these genes and how they promote fracture healing has been the focus of most studies involving the relationship between hypoxia and bone. C. Relationship to Decreased Load As previously discussed, decreased mechanical loading results in decreased bone formation due largely in part to suppressed osteoblast function. A secondary effect of unloading is diminished bone deformation including compression, fluid flow, and shear forces. One commonality of these forces is that they contribute to increasing nutrient exchange to different cell populations within the bone [232, 233, 359]. The only direct correlation demonstrated 58 between unloading and hypoxia has in fact been demonstrated in in vivo unloading conditions in turkeys. A probe visualized by immunohistochemistry was used to mark, then observe cells from unloaded turkey ulna sections [103]. As early as 24 hours after unloading, osteocyte hypoxia ensues in these unloaded limbs. Interestingly, 4 minutes of reloading following the 24 hours unloading resulted in reestablishment of normoxic osteocytes. An extension of this study demonstrated that these hypoxic osteocytes also had increased levels of HIF-Id [234]. These studies strongly implicate unloading conditions as mediators of hypoxic environment within bone. Beyond these studies, there are no in vivo studies addressing a role for hypoxia on conditions of disuse or unloading. Certainly physiologic changes under unloading conditions correlate with decreases in oxygen supply to unloaded limbs. Spaceflight results in decreased blood pressure, decreased red blood cell number, and fluid shifts away from the lower weight bearing limbs where most bone loss occurs [360- 363]. All of these physiologic changes on earth result in decreased oxygen delivery suggesting that spaceflight conditions might also result in hypoxia to the more weight bearing bones because they are deprived of normal oxygenating blood. 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Cutis, 1991. 48(4): p. 291-5. 90 CHAPTER II Simulated Microgravity Suppresses Osteoblast Phenotype, Runx2 levels and AP-1 Transactivation C Ontiveros and LR McCabe Department of Physiology. Michigan State University, 2201 Biomedical Physical Science Bldg., East Lansing, MI 48824 91 Abstract Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood but involve the suppression of osteoblast function. To elucidate the influence of mechanical unloading on osteoblasts, a rotating wall vessel (RWV) was employed as a ground based model of simulated microgravity. Mouse MC3T3-E1 osteoblasts were grown on microcarrier beads for 14 days and then placed in the RWV for 24 hours. Consistent with decreased bone formation during actual spaceflight conditions, alkaline phosphatase and osteocalcin expression were decreased by 80% and 50%, respectively. In addition, runx2 expression and AP-1 transactivation, key regulators of osteoblast differentiation and bone formation, were reduced by more than 60%. This finding suggests that simulated microgravity could promote dedifferentiation and/or transdifferentiation to alternative cell types; however, markers of adipocyte, chondrocyte, and myocyte lineages were not induced by RWV exposure. Taken together our results indicate that simulated microgravity may suppress osteoblast differentiation through decreased runx2 and AP-1 activities. 92 Introduction Increased mechanical loading associated with weight bearing exercise causes bone compression, tension, and shear stress ultimately leading to enhanced bone formation [1-7]. Given the important role for mechanical loading in the maintenance of skeletal mineralization and strength, it is not surprising that conditions of disuse result in bone loss [2, 8-10]. Decreased mechanical loading and aging are associated with decreased bone mineral density and increased fracture risk [10-12]. This phenomenon is clearly seen under space flight conditions where bone is exposed to minimal mechanical loading resulting in decreased bone mineral density in humans [11-13], monkeys [14], rats [15-17] and mice [18, 19]. The cellular and molecular mechanisms that regulate bone mineral density in response to loading or unloading remain unclear. However, suppression of bone formation under decreased loading suggests a role for osteoblasts [20-25]. Osteoblasts are derived from mesenchymal stem cells that advance to the osteoblast lineage and progressively differentiate through the upregulation of runx2, AP-1 and other transcription factors. Under normal conditions this leads to upregulation of alkaline phosphatase followed by osteocalcin expression and bone formation. However, if factors involved in this progression are altered, bone loss can occur. Two possible scenarios leading to decreased osteoblast function under unloading conditions are: 1) a decrease in pre- and/or committed- osteoblast proliferation resulting in fewer cells to form bone or 2) a reduced ability to differentiate and produce bone (either by dedifferentiation, transdifferentiation, or 93 direct effects on osteoblast function). When osteoblast histology is examined after spaceflight, an increase in less-differentiated and a decrease in more- differentiated osteoblasts can be seen [26]. This suggests that microgravity causes a block in the differentiation pathway of osteoblasts. Studies examining osteoblasts in vitro under microgravity conditions indicate that reduced gravity can directly alter osteoblast function, apart from changes in systemic factors or interactions with other cells such as osteoclasts [27-29]. For example, four days of space flight directly influences mouse osteoblast actin distribution and decreases cell growth in response to serum [27]. Space flight also suppresses human and embryonic chick osteoblast differentiation as marked by reduced alkaline phosphatase and osteocalcin activity, secretion and/or expression [28- 30]. In addition, space flight conditions suppress TGF-beta and vitamin D induction of alkaline phosphatase and osteocalcin expression [28]. In vivo models of unloading, such as hind limb suspension, also result in decreased bone mass [31, 32] and suppressed osteoblast function [33-35]. Taken together, these data implicate space flight induced decreases in mechanical force as a regulator of osteoblast function. To address the role of microgravity on cells on earth, ground based models of space flight associated microgravity conditions have been developed [36]. These units include the clinostat and rotating wall vessel (RWV), which utilize solid phase rotation to maintain cells in suspension, with or without microcarrier beads as an attachment substrate (see Figure 1). Under these conditions cells experience randomized g-vectors and minimal shear stress [37- 94 39]. This environment has been demonstrated to alter the differentiation, phenotype and gene expression patterns of several cell types including rat adrenal medullary cells (PC12) [40], tracheal epithelial cells [41], skeletal muscle [42], peripheral blood lymphocytes [43], and human prostate carcinoma cells [44]. Findings from osteoblast cultured in the RWV indicate altered cytokine expression [45], apoptosis [45, 46], and variable effects on differentiation [45, 47, 48]. Progression to a mature osteoblast phenotype is associated with three stages: growth, extracellular matrix maturation, and extracellular mineralization. Each stage is marked by the temporal expression and repression of genes [49, 50]. Nuclear run-on analyses demonstrate a significant transcriptional component to the regulated pattern of gene expression [51]. Many transcription factors are involved in regulating osteoblast gene expression and subsequent differentiation, including runx2 and AP-1. Runx2 is a homeodomain protein critical in regulating osteoblast lineage selection and differentiation [52-54]. Runx2 binds to and regulates gene expression through the osteoblast-specific cis-acting element 2 (OSE2) [55, 56] which is found in the promoter region of many major osteoblast-specific genes including alkaline phosphatase, bone sialoprotein, osteocalcin, and matrix metalloproteinase-13 [52, 57-59]. In mice, inactivation of runx2 through deletion or mutation blocks bone formation [60, 61]. In humans, a heterozygous mutation in runx2 results in cleidocranial dysplasia [62, 63], marked by delayed suture formation and the absence of clavicle formation. 95 AP-1 members have been shown to be developmentally regulated [64] and also play a role in regulating expression of genes which promote osteoblast differentiation including collagen l, alkaline phosphatase, and osteocalcin [52, 65- 67]. The AP-1 family of transcription factors is composed of seven Fos and Jun members which dimerize with each other (Fos-Jun, Jun-Jun) prior to binding DNA. Overexpression of specific family members such as delta Fos B [68, 69] or Fra 1 [70] results in increased mouse bone formation, while suppression of Fra 2 expression results in decreased bone nodule formation in tissue culture [65]. Because many of the genes regulated by AP-1 are also regulated by runx2, it is conceivable that cooperative regulation of these factors occurs at some, if not all of the sites. Work by D’Alonzo, et al. [71] shows that c-fos and c-jun physically interact with runx2 to regulate parathyroid hormone dependent MMP13 expression in osteoblasts. This suggests that runx2 and AP-1 may directly interact to activate transcription. Based on the above findings, we determined the involvement of runx2 and AP-1 in osteoblast phenotype modulation under simulated microgravity conditions in the RWV. While other studies have demonstrated long term influences of simulated microgravity on osteoblast phenotype, we focused on immediate events associated with a 24 hour exposure to simulated microgravity. Our results demonstrate that this amount of time is sufficient to down regulate markers of osteoblast differentiation and suppress runx2 expression and AP-1 transactivation. The coordinate decrease in runx2 and AP-1 implicates them in 96 the transcriptional regulation of genes responsible for decreased osteoblast differentiation under conditions of unloading. 97 Methods Cell Culture System MC3T3-E1 cells [72], subcloned for maximal alkaline phosphatase staining and mineralization, were used for all studies. Osteoblasts were seeded at 1,000 cells/mg onto gelatin coated microcarrier beads, which are 90-150 microns in diameter (Solohill, Ann Arbor, MI). Osteoblasts were cultured for 14 days and fed every two days with a-MEM containing 10% fetal calf serum (Atlanta Biologicals Norcross, GA) and supplemented to a final concentration of 2 mM B-glycerophosphate (Sigma, St. Louis, MO) and 25 pglml ascorbic acid (Sigma). On day 14, half of the cells on microcarriers were placed into the RWV to simulate microgravity, while the other half were left at unit gravity. Rotating Wall- Vessel (RWV) The RWV cell culture system (models 55ml STLV and 10ml HARV) was purchased from Synthecon, Inc. (Houston, Texas) and used for studies of simulated microgravity. The RWV bioreactor is a cylindrical vessel containing an inner oxygenator core (surrounded by a membrane) and an outer core filled with culture media into which cells grown on microcarrier beads are placed (see Figure 1). The vessel is connected to a rotator base and rotated along its longitudinal axis at a speed where cells establish a near solid phase rotation with the culture medium. The fluid dynamics in the RWV allow for colocalization of cells and aggregates of different sedimentation rates, three-dimensional spatial 98 freedom, reduced fluid shear forces, and oxygenation without turbulence by diffusion [39]. RNA Analysis Total RNA was extracted using the TRI Reagent RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH) and integrity was verified by formaldehyde-agarose gel electrophoresis (Figure 3). First strand cDNA synthesis was performed by reverse transcription with 2 pg of total RNA using the Superscript II kit with oligo d(T12-18) primers as described by the manufacturer (lnvitrogen, Carlsbad, CA). cDNA (1 pl) was amplified by PCR in a final volume of 25 pl using the SYBR Green PCR Core Reagent kit (Applied Biosystems, Warrington, United Kingdom) with 10 pmol of each primer (Integrated DNA Technologies, Coralville, IA). TBP primers were used as internal controls along with the primers of genes being analyzed, including those associated with stages of osteoblast differentiation: collagen | (Col I), alkaline phosphatase (AP), and osteocalcin (OC) (Table 1). Real time PCR was carried out for 40 cycles using the iCycler (Bio-Rad, Hercules, CA) and data were evaluated using the iCycler software. RNA-free samples, a negative control, did not produce amplicons. Melting curve and gel analyses were used to verify single products of the appropriate base pair size. Transient transfections 99 MC3T3-E1 cells were seeded onto microcarriers as described above. Twenty-four hours later, cells on microcarriers were transferred to 1% agarose/1X PBS coated 6 well tissue culture plates and cotransfected using lipofectamine (Gibco, Rockville, MD) with 900 ng of a 6XAP-1 luciferase reporter (Gibco) and 100 ng of a pSV40 B-galactosidase reporter to normalize transfection efficiency in a total volume of 1ml serum-free Opti-MEM (Gibco). Five hours after transfection, 1 ml of o-MEM containing 20% FBS was added to the transfectants. Cells were cultured for 14 days and then transferred into the RWV or unit gravity control plates for 24 hours. Cells were harvested, washed with 1X PBS, lysed for analysis of luciferase and B-galactosidase activities using the protocol provided by the manufacturers (Promega, Madison, WI; Clontech, Palo Alto, CA, respectively), and quantitated on a Turner TD 20E luminometer (Turner Designs, Sunnyvale, CA). Statistical analysis All statistical analyses were performed using Microsoft excel data analysis program for t-test analysis. Experiments were repeated at least three times. Values are expressed as a mean i SE. Images in this thesis/dissertation are presented in color. 