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I 00 O 0 0 00 .0. 0 0 0 o. 0. . 0 . . 1 I . . 0 I 0 . n . 0. 0 v 0.. 0 0 _. . .. _. . . 0 n . . .u . . o 0. V 0 I. 0 0 0 v d a c . .0 .0 'I . . A. _ 0 0 0 0 I n 0 . 0 H03F.0-.0 0 . g ..e.. O pu“ 0..... .I .I.. . 0 . 0.. ... 0 0 o Ivy. .0 . I0 0. . 0..-‘ 00.0..4 .. I .I. . ..- .. ... ...... ......u......p...._..m «.... S.3.........w...:_....u.m.....€.xé.m... xfxgmmflfiwfifififiwWank...» m. .... Hui... ...” . .....v... .....hsuuaaI; ...u... ... ....Jhxxaifua.......Hl... ...... 3.. T... .. 34.0.}... .. 2... ...... 4M ......3.u.afin..........v...-“an“.u..._fi.a..uum..§% .....fi....3.n...0.aum. 5.3. 7 .Hmwfigée ... 4....-. . ... .Y .. 0 O I 0 a. .0 .. . ' I- Il.‘ 'I This is to certify that the dissertation entitled EFFECT OF CYCLIC AMP IN MODULATING CELL DIFFERENTIATION AND SURVIVAL BEHAVIORS presented by LINXIA ZHANG has been accepted towards fulfillment of the requirements for the Doctoral degree in Materials Science and Engineering 8. Biochemistry and Molecular Biology L/ 1 V1.) Auk 6/92,... Major Professor’s Signature XZZY/jo Date MSU is an Affirmative Action/Equal Opportunity Employer LIBRARY Michigan State University 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 5108 KzlProleccaPresIClRClDateDueJndd EFFECT OF CYCLIC AMP IN MODULATING CELL DIFFERENTIATION AND SURVIVAL BEHAVIORS By LINXIA ZHAN G A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Materials Science and Engineering & Biochemistry and Molecular Biology 2010 ABSTRACT EFFECT OF CYCLIC AMP IN MODULATING CELL DIFFERENTIATION AND SURVIVAL BEHAVIORS By LINXIA ZHANG Mesenchymal stem cells (MSCs), originally identified from bone marrow, have gained popularity due to their multilineage differentiation capability and promising results from preclinical and clinical applications. In addition to their well established differentiation routes to mesodermal lineage cells, such as osteoblasts, adipocytes and Chondrocytes, MSCs are also capable of differentiating into neural lineage cells. The ubiquitous messenger cyclic adenosine monophosphate (CAMP) has been used frequently to induce neural lineage differentiation in MSCs. However, a Clear understanding of how CAMP induces MSCs into ftmctional neurons is lacking. Ongoing research employing in vitro pre-differentiated MSCs to treat neuronal diseases, including the combinatorial therapy of CAMP and MSCs for spinal cord injury repair, necessitate a better understanding of CAMP induced differentiation of MSCs to neural lineages. Accordingly, the goal of the current study is to determine the role of CAMP in MSC differentiation to neural lineages. We assessed if CAMP can enable MSCs to gain neuronal function and investigated a potential mechanism by which CAMP regulates MSC differentiation to neural cells. The results suggested that CAMP initiated neuron-like morphological Changes early and induced neural marker expression much later. These two processes are regulated differentially downstream of protein kinase A (PKA). The early-phase neuron- like morphology is the result of cell shrinkage, which gradually decreased with CAMP treatment, whereas the expression of neural markers increased with exposure time. In addition to neural marker expression, CAMP also enabled MSCs to gain some neuronal function, namely inducing a calcium rise upon stimulation by neuronal activators. Further studies suggested that CAMP response element binding protein (CREB) plays a critical role in mediating the calcium rise upon stimulation by the neuronal activators. While CREB exerts a positive affect on calcium signaling, it appears to negatively impact the adoption of a neuron-like morphology, since a dominant negative CREB promoted the appearance of a neuron-like morphology. In addition to differentiation, CAMP also participates in various other important cellular processes, such as cell death and survival. In a separate study we found that saturated free fatty acids (F FAs), i.e., palmitate, initiated cell death in hepatocellular carcinoma cells (HepGZ) cells. CAMP has been shown to be protective of liver cells from cell death due to various insults. Therefore, we also investigated the role of CAMP in palmitate-induced cell death of HepG2 cells. However, we found that CAMP enhanced palmitate-induced cell death of HepG2 cells. CAMP enhanced palmitate-induced mitochondrial fi’agmentation and mitochondrial reactive oxygen species (ROS) generation. Mitochondrial fragmentation precedes mitochondrial ROS generation and may contribute to the mitochondrial ROS overproduction. Fragmentation of mitochondria also facilitated the release of cytotoxic proteins from the mitochondria and subsequent activation of caspases. However, the cell death induced by palrnitate and CAMP was caspase-independent and predominantly necrotic. COPYRIGHT Linxia Zhang 2010 DEDICATED TO MY FAMILY ACKNOWLEDGEMENTS Many people have helped me in different ways during my doctoral study at Michigan State University in these years. I owe my deepest gratitude to all these people, although I may not be able to thank each of them individually here. I am heartily thankful to my advisor, Prof. Christina Chan. I am grateful that she took me as her student and patiently guides me to learn and grow. Without her continuous guidance, support and encouragement, this dissertation would be simply impossible. In addition, she is always enthusiastic about research, accessible, and willing to help, which has helped me to become a better researcher and person, and also has turned into a lifelong fortune for me. I would like to thank Prof. Pat Walton, for providing valuable suggestions and discussions to my research over all these years. I am also gratefirl to Prof. Bill Henry, Prof. Sarat Dass and Prof. David Amosti, to be on my committee and give me insightful suggestions. I feel lucky to have these brilliant people on my committee and learn from them. My current and former lab members made it a great place to work. It is hard to put down all of their names, because there are so many of them. I was delighted to interact with my lab members. The interactions and discussions in the lab inspired me both in research and life. Their support, and more importantly, their friendship in the past six years, has meant so much to me. Thanks. In particular, I would also like to thank the three undergraduate students who worked with me, Linsey Seitz, Amy Abramczyk, and Kendell Paweleck. They helped me with the experiments, and helped improve my oral English. My deepest gratitude goes to my parents and my sister, for always being there for me and giving me constant support and love. I am also deeply grateful to my husband, Yuxin Zhang, for his encouragement, support, love and the joy he brings to me. Last but not least, I would like to thank the Department of Chemical Engineering and Materials Science, the College of Engineering, and Michigan State University, and the funding sources from Quantitative Biology and Modeling Initiative (QBMI) at MSU, National Science Foundation and National Institutes of Health. They made my doctoral study and research life smooth and rewarding. vii TABLE OF CONTENTS LIST OF TABLES ............................... x LIST OF FIGURES ............................................................................................................ xi LIST OF ABBREVIATIONS .......................................................................................... xiii CHAPTER 1. INTRODUCTION ........................................................................................ 1 1.1 Components of the CAMP signaling pathway ............................................................... 2 1.2 CAMP in cell death and survival .................................................................................... 4 1.3 CAMP in proliferation and differentiation ..................................................................... 5 1.4 Mesenchymal stem cells and neural lineage differentiation .......................................... 7 1.5 Specific aims of the current study ............................................................................... 14 CHAPTER 2. CAMP INITIATES EARLY-PHASE NEURON-LIKE MORPHOLOGICAL CHANGES AND LATE-PHASE NEURAL DIFFERENTIATION IN MESENCHYMAL STEM CELLS .............................................................................. 17 2.1 Abstract ........................................................................................................................ 17 2.2 Introduction ................................................................................................................. 18 2.3 Materials and methods ................................................................................................. 19 2.4 Results ......................................................................................................................... 26 2.5 Discussion .................................................................................................................... 45 CHAPTER 3. CREB MODULATES CALCIUM SIGNALING ELICITED BY NEURONAL ACTIVATORS IN MESENCHYMAL STEM CELLS ............................. 51 3.1 Abstract ........................................................................................................................ 51 3.2 Introduction ................................................................................................................. 52 3.3 Materials and methods ................................................................................................. 54 3.4 Results ......................................................................................................................... 61 3.5 Discussion .................................................................................................................... 78 CHAPTER 4. SYNERGISTIC EFFECT OF CAMP AND PALMIT ATE IN PROMOTING ALTERED MITOCHONDRIAL FUNCTION AND CELL DEATH IN HepG2 CELLS .................................................................................................................. 83 4.1 Abstract ........................................................................................................................ 83 4.2 Introduction ................................................................................................................. 84 4.3 Materials and methods ................................................................................................. 86 4.4 Results ......................................................................................................................... 92 4.5 Discussion .................................................................................................................. 108 CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS ................................... 113 5.1 Conclusions ............................................................................................................... 113 5.2 Future directions ........................................................................................................ 116 5.3 Overall conclusions and impact ................................................................................. 122 viii APPENDICES ................................................................................................................. 124 Appendix 1. Isolation and enrichment of rat MSCs and separation of single colony derived MSC .............................................................................................................. 124 Appendix 2. Supplementary methods and figures for Chapter 2 .............................. 134 Appendix 3. Supplementary figures for Chapter 3 .................................................... 141 Appendix 4. Supplementary figures for Chapter 4 .................................................... 144 BIBLIOGRAPHY ........................................................................................................... 148 ix LIST OF TABLES Table 1.1 In vitro studies regarding neural lineage differentiation in MSCs .................... 11 Table 3.1 Primer sets for actin, D1, D2, D3, D4 and D5 ................................................... 56 Appendix table 1.1 Specific reagents and equipments .................................................... 133 LIST OF FIGURES Figure 1.1 Components of the Classical CAMP signaling pathway ..................................... 4 Figure 1.2 Self-renewal and multilineage differentiation ability of MSCs ....................... 10 Figure 2.1 Morphological changes upon CAMP induction ................................................ 27 Figure 2.2 Time-dependent morphological Changes and cell death induced by CAMP ....29 Figure 2.3 CAMP induced apoptosis in MSCs .................................................................. 32 Figure 2.4 Expression of neural markers, NSE, Tujl and GFAP ...................................... 34 Figure 2.5 Calcium imaging in response to neuronal activators ....................................... 36 Figure 2.6 PKA regulates both morphological Changes and neural markers expressions. 39 Figure 2.7 Morphologcial changes and neural markers are regulated differentially ......... 43 Figure 2.8 Transient activation of CREB and regulation of calcium signal by CREB ..... 49 Figure 3.1 Effect of CREB on neural marker expression and calcium signal ................... 63 Figure 3.2 Calcium imaging in response to neuronal activator dopamine ........................ 66 Figure 3.3 Calcium signaling upon stimulation by neuronal activators and ATP ............. 67 Figure 3.4 Expression of dopamine receptors ................................................................... 70 Figure 3.5 CREB affect morphology and apoptosis .......................................................... 72 Figure 3.6 Cell cycle progression, cell cycle gene expression and cell proliferation ........ 74 Figure 3.7 Effect of CREB on CCNDI and p27kipl expression ........................................ 76 Figure 3.8 Effect of CREB on cell cycle distribution ........................................................ 77 Figure 4.1 LDH release and cell death by FFAs treatment ............................................... 93 Figure 4.2 Effect of FFAs on CAMP levels ....................................................................... 94 Figure 4.3 Apoptosis and necrosis by FFA treatment in the absence or presence of F I ...95 Figure 4.4 Effect of palmitate and F1 on cell cycle distribution ........................................ 97 xi Figure 4.5 Triglyceride storage, palrnitate oxidation and cell death ............................... 100 Figure 4.6 Effect of F FAs and F1 on mitochondrial mass ............................................... 101 Figure 4.7 Mitochondrial fragmentation and release of cytotoxic protein ...................... 103 Figure 4.8 Effect of Caspase inhibition on apoptosis and necrosis ................................. 105 Figure 4.9 ROS and cell death ......................................................................................... 108 Appendix figure 1.1 Phase contrast images of rat MSCs ................................................ 130 Appendix figure 1.2 Flow cytometry analysis of MSCs for surface markers ................. 131 Appendix figure 1.3 Colony formation by MSCs and single-colony derived cells ........ 131 Appendix figure 2.1 Self-renewal and multi-lineage differentiation ability of MSCs.... 136 Appendix figure 2.2 Cytoskeleton staining for actin filaments and microtubules .......... 137 Appendix figure 2.3 Morphology of MSCs treated with staurosporine .......................... 137 Appendix figure 2.4 Morphology of MSCs treated with PI ............................................ 138 Appendix figure 2.5 mRNA levels of neural markers NSE, Tujl and GFAP ................. 138 Appendix figure 2.6 PKA regulates morphology and marker expression ....................... 139 Appendix figure 2.7 Morphology of MSCs treated with F I and paclitaxel ..................... 139 Appendix figure 2.8 CAMP levels and live cells in different treatments ........................ 140 Appendix figure 3.1 Evaluation of MSCs expressing Ml-CREB ................................... 141 Appendix figure 3.2 Time-dependent morphological changes upon Fl induction .......... 141 Appendix figure 3.3 LDH release as indication of toxicity of treatment ........................ 142 Appendix figure 3.4 CREB and GO/Gl phase cells ........................................................ 142 Appendix figure 3.5 D1 promoter and expression of ICER and c-fos ............................ 143 Appendix figure 4.1 Oil Red 0 staining for triglyceride ................................................. 144 Appendix figure 4.2 Mitochondrial morphology in different treatments ........................ 145 xii Appendix figure 4.3 Western blot of mitochondrial and cytosolic fractions .................. 145 Appendix figure 4.4 Apoptotic and necrotic labeling by PI (propidium iodide) and Alexa F luor-488 conjugated annexin V ..................................................................................... 146 Appendix figure 4.5 Mitochondrial morphology of cells treated with 0.7 mM palmitate for 12 h ............................................................................................................................ 146 Appendix figure 4.6 Mitochondrial superoxide levels .................................................... 147 (Images in this dissertation are presented in color) xiii LIST OF ABBREVIATIONS AC: adenylyl cyclase ACSF-HEPES: artificial cerebral spinal fluid with HEPES ActD: actinomycin D AKAP: A-kinase anchoring protein AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate Arp2/3: actin-related protein 2/ 3 ATF: activating transcription factor BDNF: brain-derived neurotrophic factor bFGF : basic fibroblast growth factor BHA: butylated hydroxyanisole BME: B-mercaptoethanol B-NGF: nerve growth factor'beta CaMK: Ca2+/calmodulin dependent kinase CAMP: cyclic adenosine monophosphate CCNDl: cyclin D1 CDK: cyclin-dependent kinases CHX: cycloheximide CKI: CDK inhibitor CNTF: Ciliary neurotrophic factor CRE: CAMP response element CREB: CAMP response element binding protein CRF: corticotropin-releasing factor xiv Cu—DIPS: Copper (11) 3,5-diisopr0pylsalicylate D1, D2, D3, D4 and D5: dopamine receptors 1, 2, 3, 4 and 5 db-CAMP: dibutyryl-CAMP DCFDA: dichiorofluorescin diacetate DMSO: dimethylsulfoxide DMU: N, N’-dimethylurea DRIP: dopamine receptor interacting proteins EGF: epidermal growth factor EPAC: exchange protein directly activated by CAMP ER: endoplasmic reticulum ERK: extracellular-regulated kinase ESC: embryonic stem cell FFA: free fatty acid Fl: forskolin and IBMX GFAP: glial fibrillary acidic protein GLAST: glutamate/aspartate transporter GLP-l: glucagon-like peptide-1 GLT-l: glutamate transporter-1 GPCR: G-protein coupled receptor Gia: inhibitory G protein a-subunit Gsot: stimulating G protein (Jr-subunit GSK3I3: glycogen synthase kinase 3B HepG2: hepatocellular carcinoma cells XV hiPSC: human induced pluripotent stem cell HSC: hematopoietic stem cell IBMX: isobutylrnethylxanthine iGluR: ionotropic glutamate receptor IL-6: interleukin-6 KID: kinase inducible domain KIX: interacting domain LDH: lactate dehydrogenase Ml-CREB: CREB with serine 133 site mutated to alanine MAPZ: microtubule-associated protein 2 MEK: mitogen-activated protein kinase kinase mGluR: metabotropic glutamate receptor MMP: mitochondrial membrane permeabilization MSC: mesenchymal stem cell MSK: mitogen and stress activated kinase NeuroD: neurogenic differentiation NeuN: neuronal nuclei NF: neurofilarnent; including light (L), medium (M) and heavy (H) Chain NMDA: N-methyl-d-aspartate NO: nitric oxide NSC: neural stem cell NSE: neuron-specific enolase OB: olfactory bulb xvi Op18: oncoprotein 18 PDE: phosphodiesterase PDGF: platelet-derived growth factor PGEZ: prostaglandin E2 PI: propidium iodide PBK: phosphatidylinositol 3-kinase PKA: protein kinase A PLC: phospholipase C PMP-22: peripheral myelin protein-22 Ptx: Paclitaxel RA: retinoic acid Rb: retinoblastoma protein ROS: reactive oxygen species RSKZ: ribosomal S6 kinase 2 RT-PCR: reverse transcription-polymerase Chain reaction SHP2:(SH)2-Containing phosphotyrosine phosphatase Smac: second mitochondria-derived activator of caspase SVZ: subventricular zone TNF-a: tumor necrosis or TrkA: neurotrophic tyrosine kinase receptor type 1 Tujl: B-III tubulin Shh: sonic hedgehog VA: valproic acid xvii CHAPTER I. INTRODUCTION Signaling initiated by external stimuli can greatly influence intracellular events and impinge on cell fate choices. The ubiquitous messenger cyclic adenosine monophosphate (CAMP) has been extensively studied due to its diverse roles in various important cellular processes. Ongoing research continues to reveal more aspects of CAMP that have not been appreciated before. For example, a recent study suggested that CAMP is a crucial component for maintaining human induced pluripotent stem cells (hiPSCs) in a previously undefined pluripotent state [1]. In contrast to this relatively new role in maintaining pluripotency in hiPSCs, CAMP has long been indicated to exert a role on differentiation, especially in neural lineage differentiation [2, 3]. While research using CAMP as a component for neural differentiation has been on- going for over three decades, its application of neural induction of mesenchymal stem cell (MSC) differentiation is less than a decade old [4]. The underlying mechanism leading to neural differentiation by CAMP in MSCs remains elusive. Given the emerging use of MSCs as a source for treating neuronal diseases, such as Parkinson’s disease and spinal cord injury [5, 6], a better understanding of the cellular behavior to external stimuli, such as agents that induce CAMP production is necessary. Despite its frequent use, the effect CAMP may exert on MSCs with respect to different functions, i.e., proliferation, apoptosis and functional differentiation, has not been extensively examined or investigated. Therefore, a major focus of this thesis is to study the effect and the underlying mechanism of CAMP- induced neural differentiation in MSCs. In addition, CAMP culminated in apoptosis during its induction of neural lineage differentiation in MSCs. A similar pro-apoptotic effect of CAMP was observed in hepatocellular carcinoma cells (HepG2) treated with palmitate. Given that CAMP is primarily protective in hepatocytes and islet cells according 'to the literature [7-14], we sought to determine the potential mechanism by which CAMP could be potentiating palmitate-induced cell death in HepG2 cells. 1.1 Components of the CAMP signaling pathways The universal second messenger CAMP is recognized as an important player in mediating a plethora of cellular processes including metabolism, differentiation, apoptosis and immune responses [15-17]. Generation of CAMP is mainly achieved through adenylyl cyclases, which convert ATP into CAMP [16] (Figure 1.1). At least nine adenylyl cyclases exist, and they are mainly regulated by G-protein coupled receptors (GPCRs) [18, 19]. While all the adenylyl cyclases (ACs) are activated by the stimulating G protein (it-subunit (Gsot), many of them are negatively regulated by the inhibitory G protein Ct-subunit (Get) [19]. Apart from regulation by GPCRs, calcium can also regulate the AC isoforms, either positively or negatively [20, 21]. In addition to being regulated by ACs, intracellular CAMP levels can also be controlled by phosphodiesterases (PDEs), which terminate CAMP signaling by hydrolysis of the 3’ cyclic phosphate bond [22] (F i gure 1.1). The most common downstream effector of CAMP is protein kinase A (PKA), a tetrameric holoenzyme consisting of two catalytic (C) subunits and two regulatory (R) subunits [23]. Two types of PKA holoenzymes exit: the type I holoenzyme, which contains the RI (Rla, RIB) subunits and the type 11 holoenzyme, which contains the R11 (RlIot, RIIB) subunits. The R subunit dimers associate with and inhibit the activation of the C subunits (Ca, CB, Cy) [24]. Binding of CAMP to the R subunits releases the R subunits from the C subunits and thereby enables the activation of the C subunits [16] (Figure 1.1). Regulation of PKA activity is also highly coordinated by the A-kinase anchoring proteins (AKAPs), which target PKA to distinct subcellular locations [25]. AKAPs serve as scaffolding proteins for the formation of multiple protein complexes including kinases, phosphatases and PDEs [26]. Such compartmentalization facilitates the generation of spatioternporal PKA signaling as well as integration of multivalent signaling events [25, 26]. One of the most important PKA targets is the transcription factor CAMP response element binding protein (CREB) [16, 27] (Figure 1.1). CREB is one of the CREB/activating transcription factor (CREB/ATF) family transcription factors that binds to the concensus palindromic CRE sequence TGACGTCA and the half CRE sequence CGTCA/TGACG [28]. Activation of CREB is mediated by phosphorylation at the serine 133 site in its kinase inducible domain (KID) by various kinases, such as PKA, Ca2+/Calmodulin dependent kinase (CaMK), ribosomal S6 kinase 2 (RSK2) and mitogen and stress activated kinase (MSK) [29, 30]. Phosphorylation of CREB at serine 133 helps the interaction of the KID domain with the KID interacting domain (KIX) on the transcription coactivators CBP/p300, which in turn facilitates the recruitment of the basal transcription machinery [31-33]. Transcriptional activation of CREB culminates in expression of genes involved in various events including differentiation, proliferation and survival [34,35]. O . Ligand O :.3333333:(({fififlfifit} iftjfififififitfa'-33333331.3333333333:t:($333:t:t:t:i:i:t:t:t:t:i:iXi33:0: I 0 a _, “a“m Figure 1.1 Components of the Classical CAMP signaling pathway. 1.2 CAMP in cell death and survival The ability of CAMP to influence cell death and survival has been implicated in various cell systems. Whether CAMP protects cells from cell death or promotes cell death differs in different systems and the mechanism remains elusive. CAMP has been indicated to be protective in the survival of various types of neurons, such as sympathetic and sensory neurons [36], spinal motor neurons [37] and cerebellar granule neurons [3 8]. Further studies indicate that CAMP-mediated survival in neurons may be related to the phosphorylation of glycogen synthase kinase 35 (GSK30), whose proapoptotic ability is reduced upon phosphorylation [3 9]. Several studies indicated that CAMP delays apoptosis in neutrophils [40-42], which nevertheless have a short life span and will eventually die by apoptosis [43]. Introducing CAMP to pancreatic cancer cells [44] or pancreatic B-Cells [14] also protected these cells from apoptosis. In hepatocytes, elevation of CAMP protected them fi'om bile acid-induced apoptosis [7-10], which may involve both PKA- dependent and independent mechanisms [9]. CAMP also suppressed tumor necrosis factor a (TNF-or) [11, 12] and F as-induced apoptosis in hepatocytes [l3]. Physiological stimuli that trigger CAMP production, such as prostaglandin E2 (PGE2) and glucagon, are also reported to promote cell survival in many systems [45-47]. Moreover, the protective role of CAMP was observed in hematopoietic stem cells (HSCs) [48], HSCs-derived megakaryocytes [49], epithelial cells [50], endothelial cells [51], vascular smooth muscle cells [52], thyroid follicular cells [53] and leukemia cells [54, 55]. While the majority of the studies suggest a positive role of CAMP on cell survival, a negative impact of CAMP on cell survival has also been reported. Raising CAMP levels was shown to promote apoptosis [56] and potentiate glucocorticoid induced apoptosis in thymocytes [57], enhance Fas-mediated apoptosis in T lymphocytes [58], trigger apoptosis in B-Cells [59] and enhance glucocorticoid stimulated apoptosis in lymphoblastic leukemia cells [60, 61]. Although a protective effect of CAMP has been observed in many different cell types, a deleterious effect of CAMP has been reported predominantly in immune response related cells, to which MSCs are Closely related. 1.3 CAMP in proliferation and differentiation While CAMP stimulates proliferation in some cells, it is generally considered to be growth inhibitory in most cells, especially in cells of mesenchymal origin [62]. A large body of evidence indicates that CAMP induced growth inhibition can be mediated through regulation of cell cycle regulators [63-69]. Cell cycle regulators, such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors (CKIs) and retinoblastoma protein (Rb), act in a concerted manner to control cell progression. CDK activity is positively regulated by cyclins and negatively regulated by CKIs [70]. Activation of CDK4/6 phosphorylates Rb and thereby relieves the inhibitory effect of Rh on the E2F transcription factors, promoting S phase entry [71]. The proliferation inhibitory effect of CAMP has been demonstrated to be related to the down-regulation of cyclin D1 [66, 68], upregulation of CKIs such as p27kipl and p21“ipl [63, 67], and dephosphorylation of Rb [64, 67]. It is believed that the proliferation inhibitory effect of CAMP is related also to the down-regulation of the Ras/Raf-l/MEKl/Z/ERK signaling cascade [72-76], which can be directly or indirectly mediated by PKA [75, 77-79]. CAMP not only affects proliferation, it also influences differentiation, especially of neural lineage cells such as neurons and glias. Early studies that showed CAMP can induce neuronal differentiation were performed in murine neuroblastoma cell lines and normal neuronal cells [80]. One of the most well Characterized models for studying neuronal differentiation is the rat pheochromocytoma PC12 cells [81]. CAMP is reported to induce neuronal differentiation of PC12 cells through PKA-dependent [82] and — independent [83] mechanisms. Regulation of CAMP induced neuronal differentiation of PC12 cells has also been suggested to be mediated by PI3K [82], p38 [84] and ERK [83]. Elevated intracellular CAMP levels also promote morphological differentiation in human neuroblastoma cells [85, 86], human neuronal progenitor cells [87] and neural stem cells [88]. Moreover, the CAMP signaling pathway has been demonstrated to be critical in estrogen-induced differentiation of midbrain dopaminergic neurons [89] and corticotropin-releasing factor (CRF)-induced differentiation of catecholaminergic immortalized neurons [90]. CAMP not only mediates differentiation of neurons, it also regulates differentiation of glia cells, i.e., oligodendrocytes, Schwann cells, astrocytes and microglias, which are important components of the nervous system [91]. Elevation of CAMP led to astrocytic differentiation of cortical precursor cells [92, 93] and rat glioma cells [94, 95], mediated perhaps by interleukin-6 (IL-6) [96]. The glial lineage differentiation induced by CAMP is not limited to aStrocytes; reports have suggested that CAMP can also induce differentiation of progenitor cells into oligodendrocytes [97, 98] and schwann cells [99]. Taken together, these results show that CAMP is a critical component for inducing neuronal or glial tumor cell lines as well as differentiating neural progenitor cells into neurons or glias. 