100 Results To address the influence of acute exposure to unloading, differentiating osteoblasts were transferred to a rotating wall vessel (RWV) to simulate microgravity. Figure 10 illustrates the experimental design. Osteoblasts were seeded onto microcarrier beads in agarose-coated plates to prevent attachment of cells to the plate surface. After 14 days, at which point markers of osteoblast differentiation were detectable, beads were either retained at unit gravity or placed into the RWV. The RWV was rotated at 20-25 rpm to approximate solid phase rotation. Measurement of alkaline phosphatase and osteocalcin mRNA levels revealed that 24 hours of RWV exposure dramatically suppressed these markers of osteoblast maturation compared to control cultures (Figure 11). Specifically, alkaline phosphatase and osteocalcin expression were decreased to less than 20% and 50% of control levels, respectively. This supports the hypothesis that osteoblast differentiation is suppressed under acute RWV conditions. In contrast, TATA binding protein (TBP) mRNA levels were unchanged by RWV exposure (Figure 12) demonstrating that decreases in alkaline phosphatase and osteocalcin expression are not due to changes in general cellular transcription. In addition, RNA was found to be similarly intact under both unit gravity and microgravity conditions (Figure 13). Given that runx2 is a major transcription factor involved in the regulation of osteoblast lineage commitment, osteoblast gene expression, and osteoblast differentiation [52, 53], we measured the influence of RWV exposure on runx2 101 expression in MC3T3-E1 osteoblasts. Figure 14 demonstrates that 24 hours in the RWV is sufficient to suppress runx2 expression by more than 50%. This finding is consistent with the suppression of markers of the mature osteoblast phenotype. To determine if suppression of runx2 correlates with altered expression of genes associated with early stages of osteoblast growth and differentiation, collagen l mRNA levels were measured. Figure 15 demonstrates that collagen l expression is not decreased by RWV exposure and its associated suppression of runx2 expression. In fact a slight but significant increase in collagen I was observed. This could indicate that the RWV selectively influences genes associated with specific stages of osteoblast differentiation or that 24 hours of RWV treatment initiates regression to an early differentiation phenotype. Alternatively, suppression of runx2 could signify a lineage shift. Pluripotent mesenchymal cells can give rise to multiple cell types, including osteoblasts, adipocytes, myocytes and chondrocytes. Therefore, we examined the expression of early gene markers of these lineages. We measured levels of Sox9 to examine chondrogenesis [73], myocyte enhancer factor 2A (MEF2A) for myogenesis [74], and peroxisome proliferator-activated receptor (PPAR) gamma expression for adipogenesis [75]. While MEF2A was not detectable in our samples (but was present in positive control myocytes), our findings of more than 3 separate experiments demonstrate no significant enhancement in the expression of Sox9 or PPAR gamma (0.9 +/- 0.2, 0.96 +l- 0.5, respectively; expressed as an average fold increase relative to unit gravity controls +l-SE). 102 This finding suggests that culturing MC3T3-E1 mouse osteoblasts for 24 hours in the RWV does not lead to a detectable lineage shift. The regulation of osteoblast phenotype and gene expression involves a significant transcriptional component. To address the influence of the RWV on general transcription factor activities, we measured expression of an SV40 promoter driven reporter plasmid in MC3T3-E1 cells. As shown in Figure 16, the RWV had no effect on SV40 promoter activity. In contrast, activity of an AP-1 dependent promoter reporter was suppressed by more than 50%, suggesting selective influences on AP-1 transactivation. 103 Discussion Decreased mechanical loading on the skeleton can result in suppression of osteoblast phenotype and bone formation. This has been demonstrated in humans [10, 12, 13, 25, 76], rats [20-25], and mice [18, 19] under conditions of bed rest, disuse, and spaceflight. To address the role of mechanical unloading on osteoblast phenotype we used the NASA approved RWV clinostat. After 24 hours in the RWV, markers of osteoblast differentiation, alkaline phosphatase and osteocalcin, are significantly suppressed in mouse osteoblasts. In addition, we find that mRNA levels of runx2 and AP-1 transactivation, regulators of osteoblast differentiation and bone formation [52-54, 60, 61, 68-70], are significantly suppressed compared to unit gravity controls. This finding is consistent with the suppression of markers of osteoblast differentiation. While there is some variability in results obtained from space flight, many studies demonstrate that the decrease in bone formation seen in spaceflight correlates with a suppression in osteoblast phenotype marked by decreased osteocalcin expression and secretion [12, 19, 77]. For example, space flight suppresses osteocalcin and collagen l expression in committed embryonic chick osteoblasts [30]. In addition, space flight and hindlimb suspension decreases osteocalcin and increases alkaline phosphatase and IGF-1 expression in rats [77], supporting a model where microgravity exposure suppresses the mature osteoblast phenotype and enhances an immature phenotype. Our data is consistent with this model; however, we find that alkaline phosphatase expression is also decreased. Similar to our findings, cultures of human 104 osteoblastic MG-63 cells grown for 9 days aboard the unmanned Foton 10 spaceflight exhibit a 51% and 19% decrease in alkaline phosphatase and osteocalcin, respectively [29]. Clinorotation of human osteoblast-like cells, Hu09, also leads to decreased alkaline phosphatase and osteocalcin expression [48]. Taken together human, rat, chick, and mouse osteoblasts respond to models of unloading such as spaceflight, hindlimb suspension, and clinorotation by suppressing gene expression associated with differentiation. In contrast to the above mentioned studies by ourselves and others, there are reports suggesting that osteoblast phenotype is not modified or enhanced under conditions of spaceflight [78] or RWV culturing [46, 47]. Differences may lie in within a variety of issues including species, acute versus chronic exposure, and normal versus transformed phenotypes. For example, several studies have cultured rat osteosarcoma (ROS) cells in the RWV and demonstrated an upregulation in the expression of markers of osteoblast differentiation [46, 47]. Osteosarcoma cells exhibit a proliferative phenotype and at the same time have robust expression of genes associated with differentiation. The expression of genes involved with differentiation during proliferation indicates that these genes are already able to override normal stage dependent regulation and therefore may not be affected by developmental stage regulation induced by simulated microgravity conditions. Thus the linkage between growth and differentiation observed in normal cells is lost, so the consequences of microgravity on transformed cells may not fully reflect in vivo responses. In addition, these 105 studies involved 8-10 days exposure to the RWV; adaptation to the environment over chronic periods of exposure could also result in different findings. Few studies have examined models of unloading on mice or mouse cells. Understanding mouse responses provides both fundamental knowledge and the opportunity to incorporate transgenic, knockout and mutant mice to address mechanisms of microgravity or disuse phenotypes. In vitro studies demonstrate that MC3T3-E1 osteoblasts have acute increases in fibronectin [79], exhibit cytoskeletal changes, and reduced cycloxygenase-2 expression [80] and prostaglandin synthesis [27]. These studies focused on responses of osteoblasts that have not reached later stages of differentiation, therefore expression of markers of maturation were not examined. Ex vivo studies carried out by Van Loon et al [18] demonstrate that space flight decreases mineralization of isolated fetal mouse bones, but did not address the expression of genes associated with differentiation. More recently, in vivo analysis of mice in space flight for 12 days demonstrated significant bone loss [19]. Consistent with our data, they also find a decrease in alkaline phosphatase and osteocalcin mRNA levels. In addition to suppression of osteoblast phenotypic markers, we also demonstrate that runx2 mRNA levels are suppressed. Runx2 is associated with enhanced expression of osteocalcin [56, 57, 81] and is essential for the progression of osteoblast differentiation and mouse bone development [60, 61]. Runx2 expression increases with the onset of MC3T3-E1 differentiation [82] and can be activated through post-translational modification [83]. Here we show that an acute 24 hour exposure in the RWV is enough to suppress runx2 expression. 106 Consistent with this finding, skeletal unloading has also been demonstrated to rapidly suppress osteocalcin as well as runx2 expression [33] while mechanical stretching has been demonstrated to enhance runx2 expression [84]. The suppression of runx2 expression could signal the transition of osteoblasts to a different phenotype. Differences in culture conditions, such as high density culturing, can influence chondrogenesis [85, 86]. However, we did not find an increase in Sox9 expression, a marker of the chondrocytic lineage. This suggests that osteoblasts are not transdifferentiating to chondrocytes and is consistent with the role of runx2 (which is suppressed in these cells) in hypertrophic chondrocytic differentiation [87]. Given that bone loss associated with osteoporosis and immobilization is also associated with an increase in marrow adipose tissue [33, 88, 89] we also examined expression of PPAR gamma, an early marker of adipogenesis. A 24 hour exposure to the RWV did not induce PPAR gamma, suggesting that adipogenesis or transdifferentiation to adipoctyes did not occur within the time frame we examined. Finally, we also examined MEF2A levels in our cells. Expression was undetectable, although detectable in control muscle cells. Taken together, these findings suggest that 24 hours of RWV exposure does not lead to a lineage switch. Because MC3T3- E1 cells are less pluripotent than some other cell systems such as C2012 cells or marrow cells, transdifferentiation to other cell types may not be possible. In addition, a longer time course may be required to detect lineage changes [33]. The importance of regulating transcription factor activities during the development of bone has been extensively demonstrated, although the role 107 during spaceflight related bone loss is less well understood. Though critical for normal bone development and maintenance, limited studies have examined AP-1 transcription factors in response to unloading. These studies utilized different models of unloading, but demonstrate the same phenomenon. Using A431 epidermal cells, basal c-fos expression was unchanged, while EGF induced c-fos expression was decreased in the RWV [90]. Sato et al. [91] demonstrate that MC3T3-E1 cells exposed to microgravity, on the sounding rocket TR-1A6, also exhibited depressed c-fos induction by EGF. In addition, bone marrow stromal cells isolated from 5 days unloaded rats express 50% less c-fos [35]. These findings support our results indicating a selective decrease in AP-1 transactivation as a result of RWV exposure. To our knowledge, this is the first report demonstrating the coordinate suppression of mouse osteoblast phenotype, runx2 expression, and AP-1 transactivation under conditions of decreased mechanical loading. This finding is consistent with the role of runx2 and AP-1 in the regulation of osteoblast differentiation and bone formation. 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After 14 days, half of the cells on beads are put into the RWV to simulate microgravity conditions for 24 hours, while the other half of the cells are maintained on cell culture plates to serve as unit gravity controls (4). Figure 11. Acute RWV exposure decreases alkaline phosphatase and osteocalcin expression. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or simulated microgravity (M) for 24 hours. Whole cell RNA was extracted and 2 pg of RNA subjected to reverse transcription followed by real time PCR to quantitate alterations in alkaline phosphatase (AlkPhos) and osteocalcin expression. Values are expressed relative to TBP expression, averaged and then graphed as a fold difference compared to unit gravity controls (n=4, ** p<0.01, *p<0.02). Figure 12. Acute RWV exposure does not alter RNA integrity or total cellular transcription. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or simulated microgravity (M) for 24 hours. A. Whole cell RNA was extracted and analyzed by agarose gel electrophoresis to verify RNA 118 integrity under gravity and simulated microgravity conditions. B. 2 pg of RNA were subjected to reverse transcription followed by real time PCR to quantitate alterations TBP expression. Averaged values from 5 separate experiments are expressed as fold difference relative to controls. Figure 13. Acute RWV exposure results in decreased runx2 expression. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or simulated microgravity (M) for 24 hours. Whole cell RNA was extracted and 2 pg of RNA were subjected to reverse transcription followed by real time PCR to quantitate alterations in runx2 expression. Values are expressed relative to TBP expression, averaged and then graphed as a fold difference compared to unit gravity controls (n=3, *p<0.05). Figure 14. Acute RWV exposure results in a modest increase in collagen I expression. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or simulated microgravity (M) for 24 hours. Whole cell RNA was extracted and 2 pg of RNA were subjected to reverse transcription followed by real time PCR to quantitate alterations in collagen l (Col I) expression. Values are expressed relative to TBP expression, averaged and then graphed as a fold difference compared to unit gravity controls (n=3, *p<0.05). 