1.4 Mesenchymal stem cells and neural lineage differentiation Mesenchymal stem cells (MSCs) were first identified by Friendenstein and co- workers in the 19603. When cultured in plastic culture dishes, these adherent fibroblast- like cells formed colonies that can differentiate into osteoblasts or Chondrocytes [100, 101]. This worked was later extended by other researchers in the 19808 [102, 103]. Since then, MSCs has been actively investigated and can reportedly be obtained from various sources, including bone marrow, umbilical cord, adipose tissue, skeletal muscle, synovium and postnatal organs and tissues [104-106]. Characterization of MSCs is usually based on expression of surface markers, such as Strol, CD29 (also known as Bl-integrins), CD44, CD71, CD73 (also known as SH3/4), CD90 (also known as Thyl), CD105 (also known as SH2), CD106 (also known as vascular cell adhesion molecule-1 [VCAM-1]) and CD271 (also known as low-affinity nerve growth factor receptor) [107-110]. While MSCs express the aforementioned markers, they do not express HSC-specific markers, such as CD34 and CD45 [108]. As adult stem cells, MSCs have the multipotency to differentiate into various different cell lineages [111], including osteoblasts [112-114], adipocytes [113] and Chondrocytes [115, 116] (Figure 1.2). Besides these cell types, MSCs are also capable of differentiating into hepatocytes [117], myocytes [118] and neural cells [4, 119, 120]. In addition to their multipotent differentiation ability, MSCs can secret a plethora of growth factors that favor tissue repair and regeneration [121]. Moreover, MSCs have irnmunosuppressive features and, when used allogenically, show minimal immune rejection [122, 123]. As such, a number of studies have reported the promising use of MSCs in treating bone defects [124, 125], coronary artery disease [126], osteogensis imperfecta [122, 127], hernatpoietic recovery [128], myocardial infarction [129] and grafl-versus-host disease [130]. Besides the potential therapeutic applications mentioned above, the ability of MSCs to give rise to neural lineage cells also makes them a promising source for treating neuronal diseases such as Parkinson’s disease [5]. Both in vitro and in vivo studies showed that MSCs can differentiate into neural lineage cells, including neurons [131- 139], astrocytes [131, 136, 138, 140] and oligodendrocytes [141]. The motivation to induce MSCs to differentiate into neural lineage cells in vitro before transplantation is desirable for two reasons: 1) pre-differentiation may enhance the firnctional integration of differentiation cells into the lesion site and may also enhance efficacy, and 2) pre- differentiation restricts the differentiation potential of MSCs and therefore reduces the possibility of tumor formation [142]. Various protocols have been applied to induce MSCs to differentiate into neural lineage cells in vitro. A list of these studies is summarized in Table 1.1. In particular, some studies suggest that in vitro pre- differentiated MSCs can survive and migrate to the lesion sites in animal models of Parkinson’s disease [143]. Moreover, others have suggested that transplantation of in vi tro pre-differentiated MSCs improves behavioral recovery in a rat model of Parkinson’s disease [144] and facilitates nerve regeneration in rat models of spinal cord injury [145, 146]. Therefore, MSCs appears to be a promising source for treating neuronal diseases and a better understaning of their ability to differentiate into neural lineage cells will help further their application in cell-based therapies. Ectoderm l Epithelial cell 1 Bone Bone marrow Self-renewal Neuron Connective stromal cell @Cartilage cell ',‘ X“. Fat cell I ‘ Is ‘ s '. -. ‘ \ Bone cell ‘. a _ I‘, Muscle cell Osteoblast v En d od erm Gut epithelial cell Lung cell L Mesoderm Figure 1.2 Self-renewal and multilineage differentiation ability of MSCs. MSCs primarily differentiate into mesodermal lineages such as fat cells and bone cells Transdrfferentratron of MSCs into ectoderrnal and endodermal lineage cells are also reported (dashed lines). 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E @832 mm :03 $8.88 .«o commmofixo rum a $2 mEggnog £78502 am: a Babsw 8 302ng 8m: 6m? 88. 58;»; $358858an 3 "HZ mo nommmoaxo ”MUAFM ha N2 98 mm Z JOE mo :ommmoaxo 6.83 S 93:. 13 1.5 Specific aims of the current study The use of MSCs in therapeutic applications for treating neuronal diseases necessitates a better understanding and identification of intracellular events involved in regulating the induction of MSCs into neural lineage cells. While CAMP is frequently used for in vitro induction of MSCs towards the neural lineage cells, little is known regarding whether CAMP can turn MSCs into functional neurons or how CAMP elicits neural differentiation. Therefore, two of the specific aims are: 1. Investigate the effects of CAMP on MSC neural differentiation and determine whether CAMP can turn MSCs into functional neurons. Previous studies suggested that CAMP is one of the best inducers for differentiating MSCs into neural lineages [153]. However, such evidence mostly relied on the appearance of neuron-like morphology, which may be an artifact rather than true differentiation as suggested with BHA/DMSO induced neural lineage differentiation of MSCs [159]. We observed that CAMP initiated neuron- like morphology changes quickly, which decreased with treatment time, while expression of neural lineage markers increased more gradually, which led to our first hypothesis that these two events may be differentially regulated. Therefore, the first part of this specific aim characterizes the effect of CAMP on the neuron- like morphology and neural marker errpression. The second part of the aim determines if CAMP is able to induce MSCs to achieve some neuronal function along with neural marker expression. This is discussed in Chapter 2. l4 2. Identify the intracellular signaling components that play a major role in cAMP- initiated neural differentiation of MSCs. The results of specific aim 1 suggested that CAMP enabled MSCs to obtain some neuronal fimction in terms of calcium signaling in response to neuronal activators, dopamine, glutamate and KCl. Preliminary results indicated that the CAMP activated transcription factor CREB may be a critical regulator of this process. Therefore, we hypothesized that CREB is involved in regulating calcium signaling during CAMP-induced neural differentiation of MSCs. In this specific aim, overexpression and down-regulation of CREB was performed to determine its role in calcium signaling upon stimulation by the neuronal activators. This is discussed in Chapter 3. The effect of CAMP is not limited to differentiation but also has been shown to protect many cell types from apoptosis [7-13, 36-55]. We observed that the CAMP level was down-regulated in palmitate-treated HepG2 cells, and palmitate-induced cell death. Given that the results gathered from the literature suggested CAMP is anti- apoptotic in HepGZ cells [7-13], we set out to determine if CAMP can protect HepG2 cells from palmitate-induced apoptosis and, if so, the potential mechanisms involved. Specific aim 3 is: 3. Characterize the effect of CAMP on palmitate-induced cell death in HepGZ cells. 15 Saturated free fatty acid (FFA) palmitate is lipotoxic and induces cell death in a variety of cell types [160-162]. CAMP is primarily protective in hepatocytes according to the literature, since it reduces bile acid [7-10], TNF-O. [11, 12] and Fas induced apoptosis [13]. We observed that palmitate but not the unsaturated FFAs oleate or linoleate, down-regulated intracellular CAMP levels [163]. Thus we hypothesize that CAMP may attenuate palmitate-induced cell death in HepG2 cells. However, the effect of CAMP on death and survival may differ depending on what other stimuli are occurring. For example, while CAMP reduced surface antibody-induced apoptosis in thymocytes [47], it potentiated glucocorticoid- induced apoptosis in thymocytes [57]. Therefore, it is likely that even though CAMP protectes hepatocytes from bile acid [7-10], TNF-CL [11, 12] and Fas- induced apoptosis [13], it may not necessarily ameliorate the toxicity exerted by palmitate. As such, we aim to Characterize the effect of CAMP on HepGZ cell survival during palmitate treatment and identify the potential mechanisms involved. This is discussed in Chapter 4. l6 CHAPTER 2. CAMP INITIATES EARLY PHASE NEURON-LIKE MORPHOLOGICAL CHANGES AND LATE PHASE NEURAL DIFFERENTIATION IN MESENCHYMAL STEM CELLS This work is in press of Cellular and Molecular Life Science: Zhang, L., Seitz, L.C., Abramczyk, AM. and Chan, C. CAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells. Cell Mol Life Sci (2010) [Epub ahead of print]. 2.1 Abstract The intracellular second messenger CAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role CAMP exerts on MSCs and whether CAMP can induce MSCs into functional neurons is still lacking. We found CAMP initiated neuron-like morphological Changes early and neural differentiation much later. The early-phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphological Changes occurred prior to the expression of neural markers, it is not required for neural marker expression; and the two processes are differentially regulated downstream of CAMP activated protein kinase A. CAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate and potassium chloride. However, only some of the cells induced by CAMP responded to the three neuronal activators and these cells firrther lack the neuronal morphology, suggesting that although CAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation. 17 2.2 Introduction Mesenchymal stem cells (MSCs) are adult stem cells with multipotency to differentiate into mesodermal lineage cells such as osteoblasts [114], adipocytes [113] and Chondrocytes [115]. Studies also suggested that these Cells have the potential to transdifferentiate into other lineages, such as hepatocytes [117], cardiomyocytes [164], neurons [165] and astrocytes [131]. Several recent studies indicated that the shape of the cell [166] guided by surface cues [167], and matrix elasticity [168] can influence the lineage commitment of stem cells. Thus, these studies suggest that the surface is as important, if not more so, in directing cell lineage and guiding function to follow form. In contrast, we found that cyclic adenosine monophosphate (CAMP) induced the function but not the form. CAMP is a soluble, biochemical cue that is frequently used either alone [4] or in combination with other factors [144, 153, 169] to induce neural differentiation of MSCs. CAMP initiated transient neuron-like morphological Changes that lasted only a few hours. These morphological Changes were the result of cell shrinkage and did not contribute to the later-phase neural differentiation. Similarly, studies using [3- mercaptoethanol (BME), dimethylsulfoxide (DMSO) and butylated hydroxyanisol (BI-IA) to induce neural differentiation of MSCs have attributed the neuron-like morphology to an artifact of cell shrinkage rather than neurite outgrowth [159]. However, unlike BME, which induced Changes in morphology for up to 24 hours [159], CAMP initiated a transient change in morphology for up to 3 hrs that decreased over time. 18 The Classical CAMP signaling pathway involves activation of PKA, which is composed of two catalytic subunits, PKAC, and two regulatory subunits, PKAr [16]. Binding of CAMP to the regulatory subunits dissociates PKAr fiom PKAC, thereby enabling the activation of PKAC [34]. Signaling events initiated by PKAC plays important roles in regulating cell death and survival [170, 171], cell movement and structure [172] as well as differentiation [173, 174]. We investigated whether PKA is involved in regulating both the morphological Changes and neural differentiation and function and whether neural differentiation is contingent upon the Changes in morphology. Although CAMP has been shown to induce neural marker expression [4, 153], we show CAMP also induced a calcium rise, an indicator of neural function, that persisted for at least one week. We found MSCs showed differential responses to neural activators (i.e., dopamine) despite the lack of neuron-like morphology, thus CAMP is able to facilitate neural differentiation but by itself is not sufficient to induce MSCs to terminally differentiated neurons. 2.3 Materials and methods 2.3.1 Materials Forskolin (Sigma) and isobutylmethylxanthine (IBMX) (Sigma) were used to increase intracellular CAMP levels at concentrations of 10 M and 100 M, respectively. H89 (Sigma) and Rp-CAMPS (Sigma) were used as PKA inhibitors at concentration of 2.5 M and 10 M. Actinomycin D (ACtD) (Sigma) was used to 19 inhibit transcription at concentration of l ug/ml. Cycloheximide (CHX) (Sigma) was used to inhibit translation at concentration of 10 ug/ml. Paclitaxel (Ptx) (Sigma) was used to stabilize microtubules at concentration of 0.4 M. The three neuronal activators used during calcium imaging were: 100 M dopamine (Sigma), 100 M glutamate (Sigma) and 50 mM KCl (J.T.Baker). 2.3.2 Cell isolation and culture All procedures in the cell isolation were approved by the Institutional Animal Care and Use Committee at Michigan State University. Bone marrow mesenchymal stem cells were isolated from 6-8 week old Sprague—Dawley female rats as described in Appendix 1. In brief, femurs and tibias from 6-8 week old rat were dissected and the two ends were cut open. The marrow was flushed out with DMEM using a needle and syringe. The cell suspension was filtered through 3 65pm nylon mesh to remove bone debris and blood aggregates. Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 ug/mL streptomycin (Invitrogen) and 100 U/mL penicillin (Invitrogen) and placed in an incubator with a humidified atmosphere containing 5% C02 at 37 °C. Non-adherent cells were removed on the second day after plating. Media was replaced every 3 to 4 days until the cells reach 80~90% confluence. Confluent cells were detached by 0.25% trypsin- EDTA (Invitrogen) and plated for further experiments. Primary cortical neurons were isolated as described in [175]. In brief, animal heads were decapitated from l-day old Sprague-Dawley rat pups. Cortical neurons 20 were obtained from the brain and cultured on poly-L-lysine coated plates in cortical media [DMEM (Invitrogen) supplemented with 10% horse serum (Sigma), 2 mM glutamine (Invitrogen), 100 ug/mL streptomycin (Invitrogen) and 100 U/mL penicillin (Invitrogen)] in an incubator with a humidified atmosphere containing 5% C02 at 37 °C. Cells were used within 3 days after isolation. 2.3.3 Live cell imaging Cells were cultured in 4-well Chambered glass bottom plate (Thermo Fisher Scientific). Before taking images, media was Changed to 0.5 ml Leibovitz (Liz) media (Sigma). The plate was mounted in a temperature controlled Chamber set at 37 °C on the microscope station. 0.5 ml Liz media containing 20 [AM forskolin and 200 M IBMX was added to the Chambered well to achieve a final concentration of 10 uM forskolin and 100 M IBMX. Phase contrast images were captured by confocal microscope Olympus Flroiew 1000 at intervals of 5 minutes. 2.3.4 CAMP assay Intracellular CAMP levels were measured by a competitive immunoassay from Assay Designs (Assay Designs) according to the manufacturer’s instructions. In brief, cells were lysed with 0.1M HCl and the supernatant collected. The CAMP in the samples or standards was allowed to bind to a polyclonal CAMP antibody in a competitive manner with alkaline phosphatase-conjugated CAMP. Cleavage of a substrate by the alkaline phosphatase is inversely proportional to the CAMP level in 21 the samples or standards. Colorimetric readings were taken by SPECTRAmax plus384 fiom Molecular Device at 405 um. All the readings were normalized to protein levels (pg/ml) by Bradford assay. 2.3.5 Caspase 3 activity assay Caspase 3 activity was measured by a kit from BIOMOL (BIOMOL) according to the manufacture’s instructions. Briefly, cell extracts were incubated with substrate AC-DEVD-AMC. The Cleavage of the substrate generates fluorescence which is proportional to the concentration of active caspase 3 in the cell extracts. Fluorescence was measured by Spectra MAX GEWNI EM plate reader at excitation of 360 nm and emission of 460 um. All the readings were normalized to protein levels (pg/ml) by Bradford assay. 2.3.6 Western blot Whole cell extracts lysed with CelLytiC (Sigma) were assayed for protein concentrations by Bradford assay (Bio-Rad). 15-30ug protein samples were separated by 10% Tris-HCl gel and transferred to nitrocellulose membrane. Membranes were then blocked in 5% milk and 0.05% Tween 20-TBS (tris buffered saline) (USB corporation) for one hour and incubated with primary antibodies, NSE (neuron- specific enolase) (BIOMOL), Tujl (BIB-tubulin) (Millipore), GFAP (Glial fibrillary acidic protein) (DAKO), GAPDH (Cell signaling), PKAC (R&D Systems), pPKAc (Cell Signaling) and ser133 phosphorylated CREB (pCREB) (EMD Chemicals) 22 overnight at 4 °C. Anti-mouse or anti-rabbit I-IRP-conjugated secondary antibody (Thermo Scientific) were added the second day after primary antibody incubation. The blots were incubated for one hour and then washed three times with 0.05% Tween 20-TBS. The blots were then visualized by SuperSignal west femto maximum sensitivity substrate (Thermo Scientific). 2.3.7 Immunocytochemistry For staining against Tujl, cells were fixed in PBS containing 3.7% formaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 (Research Products Internationals) for 20 minutes at room temperature. After washing with PBS three times, cells were blocked in 1% BSA (bovine serum albumine) (US Biological) for 20 min and incubated with Tujl (Millipore) antibody at room temperature for one hour. Cells were then washed with PBS three times and incubated with Alexa Fluor 488- conjugated anti-mouse IgG secondary antibody (Invitrogen) for one hour at room temperature. Stained glass coverslips were washed three times with PBS and mounted in ProLong Gold (Invitrogen). Fluorescence images were taken by confocal microscope Olympus Flroiew 1000. Triple staining for actin filaments, microtubules and nucleus was performed as previously described [176]. In brief, actin filaments were stained with Texas Red-X phalloidin (Invitrogen), microtubules were stained with a—tubulin (Invitrogen) primary antibody followed by Alexa Fluor 488-C0njugated anti-mouse IgG secondary antibody (Invitrogen), and the nucleus was stained with DAPI (4’, 6-diamidino-2- 23 phenylindole) (Invitrogen). Stained glass coverslips were mounted in ProLong Gold (Invitrogen). Fluorescence images were taken using an Olympus Flroiew 1000 confocal microscope. 2.3.8 Annexin Vand PI (propidium iodide) staining Apoptosis and necrosis were measured by the annexin V and PI (propidium iodide) staining kit (Invitrogen), respectively, according to the manufacturer’s instructions. In brief, cells were stained with Alexa Fluor 488 conjugated annexin V and P1 in 1X annexin binding buffer for 15 minutes at room temperature and then subjected to flow cytometry analysis by BD FACSVantage. Early apoptotic cells were identified as those stained by Alexa Fluor 488 but not PI, late apoptotic cells were those stained by both Alexa Fluor 488 and PI, and necrotic cells were those stained by P1 but not Alexa Fluor 488. 2.3.9 Calcium imaging Calcium imaging was performed according to the protocol described in [158]. Cells were cultured in 4-well Chambered cover-glass (Thermo Fisher Scientific). After the desired treatment, the cells were loaded with 4 M F luo-4 (Invitrogen) in ACSF- HEPES (artificial cerebral spinal fluid with HEPES: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgC12, 2.5 mM CaClz, 1 mM NaH2P04, 26.2 mM NaHCO3, 11 mM dextrose, 10 mM HEPES, pH=7.4) for 30 min at 37 °C. Excess dye was removed by washing cells with PBS twice placing into a 37 °C chamber on the stage of Olympus Flroiew 24 1000. 0.5 ml ACSF-I-IEPES was added to the well to begin imaging. Images were captured every 1.137 seconds and fluorescence intensity is represented by a spectral table (warmer colors represent higher intensity whereas cooler colors represent lower intensity). After 15~20 images, 0.5 ml ACSF-HEPES buffer containing the following drugs were added: 200 M glutamate (final concentration 100 pM), 200 [AM dopamine (final concentration 100 M), 100 mM KCl (final concentration 50 mM), or 200 M ATP (final concentration 100 uM). A total of 200~300 images were recorded and the data was analyzed by the Flroiew 100 software. Changes in the fluorescence intensity of the Ca2+ signal are represented as F/Fo. The percent of responsive cells is calculated as the number of cells with a F/Fo signal greater than 20% divided by the total number of cells. 2.3.10 Cell counting Cells were trypsinized by 0.25% trypsin—EDTA (Invitrogen) and an equal volume of media was added to inactivate the trypsin. Number of cells was determined by diluting the cell suspension 1:1 with 0.4% trypan blue (Sigma) and then counted on a hemocytometer. 2.3.11 Stable cell line expressing dominant negative CREB MSC were transfected with the empty control pCMV vector containing neomycin resistance and the dominant negative CREB mutant (serine 133 mutated to alanine) Ml-CREB (a kind gift from Dr. David Ginty) using lipofectomine 2000 (Invitrogen). 25 24 hours after transfection, cells were trypsinized and replated at low density in media containing 500 ug/ml geneticin (Invitrogen) for selection The geneticin-containing media was replaced every 3 days for two weeks. Colonies formed from surviving cells were isolated by Cloning cylinders (Sigma) and maintained in culture media containing geneticin. 2.3.12 Statistical analysis All experiments were performed at least three times and results were shown as mean 1- standard deviation. Statistical analysis were Carried out by an unpaired, two tail Student’s T-test. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001. 2.4 Results 2.4.1 CAMP induces early-phase neuron-like morphological changes Deng et al. showed that upon exposure of human MSC to CAMP elevating agents, 1 mM dibutyryl-CAMP (db-CAMP) and 0.5 mM IBMX, for two days, the cells exhibit neuron-like morphology [4]. However, we found the neuron-like morphology occurred much earlier than previously reported. Uninduced MSCs exhibited flat-like morphology (Figure 2.1A), whereas MSCs induced with 10 [1M forskolin and 100 M IBMX (abbreviated as F1) showed neuron-like morphology within an hour of induction (Figure 2.1B). These MSCs isolated from rat were characterized as described previously [177]. They have the ability to self-renew as well as undergo 26 multilineage differentiation to other cell lineages such as adipocytes and osteoblasts (Appendix figure 2.1). A recent study attributed the morphological Changes to an artifact of cell shrinkage rather than neurite outgrth [159]. We imaged live cells to determine whether the neuron-like morphology induced by CAMP was also a result of cell shrinkage. As the induction time increase, the cytoskeleton progressively retracts towards the cell center (Figure 2.1C-F). Microtubules and actin filaments staining confirmed the reorganization and retraction of the cell body towards the cell center. The retraction appears incomplete, with partial disruption of the cytoplasm in some of the cells (Figure 2.18 and Appendix figure 2.2, arrows). As with the previous study, the CAMP-induced neurite-like structure is due to a disruption in the cytoskeleton and cell shrinkage rather than neurite outgrowth. A B zo—rrm 2W1 Figure 2.1 Morphological Changes upon CAMP induction. (A) Morphology of uninduced MSCs. Green: microtubules; red: actin filaments; blue: nucleus. (B) Morphology of MSCs treated with 10 uM forskolin and 100 uM IBMX (F1) for I hr. (C-F) Live cell images of cells treated with F l for 15 minutes (C), 30 minutes (D), 45 minutes (E) and 60 minutes (F). 27 2.4.2 Neuron-like morphology decreases along time A large percentage of cells developed neuron-like morphology within an hour after FI treatment (Figure 2.2A, denoted by the arrow head), with an appreciable number still showing neuron-like morphology three hours later (Figure 2.2B and 2.2K). However, unlike the previous study with BME which showed continuous changes in morphology [159], the population of cells with altered morphology decreased with increasing treatment time (Figure 2.2K), with fewer cells showing neuron-like morphology 6 (Figure 2.2C), 12 (Figure 2.2D) and 24 hrs (Figure 2.2B) afier F 1 treatment. By the second day, even with fresh induction media, essentially no Change in morphology was observed (Figure 2.2F-G and Figure 2.2K), suggesting that the neuron-like morphology cannot be re-induced by F1. Similar neuron-like morphology was initiated by the apoptosis inducer staurosporine (Appendix figure 2.3), therefore we evaluated whether the morphological Changes induced by F1 treatment were due to apoptosis. However, the rise in apoptotic cells did not occur within the first 3 hrs after FI treatment (Figure 2.2L), during which maximal changes in morphology resulted (Figure 2.2K), suggesting the Change in morphology was not likely due to apoptosis. 28 .dea “...... .Amuev mzoo \H 5558 +5 ”.38 Nacho: 3:8 +\_ 5558 +5 3.28 38328 83 5:8 K 5358 .5 8:8 9:03.85 atom .98: vm 28 mg 6 .m J Com E ~23 Cogent 33 90 503338.. 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Disruption of the cytoskeletal structure can lead to cell rounding and even detachment, which can result in anchorage-dependent apoptosis called anoikis [180]. Since CAMP elevation induced a disruption of the cytoskeletal structure in the MSCs (Appendix figure 2.2), we assessed whether the morphological Changes led to apoptosis. Initially, FI treatment disrupted the cytoskeletal structure in a large number of cells (Figure 2.2A-C). However, most of the cells with Changes in morphology remained attached and apoptosis or necrosis was not observed within the first few hours (Figure 2.2L). As FI treatment continued, some cells that underwent morphological changes began to round up (Appendix figure 2.4, arrows) and detach from the surface, likely due to a loss in their ability to anchor (Appendix figure 2.4, arrow head). The cells that round up (Figure 2.3A, arrows) also showed positive staining against annexin V (Figure 2.3B-C, arrows), indicating that they have become apoptotic. The number of detached cells increased after 12 hours, with cells floating after 24 hours of treatment (data not shown), corresponding to the time at which the cells stained for apoptosis (Figure 2.2L). Apoptosis increased significantly after 24 hours (Figure 2.2L) and was further enhanced after 48 hours of F1 treatment (denoted as day 2), albeit not statistically (Figure 2.3D). Since additional morphological changes did not occur on the second day of F1 treatment (Figure 2.2K), i.e., very few cell rounding and detachment, correspondingly increases in apoptosis were not observed (Figure 2.3D). 31 Concomitantly, caspase-3 activity, another indicator of apoptosis, increased significantly upon Fl treatment but remained constant during the second day of treatment (Figure 2.3E). Accordingly, these results suggest that a disruption of the cytoskeletal structure, induced upon CAMP elevation, may have resulted in subsequent apoptosis of ~ 10% of the MSCs. D §10 E I; " ”$11., 3’0 5 =1 D "It 3 3 ‘ " .ma 2 .2 .. ° 4 w——,** j ‘5 6 '2 £3 .2 3 2 b «a 4 Ig 2 .2 0 § 2 .1“. I a I a ~ 0 < 0 =é-5 U Ctrl Flld F12d early apoptotic late apoptotic necrotic Figure 2.3 CAMP induced apoptosis in MSCs. (A-C) Phase contrast and fluorescence images of apoptotic cells labeled with Alexa Fluor-488 conjugated annexin V. Cells were treated with 10 uM forskolin and 100 uM IBMX (F1) for 24 hours. (A) Phase contrast image of MSCs treated with F1 for 24 hours. (B) Apoptotic staining with Alexa Fluor-488 conjugated annexin V. (C) Overlay image of (A) and (B). (D) Apoptotic and necrotic labeling by Alexa Fluor-488 conjugated annexin V and P1 (propidium iodide), respectively, of control cells and cells treated with F l for 1 day and 2 days. Early apoptotic cells: Pl- annexin V cells; late apoptotic cells: Pl+ annexin V+ cells; necrotic cells: PI+ annexin V cells (n=3). (E) Caspase-3 activity of control cells and cells treated with F1 for 1 day and 2 days. ‘: p<0.05, "z p<0.01, ""2 p<0.001. 2.4.4 CAMP induces late-phase neural differentiation 32 Unlike the early-phase Changes in morphology which occurred quickly, neural differentiation took much longer. A previous study using DMSO/BHA to induce neural differentiation of MSCs did not observe an increase in mRN A level of neuron- specific enolase (NSE) [159], while we observed an increase in both mRNA (Appendix figure 2.5) and protein levels (Figure 2.4A) of neural markers. Fl treatment increased the expression of neuron markers, NSE and neuron-specific class B-III tubulin (Tujl), as well as astrocytic marker, glial fibrillary acidic protein (GFAP) (Figure 2.4A and 2.4C), but not within the first 6 hours (Figure 2.4B and 2.4D), whereas the Changes in morphology peaked within the first 3 hours (Figure 2.2K). An increase in the expression of NSE and Tujl was detected 12 hours after FI treatment (Figure 2.4D) whereas an increase in GFAP was not detected until well after 12 hours (Figure 2.4A), suggesting that neural marker expression is a late-phase response as compared with the morphological Changes. Immunostaining for the neuronal marker Tujl further support the gradual increase in the neural marker expression. Untreated cells do not express Tujl (Figure 2.4B), but a few cells gained Tujl expression after two days of F1 treatment (Figure 2.4F), with more cells expressing Tujl a week after FI treatment (Figure 2.4G). Nonetheless, the cells that expressed Tujl look distinctly different from primary neurons (Figure 2411'). 33 A Ctrl Flld F12d 3 Fl 0h 1h 3h 6h 12h NSE «.... d NSE .- .....- ....- ...... ..- Tujl ...... .. Tujl GFAP -- '- - GFAP —- -- -- -' ~— GAPDH ——- GAPDH -..-u...- C D 9.3 $ 5 g .r: 2.5 f. 7 ° “#55 * NSE a: E Iujl E. 6 Emir .53 2'0 IGFAP a .2 I61‘ P m l ‘3 w 15 U) * a t; 4 f if 3 *** g 21 :3 E.” 0 Z r inflin_ 12h CE. ctrl FIld FIZd 'l—> 0h lh H Figure 2.4 Expression of neural markers, NSE, T ujl and GFAP. (A) Protein levels of NSE, Tujl, GFAP and GAPDH of uninduced cells (ctrl), cells treated with 10 [1M forskolin and 100 M IBMX (F1) for 1 day (FIld) and 2 days (FIZd). (B) Protein levels of NSE, Tujl, GFAP and GAPDH of cells treated with F1 for 0, l, 3, 6 and 12 hours. (C) Quantification of NSE, Tujl and GFAP levels of the blots shown in (A) (n=3). (D) Quantification of NSE, Tujl and GFAP levels of the blots shown in (B) (n=3). ‘: p<0.05; *“z p<0.001. (E-H) Tujl staining of uninduced MSCs (E), MSCs treated with F l for 2 days (F) and 7 days (G) and primary neurons (H). The induced cells also gained some neuronal function despite their distinct lack of neuronal morphology and features. Neuronal function was assessed with several neuronal activators, 1) dopamine, which stimulates Ca2+ signal through the dopamine receptors [181] or through potentiation of the N-methyl-D-aspartic acid (NMDA) receptors [182], 2) KCl, which stimulates Ca2+ signal through voltage-gated ion Channels [183], and 3) glutamate, which induces Ca2+ signal through the glutamate receptors [184]. Figures 2.5A-D are representative images of F1 treated cells upon 34 stimulation with 100 uM dopamine, with time-dependent fluorescence intensity profiles of four individual cells shown in Figure 2.5B. Intracellular CaZ+ levels remained fairly constant before stimulation (Figure 2.5A and 2.5E, position denoted by A). Shortly afier dopamine supplementation (Figure 2.5E, arrow), some cells showed a Ca2+ signal within 10 seconds, and others peaked more slowly, taking up to 34 seconds (Figure 2.5E). The pattern of Ca2+ signaling also varied, with some cells responding with a single Ca2+ peak (Figure 2.5E, Cell-1 and Cell-3) and other cells being repeatedly excited (Figure 2.5E, Cell-2 and Cell-4). Uninduced cells and cells treated with F I for 1, 2 and 7 days were quantified for their neuronal function upon stimulation by dopamine, KCl and glutamate (Figure 2.5F). Around 40% of the uninduced MSCs showed a response to dopamine, which increased to 60% after one day and 80% after 7 days of F1 treatment (Figure 2.5F). Only 12% of the uninduced MSCs responded to KC] stimulation, which increased to 40% after Fl treatment (Figure 2.5F). Few uninduced cells responded to glutamate, with about 20% of the cells glutamate responsive after a week of F1 induction (Figure 2.5F). These results suggest that PI induction enabled MSCs to gain some neuronal function. However, the heterogeneity in their response to neuronal activators (Figure 2.5F), and the lack of neuronal morphology (Figure 2.4G) indicate that the cells are not terminally differentiate 35 .88.on ”...... .dea H.... 21.5 60. 