119 Figure 15. Acute RWV exposure results in decreased AP-1 transactivation but does not alter general cellular transcription. MC3T3 E1 osteoblasts were seeded onto microcarrier beads and transfected with a 6X AP-1 luciferase reporter along with a constitutively active SV40 Bgal expression vector to normalize for transfection efficiency. Transfected cells were grown on microcarrier beads for 14 days then left at unit gravity (G) or simulated microgravity (M) for 24 hours. Cell lysates were analyzed for luciferase activity indicative of AP-1 activity, and normalized to SV40 Bgal expression. Values represent the average fold difference, relative to unit gravity controls, of 3 separate experiments (p<0.01). 120 Figure 10 Comblno cells wlth mlaocarrlors In modla / Solld phase rotation (slmlahd mlcromtvlty) Grow cells on bad. for 14 days 121 Figure 11 Osteocalcin cyclophllin .m ‘“ ohn. ho Pl in Me m M G .. M G 8.54.20 0000 2 1 a o. A 2 o 2. 1 1 0 0 0 0 1 3:235:22 00:20:.qu mmtmozmxz mmtEofiooofiO 122 Figure 12 123 Figure 13 runx2 G ” g" cyclophllln q 2. 1 1 _ _ — 3 c 4. o o 0 70:05.50 29: mmtuxcsm — 2. 0 124 Coll/TBP (fold difference) Figure 14 125 Figure 15 — fl _ _ _ _ _ 21354.20 1 0 0 0 0 3822.3 22. .53 8535A? 1 l— 1 a 1 s .. ._.. .... m. 0 0 0 0 8835.. 29. Ema 3.5 126 CHAPTER III Hypoxia suppresses runx2 independent of modeled microgravity 1 Christopher Ontiveros, 1Regina Irwin, 1Robert W. Wiseman and 1Laura R McCabe 1Molecular Imaging Research Center 1Departments of Physiology and Radiology 2201 Biomedical Physical Science Building Michigan State University, East Lansing, MI 48824 127 Abstract Disuse associated bone loss is a consequence of conditions such as bed rest and space flight where the skeleton is exposed to decreased load and oxygenation. Under these conditions, osteoblast differentiation and function are suppressed. Previously, we have shown that the RWV reproduces these effects and decreases runx2 expression, a major regulator of osteoblast differentiation. To identify mechanisms accounting for runx2 suppression, we simulated disuse in vitro by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV) which reproduces at least two aspects of disuse: model microgravity and hypoxia. Hypoxia is indicated by reduced oxygen tension and increased GAPDH and VEGF expression. Uncoupling modeled microgravity from hypoxia is obtained by rotating the RWV in the vertical direction. In the vertical orientation osteoblasts are not under modeled microgravity conditions but are exposed to hypoxia. Modulation of ruxn2 was determined by real time PCR, DNA binding and promoter activation assays. Consistent with a role for hypoxia in suppression of runx2, expression, DNA binding and promoter activity of runx2 is decreased under horizontal and vertical RWV rotation. To distinguish hypoxic effects from other RWV associated conditions, medium oxygen tension in the RWV was titrated to control levels. Runx2 was not suppressed under this normoxic RWV condition. Finally, we demonstrate that direct exposure to hypoxia, independent of the RWV, is sufficient to 128 suppress runx2 expression in standard tissue culture. Here we identify that differentiating osteoblasts cultured in the RWV are under hypoxic conditions, which may contribute to phenotype changes seen in other cell types. Furthermore, our findings indicate that hypoxia is a key factor responsible for suppression of runx2 expression and promoter activity in our culture system. By extrapolation, we propose that the hypoxic microenvironment associated with unloading leads to suppression of runx2 expression in vivo and ultimately suppression of osteoblast differentiation and bone formation. 129 Introduction Mechanical loading is a key stimulator of bone formation thereby allowing bone to adapt and strengthen at areas where increased force is sensed [1-3]. Correspondingly, disuse/unloading conditions such as bed rest, spinal cord injury and space flight, where strong bone is no longer required, result in bone loss [4, 5]. Several hypotheses account for mechanoregulation of bone formation [6, 7] including direct sensing of bone deformation [8-10], direct responses to load incurred microdamage [11-13], and transduction of small mechanical deformations into larger signals as seen with increased canalicular fluid flow and intramedullary pressure [14-18]. While the above hypotheses focus on a mechanical effect (compression, tension or shear) on bone cells, loading also facilitates oxygenation in bone tissue. Consequently, unloading can result in bone tissue hypoxia. Given the high metabolic needs of differentiating osteoblasts and their increased oxygen consumption [19], oxygenation in bone could be critical for proper cell function. In support of this hypothesis, Dodd et al. demonstrate that 24 hours of unloading increases the number of hypoxic osteocytes in avian ulna [20]. Furthermore, hypoxia suppresses alkaline phosphatase activity in rat primary osteoblasts [21] and expression of differentiation markers in human osteoblast-like MG-63 cells [22]. 130 To study the influence of disuse on osteoblast function, we culture mouse osteoblasts (MC3T3-E1 cells) in horizontal rotating wall vessels (hRWV) to model microgravity and/or hypoxic conditions. The hRWV models microgravity by placing osteoblasts on microcarriers into solid phase rotation (in suspension culture) to obtain a net gravitational vector of near zero [23]. Previously we demonstrated that transferring mature/differentiating osteoblasts into the hRWV for a 24-hour period decreases expression of genes associated with osteoblast differentiation. Specifically, alkaline phosphatase and osteocalcin expression are decreased to less than 20% and 50% of control levels, respectively [24]. Runx2 expression, a transcription factor critical for osteoblast differentiation and ultimate bone formation, is also suppressed [24]. This is consistent with the lack of bone formation seen with inactivation of runx2 by deletion or mutation in vivo and decreased osteoblast differentiation in vitro [25-28]. Here we address the roles of hypoxia and modeled microgravity as mechanisms accounting for the suppression of runx2 expression in osteoblasts cultured under hRWV conditions. Hypoxia was uncoupled from modeled microgravity effects using three separate approaches. In the first approach, hypoxia is present and modeled microgravity is absent (through rotation of the RWV in the vertical orientation, vRWV). Under these conditions runx2 suppression still occurred. In the second 131 approach, hypoxia is absent and modeled microgravity is present. This prevented the suppression of runx2 expression. Finally, hypoxia alone (independent of the RWV) is sufficient to suppress runx2 expression. Taken together, our findings indicate that hypoxia is a major suppressor of runx2 expression. Consequently, bone tissue hypoxia associated with conditions of disuse or unloading, could contribute to osteoblast dysfunction and bone loss. 132 Methods Cell Culture System MC3T3-E1 cells [29], subcloned for maximal alkaline phosphatase staining and mineralization, were used for all studies. Osteoblasts were seeded at 1,000 cells/mg onto gelatin coated microcarrier beads, which are 90-150 microns in diameter (Solohill, Ann Arbor, MI). Osteoblasts were cultured for 14 days and fed every two days with a-MEM containing 10% fetal calf serum (Atlanta Biologicals Norcross, GA) and supplemented to a final concentration of 2 mM B-glycerophosphate (Sigma, St. Louis, MO) and 25 pg/ml ascorbic acid (Sigma). On day 14, half of the cells on microcarriers were placed into the horizontal RWV (hRWV) while the other half were left at unit gravity. In some experiments cells were put into a vertical RWV (vRWV), which did not maintain cells in suspension, thereby allowing dissection of suspension (modeled microgravity) versus hypoxia and other RWV effects. For studies incorporating direct exposure to hypoxia, osteoblasts were seed onto standard tissue culture dishes at a density of 2,500 cells/cm2. Osteoblasts were cultured for 14 days as described above. On day 14, half of the dishes were transferred to a hypoxic incubator maintained at 2% oxygen by regulating nitrogen intake. Rotating Wall- Vessel (RWV) 133 The RWV cell culture system (models 55ml STLV and 10ml HARV) was purchased from Synthecon, Inc. (Houston, Texas) and used for osteoblast suspension cultures. The RWV bioreactor is a cylindrical vessel containing an inner oxygenator core (surrounded by a membrane) and an outer core filled with culture media into which cells grown on microcarrier beads are placed. The vessel is connected to a rotator base and rotated along its longitudinal axis at a speed where cells establish a near solid phase rotation with the culture medium. The fluid dynamics in the hRWV allow for three-dimensional spatial freedom, reduced fluid shear forces, vector averaged gravitational force (to model microgravity conditions) and oxygenation without turbulence [30-32]. For oxygenation studies, oxygen was pumped into the RWV through an air filter port in the back of the rotating cell culture system. Oxygen concentration was regulated to maintain normoxia in the medium of the hRWV. Oxygen Tension Oxygen concentration was determined using a Dual Digital Model 20 oxygen sensor (Rank Brothers, LTD, Cambridge, England) and a Lauda Super K-2lR water bath (Brinkman Instruments, Westbury, NY). The oxygen sensor was normalized to 100% atmospheric oxygen saturation using cell culture media kept at 37°C and open to room atmosphere for 30 minutes with periodic shaking. Samples were injected 134 into the sensor and read relative to 100% atmospheric oxygen saturation and expressed here in terms of normal atmospheric oxygen (20%) or the partial pressure of oxygen (p02). Data from the oxygen sensor was collected using Logger Pro Version 1.0 software (Vernier Software, Beaverton, OR). RNA Analysis Total RNA was extracted using the TRI Reagent RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH) and integrity was verified by formaldehyde-agarose gel electrophoresis (Figure 3). First strand cDNA synthesis was performed by reverse transcription with 2 pg of total RNA using the Superscript II kit with oligo d(T12-18) primers as described by the manufacturer (lnvitrogen, Carlsbad, CA). cDNA (1 pl) was amplified by PCR in a final volume of 25 pl using the SYBR Green PCR Core Reagent kit (Applied Biosystems, Warrington, United Kingdom) with 10 pmol of each primer (Integrated DNA Technologies, Coralville, IA). Runx2 was amplified using the following primers: 5’-GAC AGA AGC TTG ATG ACT CTA AAC C-3’ and 5'-TCT GTA ATC TGA CTC TGT CCT TGT G-3’. Cyclophilin was used as an internal PCR control that is not modulated [33, 34] (primers: 5’-ATT CAT GTG CCA GGG TGG TGA C-3’ and 5’-CCG 'lTl' GTG TI'T GGT CCA GCA-3'). Genes associated with hypoxia were also examined, glyceraldehyde phosphate dehydrogenase (GAPDH) (primers: 5’-GTG TAC ATG GTT CCA GTA TGA CTC C-3’ and 135 5’-AGT GAG TTG TCA TAT TTC TCG TGG T-3’) and vascular endothelial growth factor (VEGF) (primers: 5’-ATA TCA GGC TTT CTG GAT TAA GGA C-3’ and 5’-CAG ACG AAA GAA AGA CAG AAC AAA G-3’). Real time PCR was carried out for 40 cycles using the iCycler (Bio-Rad, Hercules, CA) and data were evaluated using the iCycler software. RNA- free samples, a negative control, did not produce amplicons. Melting curve and gel analyses (sizing, isolation and sequencing) were used to verify single products of the appropriate base pair size. Transient transfections MC3T3-E1 cells were seeded onto microcarriers as described above. After 7 days osteoblasts were transfected using lipofectamine (Gibco, Rockville, MD) with 900 ng of a runx2 P1 promoter-luciferase reporter (generously provided by Dr. Gary Stein, [35]) containing a 3000 base pair region upstream of the runx2 transcriptional start site. Cells were also cotransfected with 100 ng of a constitutively active pSV40 8- galactosidase reporter to normalize transfection efficiency in a total volume of 1ml serum-free Opti-MEM (Gibco). Five hours after transfection, 1 ml of o-MEM containing 20% FBS was added to the transfectants for 24 hours. The following day, cells were transferred into the RWV or unit gravity control plates for 24 hours. Cells were harvested, washed with 1X PBS, lysed for analysis of luciferase and B—galactosidase activities using the protocol provided by the manufacturers (Promega, 136 Madison, WI; Clontech, Palo Alto, CA, respectiveIY). and quantitated on a Turner TD 20E luminometer (Turner Designs, Sunnyvale, CA). Electrophoretic Moblity Shift Assay Nuclear extracts were obtained by hypotonic lysis as previously described [36]. Nuclear extracts (4 ug) were incubated for 20 minutes at room temperature with 100,000 cpm 32P-end labeled (T4 polynucleotide kinase, Invitrogen, Carlsbad, CA) OSE2 oligonucleotide (5’- AGCTGCAATCACCAACCACAGCA—3’) [37, 38]. Samples were loaded onto a prerun (1 hour, 200 V) 5% polyacrylamide gel and run for 2 hours at 200 V at 4°C. Gels were dried and exposed to film at -80°C. Statistical analysis All statistical analyses were performed using Microsoft excel data analysis program for t-test analysis. Experiments were repeated at least three times. Values are expressed as a mean i SE. 137 Results Previously we have shown that expression of markers of osteoblast differentiation, including runx2, alkaline phosphatase and osteocalcin, are suppressed following a 24-hour culture period in an hRWV compared to static controls [24]. In these studies osteoblasts grown on microcarriers for 14 days are exposed to at least two major parameters of disuse: modeled microgravity (through vector averaged G force) and hypoxia. Hypoxic conditions were verified in the hRWV by measuring media oxygen concentrations after 24 hours of rotation. Media from control cultures had an oxygen saturation of 96.5% +l- 0.2 (mean of 3 experiments +l- SE) which is equivalent to a 19% atmospheric oxygen concentration (normoxia; p02 of 151 +l- 0.3 mmHg), while media sampled from hRWV conditions had an oxygen saturation of 48.5% +/- 1.8 which is equivalent to roughly a 9.7 % atmospheric oxygen concentration (hypoxia; p02 of 76.4 +I- 2.8 mmHg). Here we set out to determine the importance of modeled microgravity versus hypoxic conditions in suppressing runx2 expression in the hRWV. To do this we took several approaches. First, we uncoupled the effects of modeled microgravity from hypoxia by rotating the RWV in the horizontal or vertical direction (see Figure 16). In the vertical orientation osteoblasts are no longer in suspension and are not