28 om 58 085823 21 o3 65538 21 03 8 55%“: 85 38 A. 58 88 N S8 _ cow mom—2 8265 E .mDmE 826:5: Co 85888 05 .«o :ocmoczasz CV .838; 5 885 05 9 mwcommotoo D .U .m .< Co 85303 .8528 8588 .«o “58 05 88865 38.2.. .05: 8>o Amv 5 n32? 230 888—8 58 mo 8an .5885 8888875 at .8855? 8588c 8am 8288 mm 58 em .2 .5 855253 055858 288 85598 683 .8me— 85825 cocoomoaoam 832 2865 528 838 58 85885 888225 83.5 8855 228 883 638 _g8mm m E 888888 885 880885..— .05885 21 co“ 553 85253 58 >8 _ no.“ out NEE 21 o2 28 56558 21 2 .53 8265 63.4 5-5 ADM 58 085835 £5588 £85588 6:058 05 9 080mm?— 5 m5w§5 55280 m.~ 955% 36 COODm OE— 20 Ex 80 02 2:8 V pom o o od om mo 00 S . d m 3 m ov u . 0 0..... m. m — m: a . m 2:- AL 8 m 0.0 m. BEN. m m N m 2:.” F 3 % . a Eu 0 .12.. , ( N-:0o|1 _ o m i... [:HL 2: n. 7:8... m Wm m < m «mug m 3H“ 0 St 05838 9528 08.8m— 058320 w528 8o¢ 0.5% 37 2.4.5 PKA regulates both the changes in morphology and neural marker expression As shown above, the percentage of cells with changes in morphology peaked an hour afier FI treatment and decreased with treatment time (Figure 2.2K). The intracellular cAMP levels paralleled the changes in morphology, peaking also an hour after FI treatment and decreasing over the next few hours to a constant level that was maintained for the next 12 hrs (Figure 2.6A). Although fresh media containing F1 was added on the second day, the intracellular cAMP level did not increase (Figure 2.6A), perhaps, because of desensitization of the agonist-induced receptor [185]. Since cAMP can trigger a variety of intracellular events through the activation of the ' classical downstream component protein kinase A (PKA) [16], we therefore assessed whether PKA activation is involved in regulating the changes in morphology and neural marker expressions. The activity of PKA can be determined by the level of phosphorylation of threonine 197 on the PKA catalytic subunit (PKAC) [186, 187]. Upon FI treatment, phosphorylated PKA (pPKAc) increased transiently and then returned to below basal level, whereas the total PKAC level dropped continuously, with both remaining stable after one day (Figure 2.6B-C). Inhibiting PKA activity with H89 prevented the FI-induced morphological changes (Figure 2.6D-E) as well as the neural marker expressions (Figure 2.6F-G). Similar results were obtained using another PKA inhibitor Rp-cAMPS (Appendix figure 2.6), suggesting that PKA mediates the onset of both the morphological changes and neural marker expression. 38 Figure 2.6 PKA regulates both morphological changes and neural markers expressions. (A) Intracellular cAMP levels of control MSCs, MSCs induced with 10 M forskolin and 100 M IBMX (F1) for 1, 3, 6, 12 and 24 hours (Day 1), and MSCs induced again with F1 for l, 3, 6, 12 and 24 hours, following a 24-h FI induction period (Day 2) (n=3). (C) Western blotting results of PKAC and pPKAc levels of control MSCs, MSCs induced with F1 for 1, 3, 6, 12 and 24 hours, and MSCs induced with F1 for 1, 6 and 24 hours following a 24-h FI induction period. (C) Quantification of PKAC and threonine 197 phosphorylated PKAC (pPKAc) levels as in (B) (n=4). *: p<0.05, **: p<0.01, ***: p<0.001 as compared with the corresponding control condition. (D) Phase contrast image of MSCs induced with F1 for 1 hour. (E) Phase contrast image of MSCs induced with F1 in the presence of 2.5 uM PKA inhibitor H89 for 1 hour. (F) Quantification of NSE, Tujl and GFAP expression for control MSCs, MSCs induced with PI for 1 day in the absence or presence of PKA inhibitor H89 (n=3). (G) Expression of neural markers, NSE, Tujl and GFAP, for control MSCs, MSCs induced with F1 for 1 day in the absence or presence of H89. 39 £3 E 700 \ “g 600 c: 500 M <' 400 .. O S 300 2 E 200 -- —- 100 i D1 2 0 _ 7 - i.‘ - - --— 5 Oh lh' 3h 6h 12h 2411' lb 3h 6h 12h 24h cn'l Day] Fl treatment 3. Day2 F 1 treatment B F 00 83.5. . c_trl Dayl Fl Day2 Fl E30: acul . . H-H89 Oh 1b 3b 6h 12h 24h 1h 6h 2411.325; ... 2%? p-PKAcu-n an an m - w' W W "‘- “32.0; 'FHH89 D '. A4 7 PKAC ~~-w-~ ,. . 31.53.---“ 5103—.- GAPDHHHWI—nmflmuw E0593, 20.03 C E, 1.6 ..... G g 1.4 1 2 3 4 2g, 1.2 Fl - - + + '13 1.0 ”39 ' + ' + Q) :2 0.6 i E 0,4 Tujl k 2w G. 8 0-2 GFAP "' “- - .... § 0.0 3 °- 0h 1h 3h 6h 12h 24h lh 6h 24h GAPDH"""“"" ctrl Dayl Fl treatment Day2 F 1 treatment 40 2.4.6 Morphological changes and neural marker expression are differentially regulated Although the changes in both morphology and neural marker expression are regulated by PKA, the neuron-like morphology occurs early and very quickly, whereas the neural marker expressions appear much later and more slowly. Since the morphological changes appear prior to changes in neural marker expression (Figure 2.1 and Figure 2.4), we assessed whether the morphological changes are required and thus precede the gain of neural marker expression. The microtubule stabilizer paclitaxel (Ptx) was used to inhibit the changes in morphology. Microtubules are dynamic polymers that undergo polymerization and depolymerization powered by energy from GTP hydrolysis [188]. Binding of Ptx to the microtubules prevents the depolymen'zation and stabilizes the microtubules [189]. Since dynamic polymerization and depolymerization are required for the assembly of the mitotic spindles, applying Ptx may inhibit mitosis and cause subsequent apoptosis [190, 191]. To minimize the toxicity induced by Ptx while still inhibiting the onset of morphological changes, the cells were treated with F1 in the presence of 0.4 uM Ptx for only 3 hours and the media was subsequently changed to F I media for another 21 hours. Stabilized cytoskeletal structure was maintained upon removal of Ptx from the FI media (Appendix figure 2.7). This could be due to the inability of the media to initiate another intracellular cAMP peak (Appendix figure 2.8A) required for regulating the morphological changes (Figure 2.2K and 2.6A). The number of viable cells 21 hours after replacing the induction media (a total of 24 hrs including the 3 41 hour pretreatment) remained constant, thus Ptx exposure did not adversely affect cell viability (Appendix figure 2.8B). Although Ptx inhibited changes in morphology (Figure 2.7A-B), it did not prevent the increase in neural marker expression (Figure 2.7C-D), suggesting neuron-like morphology is not required for neural markers expression. The morphological changes occur very quickly (beginning minutes after F1 treatment), thus is unlikely that gene or protein synthesis is required for this event. Indeed, inhibiting transcription with actinomycin D (ActD) and translation with cycloheximide (CHX) did not impact the morphological changes (Figure 2.7E-F), but did reduce the neural marker expression upon treatment with FI (Figure 2.7G-H). Thus transcription and translation are required for neural marker expression. ActD and CHX affect transcription and translation on a global level and treatment for a long period may cause cell death. Therefore as with the paclitaxel treatment, the media supplemented with ActD or CHX was replaced after 3 hours and the viability of the cells was assessed after 24-hours of treatment. Supplementing the F I treatment with ActD or CHX did not significantly change the total number of viable cells (Appendix figure 2.8B). 42 Figure 2.7 Morphological changes and neural markers are differentially regulated. (A) Appearance of neuron-like morphology of MSCs induced with 10 M forskolin and 100 M IBMX (F1) for 1 hour. (B) Cell morphology of MSCs induced with F1 in the presence of 0.4 uM microtubule stabilizer paclitaxel (Ptx) for 1 hour. (C-D) Expression of neural markers, NSE, Tuj l and GFAP, in control cells, cells induced with F1 for 1 day in the absence or presence of Ptx (n=3). For the FI treatment, cells were treated with F1 or F1 plus Ptx for 3 hours and then changed to fresh F1 media to reduce toxicity from Ptx treatment. (B) Appearance of neuron-like morphology of MSCs induced with F1 for 1 hour in the presence of l ug/ml Actinomycin D (ActD). (F) Appearance of neuron-like morphology of MSCs induced with F1 for 1 hour in the presence of 10 ug/ml Cycloheximide (CHX). (G-H) Expression of neural markers, NSE, Tujl and GFAP, in control cells, cells induced with F1 for 1 day, in the presence or absence of ActD or CHX. For the F I treatment, cells were treated with F I or F1 plus ActD/CHX for 3 hours and then changed to fresh Fl media to reduce toxicity fi'om ActD/CHX treatment *: p<0.05, **: p<0.01, *"z p<0.001. ' 43 Fl ' PTX - + + - + NSE , dd Tujl h? GFAP - ~- GAPDH- -... G Fl - + + + ActD - - + - CHX - - - + Tujl ~~ .. GFAP -~- GAPDH-o — - ’ a. 4:0 0 Ct” 5 35 71ml EJFl-ld .2 o :9“ 2.5 g 2.0 “E; 1.5 E 1.0 :6 § 0.5 I: I I z 0.0 NSE Tujl GFAP F H 40 Uctrl 35 FE" DFl-ld 30 . J. "‘ BFl-ActD—ld 2'5 i IFl-CHX-ld ' . '1' C M r 2i 1‘ Neural markers fold change 3 23’ .9 o Taken together, the early-phase neuron-like morphological changes do not rely on gene and protein synthesis whereas the late-phase neural marker expressions do. Correspondingly, changes in morphology occur prior to but are not required for the expression of neural markers. Therefore, although both the morphological changes and the neural marker expressions are regulated by PKA, they are differentially regulated downstream of PKA. 2.5 Discussion cAMP has been shown to play a positive role in the regeneration of the central nervous system (CNS) [36, 192, 193]. Decrease in neuronal cAMP level has been associated with loss in neuronal regenerative capacity [192], whereas an increase in cAMP levels can promote axonal regeneration [194]. In addition to directly stimulating neuronal regeneration for neuronal ,repair, cAMP also can indirectly facilitate neuronal repair by inducing adult stem cells, such as MSCs, to differentiate into neural lineage cells [4]. cAMP has been suggested to be one of the best inducers of MSCs neural differentiation [153]. However, the assessment of neural differentiation has been largely based on the appearance of neuron-like morphology and neural marker expression [4, 153]. MSCs treated with cAMP increasing agent (FI) achieved neuron-like morphology (Figure 2.1B) which was due to disruption of the cytoskeleton and cell shrinkage (Figure 2.1C-F) rather than neurite outgrowth. Thus, the morphological changes are not always a reliable assessment of neural differentiation. In contrast to the early onset of changes in morphology, expression of neural markers occurred much later. An increase in the neural markers, such as NSE, 45 Tujl and GFAP became apparent a day after induction (Figure 2.4A and 2.4C) with some degree of neuronal function, i.e. rise in calcium signaling in response to neuronal activators (Figure 2.5). The gain of neuronal function by MSCs upon induction with cAMP alone has not been previously reported. Although our results showed that cAMP enabled MSCs to obtain neuronal frmction, the cells are at different stages with respect to their ability to respond to the different neuron activators. Some cells show a calcium rise upon stimulation by all three neuronal activators, while others respond to only one or two, or even none of the activators (Figure 2.5F). Since these cells do not show the morphology of primary neurons (Figure 2.40), cAMP alone is unable to terminally differentiate the MSCs into neural lineage cells. The argument whether MSCs can differentiate into flmctional neurons in vitro and in vivo is rarely addressed. A couple of transplantation studies indicated that MSCs can differentiate towards neural lineage cells in vivo [13], 134, 195-197] and some of the differentiated cells were able to gain neuronal functionality [197]. However, whether these functional neurons are generated by MSCs through differentiation or fusion of MSCs with existing neurons remains unclear [198]. Our results point to differential regulation of the morphology and neural marker expression downstream of PKA. With respect to morphology, PKA can activate the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHPZ) [199], which can then dephosphorylate the focal adhesion protein paxillin and result in disassembly of the focal adhesion and subsequent loss of actin stress fibers and cell rounding [199, 200]. Alternatively, the cAMP-PKA signaling pathway may disrupt 46 the cytoskeleton by inhibiting the Rho family GTPases, which play important roles in modulating the cytoskeletal structure. Rho GTPases can promote actin polymerization and stress fiber formation by activating the polymerization factors actin-related protein 2/3 (Arp2/3) and forrnin, and inhibiting the depolymerization factor cofilin [201]. They can also regulate microtubule stability by inhibiting the microtubule disassembly factor oncoprotein l8 (Opl8) and activating the mammalian Diaphanous- related (mDia) formins [201, 202]. cAMP can promote cytoskeleton disruption in melanocytes [203] mediated by RhoA inhibition [204], through phosphorylation of RhoA at serine 188 by PKA [205]. Therefore, the cAMP-PKA pathway could modulate the cytoskeleton by altering the phosphorylation status of focal adhesion proteins or the Rho GTPases. Signaling events are rapid as compared to gene or protein synthesis. Thus in contrast, expression of neural markers takes longer and requires transcription and translation (Figure 2.7G-H), suggesting that transcription factors activated by the cAMP-PKA signaling pathway may be involved. One such downstream factor is the cAMP response element binding protein (CREB), which can be phosphorylated by PKA at serine 133 [16]. F1 treatment transiently increased nuclear PKAC and pCREB levels (Figure 2.8A-B). Phosphorylation of CREB regulates neurogenesis by promoting survival and differentiation of newborn neurons [206, 207]. Over- expressing the dominant negative form of CREB, Ml-CREB (the serine 133 residue is mutated to alanine, therefore it can no longer be phosphorylated) in MSCs led to loss in their response to neuronal activators, such as dopamine (Figure 2.8C-D). This 47 suggests that phosphorylation of CREB plays a critical role in regulating MSC differentiation towards the neural lineage cells. Interestingly, MSCs stably expressing Ml-CREB were much smaller than the normal MSCs after a week of F1 induction (Figure 2.8D), indicating that activated CREB is an important determinant in regulating cell size. Indeed, mice expressing dominant negative CREB are reduced in size during embryonic development [208]. Therefore, although the early onset of morphology changes induced by F1 do not appear to require transcription (Figure 2.7A), the transcription factor CREB is still required for regulating cell shape during MSC differentiation (Figure 2.8D), hinting as possible cross-talk downstream of PKA. The present study showed that cAMP induced changes in morphology early and neural differentiation and function much later and these events are regulated by cAMP activated PKA, but diverge in their regulation downstream. While the morphological changes are likely due to cell shrinkage, which rendered some cells to be subsequently apoptotic, nonetheless the neural differentiation induced by cAMP enabled the MSCs to gain neuronal function. 48 Eggs 21 2: 55 83385 Ea V283 2 é E a? 883 5.56;: message .2822: are 2 09 65838 21 o2 55 38—253 28 :83 ~ .89 E at.» 3965 @882, Sheen wfimmoaxov mum: sin 9 79 05838 8:258 3955: 3 328%“: 5 Emma 8338 me 29:38 o>tfiaomoa2 < av docdvm ”...: .muav 996 h com E at.» 826E 8 «:58 3.280 E .8220 895 855-32 mammoaxov «Um—2-3.4 28 C903 3.980 mammofixov mDmE .ofifiaov 212: £15 Bum—=85 £3 03833— mo mouse—manna ADV £53980 ho. wgaonmoboo 05 ~23 339:8 ma 5.on ”...... ”modvm ”... .Amuev 20>“: mmmu Ea mmmog .303 320:: we nouaomaawsO av .52 em 23 S a .m ._ 5 ca x28 21 2: 2a geese :1 S a? Base 28 2a 28 688 a $20 8a Ammmu Bfibofimofi 92 2:33 mmdoa .955 me £32 £882 .8282 $3 .mmMU 3 Emma 8323 we :ouflamoc Ea mmmo mo coagug Bough QN «...—ME 49 3.....- “Um—2-32 31:- Um: mwvuu menu“ awn“ 1 0:538 363 ES... 08:. 