under modeled microgravity conditions, but they are still exposed to hypoxic conditions. Osteoblast expression of markers of hypoxia, namely GAPDH 138 and VEGF, are significantly increased 4-6 fold after 24 hours in the RWV under either horizontal or vertical rotation (Figure 17). In contrast to GAPDH and VEGF expression, runx2 expression is significantly decreased in the RWV under both vertical and horizontal rotation (Figure 18). This suggests that hypoxia, rather than vector averaged gravity (modeled microgravity), may be sufficient for RWV mediated suppression of runx2 expression. Consistent with this finding, we observed a decrease in runx2 DNA binding activity at an OSE2 element in both RWV orientations (Figure 19). Runx2 expression and activity can be regulated at several levels, therefore we examined runx2 promoter reporter activity in osteoblasts grown on microcarriers to determine the role of transcriptional regulation in runx2 suppression. Figure 20 demonstrates that runx2 promoter activity is decreased in osteoblasts grown for 24 hours under either horizontal or vertical rotation by 40% and 30%, respectively. Constitutive activity of a control reporter indicates specificity of transcriptional effects. Thus, these findings suggest that the hypoxic hRWV environment itself may be involved in suppression of runx2 transcription. A second approach to uncouple the effects of hypoxia from modeled microgravity involved maintaining normoxic conditions in an RWV under horizontal rotation (see Figure 16). By directly oxygenating the hRWV, modeled microgravity is maintained and hypoxic conditions are no longer present. This is evidenced by the maintenance of normoxic 139 conditions marked by an oxygen saturation of 94.0% +/- 1.0 in the unit (19% atmospheric oxygen concentration; p02 of 148 +l- 1.6 mmHg) and by inhibition of GAPDH and VEGF induction (Figure 21). Under these modified RWV conditions, runx2 mRNA was no longer suppressed (Figure 22). These findings further support our hypothesis that the changes in GAPDH, VEGF and runx2 expression, initially observed under horizontal rotation, are due to hypoxic conditions and not some still undefined characteristic of the RWV. To address if direct exposure to hypoxic conditions could induce the responses that we obtained in the RWV, osteoblasts were maintained in standard tissue culture dishes under normoxic conditions for 14 days then transferred into a hypoxic incubator containing 2% oxygen for 24 hours. As expected GAPDH and VEGF expression was significantly increased 6-8 fold above control levels (Figure 8). In contrast, runx2 expression was markedly suppressed to less than 60% of control levels (Figure 24). This response mimicked the response we saw in the RWV. 140 Discussion Runx2 is a critical transcriptional regulator of osteoblast differentiation and bone formation. Consistent with disuse induced suppression of osteoblast differentiation we have previously demonstrated that 24 hours under hRWV conditions suppresses expression of runx2 as well as other markers of osteoblast differentiation, namely alkaline phosphatase and osteocalcin [24]. Our current studies indicate that hypoxia is the cause of runx2 suppression in the hRWV. While hRWV conditions model microgravity there are additional components that are altered in the hRWV, including the hypoxic environment that occurs when mature osteoblasts are grown in it. We distinguished that hypoxia is the critical component of the runx2 response by demonstrating that 1) it is suppressed under both horizontal and vertical RWV orientations, 2) it is suppressed in standard tissue culture dishes grown under hypoxic conditions and 3) it is not suppressed under normoxic hRWV conditions. To our knowledge, this is the first demonstration that hRWV associated hypoxia causes the suppression runx2 expression and increase in GAPDH and VEGF in nontransformed osteoblasts. Indeed, conditions of unloading cause hypoxia in bone. Specifically, Dodd et al. observed an increase in hypoxic osteocytes in unloaded avian ulna compared to normally loaded controls [20]. Subsequent studies demonstrated that hypoxia inducible factor-1 (HIF-1) alpha levels were also increased in unloaded compared to normally loaded osteocytes in 141 vivo and in hypoxic osteocytes in vitro [39]. We propose that the hypoxic microenvironment associated with unloading leads to suppression of runx2 expression and ultimately osteoblast differentiation and bone formation. In support of our findings demonstrating a role for hypoxia in the suppression of runx2 expression and osteoblast differentiation, Tuncay et al. report decreased alkaline phosphatase activity in primary fetal rat calvarial osteoblast cultures exposed to hypoxia (10% oxygen) [21]. Furthermore, Park et al [22] report a decrease in alkaline phosphatase and osteocalcin expression in human osteoblast-like MG63 cells under hypoxic conditions, however GAPDH levels did not change. Longer incubations under hypoxic conditions have been demonstrated to suppress runx2 mRNA levels in MG-63 cells [22]. Differences may result from the use of a transformed cell line, MG-63, which could exhibit different energy requirements and altered signaling pathways and therefore responsiveness. Interestingly, hyperoxia (90% oxygen) has been reported to increase osteoblast alkaline phosphatase activity [21]. This further suggests that modulation of oxygen levels (between low and high levels) could lead to the differential regulation of bone cell phenotypes and thereby influence skeletal homeostasis. While our RWV studies demonstrate suppression of runx2, alkaline phosphatase and osteocalcin, there are reports suggesting that the RWV does not influence or can increase expression of osteoblast markers. For 142 example, rat osteosarcoma cells grown in an RWV have been shown to have no change or an increase in alkaline phosphatase and osteocalcin expression [40, 41]. Differences between our findings could be related to cell types used. Osteosarcoma cells exhibit an abnormal pattern of gene expression marked by simultaneous expression of genes associated with proliferation and differentiation, whereas MC3T3-E1 mouse osteoblasts undergo stage specific gene expression similar to osteoblasts in vivo [42]. This difference may make the osteosarcoma cells less respondent to phenotypic effects of hypoxia since they already exhibit an altered phenotype. In addition, oxygen requirements of ROS cells may be lower than what is required for maturing MC3T3-E1. If this is the case, then ROS cells may not be under hypoxic stress in the RWV. Our studies also demonstrate that osteoblast expression of both VEGF and GAPDH is induced under hypoxic conditions, consistent with observations in other tissues [43]. This upregulation involves hypoxia inducible factors (HlFs) binding to a hypoxia response element (HRE) located in the promoter of VEGF as well as several other hypoxia inducible genes [44]. GAPDH expression is also regulated by a HRE in its promoter and has been demonstrated to be increased under hypoxic conditions in a variety of tissues including endothelial cells [45]. Consistent with our findings of upregulation of VEGF in osteoblasts, other studies have also shown that hypoxia stimulates VEGF mRNA and protein levels in several osteoblastic cell lines [46, 47]. 143 Decreased mechanical loading of the skeleton leads to bone loss. While several hypotheses incorporate a role for direct mechanical stimulation of bone cells under load, our studies suggest that oxygenation associated with increased blood flow under mechanical loading can be an important contributor to the maintenance of bone cell function. Certainly the flow of blood within bone is altered under conditions of disuse as seen in models of unloading [48, 49]. Culturing osteoblasts in the hRWV has allowed us to uncouple two potential mechanisms associated with skeletal unloading: modeled microgravity and hypoxia. Our findings demonstrate that hypoxia is a major inhibitor of runx2 expression, DNA binding activity and promoter activity in differentiating osteoblasts. Given that hypoxia is associated with unloading in vivo, it is possible that hypoxia contributes to bone loss under conditions of disuse by suppressing runx2 expression. Understanding the mechanisms of hypoxia induced suppression of osteoblast differentiation and bone formation will contribute to further understanding skeletal adaptation to altered mechanical loading. Acknowledgements We thank Dr. J Lapres (Dept. of Biochemistry, MSU) for insightful discussions and S Botolin, M Hossaln, J Xia, and S Kinser for their helpful suggestions. Funded by grants from NASA (NAG8-1575) and NIH (DK061184) to LRM and NSBRI (MA00210) to RWW. 144 References 1. 10. 11. 12. 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Graven, K.K., et al., Regulation of endothelial cell glyceraldehyde-3- phosphate dehydrogenase expression by hypoxia. J Biol Chem, 1994. 269(39): p. 24446-53. Akeno, N., et al., Hypoxia induces vascular endothelial growth factor gene transcription in human osteoblast-like cells through the h ypoxia-inducible factor-Zalpha. Endocrinology, 2001. 142(2): p. 959-62. Steinbrech, D.S., et al., VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism. Am J Physiol Cell Physiol, 2000. 278(4): p. C853-60. 148 48. 49. Knothe Tate, ML, et al., In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation. J Exp Biol, 2000. 203 Pt 18: p. 2737-45. Colleran, P.N., et al., Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling. J Appl Physiol, 2000. 89(3): p. 1046-54. 149 Figure Legends Figure 16. Three RWV experimental approaches are used to uncouple modeled microgravity from hypoxic conditions. Osteoblasts were cultured on microcarrier beads (light grey circles in cylinder). After 14 days, cells were transferred into RWV conditions of modeled microgravity and hypoxia (hRWV), microgravity alone (hRWV + 02) or hypoxia alone (vRWV). Figure 17. GAPDH and VEGF mRNA levels are increased in osteoblasts grown in horizontal or vertical rotating RWV. Day 14 MC3T3-E1 osteoblasts grown on microcarriers were transferred to RWVs in the vertical or horizontal orientation (vRWV or hRWV, respectively) or to static control tissue culture dishes (G). After 24 hours cells were harvested, RNA extracted and GAPDH and VEGF mRNA levels determined by real time RT-PCR. Levels were expressed relative to cyclophilin mRNA levels. Values are expressed as an average fold increase +l- SE relative to static control cultures (set at 1) and represent an n of 7 separate experiments; * p<0.05. Insert is a representative agarose gel of PCR products obtained from the linear phase of amplification. Figure 18. Runx2 mRNA levels are decreased in osteoblasts grown in horizontal or vertical rotating RWV. Day 14 osteoblasts grown on 150 microcarriers were transferred to RWVs in the vertical or horizontal orientation (vRWV or hRWV, respectively) or to static control tissue culture dishes (G). After 24 hours cells were harvested, RNA extracted and runx2 mRNA levels determined by real time RT-PCR. Levels were expressed relative to cyclophilin mRNA levels. Values are expressed as an average fold increase +l- SE relative to static control cultures (set at 1) and represent an n of 7 separate experiments; * p<0.05. Insert is a representative agarose gel of PCR products obtained from the linear phase of amplification. Figure 19. OSE2 DNA binding is decreased in osteoblasts grown in horizontal or vertical rotating RWV. Day 14 osteoblasts grown on microcarriers were transferred to RWVs in the vertical or horizontal orientation (vRWV or hRWV, respectively) or to static control tissue culture dishes (G). After 24 hours cells were harvested and nuclear extracts isolated and used for electrophoretic mobility shift assays. Nuclear extracts (4 ug) were incubated with a consensus runx2 binding site probe (OSE2). A representative autogradiograph of 3 experiments is shown. The arrow points to the shifted runx22DNA complex as determined by competition with cold probe. Figure 20. Horizontal or vertical RWV rotation suppresses runx2 promoter activity. Day 10 osteoblasts transfected with a runx2 promoter- 151 luciferase reporter were transferred to RWVs in the vertical or horizontal orientation (vRWV or hRWV, respectively) or to static control tissue culture dishes (G). After 24 hours cells were harvested and luciferase activity measured. Expression was calculated relative to constitutive SV40 beta- galactosidase levels (to control for transfection efficiency). Values are expressed as an average fold increase +/- SE relative to static control cultures (set at 1) and represent an n of 3 separate experiments; * p<0.05. Figure 21. Normoxic conditions in the RWV prevent GAPDH and VEGF mRNA Induction. Osteoblasts were cultured for 14 days. Cells were then left at unit gravity (G), subjected to normal horizontal RWV rotation (hRWV) or horizontal RWV rotation supplemented with oxygen to normoxic conditions (hRWV+Oz). After 24 hours cells were harvested, RNA extracted and GAPDH and VEGF mRNA levels determined by real time RT-PCR. Levels were expressed relative to cyclophilin mRNA levels. Values are expressed as an average fold increase +l- SE relative to static control cultures (set at 1) and represent an n of 3 separate experiments; * p<0.05. Insert is a representative agarose gel of PCR products obtained from the linear phase of amplification. Figure 22. Normoxic conditions in the RWV prevent runx2 mRNA suppression. Osteoblasts were cultured for 14 days. Cells were then left at unit gravity (G), subjected to normal horizontal RWV rotation (hRWV) or 152 horizontal RWV rotation supplemented with oxygen to normoxic conditions (hRWV+Oz). After 24 hours cells were harvested, RNA extracted and runx2 mRNA levels determined by real time RT-PCR. Levels were expressed relative to cyclophilin mRNA levels. Values are expressed as an average fold increase +/- SE relative to static control cultures (set at 1) and represent an n of 3 separate experiments; * p<0.05. Insert is a representative agarose gel of PCR products obtained from the linear phase of amplification. Figure 23. Hypoxia alters GAPDH, VEGF and Runx2 mRNA levels in osteoblasts. Day 14 osteoblasts seeded into standard tissue culture dishes were maintained in a normoxia incubator (20% oxygen) or transferred to a hypoxic incubator (<2% oxygen). After 24 hours cells were harvested, RNA extracted and runx2 mRNA levels determined by real time RT-PCR. Levels were expressed relative to TBP mRNA levels. Values are expressed as an average fold increase +l- SE relative to static control cultures (set at 1) and represent an n of 3 separate experiments; * p<0.05. 