2:638 waved. 280m 0 .41 . 2 $5 $qu om? womfi 0&7; om? , . m - ..- -. i - ..E 9%. o9 46 6.0 m _U o w m s . 35 d . - . I . E a m 5&- m a 8w. 2.20 m. . ..l'..mmxu m. ,6: w. o0 W 5mm. . ...... W Ill..- . u<¥m 8m in m. :3 .5 so 5.. E 51: _.:.:._ . .....i 8 1W :00 mm 9 U m < 50 CHAPTER 3. CREB MODULATES CALCIUM SIGNALING ELICITED BY NEURONAL ACTIVATORS IN MESENCHYMAL STEM CELLS This work is in review: Zhang, L. and Chan, C. CREB modulates calcium signaling elicited by neuronal activators in mesenchymal stem cells (In review). 3.1 Abstract Various studies have suggested that activation of CREB facilitates maturation and survival of immature neurons; however the role of CREB in transdifferentiation of mesenchymal stem cells (MSCs) into neural lineage cells remains unclear. In particular, whether and how CREB affects calcium signaling upon neuronal activator stimulation has not been investigated. In our previous study we observed that cAMP enabled MSCs to generate intracellular calcium signaling in response to several neuronal activators. In an effort to uncover the role of CREB in calcium signaling, we knocked down CREB activity both by siRNA and introducing a dominant negative form of CREB. Our results suggest that knock-down of CREB activity in MSCs greatly reduced or abolished the cAMP-induced calcium response to neuronal activators, whereas reintroducing a constitutively active CREB partially restored the calcium response. The reduction in the calcium response to the neuronal activators appears to involve CREB regulated membrane receptor expression, i.e., dopamine receptor expression. In contrast to the positive effect of CREB on the calcium response, CREB appears to exert a negative effect on the neuron-like morphology, suggesting a complex role of CREB during differentiation of MSCs into neural lineages. 51 3.2 Introduction The transcription factor cAMP response element binding protein (CREB) is activated by various extracellular stimuli and serves as a hub for many cellular processes including metabolism, survival, immune response as well as learning and memory [34, 209]. Phosphorylation of CREB at the key serine 133 site by kinases such as protein kinase A (PKA) is required for its transcriptional activity [30, 33]. Recent studies suggest that serine 133 phosphorylated CREB (pCREB) colocalizes in immature neurons of adult hippocampus [206] and enhances maturation and survival of these cells [206, 207]. Activation of CREB was also shown to facilitate the migration of neuronal progenitor cells from the subventricular zone (SVZ) into the olfactory bulb (OB) and enhanced survival of the differentiated neurons in the OB [210]. Nevertheless, understanding how CREB affects neural stem cells function is still at an early stage. The impact of CREB on other stem cells and, in particular, their differentiation towards neural lineages has not been investigated. Although activated CREB appears to co-localize with immature neurons and to be required for neuronal maturation, its role in regulating neuronal functionality such as calcium rise in response to neuronal activators is not known. Elevation of cAMP level, which stimulates CREB activity, has been shown to induce neural stem cells (N SCs) to exhibit neuronal phenotypes [211], and mesenchymal stem cells (MSCs) to differentiate into neural lineages [4]. In a previous study, we observed that cAMP, accompanied by transient activation of CREB, enabled MSCs to produce a calcium rise upon stimulation by several neuronal activators [212]. We set out in this study to 52 determine the role played by CREB on inducing a calcium rise in cAMP activated MSCs. Neuronal activators stimulate intracellular calcium rise through voltage-gated ion channels or receptor-gated channels which are predominantly expressed in excitable cells [213]. For example, KCl induces membrane depolarization and calcium influx through voltage-gated calcium channels [214]. Glutamate elicits intracellular calcium rise through ionotropic and metabotropic glutamate receptors [215, 216]. Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels which can be classified into N—methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole- propionate (AMPA) and kainate (Ka) receptors [215]. Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors that signal calcium rise through the generation of second messengers, which includes Group I (mGluR 1, 5), Group H (mGluR 2 and 3) and Group III (mGluR 4, 6-8) mGluRs [216, 217]. The mechanism by which dopamine induces calcium rise is complex and can occur through a variety of mechanisms, such as through binding to the Dl-like (including D1 and D5) and D2-like dopamine receptors (including D2, D3 and D4) [218, 219] and through potentiation of NMDA receptors [182]. Previously, we observed that cAMP induction enabled MSCs to produce a calcium rise in response to neuronal activators, dopamine, glutamate and KCl [212]. Although it is well known that CREB activity can be modulated by the Ca2+/calmodulin dependent kinases (CaMKs) [30], to our knowledge, there is no study that has investigated the role of CREB in calcium signaling. Therefore, we set 53 out to determine if cAMP induced CREB activation is involved in regulating calcium signaling in MSCs. Our results suggested that CREB plays a critical role for MSCs to adopt neuronal function, namely to elicit a calcium rise in response to neuronal activators. In contrast to its role in facilitating the neuronal function, CREB appears to have a negative impact on the production of a neuron-like morphology. Knock-down of CREB activity in a dominant negative CREB cell line promoted the appearance of neuron-like morphology. We also examine if CREB can regulate MSCs proliferation and survival. Our results suggest that while CREB activation, induced by cAMP, does not affect G1 phase lengthening, it is required to protect MSCs from cAMP induced apoptosis. Taken together, CREB regulates several aspects of cellular behavior in MSCs during cAMP induced neural differentiation. 3.3 Materials and methods 3.3.1 Cell culture and materials All procedures in the cell isolation were approved by the Institutional Animal Care and Use Committee at Michigan State University. Bone marrow mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rats as previously described [177]. In brief, femurs and tibias were taken from the hind legs of 6-8 week old rats. The marrow was flushed outwith DMEM and filtered through a 65 um nylon mesh to remove bone debris and blood aggregates. Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 ug/mL streptomycin (Invitrogen) and 100 U/mL penicillin (Invitrogen) and placed in an 54 incubator with a humidified atmosphere containing 5% C02 at 37 °C. Non-adherent cells were removed on the second day after plating. Medium was replaced every 3 to 4 days until the cells reach 80~90% confluence. Confluent cells were detached using 0.25% trypsin-EDTA (Invitrogen) and plated for further experiments. To induce neural differentiation, forskolin (Sigma) and isobutylmethylxanthine (IBMX) (Sigma) were used to increase intracellular cAMP levels at concentrations of 10 14M and 100 M, respectively. 3.3.2 Cell cycle analysis Cell cycle analysis was performed as described previously [177]. In brief, harvested cells were fixed with 70% ethanol on ice for 2 hours. RNA was digested by RNase and then DNA was labeled with PI (propidium iodide) for 30 minutes at room temperature. labeled samples were analyzed by flow cytometer BD FACSVantage. 3.3.4 Quantitative real time polymerase chain reaction (RT-PCR) Cells were treated as desired and mRNA was extracted by the RNA extraction kit from Qiagen according to the manufacture’s instruction. mRNA was then reverse transcribed to cDNA using the cDNA synthesis kit from Bio-Rad. The primer sets for actin, dopamine receptors D1, D2, D3, D4 and D5 were obtained from Eurofins MWG Operon and are shown in Table 3.1 were used for PCR. Amplification of the cDNA templates were detected by SYBR Green Supermix (Bio-Rad) using Real- 55 Time PCR Detection System (Bio-Rad). The cycle threshold (CT) values for each condition were determined by the Mle software. Table 3.1 Primer sets for actin, D1, D2, D3, D4 and D5. Gene Primer sets actin 5’-CTCTTCCAGCCTTCCrrccr-3’ 5,-AATGCCTGGGTACATGGTG-3’ D1 5,-GCCATAGAGACGGTGAGCAT-3’ 5,-A'ITCCACCAGCC’I‘CT1‘CCTT-3’ D2 5,-TATGGCTTGAAGAGCCGTGCCA-3’ 5’-TACAGCCATGCACACCAGCACA-3’ D3 5’cAGCCGCAmocroroACGTT-3’ 5’-AGCAAAAGCCAGCACCCACACA-3’ D4 5,-TGCTGCTCATCGGCATGGTGT-3’ 5,-AGCCACAAACCTGTCCACGCT-3’ D5 5,-TGGAGCCTATGAACCTGACC-3’ s’cAAGAAAGGCAACCAGCAAc-s’ 3.3.5 Nuclear extraction Nuclear extraction was canied out according to a protocol described in [220]. In brief, cells were suspended in buffer A (10 mM HEPES (pH=8.0), 1.5 mM MgC12, 10 mM KCl, protease and phosphatase inhibitor cocktail) and allow to swell on ice for 15 minutes. After swelling, the cells were lysed with a 25-gauge 5/8 inch needle and lysates were spinned down to collect the nuclear pellets. Nuclear pellets were re- suspended in buffer C (20 mM HEPES (pH=8.0), 1.5 mM MgC12, 25% (v/v) glycerol, 420 mM NaCl, 0.2 mM EDTA (pH=8.0, protease and phosphatase inhibitor cocktails) and incubated on ice for 30min, then centrifuged at 12,000g for 5 minutes to obtain the nuclear extracts. 3.3.6 Western blot Nuclear extracts or whole cell extracts lysed with CelLytic (Sigma) were assayed for protein concentrations by Bradford assay (Bio-Rad). 15-30 pg protein samples 56 were separated by 10% Tris-HCl gel and transferred to nitrocellulose membrane. Membranes were then blocked with 5% milk in 0.05% Tween 20-TBS (Tris buffered saline) (U SB Corporation) for one hour and incubated with primary antibodies, NSE (neuron-specific enolase) (BIOMOL), Tujl (BIII-tubulin) (Millipore), GFAP (Glial fibrillary acidic protein) (DAKO), GAPDH (Cell signaling), CREB (Cell signaling), ser133 phosphorylated CREB (pCREB) (EMD Chemicals), CDK4 (Cell signaling), CDK6 (Cell Signaling), cyclin D1 (Cell Signaling), 1327‘“pl (Cell Signaling), dopamine receptor 1 (D1) (Novus Biologicals), ICER (kinly provided by Dr. Carlose Molina), c-fos (Cell Signaling) and actin (Sigma) overnight at 4 °C. Afier removing excessive primary antibodies, anti-mouse or anti-rabbit HRP—conjugated secondary antibodies (Thermo Scientific) were added and the blots were incubated for one hour at room temperature. The blots were then washed three times with 0.05% Tween 20- TBS and visualized by SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific). 3.3.7 Immunocytochemistry Triple staining for actin filaments, microtubules and nucleus was performed as previously described [176]. In brief, actin filaments were stained with Texas Red-X phalloidin (Invitrogen), microtubules were stained with or-tubulin (Invitrogen) primary antibody followed by Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (Invitrogen), and the nucleus was stained with DAPI (4’, 6—diamidino-2- phenylindole) (Invitrogen). Stained glass coverslips were mounted in ProLong Gold 57 (Invitrogen). Fluorescence images were taken using an Olympus Flroiew 1000 confocal microscope. For staining against dopamine receptor 1, Tujl or synapsin, cells were fixed in PBS containing 3.7% formaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100 (Research Products Internationals) for 10 minutes at room temperature. After washing with PBS three times, cells were blocked in 1% BSA (Bovine Serum Albumin) (US Biological) for 20 min and incubated with dopamine 1 receptor (Novus Biologicals) antibody, Tujl antibody (Cell Signaling) or synapsin antibody (Cell Signaling) at room temperature for one hour. Cells were then washed with PBS three times and incubated with Alexa F luor 488-conjugated anti-mouse IgG secondary antibody (Invitrogen) or Rhodamine-conjugated anti-rabbit secondary antibody (Calbiochem) for one hour at room temperature. Stained glass coverslips were washed three times with PBS and mounted in ProLong Gold (Invitrogen). Fluorescence images were taken by an Olympus Flroiew 1000 confocal microscope. 3.3.8 Annexin Vand PI (propidium iodide) staining Apoptosis and- necrosis were measured by the annexin Vand PI (propidium iodide) staining kit (Invitrogen), respectively, according to the manufacturer’s instructions. In brief, cells were stained with Alexa Fluor 488 conjugated annexin V and PI in 1X annexin binding buffer for 15 minutes at room temperature and then subjected to flow cytometry analysis by BD FACSVantage. Early apoptotic cells were identified as those stained by Alexa Fluor 488 but not PI, late apoptotic cells were those stained by 58 both Alexa Fluor 488 and PI, and necrotic cells were those stained by P1 but not Alexa F luor 488. 3.3.9 Calcium imaging Calcium imaging was performed according to the protocol described in [158]. Cells were cultured in 4-well chambered cover-glass (Thermo Fisher Scientific). After the desired treatment, the cells were loaded with 4 M F luo-4 (Invitrogen) in ACSF- HEPES (artificial cerebral spinal fluid with HEPES: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgC12, 2.5 mM CaClz, 1 mM NaHzPO4, 26.2 mM NaHCO3, 11 mM dextrose, 10 mM HEPES, pH=7.4) for 30 min at 37 °C. Excess dye was removed by washing the cells twice with PBS and placing them into a 37 °C chamber on the stage of an Olympus Flroiew 1000 microscope. 0.5 ml ACSF-HEPES was added to the well to begin imaging. Images were captured every 1.137 seconds and fluorescence intensity is represented by a Spectral table (warmer colors represent higher intensity whereas cooler colors represent lower intensity). Afier 15~20 images, 0.5 ml ACSF-HEPES buffer containing the following compounds were added: 200 M glutamate (final concentration 100 uM), 200 M dopamine (final concentration 100 pM), 100 mM KCl (final concentration 50 mM), or 200 pM ATP (final concentration 100 M). A total of 200~300 images were recorded and the data was analyzed by the Flroiew 100 software. Changes in the fluorescence intensity of the Ca2+ signal are represented as F/Fo. The percent of responsive cells is calculated as the number of cells with a F/Fo Signal greater than 20% divided by the total number of cells. 59 3.3.10 Transfection For siRNA Silencing, siRNAs targeting CREB mRNA as well as a scramble siRNA (negative control) were purchased from Ambion. In brief, the transfection reagent Lipofectamine RNAiMAX (Invitrogen) was diluted in Opti-MEM (Invitrogen) reduced media The siRNAs were also diluted in Opti-MEM and then mixed with RNAiMAX to allow the formation of siRNA-RNAiMAX complex at room temperature for 20 minutes. Cell culture medium was replaced with media without antibiotics. The siRNA-RNAiMAX complexes were then added to the corresponding wells to reach a final concentration of 10 nM siRNA. Medium was replaced after 4~6 hours incubation in a 5% CO; incubator at 37 °C. Treatments were performed 24 hours from the start of silencing. The constitutively active form CREB (VPl6-CREB) and the dominant negative form CREB (serine 133 mutated to alanine, named Ml-CREB) and the empty vector pCMV are kind gifts from Dr. David Ginty. In brief, cells were transfected with 1.5 llg pCMV, VPl6-CREB or Ml-CREB using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction Medium was replaced after 4~6 hours and cells were incubated in fresh culture medium for up to 24 hours until treatment was carried out. To establish a stable cell line expressing Ml-CREB, MSCS that are transiently transfected with Ml-CREB for 24 hours were trypsinized and replated at low density in media containing 500 ug/ml geneticin (Invitrogen) for selection. For control, MSCS were also transfected with an empty pCMV vector containing neomycin resistance to establish a control cell line. The geneticin-containing media 60 was replaced every 3 days for two weeks. Colonies formed from surviving cells were isolated by cloning cylinders (Sigma) and maintained in culture media containing geneticin. 