153 Figure 16 lg vector ROTATION: horizontal vertical horizontal MODELED MICROGRAVITY: Yes "0 Yes HYPOXIA: yes yes no 154 Figure 17 cyclophilin VEGF cyclophllln GAPDH * * LII W G hRWV vRWV '1: * -u M _ 2 654321 5 4 3 2 1 0 8:246 20: 8:28.... 2.5 5.28.0 2:36 =____ao_o>otom> vRWV hRWV 155 Figure 18 cyclophilin 1.2 - d u 1 853.5“. 2o: :__an_o>2mx::._ hRWV vRWV G 156 Figure 19 4" runx2 ‘— free probe 157 Figure 20 — - - q - 2. 1 3 6. o 1 0 0 04- 0.2- 8596 2a: Emu o¢>m32 .80an «5.5.. hRWV vRWV G -3000 l Runx2 promoter]— { luciferase +1 I” 158 GAPDH/cydophlll (fold difference) OJNQ&OOV VEGF/cyclophlllu (fold difference) .I Figure 21 GlhRWVlm G hRWV hRWV+02 4 3 2 G unwvl e02 VEGF 0 cyclophllln hRWV hRWV+02 GAPDH cyclophilin 159 an. . -| N 1 I .° “ 1 runx2/cyclophilin (fold difference) P N 1 O 1 G Figure 22 hRWV e |thv| 00: hRWV runx2 cyclophilin hRWV+O2 Figure 23 _ all — 0 2 _ _ _ 5 0 5 1 1 8:285 20: 5.28.051926 20- < _ a q _ _ I 8 6 4 2 0 5 0 5 . . . . 1 1 0 0 0 0 853:... 2o: _Eno_o>ofix::m 0:93.56 .20: _:eo_o>oEom> 24 hour hypoxia Control 161 CHAPTER IV RWV associated hypoxia suppresses AP-1 in osteoblasts 1Ontiveros C and 2LR McCabe Michigan State University 1Department of Microbiology and Molecular Genetics 2Departments of Physiology and Radiology Molecular Imaging Research Center 2201 Biomedical Physical Science Bldg East Lansing, MI 48824 162 Abstract While mechanical stresses associated with skeletal loading increase expression of markers of osteoblast differentiation, conditions of unloading and decreased stress have the opposite effect. Interestingly, not much is known about the role of transcription factors involved in these responses. The AP-1 family of transcription factors influence osteoblast phenotype and subsequent bone formation. To test the effects of decreased load, as seen during spaceflight, on AP-1 member expression in osteoblasts we used the NASA designed rotating wall vessel (RWV). The RWV exhibits two characteristics associated with unloading: modeled microgravity and hypoxia. Culturing differentiating osteoblasts in the RWV for 24 hours suppressed basal expression of c-fos and c- jun whereas mRNA expression of all other AP-1 family members remained unchanged. The decrease in AP-1 member expression correlated with decreased AP-1 binding and transactivation that was maintained to 48 hours and was reversible. Normoxic RWV conditions blocked c-fos and c-jun suppression demonstrating that hypoxia rather than modeled microgravity is a critical regulator of AP-1 expression. While a close association between AP-1, cell growth and apoptosis exists, no apparent alterations were seen in these parameters. These findings suggest that hypoxia could be an important factor contributing to suppression of AP-1 expression under conditions of skeletal unloading resulting in bone loss. 163 Introduction Mechanical stress is a key regulator of skeletal phenotype. Shear, vibration and loading stresses promote new bone formation [1-3] to withstand normal daily stresses. In contrast, decreased mechanical stress causes bone loss [4-11]. The mechanisms responsible for this process are not fully understood. While the actual loading stress is important for bone formation, another factor common among all conditions of loading is the facilitated oxygen and nutrient delivery to cells within bone [12, 13]. Animal models demonstrate that under unloading conditions bone mineral density is decreased [4-11] and cells within bone are under hypoxic stress [14, 15]. Regulation of osteoblast gene expression and differentiation involves changes in transcription factor activities, including those of the AP-1 family. AP-1 transcription factors are composed of Fos (c-fos, Fos B, Fra 1, and Fra 2) and Jun (c-jun, Jun B, and Jun D) members. FoszJun and Jun;Jun dimers bind to AP-1 consensus and AP-1-like sites to activate transcription [16, 17]. A role for AP-1 in the regulation of osteoblast phenotype has been suggested by the coordinate changes in AP-1 associated with hormones and conditions (including differentiation, stress), which alter osteoblast gene expression and differentiation [18-29]. A more direct role in modulating skeletal phenotype is indicated by AP-1 overexpression and gene ablation studies [30-37]. Taken together, these findings suggest that regulation of Fos and Jun family member expression is important for normal development and maintenance of bone. 164 Given the important role of AP-1 in regulating skeletal homeostasis, it is not surprising that alterations in mechanical load influence AP-1 in bone [19, 23, 24, 28, 33, 38-41]. Specifically, AP-1 is induced by bending (c-fos and c-jun) and hypergravity (c-fos) in osteoblasts [42-44]. c-fos is further implicated as a target of mechanical loading by the presence of a consensus shear stress response element in its promoter [45, 46]. While studies regarding unloading and its effects on AP-1 are limiting, they suggest that AP-1 activity could be suppressed. For example, hindlimb unloading suppresses c-fos expression in isolated osteoprogenitor cells compared to normally loaded controls [47]. Induction of c- fos is suppressed under simulated microgravity conditions [48, 49] as well as under acute real microgravity conditions in sounding rocket experiments [48]. Effects on other family members were not examined. Mechanisms of decreased loading on osteoblast phenotype can be studied in the NASA designed rotating wall vessel (RWV) to model microgravity and hypoxia. In the RWV cells are cultured in solid phase rotation such that they experience randomized g-vectors and minimal shear stress [50-52]. We have previously demonstrated that osteoblasts cultured in the RWV have decreased expression of markers of differentiation such as runx2, alkaline phosphatase and osteocalcin [53]. Further analyses demonstrated transcriptional suppression of runx2 expression (Ontiveros et al., submitted). In addition, we identified that culturing of mature MCST3 E1 osteoblasts under RWV conditions creates a hypoxic environment (Ontiveros et al., submitted). 165 To address the effects of decreased loading on AP-1 in osteoblasts, we cultured differentiating osteoblasts under RWV conditions for 24 hours. RWV conditions caused a reversible decrease in c-fos and c-jun mRNA levels, consistent with RWV mediated decreases in AP-1 DNA binding and transactivation activities. Furthermore, we determined that hypoxia and not modeled microgravity is responsible for the AP-1 suppression in the RWV. Taken together our findings indicate that hypoxia associated with skeletal unloading could contribute to suppression of osteoblast differentiation and bone formation through the downregulation of activities of key transcription factors such as AP-1. 166 Methods Cell Culture System MC3T3-E1 cells [54], subcloned for maximal alkaline phosphatase staining and mineralization, were used for all studies. Osteoblasts were seeded at 1,000 cells/mg onto gelatin coated microcarrier beads, which are 90-150 microns in diameter (Solohill, Ann Arbor, MI). Osteoblasts were cultured for 14 days and fed every two days with a-MEM containing 10% fetal calf serum (Atlanta Biologicals Norcross, GA) and supplemented to a final concentration of 2 mM B-glycerophosphate (Sigma, St. Louis, MO) and 25 pg/ml ascorbic acid (Sigma). On day 14, half of the cells on microcarriers were placed into the RWV to simulate microgravity, while the other half were left at unit gravity. Rotating Wall- Vessel (RWV) The RWV cell culture system (models 55ml STLV and 10ml HARV) was purchased from Synthecon, Inc. (Houston, Texas) and used for studies of simulated microgravity. The RWV bioreactor is a cylindrical vessel containing an inner oxygenator core (surrounded by a membrane) and an outer core filled with culture media into which cells grown on microcarrier beads are placed (see Figure 1). The vessel is connected to a rotator base and rotated along its longitudinal axis at a speed where cells establish a near solid phase rotation with the culture medium. The fluid dynamics in the RWV allow for colocalization of cells and aggregates of different sedimentation rates, three-dimensional spatial 167 freedom, reduced fluid shear forces, and oxygenation without turbulence by diffusion [52]. Th ymidine Incorporation Osteoblasts grown on microcarrier beads for 14 days were subjected to RWV conditions. After 48 hours, cells on microcarriers were transferred to normal tissue culture plates and pulsed with 4pCi 3H-thymidine per milliliter of cell culture media. Cells were then allowed to continue incubation with 3H-thymidine under normal 37°C and 5% 002 incubator conditions. After two hours, cells were washed twice with ice cold PBS then twice with 10% TCA for two minutes on ice. Finally, 10% SDS was added to solubilize TCA precipitates on the plate. Aliquots of the 10% SDS samples were analyzed using a Tri-Carb 2100TR scintillation counter (Perkin Elmer, Boston, MA) for determination of 3H-thymidine incorporation. Fluorometric DNA Quantitation Cells collected for thymidine incorporation were brought to neutral pH by addition of HCI. Once neutralized, samples were mixed in a 10 mM EDTA buffer pH 12 brought to neutral pH with 1M KH2PO4. A 4.8 pglml solution of Hoechst Dye (Molecular Probes, Eugene, OR) was prepared by mixing 24 ul of a 200 pglml Hoechst Dye stock in HzO to 1 ml neutralized 10 mM EDTA solution. A salmon sperm DNA (Stratagene, La Jolla, CA) standard was prepared which ranged from 0 ng to 3200 ng total DNA. The final volume of standard DNA was 168 brought to a final volume of 170 pl with the neutralized 10 mM EDTA solution. To each standard, 30 pl of the 4.8 pglml Hoechst Dye solution was added. 150 pl of each sample to be determined was mixed with 20 pl 10 mM neutralized EDTA and 30 pl of the Hoechst Dye solution. Standards and samples were mixed in 96 well microtiter (Fisher, Itasca, IL) plates and read using a CytoFluor ll Microplate Fluorescence Reader (Biosearch, Bedford, MA) to determine the amount of DNA present in each sample. RNA Analysis Total RNA was extracted using the TRI Reagent RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH) and integrity was verified by formaldehyde-agarose gel electrophoresis (Figure 3). First strand cDNA synthesis was performed by reverse transcription with 2 pg of total RNA using the Superscript II kit with oligo d(T12-13) primers as described by the manufacturer (Invitrogen, Carlsbad, CA). cDNA (1 pl) was amplified by PCR in a final volume of 25 pl using the SYBR Green PCR Core Reagent kit (Applied Biosystems, Warrington, United Kingdom) with 10 pmol of each primer (Integrated DNA Technologies, Coralville, IA). Cyclophilin primers were used as internal controls along with the primers of genes being analyzed: c-fos, fos B, fra 1, fra 2, c-jun, jun B, jun D, alkaline phosphatase (AP), and osteocalcin (00) (Table 2). Real time PCR was carried out for 40 cycles using the iCycler (Bio-Rad, Hercules, CA) and data were evaluated using the iCycler software. RNA-free samples, a 169 negative control, did not produce amplicons. Melting curve and gel analyses were used to verify single products of the appropriate base pair size. Whole Cell Protein Extraction For whole cell protein isolation, MC3T3-E1 cells were washed with chilled 1x phosphate-buffered saline, left on ice for 3min, centrifuged at 800 x g for 5 min at 4° C, and resuspended in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 10% glycerol). A mixture of protease and phosphatase inhibitors (1 mM sodium orthovanadate, 2 mM phenylmethylsulfonylfluoride, 5 pglml aprotinin, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, and 0.1 mM B-glycerophosphate) was added to lysis buffer. Samples were centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant protein concentration was quantitated by the Bio-Rad DC protein detection system. Western Blot Analysis Whole cell extracts were loaded (50 pgllane) on a mini-PAGE system. Following electrophoreses, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) using a semidrytransfer system. Protein transfer and size determinations were verified using prestained protein markers. Membranes were then blocked with 5% nonfat dry milk in TTBS (10 mM Tris-HCI, pH 8,150 mM NaCl, 0.05% Tween-20) and subsequently incubated with antibodies directed against c-jun protein (Santa Cruz Biotechnology). Signals were detected using a 170 horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL, Amersham Biosciences). TranSignal Array Nuclear extracts of cells incubated under control and RWV conditions for 24 hours were obtained using the nuclear extraction kit from Panomics (Redwood City, CA). Briefly, cells are allowed to swell in a hypotonic buffer and disrubted to separate nuclei from cytoplasmic component. The cytoplasmic component is removed and the nuclear proteins are removed from the nuclei using a high salt buffer. The TranSignal Protein/DNA Array l kit from Panomics was then used to obtain transcription factor array data. Briefly, Nuclear extracts from cells were preincubated with a mix of biotin labeled transcription factor binding site oligonucleotide sequences. The protein/DNA complexes are separated from the unbound oligonucleotides by agarose gel electrophoresis then purified by gel purification. The oligonucleotides are then hybridized to the TranSignal Array and detected by chemiluminescent imaging. Nuclear Extraction and Electrophoretic Mobility Shift Assay (EMSA) During nuclear extract preparation, a protease inhibitor cocktail (Calbiochem, San Diego, CA) with broad specificity was used in all sample preparations. Cells grown on microcarriers are washed twice with ice cold PBS then resuspended in NP40 lysis buffer (10 mM Tris HCI pH 7.4, 10 mM NaCl, 3 mM M9012, 0.5% NP40, 0.56M sucrose) containing protease inhibitors. Cells 171 were dounce homogenized with a loose pestle then allowed to incubate on ice for 5 minutes. Cells were spun at 3000 rpm in a model 5417R Eppendorf refrigerated centrifuge (Westbury, NY) at 4°C and the supernatant is removed. The pellet is then resuspended in 3X volume of hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgClz, 10 mM KCI) containing protease inhibitors. Samples were spun at 6000 rpm for 5 minutes and the supernatant was again discarded. The nuclear pellet is resuspended in extraction buffer (20 mM HEPES pH 7.9, 20% glycerol, 600 mM KCI, 1.5 mM MgCl2, 0.2 mM EDTA) containing protease inhibitors. Samples were vortexed vigorously for 30 minutes at 4°C then spun at 14,000rpm for 10 minutes. The supernatant was aliquoted into several tubes then stored at -80°C to be used for EMSA analysis. Nuclear extracts (4 pg) were incubated for 30 min at 24°C, with 50,000 cpm 32P-end labeled (T4-polynucleotide kinase; Invitrogen, Carlsbad, CA) AP-1 oligonucleotide (CGCTTGATGAGTCAGCCGGAA). The samples were loaded on prerun (2 hour; 200V) 5% polyacrylamide gels and run for 3 h at 150V at 4 C. Gels were dried and exposed to film at -80 C. Transient transfections MC3T3-E1 cells were seeded onto microcarriers as described above and grown for 7 days. On day 7, microcarriers were transferred to 1% agarose/1X PBS coated 6 well tissue culture plates and cotransfected with 900 ng of a 6XAP- 1 luciferase reporter (Gibco) and 100 ng of a pSV40 B-galactosidase reporter to normalize transfection efficiency in a total volume of 1ml serum-free Opti-MEM 172 (Gibco) using lipofectamine (Gibco, Rockville, MD). Five hours after transfection, 1 ml of a-MEM containing 20% FBS was added to the transfectants. Cells were cultured for 2 additional days then transferred into the RWV or unit gravity control plates for 24 hours. Cells were harvested, washed with 1X PBS, lysed in Reporter Lysis Buffer (Promega, Madison, WI) for analysis of luciferase. B- galactosidase activity was determined using the Luminescent B-galactosidase Detection Kit (Clontech, Palo Alto, CA) and quantitated on a Turner TD 20E luminometer (Turner Designs, Sunnyvale, CA). Oxygen Tension Oxygen concentration was determined using a Dual Digital Model 20 oxygen sensor (Rank Brothers, LTD, Cambridge, England) and a Lauda Super K-2lR water bath (Brinkman Instruments, Westbury, NY). The oxygen sensor was normalized to 100% atmospheric oxygen saturation using cell culture media kept at 37°C and open to room atmosphere for 30 minutes with periodic shaking. Samples were injected into the sensor and read relative to 100% atmospheric oxygen saturation and expressed here in terms of normal atmospheric oxygen (20%) or the partial pressure of oxygen (p02). Data from the oxygen sensor was collected using Logger Pro Version 1.0 software (Vernier Software, Beaverton, OR). Caspase 3 Activity 173 Caspase 3 activity was determined using the BD ApoAlert Caspase-3 Colorimetric Assay Kit according to the manufacturers protocol (BD Biosciences, Palo Alto, Ca). Briefly, MC3T3-E1 osteoblasts were cultured on microcarriers for 14 days then subjected to unit gravity or RWV conditions for 24 hours. Cells were collected, subjected to lysis buffer treatment, then centrifuged to remove cell debris. 50 pl of supernatant was mixed with 50 pl of a 2X reaction mix containing the caspase 3 substrate then incubated at 37°C for 1 hour. Samples were read at 405 nm in a 100 pl quartz cuvette using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ). Statistical analysis All statistical analyses were performed using Microsoft excel data analysis program for t-test analysis. Experiments were repeated at least three times. Values are expressed as a mean i SE. 174 Results Previously, we demonstrated that markers of osteoblast phenotype, alkaline phosphatase, osteocalcin and runx2, are suppressed following 24 hours in the RWV. To test whether modeled microgravity alters osteoblast AP-1 member expression, osteoblasts were similarly cultured for 14 days then subjected to RWV conditions for 24 hours. Of the seven AP-1 family members, only c-fos and c-jun mRNA expression was altered. Specifically, c-fos and c-jun mRNA levels were significantly decreased to 23% and 55% of unit gravity controls, respectively (Figure 24). To determine if decreases in c-fos and c-jun mRNA correlated with decreased c-fos and c-jun protein levels, whole cell protein extracts were subjected to western blot analysis (Fig 25). As with decreased levels of c-jun mRNA, protein levels were also dramatically decreased in RWV samples compared to controls. Unexpectedly, there was no difference in c-fos protein levels in RWV treated cells when compared to controls (data not shown). To determine if decreases in c-fos and c-jun mRNA correlated with alterations in DNA binding, we used two separate assays. Firstly, we performed the TranSignal DNA binding assay, which allows for the profile of multiple transcription factor activities simultaneously. This approach demonstrated that AP-1 DNA binding was decreased in cells grown under RWV conditions relative to unit gravity controls while internal SP-1 DNA binding remained unchanged (Figure 263). This was confirmed using, as a second approach, conventional EMSA. Figure 26b demonstrates that DNA binding to a consensus AP-1 site 175 was decreased in nuclear extracts from RWV grown cells relative to unit gravity controls supporting the TranSignal array data. To determine the role of c-fos and c-jun in decreased AP-1 DNA binding, antibody supershifts using anti-c-jun and anti-c-fos antibodies determined that both c-jun and c-fos DNA binding were both decreased after 24 hours in the RWV (Figure 27). Consistent with a decrease in AP-1 DNA binding activity, AP-1 transactivation is also decreased after 24 hours of RWV conditions, as we have previously reported [53]. Here we further examined whether RWV induced suppression of AP-1 transactivation is maintained following longer durations in the RWV and whether this suppression is reversible. Figure 28 demonstrates that 48 hours in the RWV results in continued suppression of AP-1 transactivation to 38% of control levels. However, AP-1 transactivation is restored to control levels when osteoblasts in the RWV for 24 hrs are allowed to recover under normal culture conditions for an additional 24 hours (Figure 28). Culturing differentiating osteoblasts in the RWV system puts them under both modeled microgravity and hypoxic conditions. Specificially, cells grown under normal unit gravity conditions have media with a partial pressure of oxygen of 151 mmHg (normoxia) while cells grown in the RWV for 24 hours have media with a partial pressure of 76.4 mmHg (hypoxia). Furthermore, osteoblast expression of GAPDH and VEGF, genes induced by hypoxia, was increased in RWV cultured cells (data not shown). To separate modeled microgravity from the hypoxic component of the RWV, MC3T3 E1 osteoblasts were grown in the RWV supplemented with external oxygen. This maintained modeled microgravity 176 conditions but prevented hypoxic conditions as determined by a partial pressure of oxygen of 148.1 mmHg (normoxia) and the lack of VEGF and GAPH upregulation (data not shown). Under these normoxic RWV conditions, suppression of c-fos and c-jun was prevented indicating that the initial suppression of c-fos and c-jun expression is due to hypoxia in the RWV rather than modeled microgravity conditions (Figure 29). Given the important role of AP-1 family members in the regulation of cell cycle control and apoptosis, we examined whether RWV induced suppression of c-fos and c-jun expression influenced these parameters. MC3T3-E1 cells were grown for 14 days then transferred to the RWV. 3H-thymidine incorporation was subsequently measured after 24 hours (Figure 30) of RWV conditions. Cell growth and total DNA content was not statistically different compared to unit gravity controls. Nuclear staining by propidium iodine (not shown) as well as measurement of caspase 3 activity at 24hrs (Figure 31) and the absence of DNA Iaddering (data not shown) indicated that the cells were not undergoing apoptosis, consistent with unmodulated DNA levels. From this we conclude that 24 hours of RWV conditions do not alter cellular proliferation or apoptosis relative to unit gravity controls even though AP-1 activity is suppressed. 177 Discussion It is well documented that conditions of disuse and unloading result in decreased bone formation [8, 55-59]. Areas of the skeleton most susceptible to bone loss are weight bearing bones, implicating load as an important mediator of normal bone homeostasis [10, 60-62]. To understand the role of loading on bone formation, we used the NASA designed RWV to model microgravity conditions and hypoxia. Because the family of AP-1 transcription factors plays a role in normal bone formation, we wanted to determine whether RWV induced alterations in osteoblast phenotype correlated with alterations in AP-1. Here we demonstrate that c-fos and c-jun expression decreases to 23% and 55% of unit gravity control levels, respectively in differentiating MC3T3-E1 osteoblasts after 24 hours of RWV conditions. We also showed that c-jun and not c-fos protein levels were decreased after 24 hours of RWV conditions. The effect of c-fos during and AP-1 response is usually transient because of the short half life of the protein. It is possible that the reason we do not detect a difference is because by the 24 hours we analyzed our samples, any c-fos effect may have already occurred and is no longer detectable at the protein level. This decrease in c-fos and c-jun in turn also correlated with a decrease in c-jun and c-fos DNA binding and transactivation, however growth in the RWV was unaltered. When we uncoupled the effect of hypoxia from the RWV, we found that suppression of c- fos and c-jun mRNA expression in the RWV was due to hypoxia and not modeled microgravity. 178 7}. 2..A‘ Our finding that AP-1 is suppressed in the RWV is supported by several studies. One in vivo study demonstrated that c-fos mRNA was decreased by 50% in isolated bone marrow stromal cells after 5 days of hindlimb unloading [47]. Similarly, c-fos expression is impaired in periosteal cells in 14 day hindlimb unloaded rats [63]. Models of decreased loading also demonstrate attenuation of a normally activating AP-1 response. Activation of c-fos mRNA expression by vitamin D3 was suppressed in rat osteoblasts cultured under true microgravity conditions compared to ground controls [64]. EGF induced c-fos expression under clinostat growth is depressed in MC3T3-E1 osteoblasts as well as other cells [65, 66] compared to non-rotated controls [48]. A431 epithelial cells treated with EGF under microgravity conditions also exhibited decreased c-fos and c-jun expression compared to gravity controls [67]. To our knowledge, oxygen tension was not measured in any of these just described experiments. We demonstrate here that differentiating osteoblasts are under hypoxic stress when cultured in the RWV. Supplementation of oxygen results in normoxic conditions in the RWV, thereby allowing us to uncouple the hypoxic component of the RWV from the modeled microgravity component. This uncoupling demonstrated that suppression of c-fos and c-jun expression is due to hypoxia and not modeled microgravity. Knowing this, it would be interesting to determine whether the in vitro and in vivo findings described above are related to the hypoxic environment of unload bone and clinostat culturing rather than direct effects of mechanical unloading. Interestingly, it has been demonstrated that hypoxic conditions can suppress differentiation of several cell types including 179 myeloid, hematopoietic, osteoblastic, and preadipocytic cells, though the role of AP-1 in this hypoxic induced suppression is not known [68-72]. In contrast to the above studies and ours, there are a few studies suggesting that microgravity may enhance AP-1 expression. For example, in true spaceflight experiments, serum induction of c-fos mRNA levels is increased compared to ground controls [73]. Differences between studies could result from differences in the degree of cell differentiation [74-79] and/or extent of serum starvation (quiescence versus proliferation) [79-82]. In osteoblastic ROS17/2.8 cells, expression of all AP1 members except fos B mRNA increased within the first 2 hours of clinorotation and correlated with translocation to the nucleus [83]. While this study addressed the immediate effects, chronic effects of clinorotation were not examined. It is likely that acute responses will differ from chronic responses. In addition, the ROS17/2.8 cell line is a transformed osteoblast cell line which exhibits an inherent alteration in transcriptional regulation as demonstrated by the constitutive expression of alkaline phosphatase and osteocalcin mRNA without regulated proliferation. Thus, these cells may have altered basal and activated AP-1 responses when compared to a nontransformed phenotype. Our data supports the finding that effects from conditions of decreased loading as seen in spaceflight are not permanent, at least after a short exposure of 24 hours. While we are not aware of any papers which address recovery of altered AP-1 in response to decreased loading conditions, several support that restoration to a normal phenotype occurs following alleviation of unloading 180 conditions. For example, rats flown aboard the Soviet mission COSMOS 1129 for 18.5 days were found to have a marked reduction in total bone mineral upon return to Earth which was not completely restored after 29 days postflight [84]. Similarly, cosmonauts from the Russian space station MIR exhibited bone loss within the first month in space which continued to worsen [11]. Upon return to earth, bone mineral density had increased, though was still decreased compared to preflight measurements even after 6 months. Correspondingly, gene expression studies follow patterns of restoration to a normal phenotype [85, 86]. We report here that RWV suppression of AP-1 transactivation is restored to control levels when osteoblasts in the RWV are allowed to recover under normal culture conditions for an additional 24 hours. Most studies demonstrate a partial recovery following unloading conditions, contrary to the full recovery we see with AP-1 transactivation. This suggests