3.3.11 Statistical analysis All experiments were performed at least three times and results were shown as mean :1: standard deviation. Statistical analysis were carried out by an unpaired, two tail Student’s t-test. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001. 3.4 Results 3.4.1 CREB regulates expression of some neural markers and dopamine response CREB has been shown to be an important player in maturation and survival of newborn neurons in the central nervous system [206, 207]. Previously we observed that CREB was transiently activated during cAMP induced neural differentiation of MSCS [212], therefore, we examined whether CREB can regulate neural differentiation of MSCS. Neural induction by forskolin and IBMX (abbreviated as FI, two agents used to increase cAMP levels) increased the expression of neural markers, i.e. neuron-specific enolase (N SE), B-III tubulin (Tujl) and glial fibrillary acidic protein (GFAP) (Figure 3.1A-B). Silencing of CREB reduced the expression of neuron marker NSE but not the neuron marker Tujl or the astrocyte marker GFAP (Figure 3.1A-B), indicating that cAMP-activated transcription factor, CREB, is 61 involved in regulating some of the neural markers. In addition to neural marker expression, Fl also enabled MSCS to gain some neuronal function, such as calcium rise in response to neuronal activators dopamine, glutamate and KCl [212]. It is unclear how cAMP enabled MSCS to gain this function. To determine if CREB plays a role in this process, we silenced CREB in MSCS and assessed their dopamine response. After silencing with either a scramble siRNA (control) or CREB siRNA, MSCS were induced with F1 for one day and their calcium rise in response to dopamine was measured as described in [212]. Although the CREB siRNA silenced cells were able to respond to dOpamine as compared to the control cells (Figure 3.1C), the response was muted. Quantifying the percentage of dopamine responsive cells suggested that silencing CREB significantly reduced the dopamine response (Figure 3.1D), suggesting that CREB may influence the dopamine response elicited by cAMP. 62 I O + + U .h C Fl . & DScbsi w/o Fl 3 3 5 NS DCREBsi w/o Fl .. .. J: _ .8 m .8 0 3,0 BScbsr w/ Fl 0 M 0 2 -o - m U m U E ... ICREBSI vg Fl CREB ‘ ”l E 2.0 NSE - w - at 'E Tujl .5 Lo GFAP " ' *- -- £300 GAPDH "" "' "" ' NSE Tujl GFAP C Time from adding dopamine Before adding dopamine t=185 F635 D $3 Scbsi :lm—T—_ -Flld E 80. I I E, 601 '5 40 r: 8. 20 3 0 m v b as e" CREBSI a?" g -Flld C) Figure 3.1. Effect of CREB on neural marker expression and calcium rise in response to dopamine. (A) Protein levels of neural markers NSE, Tujl and GFAP. MSCS were transfected with the control scramble siRNA or CREB siRNA and incubated in the absence or presence of 10 uM forskolin and 100 uM IBMX (F1) for 1 day. (B) Quantification of protein levels fold change for NSE, Tujl and GFAP (n=3). *: p<0.05; **: p<0.01; ***: p<0.001; NS: not Significant. (C) Calcium imaging in response to neuronal activator dopamine. (C-l to C-3) MSCS transfected with a scramble siRNA (control) and induced by F1 for 1 day responded upon stimulation with 100 uM dopamine. (O4 to C-6) MSCS transfected with CREB siRNA and induced Fl for 1 day stimulated with 100 uM dopamine. Images were captured before dopamine stimulation or 18 and 63 seconds after dopamine stimulation. Fluorescence images represented by a spectral table; warmer colors indicate higher fluorescence intensities and cooler colors indicate lower fluorescence intensities. (D) Quantification of the percentage of cells responsive to dopamine (n=4). **: p<0.01. 3.4.2 CREB regulates calcium rise in response to several neuronal activators To assess further the long term effect of CREB on neuronal function, we engineered MSCS to stably express the dominant negative form of CREB (M l -CREB, whose serine 133 site is mutated to alanine) and compared their response to MSCS 63 stably transfected with a control vector that contains neomycin-resistance. MSCS stably expressing Ml-CREB, denoted as Ml-MSCS, have higher total CREB expression than the control MSCS (Appendix Figure 3.1A). Ml-MSCS grew slightly Slower as compared with MSCS expressing the control vector (Appendix Figure 3.1B) and are able to form colonies (Appendix Figure 3.1C). To assess for neuronal function, we used three neuronal activators: dopamine, glutamate and KC] as described previously [212]. The response of Ml-MSCS to dopamine is Similar to MSCS (Figure 3.2A and 32C). After FI induction for one week, however, Ml-MSCS lost their ability to respond to dopamine as compared to MSCS (Figure 3.23 and 3.2D). Quantification of the results suggests that the uninduced Ml-MSCS and uninduced MSCS respond similarly to dopamine, KCl and glutamate (Figure 3.3A). Afier a week of F1 induction however, the response to dopamine, KCl and glutamate dramatically reduced or disappeared in Ml-MSCS (Figure 3.3A). When a constitutively active form of CREB (VPl6-CREB) was transiently introduced into Ml-MSCS, their response to dopamine, KCl and glutamate partially recovered (Figure 3.3A), suggesting a role of CREB in the calcium rise in response to the neuronal activators. Generation of calcium signals can be achieved through both internal and external sources of Ca2+ [221]. The influx of external Ca2+ into cells is mainly regulated by membrane receptor—operated channels and voltage-operated channels, while release of internal Ca2+ from endoplasmic reticulum (ER) can be controlled by second messengers, such as inositol-l,4,5-trisphosphate (1P3) and Ca2+ itself [222]. Dopamine, glutamate and KCl can generate Ca2+ signals through membrane receptor- 64 operated channels and voltage- operated channels, and Ca2+ influx through these membrane channels can further stimulate the release of calcium from the intracellular ER stores [214—216, 218, 219]. Since the response of Ml—MSCS to dopamine, glutamate and KCl was compromised (Figure 3.3A), we determined whether the decrease or loss of response to the neuronal activators is related to a general damage of the membrane receptors and intracellular ER stores. We applied a positive control, ATP, which is able to elicit calcium Signals from the external and internal calcium sources through the membrane bound P2X inotropic receptors and P2Y receptor- operated channels [223, 224]. Ml-MSCS induced with F1 for a week can still respond to ATP (Figure 3.3B), suggesting that the reduction or loss of calcium rise in response to the neuronal activators may not be due to a general damage of the plasma membrane or ER. 65 Before adding mine _ _ Time from adding dopamine _ t=12 sec t=34 sec Figure 3.2 Calcium imaging in response to neuronal activator dopamine. (A-l to A-4) Uninduced MSCS (expressing control vector) before dopamine stimulation and 12, 34 and 56 seconds after 100 uM dopamine stimulation. (B-l to B-4) MSCS (expressing control vector) induced with 10 uM forskolin and 100 llM IBMX (PI) for 7 days before and after dopamine stimulation. (C-l to C-4) Uninduced Ml-MSCS (expressing Ml-CREB) before and after dopamine stimulation. (D-l to D-4) Ml- MSCS induced with Fl for 7 days before and after dopamine stimulation. Fluorescence images represented by a spectral table; warmer colors indicate higher fluorescence intensities and cooler colors indicate lower fluorescence intensities. 66 > '5 o F10 $3 80 ; g /: Int Cl MSC ctrl 8 6O / _‘ UMl-MSC ctrl _f>_’ 6 [21 MSC Fl7d g 40 // llMl-MSC Fl7d 3 j ” m E] M l -MSC+pCMV Fl7d a: 4 I M l -MSC+CREB Fl7d 20 ,2 F3 : a» [a / 0 fl _ - B Dop Glut Time from adding ATP Before Adding ATP t=8s t=205 t =4OS I... Figure 3.3 Calcium Signaling upon stimulation by neuronal activators and ATP. (A) Quantification of the percentage of cells that respond to neuronal activators, dopamine, KCl and glutamate. MSCS (expressing control vector) and Ml-MSCS (expressing Ml- CREB) were either uninduced or induced with 10 uM forskolin and 100 uM IBMX (F I) for 7 days. To assess whether reintroducing CREB restores the response of the Ml-MSCS to neuronal activators, either pCMV or the constitutively active VPl6- CREB was introduced and the cellular calcium response was examined (n=4). *: p<0.05; **: p<0.01, "*z p<0.001; NS: not Significant. (B) Calcium imaging in response to the positive control ATP for Ml-MSCS induced with F1 for 7 days. Images were captured before ATP stimulation and 8, 20 and 40 seconds afier ATP stimulation. Fluorescence images represented by a spectral table; warmer colors indicate higher fluorescence intensities and cooler colors indicate lower fluorescence intensities. 3.4.3 CREB regulates expression of certain dopamine receptors To investigate the possibility that CREB modulates the calcium response upon stimulation by the neuronal activator, and Since dopamine elicited a robust calcium response in MSCS (Figure 3.3A), we used dopamine. Dopamine modulates calcium Signal mainly by binding to the dopamine receptors, which consists of the Dl-like (include D1 and D5) and the D2—like (include D2, D3 and D4) families of receptors 67 [218]. Promoter analysis revealed that these dopamine receptors contained a half CRE sequence (TGACG/CGT CA) [34] and may be regulated by CREB [225]. Thus we hypothesized that CREB modulates the expression of these dopamine receptors to impact calcium Signaling. No prior studies examined the expression of all the dopamine receptors in MSCS, and only one study indicated that the expression of D1 is detected in uninduced human MSCS [226]. In this study, we observed that mRNA levels of dopamine receptor D3 and D5 were not affected by CREB silencing (Figure 3.4A), and the expression of dopamine receptor D2 mRNA level was undetectable in MSCS (data not shown). To our surprise, knock down of CREB activity increased rather than decreased mRNA levels of dopamine receptor D1 and D4 afler F I induction for one week (Figure 3.4A). While dopamine receptor D1 positively regulates calcium signaling, dopamine receptor D4 can either positively or negatively regulate calcium Signaling in a context— and cell type-dependent manner [218]. Moreover, D1 and D2 are the most abundant dopamine receptors in mature brain [227]. Due to a more definitive role of D1 in regulating calcium signaling and its predominant expression in mature brain, we therefore measured Dl protein expression using both immunoblotting and immunostaining. Immunoblotting results suggest that Ml-MSCS treated with F1 for 7 days have much lower D1 expression than MSCS treated with F1 for 7 days (Figure 3.43). The immunostaining results confirmed the immunoblotting results, i.e. down-regulating CREB activity did not increase but rather decreased the dopamine receptor Dl protein level. Fluorescence intensity of individual Ml-MSCS (expressing Ml-CREB) appears to be lower than of 68 the control MSCS (Figure 3.4C-D). Although knock down of CREB activity in the Ml-CREB cells increased dopamine receptor D1 mRNA level, it did not translate to an increase in the D1 protein level, but rather to a reduction in the D1 protein level which may contribute, in part, to the reduced dopamine response. The disagreement in mRNA and protein levels of D1 may due to post-transcriptional regulation. Indeed, a previous study showed that an increase in the D1 receptor mRN A level did not result in an increase in the D1 protein level in a mouse neuronal cell line [228]. While a downregulation in the protein level of D1 receptor may play a role in diminishing calcium signaling in Ml-CREB cells, this does not preclude other mechanisms that contribute to the CREB-mediated calcium response elicited by dopamine [182, 218]. 69 A B ,\ , \ $0 '1, 80 C' x5 c; x?) o 6 I"; §$ ‘A\ ‘r‘$ ‘r‘\ 9:0 — MSC-Fl7d DI . ,. ___; g 5‘ =a Ml-MSC-Fl7d "" "' ‘- é“ GAPDH ~ and d d t... it! 3 3 r w I 2 "I E E IO. 2 2 2 08 024 |< '5 06‘ r E L E on o- E 0.2. D3 D4 D5 D 0.0 MSC MI-MSC Figure 3.4 Expression of dopamine receptors. (A) mRNA levels of dopamine receptors D1, D3, D4 and D5 for MSCS (expressing control vector) and Ml-MSCS (expressing Ml-CREB) treated with 10 uM forskolin and 100 M IBMX (F1) for 7 days (n=3). ”z p<0.01, "*z p<0.001. (B) Protein levels of dopamine receptor 1 (D1) for MSCS and Ml-MSCS treated with F1 for 7 days (n=3 for quantification). (C) Dopamine receptor D1 staining for MSCS. (D) Dopamine receptor Dl staining for Ml-MSCS. Green: Dl staining; blue: nucleus staining. 3.4.4 CREB participates in regulating morphology and survival Previously, we showed that F 1 induced neuron-like morphology changes due primarily to cell Shrinkage [212]. The neuron-like morphology decreased as the FI induction continued and nearly disappeared after one week of induction (Figure 3.5A). In contrast, one week of F1 induction of Ml-MSCs appeared to promote a neuron-like morphology with long threads resembling neurites radiating from the cell center (Figure 3.5B). The morphology changed gradually; the Ml-MSCS initially appeared similar to the MSCS, but continuous remodeling of the cytoskeleton during Fl 70 induction resulted in neurite-like extensions (Appendix Figure 3.2). However, these neurite—like extensions lack the expression of the pan neuronal marker, Tujl (Figure 3.5C, arrow). Immunostaining with the synaptic marker, synapsin, indicates that synapsin is found near the body but not within the neurite-like structures (Figure 3.5C, arrow head). There are a few cells that have Tujl staining within the neurite-like structures (Figure 3.5D, arrow); however, they do not stain for synapsin within these structures. These results indicate that although knock-down of CREB promoted the adoption of neurite-like structures, these structures are still quite different from mature neurites. Nevertheless, it is surprising that CREB is required to gain neuronal firnction yet it appears to prohibit the adoption of a neuron-like morphology. Similar contrasting effects between function and morphology have been reported in retinoid- mediated differentiation of human monoblastic U937 cells, where CREB facilitated functional maturation but inhibited morphological differentiation of these cells [229]. However, it remains a mystery how CREB manages this. 71 DMSCctrl 3214 .g DMt-MSC ctrl 812 ... El MSC Fltd 10 I M1-MSC Fltd a 6 4 2 0 Cl: Dir:- early apoptotic late apoptotic necrotic Figure 3.5 CREB affects morphology and apoptosis. (A) Morphology of MSCS (expressing control vector) induced with 10 uM forskolin and 100 uM IBMX (F1) for 7 days. Green: microtubules; red: actin filaments; blue: nucleus. (B) Morphology of Ml-MSCS (expressing Ml-CREB) induced with F1 for 7 days. (C-D) Ml-MSCS induced with F1 for 7 days stained with antibody against the pan neuronal marker, Tujl (green), and the synaptic marker, synapsin (red). Nucleus is counterstained with DAPI. (E) Apoptotic and necrotic labeling by Alexa Fluor-488 conjugated annexin V and PI (propidium iodide), respectively, of control cells and cells treated with F1 for 1 day and 2 days. Early apoptotic cells: PI- annexin V cells; late apoptotic cells: Pl+ annexin V+ cells; necrotic cells: PI+ annexin V cells (n=3). *"z p<0.001. Furthermore, CREB is required for maintaining cell viability. Since fewer Ml- MSCS remained attached upon Fl induction as compared to the control MSCS (Figure 3.5A-B), we assessed whether knock down of CREB activity caused apoptosis. Fl induction itself resulted in significant increases in both early apoptotic and late- apoptotic cells for both Ml-MSCS and control MSCS (Figure 3.5B). Fl induced apoptosis is not likely due to the concentration of F1 used, Since 10 llM forskolin and 100 uM IBMX only caused a Slight but insignificant increase in toxicity as indicated by lactate dehydrogenase (LDH) release (Appendix Figure 3.3). Knock down of 72 CREB activity did not increase apoptosis or necrosis in the control medium, however, it greatly enhanced apoptosis upon FI induction (Figure 3.5E), suggesting that CREB plays a protective role against Fl induced apoptosis in MSCS. 3.4.5 CREB does not affect G1 phase lengthening Two important features of stem cells are: unlimited self-renewal to generate daughter stem cells and differentiation into multiple cell lineages [230]. When stem cells differentiate, the G1 phase is lengthened, and proliferation is Slowed to allow sufficient time for differentiation [231, 232]. We observed that cAMP rendered MSCS to accumulate in the GO/Gl phase and resulted in a lengthened G1 phase. Uninduced MSCS progressed normally into the S and G2/M phases after release from cell cycle synchronization (Figure 3.6A-C). When MSCS were induced with FI, the cells were retained in the GO/Gl phase without progressing into the S and GZ/M phases (Figure 3.6D-F), lengthening the G1 phase or arresting cell cycle in G1. CDK4/6 are the first to be activated during Gl/S transition [71], and their activities is positively regulated by cyclin D and negatively by CDK inhibitors (CKIs), such as p27kipl [70]. Although the expression level of CDK4/6 did not changed, the expression level of the positive cell cycle regulator cyclin D1 (CCNDl) decreased (Figure 3.6G) while the negative regulator p27kipl increased upon FI treatment (Figure 3.6G). Both down-regulation of CCNDl and up—regulation of p27kipl may contribute to lengthening of the G1 phase and thereby Slowing proliferation. In support, a proliferation assay indicated that the 73 number of cells in the FI treated condition was significantly lower as compared to control, both on day l and day 2 (Figure 3.6H). A B C § § GO/Gl 8 E : ”2° 5%. :3 3 4 28 z . s Vt 8; GZ/M 8 N: l N 4 o; ‘ ‘ T 0 50 100 150 200 250 o 50 I00 150 200 250 0 50 IOO 150 200 250 D DNA content E DNA content F DNA content _8 00/01 .