that recovery might be a function of the duration of unloading. It is widely demonstrated that AP-1 transcription factors regulate cellular proliferation [87-89]. RWV conditions have also been shown to alter normal cellular proliferation [90-93]. When we subjected osteoblasts to RWV conditions for 24 hours and compared them to unit gravity controls, there was no difference in proliferation. In support of our findings, modeled microgravity conditions had no effect on the growth of human osteoblast-like cells [94]. Similarly, jejunal mucosal cells and hamster kidney cells from space flight missions did not exhibit altered growth compared to ground controls [95, 96]. Contrary to these findings, many cell types (lymphocytes, erythroleukemia cells, skeletal muscle satellite 181 cells, and hematopoietic stem cells) demonstrate a decrease in proliferation following exposure to both true and modeled microgravity conditions [90, 93, 97- 99]. There is also a report that osteoblasts exhibit suppressed proliferation in response to serum induction aboard the STS-56 shuttle flight [100]. While we did not examine serum induced proliferation, a proliferative response would not be expected since our cells are in a stage of differentiation and not proliferation. This is consistent with our results. We also found that 24 hours of RWV conditions did not lead to caspase 3 activation, changes in nuclear morphology or DNA Iaddering suggesting apoptosis was not induced by 24 hours of RWV conditions, consistent with studies by other labs [93, 99]. In fact, some studies demonstrate that culture under true or modeled microgravity conditions prevent or even decrease apoptosis. Human pancreatic carcinoma cells grown under rotary cell culture conditions for seven days showed less apoptotic cells compared to static controls [101]. Radiation induced apoptosis in peripheral blood mononuclear cells is less under modeled microgravity conditions than controls [102]. On the other hand, increases in apoptosis following true and modeled microgravity conditions have been reported in several cell types including osteoblasts, human follicular thyroid carcinoma ML-1 cells, Iuteal cells, Jurkat T cells, and human MlP-101 colorectal carcinoma cells [90, 103-108]. Because our data suggests that apoptosis does not increase in differentiating MC3T3-E1 cells, we considered the possibility that they could be more refractory to RWV induced apoptosis. This could be due to differentiation/stage specific responsiveness of osteoblasts to the RWV 182 conditions or could also be related to the differences in cell lines (osteosarcoma versus nontransformed cells) or differences in RWV culture conditions (high versus low rotation speeds of the RWV). Here, we demonstrate that expression of c-fos and c-jun is suppressed in the RWV due to the hypoxic environment rather than modeled microgravity. Consistent with this finding, AP-1 DNA binding and transactivation were also suppressed. Transactivation was maintained up to 48 hours and was reversible. Previously, we reported that 24 hours of modeled microgravity conditions lead to decreased markers of osteoblast differentiation, namely alkaline phosphatase and osteocalcin [53]. Promoters of these genes contain AP-1 sites which in the case of osteocalcin have been demonstrated to be involved in gene regulation [25, 109-111]. This suggests a potential role for decreased AP-1 in the RWV mediated suppression of osteocalcin and alkaline phosphatase. 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Mol Med, 2001. 7(1): p. 68-78. 194 Table 2-PCR Primers mRNA Cyclophilin cdun jun B jun D c-fos fos B fra 1 Ira 2 Left primer (5'-3') ATT CAT GTG CCA GGG TGG TGA C GAC CTA AGA 'I'I'C GAT CTC ATT GAG T GGA ACA GCG 'ITI' CTA TCA CG CCT ATT TAT GTT TCT ACT CGG GAA C CAT GGT CAC AGA GCT GGA G ACG CCT 'I'I'A GAG AGC ATT ACT GTG AAA ATC CCA GAA GGA GAC AAG AAG CCT TCA TCC CCA CAA TCA AC 195 Right primer (5'-3') CCG TTT GTG TTT GGT CCA GCA 'ITI’ GTG TTA AGG AGC ACT AGA GAA G GGT GGG T'l'l' CAG GAG TTT GT GCG TAA CGA AGA C'IT TAC TGA AAA C TCA GCT TCA GGG TAG GTG AA CTA GGC GTG CTC TCT CTG TAT TAA G ACT CGG AGT AAA AGG AGT CAG AGA G CGT GGA TAG GGA TTG GAC AT f Figure Legends Figure 24. c-fos and c-jun mRNA levels decrease 24 hours following RWV culture. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or subjected to RWV conditions for 24 hours. Whole cell RNA was extracted and 2pg of RNA was subjected to reverse transcription followed by real time PCR to quantitate alterations in Fos and Jun family member expression. Values are expressed relative to cyclophilin expression, averaged, then graphed as a fold difference compared to unit gravity controls (n=4, *p<0.05). A 2% TBE agarose gel was run using samples subjected to real time PCR and stopped during the linear phase of amplification as a representation of graphical data (inserts). Figure 25. c-jun protein levels decrease 24 hours following RWV culture. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or subjected to RWV conditions for 24 hours. Whole cell protein was extracted and subjected to western blot analysis. Figure 26. AP-1 DNA binding is decreased In the RWV. MC3T3 E1 osteoblasts were cultured for 14 days then left at unit gravity conditions (G) or subjected to RWV conditions. After 24 hours, nuclear protein extracts were obtained for TranSignal array analysis or conventional EMSA as described above. TranSignal data demonstrated decreased binding to AP-1 consensus sites in RWV samples compared to controls, while SP1 binding was unaltered (Fig 23). 196 2pg of nuclear extract were also subjected to EMSA to determine alterations in binding of AP-1 to an AP-1 consensus element (Fig 2b). Radiolabeled DNA containing the AP-1 element and nuclear extract protein samples were mixed to allow binding then run on a 1% agarose gel. Gels were dried then exposed to film for visualization of DNA/protein interactions (n=3). Figure 27. c-fos and c-jun DNA binding Is decreased in the RWV. MC3T3 E1 osteoblasts were cultured for 14 days then left at unit gravity conditions (G) or subjected to RWV conditions. After 24 hours, nuclear protein extracts were obtained and DNA/protein complexes were supershifted using c-fos and c-jun specific antibodies. Figure 28. AP-1 transactivation is decreased and reversible up to 48 hours in the RWV. MC3T3 E1 osteoblasts were seeded onto microcarrier beads and cultured for 7 days. On day 7, cells were transfected with an AP-1 dependent luciferase reporter. This reporter was cotransfected with a constitutively active SV40 Bgal expression vector to normalize for transfection efficiency. Transfected cells were either left at unit gravity G) or subjected to RWV conditions for 24 and 48 hours. After the initial 24 hours, one sample was removed and transferred to normal unit gravity culture conditions (Recovery). Cell lysates were analyzed for luciferase activity indicative of runx2 promoter transactivation, and normalized to SV40 Bgal expression. Values represent the average fold difference, relative to unit gravity controls (n=3, *p<0.05). 197 Figure 29. Oxygenation of the RWV to normoxic conditions prevents c-fos and c-jun mRNA suppression. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G), RWV conditions, or RWV conditions supplemented with oxygen (RWV-+02) for 24 hours. Whole cell RNA was extracted and 2pg of RNA was subjected to reverse transcription followed by real time PCR to quantitate alterations in c-fos and c-jun mRNA levels. Values are expressed relative to cyclophilin expression, averaged, then graphed as a fold difference compared to unit gravity controls (n=3, *p<0.05). A 2% TBE agarose gel was run using samples subjected to real time PCR and stopped during the linear phase of amplification as a representation of graphical data (inserts). Figure 30. RWV conditions do not alter osteoblast proliferation. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or RWV conditions for 24 hours. Samples were pulsed with 3H thymidine for four hours and quantitated for 3H incorporation by scintillation counts as a measure of proliferation. Total DNA was also quantitated using DNA fluorometry to determine the total DNA present in G and RWV samples. 3H thymidine incorporation is expressed relative to total cellular DNA in each condition, then graphed as a fold difference relative to unit gravity controls (n=3, *p<0.05). Total DNA is expressed for each condition. 198 Figure 31. RWV conditions do not induce osteoblast apoptosis. MC3T3 E1 osteoblasts were cultured for 14 days and left at unit gravity (G) or RWV conditions for 24 hours. Samples were collected and analyzed for caspase 3 activity as a marker for apoptosis then expressed relative to unit gravity controls (n=3). 199 Figure 24 32310 .851... :8 5.283382 q ..J 1 ... 1 ...fi L. ... Al 0000 .8338 fiscal...» Fn1 cyclophilin rrrunuu 1 o o o o .852... :8 5885.5 200 Figure 24 - continued MB n u .u. M cyclophilin 52326233 D n u I. m b m JunD 2528.40 1100 70:28:“. .30: pissniuacsw 201 Figure 25 (3 FUAHV (2 ciun ~Illflaiggif1acfin 202 Figure 26 26a. AP-1 sp1 G I. 1x 0 e O O 10x e e va - a 1x . . Ie1oxe. 26b. ‘— AP-1 free ‘— probe 203 Figure 27 GRWV *— c-Fos free probe free probe 204 Figure 28 RWV Recovery G RWV - . n q q 2 1 0 883...... 2.... ...am 8532...? 0-1 205 cFoslcyclophlIln cJun/cyclophilin (fold difference) (fold difference) Figure 29 cJun cyclophilin G RWV RWV+02 RWV G RWV +0, cFos cyclophilin G RWV RWV+02 Figure 30 218.542 1 0000 8:28.... 2.... 2.. 3525...... :n . . . . . 5 4 3 2 1 a... <29 .33 207 Figure 31 u - u u u 1 2. 1 3 5 4. 2 0 0 0 0 0 $822.... 2.... >=>=on m ommnmno RWV 208 CHAPTERV Discussion In these studies, the NASA designed RWV was used to model microgravity conditions, decrease loading, and examine effects on MCBT3 E1 osteoblasts. As with other conditions of decreased load, osteoblast gene expression profiles in the RWV suggest suppression of differentiation. Culturing for 24 hours in the RWV is sufficient to suppress markers of osteoblast differentiation, namely alkaline phosphatase and osteocalcin expression. Moreover, transcription factors associated with regulating expression of these genes and progressive osteoblast differentiation were also decreased. Specifically, 24 hours of RWV exposure was enough to suppress runx2 and AP-1 expression, DNA binding, and transactivation. Of the seven AP-1 members, c-fos and c-jun expression significantly decreased following 24 hours of growth in the RWV, while expression of other members did not change. Two separate approaches, use of transcription factor binding arrays and conventional EMSAs, revealed that AP-1 DNA binding was decreased in the RWV. Furthermore, AP-1 dependent luciferase reporters showed that while 24 and 48 hours of RWV conditions resulted in decreased AP- 1 response element transactivation. Removal of osteoblasts cultured for 24 hours under RWV conditions resulted in complete recovery of AP-1 transactivation after 24 hours in control culture conditions. This suggested that while the RWV suppresses AP-1 transactivation, these effects are reversible. A role for AP-1 in cell growth is well established. For this purpose, we tested 209 whether proliferation and/or apoptosis were altered following RWV conditions. Examination of proliferation by 3H thymidine incorporation and apoptosis by caspase 3 activation determined no difference between both culture conditions. Runx2 expression exhibited a similar response to that observed for c-fos and c-jun. Initial studies showed a decrease in runx2 expression as early as 24 hours exposure to RWV conditions. Consistent with this finding, runx2 DNA binding and promoter activity were also suppressed after 24 hours of RWV exposure. Other reports demonstrate runx2 functions through a negative feedback autoregulatory mechanism whereby it regulates expression from its own promoter [1, 2]. Therefore, it is not surprising to expect a decrease in runx2 promoter activity when runx2 DNA binding is decreased. An unexpected finding regarding RWV culture conditions, was that oxygen tension was altered compared to cells grown outside the RWV. Specifically, when cells were grown under control/unit gravity conditions, the media in which they were cultured contained 96.5% atmospheric oxygen. This was in contrast to media from cells grown in the RWV where the percent atmospheric oxygen was only 48.5%. This suggested that in addition to being exposed to randomized g vectors, osteoblasts were also under hypoxic stress. The hypoxic effect was underscored by the finding that cells grown in the RWV also increased expression of the hypoxic responsive genes, GAPDH and VEGF. Because we demonstrated hypoxic conditions within the RWV, we tested whether the c-fos and c-jun suppression was due to hypoxia and not randomization of g vectors. When we cultured cells in the RWV with external oxygen to raise media to 210 normoxic levels, suppression of both c-fos and c-jun expression did not occur. This demonstrated that the c-fos and c-jun suppression we initially observed in the RWV was due to hypoxia and not modeled microgravity. To determine whether runx2 mRNA expression was due to randomized g vectors of the RWV or hypoxia, runx2 expression was analyzed from cells grown under normoxic conditions in the RWV. As with AP-1, runx2 suppression was prevented under normoxic RWV conditions demonstrating, that as for c-fos and c-jun, expression of runx2 was due to the hypoxic environment and not modeled microgravity of the RWV. Growth of cells under normoxic conditions in the RWV is shown here to uncouple expression of runx2 and AP-1 from gene markers of differentiation, namely alkaline phosphatase and osteocalcin. Surprisingly, the suppression of alkaline phosphatase and osteocalcin observed under hypoxic RWV conditions was not prevented under normoxic RWV conditions. Yet suppression of AP-1 and runx2 expression was prevented. This suggests that normoxia is not sufficient for maintaining alkaline phosphatase and osteocalcin expression and factors in addition to runx2 and AP-1 are involved in RWV suppression of osteoblast differentiation and phenotype. This is particularly interesting given that osteocalcin expression is a major downstream target of runx2 activation. An important implication of these findings is that some cell types grown in the RWV may be subjected to hypoxic conditions if their oxygen consumption is greater than the amount supplied by the oxygenating membrane. This must be considered for the interpretation of data derived from RWV experiments. 211 Because some of the effects of RWV conditions on different cell types are also seen under conditions of hypoxia, it is necessary to differentiate between RWV and hypoxic effects. An example of this is the observation that RWV conditions induce expression of nitric oxide in a variety of cell types [3, 4]. Interestingly, hypoxia also induces nitric oxide in these same cells [5-8]. Similarly, it has been reported that RWV conditions induce apoptosis [9]. Again, hypoxia is able to induce apoptosis of these same cell types [10]. Lastly, several reports suggest the RWV is a good system in which cartilage can be grown to be used for implants [11-14]. Coincidently, in addition to being an avascular tissue, in vivo chondrogenesis occurs under low oxygen tension [15—17]. These studies shed light on the possibility that the effects observed by other labs could be due in part or completely to a hypoxic environment and not the RWV itself or modeled microgravity. Beyond the finding that the RWV does not adequately supply sufficient oxygenation for culture conditions, this data has biological implications for both modeled microgravity and hypoxic effects. This work suggests that RWV effects can be separated to at least two components: modeled microgravity and hypoxia. Effects due to modeled microgravity can be further uncoupled from other possible inherent effects of the RWV by growing cells in the RWV in the vertical orientation. Under these conditions, cells experience the same effects of horizontally grown cells except randomized g vectors allowing for the removal of the 9 vector component from the RWV. 212 We have demonstrated that hypoxia is certainly a strong player in the regulation of expression of genes important for normal osteoblast differentiation. We demonstrate here that c-fos, c-jun, and runx2 are all responsive to changes in oxygen tension in osteoblasts. To our knowledge, this is the first demonstration that hypoxia can suppress c-fos and c-jun transcription in osteoblasts. Indeed, several genes important for normal bone growth and development including osteopontin, collagen, and bone sialoprotein among others contain binding sites for AP-1 [18-22]. From this understanding, it is conceivable that osteoblast differentiation under hypoxic conditions could be through suppression of these or other AP-1 regulated genes. Runx2 also displays similar characteristics of c-fos and c-jun. The most popular thought regarding the relationship between runx2 and gene markers of differentiation is that they are directly related. That is increased runx2 expression directly correlates with increased osteoblast differentiation and bone formation, while decreased runx2 expression directly correlates with decreased osteoblast differentiation and bone formation. This is further supported by the idea that the osteocalcin promoter is directly regulated by runx2. These studies demonstrate that these relationships are not always the circumstance. Though we prevent the suppression of runx2 expression in the RWV under normoxic conditions, expression of osteocalcin remains suppressed. It is possible that continued suppression of osteocalcin occurs through the presence of some yet identified transcriptional repressor which increases during conditions of hypoxia. Certainly there are known trans acting repressors of osteocalcin expression including 213 AEF 1, TLE, and MSX2 which could be responsible for directly suppressing osteocalcin expression [23-25]. Runx2 itself has been demonstrated to mediate both activation and repression of promoter expression and it is conceivable that the osteocalcin promoter is likewise regulated [1, 26-28]. Based on this work, we propose that conditions of disuse/unloading as seen in limb immobilization, spaceflight conditions, and chronic bed rest may involve a hypoxic component. While the finding of the hypoxic component of the RWV was unexpected, a hypoxic component to unloading has been demonstrated. In the unloaded turkey ulna studies, increased hypoxic osteocytes and increased expression of HIF-1o in these cells was demonstrated [29]. Furthermore, in vivo lacuno-canalicular fluid flow studies demonstrate that unloading of bone suppresses the delivery of nutrients including oxygen to surrounding bone forming cells [30, 31]. In our studies, the decreased oxygen tension results in decreased runx2, c-fos, and c-jun expression. This hypoxic effect may contribute at least in part to decreased bone formation in vivo. In addition, we demonstrate that decreased load, independent of hypoxic conditions, leads to suppression of alkaline phosphatase and osteocalcin expression which can only be explained by randomization of g vectors or some other component of the RWV that is still undefined. As previously discussed, there is a role for both AP-1 and runx2 in the regulation of alkaline phosphatase and osteocalcin. Because we found the suppression of these transcription factors in the RWV is due to hypoxia, we tested whether the initial suppression of alkaline phosphatase and osteocalcin 214 may likewise be regulated by hypoxia. Differentiating osteoblasts were subjected to 24 hours normoxic RWV conditions then analyzed for changes in alkaline phosphatase and osteocalcin expression. Surprisingly, unlike AP-1 and runx2, normoxic RWV conditions did not alleviate the suppression of alkaline phosphatase and osteocalcin expression(Figure 32). The continued suppression of alkaline phosphatase and osteocalcin expression in the RWV under normoxia was unexpected since AP-1 and runx2 are major players in the regulation of their expression. One possible explanation for this observation is that RWV conditions activate a yet unknown repressor of alkaline phosphatase and osteocalcin expression that is not relieved by normoxia. AEF1, TLE, and MSX2 all mediate suppression of osteocalcin expression [23-25]. It is also conceivable that a yet identified transcriptional activator mediating normal expression of alkaline phosphatase and osteocalcin independent of AP-1 and runx2 is suppressed by RWV conditions and not alleviated by normoxia of the RWV. Factors including CIEBPB, p300 and vitD increase expression of osteocalcin [24, 32, 33]. It is possible that regulation of alkaline phosphatase and/or osteocalcin expression by these or factors such as these may be suppressed which would in turn lead to alkaline phosphatase and osteocalcin suppression. While it is now determined that the hypoxic component of the RWV is not completely responsible for the suppression of alkaline phosphatase and osteocalcin, their continued suppression could contribute to unloading (as in microgravity) induced bone loss not through suppression of AP-1 or runx2, rather through suppression of alkaline phosphatase and osteocalcin directly or indirectly. 215 It is quite interesting that when people think about the function of runx2, it is always thought about in terms of being a “master switch” in mediating osteoblast differentiation and associated alterations in gene expression associated increased bone formation. Our studies demonstrate that runx2 expression can be uncoupled from expression of genes necessary for proper osteoblast differentiation. This in turn would suggest that while runx2 may be a target for loading, additional signals induced by loading may regulate alkaline phosphatase and osteocalcin expression. In turn, these targets which mediate normal bone formation may be more important than runx2 in mediating normal bone homeostasis. With regards to spaceflight in particular, several physiologic alterations occur consistent with hypoxic conditions which could further enhance unloading induced hypoxia and decreased bone formation. In spaceflight conditions, there is decreased flow of blood to lower extremities (sites where decreased bone formation predominates), blood volume and pressure are decreased, and there appears to be a decrease in the number of red blood cells [34]. These factors alone are sufficient to create a hypoxic environment. When the component of loading induced hypoxia is added, spaceflight may induce an even greater decrease in oxygen than in unloading conditions alone. This combination of factors may contribute to the even greater degree of bone loss observed in astronauts compared to unloading conditions alone. While these studies determined hypoxia to be a regulator of c-fos, c-jun, and runx2 expression, the exact molecular mechanism of hypoxic mediated 216 suppression remains to be elucidated. Because the effects of hypoxia alter expression, a greater understanding of transcription of these genes under hypoxia should be increased. One approach is to use c-fos, c-jun, and runx2 promoter reporter constructs containing progressive 5’ deletions. Once a region of the promoter is isolated which is no longer suppressed under hypoxic conditions, known and putative binding proteins necessary for hypoxia responsiveness can be identified by mutaginizing these sites within the promoter. This will allow for the eventual elucidation of the hypoxic regulated factor and/or factors important for mediating the suppression under hypoxia. Furthermore, understanding the transcription factor or factors may lead to understanding the intracellular signaling pathways and eventually the hypoxic sensor in osteoblasts. Similarly, promoter reporter constructs containing progressive 5’ deletions to localize the RWV responsive regions for alkaline phosphatase and osteocalcin can also be performed. It would also prove beneficial to look at the mechanism of osteocalcin suppression without altered runx2 expression. As previously stated, osteocalcin expression is largely thought to be directly regulated by runx2. One of the first things that should be analyzed is the runx2 protein levels. While our studies demonstrate a decrease in runx2 mRNA expression, we have not addressed whether runx2 protein levels are changed. If decreased runx2 protein levels were responsible for the decrease in osteocalcin expression, it would mean that while runx2 protein levels are regulated by randomized g vectors, transcription of runx2 would be regulated by hypoxia. Protein modification might also be 217 responsible for suppression of runx2. While phosphorylation of runx2 on different residues has been shown to activate or suppress runx2 activity, other posttranslational modifications might also mediate repression of runx2 activity at runx2 dependent promoters [35-38]. This could be determined by immunoprecipitating runx2 from nuclear extracts followed by 2D gel electrophoresis to separate and identify modified isoforms. Coimmunoprecipitation studies can also be performed to determine whether corepressors might be responsible for runx2 mediated suppression of osteocalcin. An extension of the understanding of these in vivo results can also be addressed in in vitro systems. The best test of whether true microgravity conditions promote a hypoxic state and associated suppression of c-fos, c-jun, and runx2 expression would be to analyze bone cells from animals flown aboard space flight missions. According to previous data, 24 hours of space flight conditions might be enough to observe suppression of runx2 expression. Because of the lack of feasibility of space flight missions, ground based models of decreased load can be used. As previously described, hindlimb unloaded animals experience decreased loading of suspended limbs. In addition, there is only one in vivo study in avian ulna which demonstrates immobilization (and thus unloading) resulting in hypoxic cells within bone. It would be interesting to see whether different animal models also experience hypoxia and have correlative suppression of runx2 and AP-1. It may be possible to determine whether unloading induced bone loss can be overcome by overexpression of runx2 and/or AP-1 members. While our data suggests that preventing the suppression 218 of AP-1 and runx2 is not sufficient to prevent alkaline phosphatase and osteocalcin suppression, overexpression of AP-1 and/or runx2 may be sufficient to overcome hindlimb unloaded bone loss. A similar approach would be to hindlimb unload transgenic mice overexpressing alkaline phosphatase and osteocalcin and determine whether expression of these factors is sufficient to overcome hypoxia mediated bone loss. Furthermore, it might be worthwhile to measure oxygen tension within bone from people under different conditions of immobilization as in spinal injury and bed rest. Accordingly, targeted angiogenic drugs to bone may provide to prevent bone loss or promote bone formation for people who suffer from hypoxia induced osteoporosis. It might also be interesting to determine whether hyperbaric treatments to conditions of osteoporosis may help in curbing bone loss. 219 REFERENCES 1. 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Franceschi, R.T., et al., Multiple signaling pathways converge on the bea1/Runx2 transcription factor to regulate osteoblast differentiation. Connect Tissue Res, 2003. 44 Suppl 1: p. 109-16. 223 Figure Legend Figure 32. Normoxic RWV conditions do not prevent suppression of alkaline phosphatase and osteocalcin expression. Osteoblasts were cultured for 14 days. Cells were then left at unit gravity (G), subjected to normal horizontal RWV rotation (hRWV) or horizontal RWV rotation supplemented with oxygen to normoxic conditions (hRWV+02). After 24 hours cells were harvested, RNA extracted, and runx2 mRNA levels were determined by real time RT-PCR. Levels were expressed relative to cyclophilin mRNA levels. Values are expressed as an average fold increase +l- SE relative to static control cultures (set at 1) and represent an n of 3 separate experiments; *p<0.05. Insert is a representative agarose gel of PCR products obtained from the linear phase of amplification. 224 Figure 32 6 RM +o2 Alk phosphatase cyclophilin Alkphoslcyclophilin (fold difference) P P P P r‘ O N uh a: on d N G hRWV hRWV+02 6 RM +o2 osteocalcin -I die “I" W" W cyclophilin * OCIcyclophilin (fold difference) P P P P O N 1h O on G hRWV hRWV+02 225