- 00/0] 8 15 / E§ E :3 3 z z Vt 8 N 0 50 mo 150 200 250 0 50 100 ISO 200 250 0 50 '00 '50 200 250 NA content DNA content DNA content 6 Id 2d H Fl - + + 0 I.2 .. co actrl CDK4 s q..— Gill: 2 1.0 I I I ’ lFl O CDK6 “ * - " Pg 0-8 “-4 CCNDI ' . E 0'6 N ov- ‘a “’ ‘ ‘ ‘* E :1 0.4 I: p27 - - --- § 0.2 . 0.0 actln — — — 1d 2d Figure 3.6 Cell cycle progression, cell cycle gene expression and cell proliferation of MSCS and MSCS induced with 10 uM forskolin and 100 uM IBMX (Fl). (A-C) MSCS in control media for 5, 15 and 20 hours after release from cell cycle synchronization. (D-F) MSCS induced with F I for 5, 15 and 20 hours after release from cell cycle synchronization. (G) Expression of cell cycle regulators CDK4, CDK6, kipl cyclin D1 (CCNDl) and p27 of control cells and cells treated with F] for l and 2 days. (H) Proliferation of untreated MSCS and cells treated with F I for 1 and 2 days (n=3). *: p<0.05, "z p<0.01. 74 Many of the cell cycle regulators are potential CREB target genes. CCNDI is a known CREB target gene [34] while p27kipl is predicted to have half CRE sequences and therefore is a putative CREB target gene [225]. Therefore, we set out to determine if CREB can impact cell cycle progression by regulating genes, such as CCNDI and p27kipl, and subsequently contribute to lengthening of the G1 phase. Overexpression of CREB by introducing the constitutively active form of CREB increased expression of the exogenous VP16-CREB protein near 60kD, whereas CREB knock-down by siRNA nearly abolished endogenous CREB protein level (Figure 3.7A). Overexpression of CREB increased CCNDI protein levels significantly in control media, however, it did not increase CCNDI protein significantly in the FI condition (Figure 3.7A-B). Protein level of p27kipl was essentially unaffected by CREB (Figure 3.7A—B). Since the protein levels of CCNDI and p27kipl were affected minimally by F1 treatment, it is likely that CREB may not affect 61 phase lengthening induced by cAMP. Indeed, the percentage of cells in the Gl/GO phase was unaffected by CREB overexpression or silencing (Figure 3.8 and Appendix Figure 3.4), suggesting that G1 phase lengthening initiated by cAMP is not regulated by CREB. 75 A FI + + + + B a an o > 6 > g _ a _ 5i :98 Dw/oFl Eager-r3543? 36"“ ' H ..., a... *VP—CREB “34 C T; a u . ‘ R53 [_ , .._.,.._. .. +endo-CREB .22 ET. ”1 1‘1 7"] TBP .. - we... ......a-w M "go D- U- [l E]- iiE-;:C.';'.-z;- :14 1".21 “:1? a > m: ”- .6; E m '- a CCNDI ”T“ .;.;.:.-:;2;W.m .. '-.= 2 d‘m' '8 m mg 3 m u>€§ié§ 9.3 %>U‘g 2 p27 '1" w “i "" a U 0 O 9 E 'E. q GAPDH bw~-~~U-fl figure 3.7 Effect of CREB on CCNDI and p27kipl expression. (A) CREB overexpression is achieved by transfection of the constitutively active VP16-CREB and Silencing is achieved by CREB siRNA. pCMV and scramble siRNA are used as controls. Transfected cells were incubated in the absence or presence of 10 M forskolin and 100 M IBMX (F1) for 1 day. Western blotting is carried out for nuclear extracts and whole cell extracts. TBP and GAPDH are used as loading controls. (B) Quantification of CCNDI and p27kipl protein levels (n=3). *: p<0.05; "z p<0.01; ***: p<0.001. 76 .eoufieenofim ace 0322 see anon om é EV x28 21 2: nee geese :1 2 5? Been £8 vouooumaflh 9+8 .aotmflaoflogm Sch 0322 has anon on 8:608 75:8 5 $3 380.35; 5-3 @3on 3 com: 93 <56 03883. 93 Eva {72% $20 55 308mm 28 mam—duet; 026a 32,333.80 05 wfimmoaxobg 38 SM 8335va 2vo :00 ad ennui E880 <20 .583 <20 28:8 >‘ m $15 50 can 3?" aunt 340 l 3 ' 8., ,2 10 0 ’5 330 g a C1 :20 93 5 , _.,,. D .2 410 1‘9 0 — — - = g 0 . -... ctrl ole lin palm <2 __ . early apoptotrc late apoptotnc necrotic Figure 4.1 LDH release and cell death by FFAs treatment. (A) LDH release from cells in medium/BSA (ctrl), 0.7 mM oleate, linoleate and palmitate for 24 h. (B) Apoptotic and necrotic labeling by P1 (propidium iodide) and Alexa Fluor-488 conjugated annexin V for cells in medium/BSA (ctrl), 0.7 mM oleate, linoleate and palmitate for 24 h. Early apoptotic cells: PI_ annexin V+ cells; late apoptotic cells: PI+ annexin V” cells; necrotic cells: P1+ annexin v’ cells (n = 3). *: p < 0.05; **: p < 0.01; ***: p < 0.001. 4.4.2 Palmitate dose dependently reduces intracellular cAMP levels Previously we showed that intracellular cAMP levels were reduced by palrrnitate but not oleate or linoleate in HepG2 cells [163]. The effect of palrrnitate on cAMP levels was also dependent on the palrrnitate concentration. cAMP level was increased slightly by 0.2 mM palmitate and significantly by 0.4 mM palmitate, whereas a high concentration of palmitate (0.7 mM) decreased cAMP level significantly (Figure 4.2A). Since the high concentration palmitate caused significant cell death, we assessed whether restoring cAMP level to near control level would prevent cell death. Intracellular cAMP levels were restored by IBMX (phosphodiesterase inhibitor), forskolin (adenylyl cyclase agonist), 8CPT-2Me-cAMP (cell-permeant cAMP analog) and glucagon (a hormone that activates adenylyl cyclase). 100 M IBMX, 1 uM forsolin, 1 uM 8CPT-2Me-cAMP and 100 nM glucagon restored cAMP levels to near control level in the 0.7 mM palmitate 93 condition (Figure 4.23). A combination of 10 pM forskolin and 100 pM IBMX was also evaluated, which achieved an even higher level of cAMP (Figure 4.23). A B 8 o 2.0 8’0 um OD . l: 5 .2 6 I 31.5; I! ' ,3 "“ O :9. c... E 4 an €1.03 ‘2 u 3 .9- 2 ..., aunt I I "‘ a. E 0.5 E l———;_l—,—-; S0.0 Q0..-I!- : ctrl 0.2? 0.4? 0.7? 0.70 0.-7L 059 s 9’9 «2&0? 9x9 .2)» Figure 4.2 Effect of FFAs on cAMP levels. (A) Intracellular cAMP levels of cells in control, 0.2 mM palnnitate (0.2-P), 0.4 mM palmitate (0.4-P), 0.7 mM palmitate (0.7-P), 0.7 mM oleate (0.7-O) and 0.7 mM linoleate (0.7-L) for 24 h (n = 4). (B) Intracellular cAMP levels of cells in control, 0.7 mM palmitate (P), 0.7 mM palrrnitate supplemented with 100 uM IBMX (P + I), 0.7 mM pahnitate supplemented with 1 uM forskolin (P + F), 0.7 mM pahnitate supplemented with 1 uM 8CPT-2Me-cAMP (P + 8CPT), 0.7 mM palnnitate supplemented with 100 nM glucagon (P + G) and 0.7 mM palmitate supplemented with 10 [1M forskolin and 100 M IBMX (P + F1) for 24b (71 = 3). *2 p < 0.05; “‘2 p < 0.01; ***: p < 0.001. 4.4.3 cAMP and palmitate synergistically promote cell death at high concentrations Studies in pancreatic B-cells indicated that apoptosis was reduced upon supplementation with cAMP increasing agents [14]. However, another study suggested that although apoptosis was reduced, the mode of cell death was switched to necrosis upon cAMP elevation in the palmitate condition [247]. When we used IBMX, forskolin, 8CPT-2Me-cAMP or glucagon to restore cAMP levels to the control level, the mode and level of cell death remained unchanged in the 0.7 mM palmitate conditions (Figure 4.3A). However, when cAMP was increased to a high level by supplementing with forskolin and IBMX (abbreviated as FI), it caused a significant increase in the necrotic cell population (Figure 4.3A). When the same concentration of F1 was used to co-treat cells in control, 94 0.4 mM palmitate or 0.7 mM oleate, the synergistic increase irn necrotic cell death was not observed (Figure 4.33). A j—n U1 El ctrl P E] P+l I P+F E3 P+8CPT . ll P+G I P+F] .—n O LII O Apoptotic & necrotic cells (%) early apoptotic late apoptotic necrotic B 23 g D ctrl 8 no ctrl+Fl g 1 L2! 0.4? .3, I 0.4P+FI 0; El 0.7P .0 5 ‘ [I 0.7P+Fl § D 0.70 D. o I 0.70+FI g 0 ii . early apoptotic late apoptotic necrotic Figure 4.3 Apoptosis and necrosis by FFA treatment in the absence or presence of F1. (A) Apoptotic and necrotic labeling by P1 (propidium iodide) and Alexa Fluor-488 conjugated annexin V for cells in control, 0.7 mM palmitate (P), 0.7 mM palnnitate supplemented with 100 M IBMX (P +1), 0.7 mM palmitate supplemented with 1 uM forskolin (P + F), 0.7 mM palmitate supplemented with 1 uM 8CPT-2Me—cAMP (P + 8CPT), 0.7 mM palrrnitate supplemented with 100 nM glucagon (P + G) and 0.7 mM pahnitate supplemented with 10 M forskolin and 100 uM IBMX (P + F I) for 24 h. Early , - . + _ + . + , apoptotic cells: PI annexin V cells; late apoptotzc cells: PI annexnn V cells; necrotic cells: PI+ annexin V_ cells (n = 3). (B) Apoptotic and necrotic labelirng by P1 (propidium iodide) and Alexa Fluor-488 conjugated annexin V for cells in control, control supplemented with Fl (ctrl + F1), 0.4 mM palmitate (0.4P), 0.4 mM palrrnitate supplemented with FI (0.4P + F1), 0.7 mM palmitate (0.7P), 0.7 mM palmitate supplemented with FI (0.7P + F1), 0.7 mM oleate (0.70) and 0.7 mM oleate supplemented with F] (0.70 + F1) for 24 h (n = 3). *: p < 0.05. 95 The increase in the sub-GI population attests further to the synergistic effect of elevated palrrnitate concentration and cAMP on cell death. Small fragnented DNA can be washed out fi'om cells, leaving a sub-G1 peak to the left of the GO/Gl peak [262]. Compared with the control cells (Figure 4.4A), cells treated with 0.7mM palmitate had a small population of sub-G1 cells (Figure 4.4C). The addition of F1 increased the sub-G1 population (Figure 4.4D), suggestive of further DNA fragnentation, in palmitate but not in the control cells (Figure 4.43). Cells in different cell cycle phases, including sub-G1 cells, were quantified and shown in Figure 4.43 Significant increase in sub-G1 phase cells was already observed 12 hours after 0.7mM palmitate treatment. The synergistic or additive effect of cAMP in promoting DNA fragnentation in the palnnitate culture was not observed at 12 hours, (Figure 4.43). However at 24 hours, cAMP increased the sub- Gl fiaction significantly in the palmitate condition (Figure 4.43). Altlnough palmitate alone decreased the G2/M phase cells, the addition of F I did not reduce the S phase or G2/M phase cells but rather increased the G2/M phase cells at 12 hours (Figure 4.4E). Since cAMP did not prevent cell cycle progession, the increase in cell death caused by cAMP in the palmitate condition is not likely the result of DNA-damage induced cell cycle arrest at the checkpoint. 96 83 83 \0‘ V0: 3 83 383 S V: 5"; z : Z 1 81 8‘ N4 Ni O: o.‘ 0 50 100 150 200 250 0 50 100 150 200 250 DNA content DNA content C D o o 00 .— 8 3 o E o g e 8 subGl ... i 0 Mr O 0 50 l00 150 200 250 0 50 100 150 200 250 E DNA content DNA content 25 u Dctrl '1 Dctrl+Fl A 20 IP 2". IP+F| 'm 8 un- u- l—' E8 15 a. W L E ‘9‘ 10 I A 6 E **‘ U 0 L ._ sub-G1 S GZIM sub-G l S GZ/M 12h 24h Figure 4.4 Effect of palmitate and F1 on cell cycle distribution. (A) ln control for 24 h (ctrl). (B) In control supplemented with 10 M forskolin and 100 M IBMX (F I) for 24 h (ctrl + F1). (C) In 0.7 mM palrrnitate for 24 h (P). (D) In 0.7 mM palmitate supplemented with F1 for 24 h (P + F1). (E) Quantification of cells in sub-G1, S and GZ/M phases 12 h and 24 h after treatment (71 = 3). *z p < 0.05; “z p < 0.01; "*z p < 0.001. 4.4.4 cAMP and palmitate induced cell death is not caused by palmitate B-oxidation Mitochondria are the main sites for fatty acid B-oxidation, with acetyl—CoA as one of the primary products. The majority of acetyl-CoA enters the TCA cycle, whereas a small 97 proportion will undergo ketogenesis in the mitochondria matrix [263]. Unused FF As can be stored in the form of triglyceride in the cytosol. The capacity of non-adipose cells to store FFAs as triglyceride is limited. A previous study indicated that storage of oleate as triglyceride protected non-adipose cells from lipotoxicity, whereas palmitate was poorly incorporated into triglyceride and tlnerefore led to apoptosis [264]. When we measured the level of triglycerides in HepG2 cells, we found that oleate and linoleate were more likely to be stored as triglyceride than palmitate (Figure 4.5A-D). This is in accordance with our previous finding that oxidation is higher with palrrnitate than with the unsaturated FFAs [163]. Given that the mitochondria serve as primary sites for F FA [3- oxidation, enhanced palmitate oxidation may be related to enhanced mitochondrial biogenesis. Therefore we measured the mitochondria mass and found a significant increase in the 0.7 mM palrrnitate condition (Figure 4.6A). Altlnough 0.7 mM palmitate geatly enhanced mitochondrial bio genesis in the palmitate treatment, this effect was not observed in the oleate treatment (Figure 4.6A). This could be due to the higher ability of oleate to be stored as triglycerides [37]. However, the addition of PI stimulated mitochondrial biogenesis, regardless of the treatment condition (Figure 4.6A). This effect was most obvious in the co-treatment of F1 with 0.7 mM palmitate (Figure 4.6A). Since mitochondria are the primary sites for FFA B-oxidation and cAMP promoted mitochondrial biogenesis to a geater extent in palmitate, we expected that the addition of F1 would enhance FFAs B-oxidation and reduce triglyceride storage. However, supplementing F I in the control and the different F F As did not appear to give a visual difference in triglyceride levels according to Oil Red 0 staining for triglycerides (Figure 4.5E-H). For finrther confirmation, we quantified the triglyceride levels. Indeed, 98 supplementing with F I did not change the triglyceride levels (Figure 4.51). It appears that mitochondrial biogenesis did not furtlner enhance FFA B-oxidation. The lower triglyceride level in the pahnitate condition was already apparent at 5 and 12 hours after treatment (Appendix figure 4.1), whereas mitochondrial bio genesis occurred at a much later time point (Figure 4.63), indicating that B-oxidation occurred prior to rrnitochondrial biogenesis. Palmitate was oxidized to a higher extent than oleate and caused significant cell death in HepG2 cells, therefore we assessed whether inhibiting palmitate B-oxidation would reduce or prevent cell death. When the FFAs oxidation inhibitor etomoxir was employed, it did not affect the cell death observed in palmitate, as reported previously [265]. Etomoxir decreased the necrotic population only slightly in the palmitate supplemented with F I condition (Figure 45]), suggesting that cell deatln induced by palmitate and palnnitate supplemented with F1 was not due to B-oxidation. 99 04.0 2‘33 up c303.5 nw/o Fl :3- n P+Eto E30 Iw/Fl 812 aP+Fl o ' .2 .‘P. 2.5 § 10 IP+FI+EtO ‘9 2.0 3 8 0 t: 3’ 1.5 °3 6 , U . bl.o D .g 4 y .0005 2 r: n— ... g» o CCZ. ctrl ole palm early apoptotic late apoptotic necrotic Figure 4.5 Triglyceride storage, palmitate oxidation and cell death. (A—H) Triglyceride storage by Oil Red 0 staining for cells in (A) control, (B) 0.7 mM oleate, (C) 0.7 mM linoleate, (D) 0.7 mM palrrnitate, (E) control supplemented with 10 M forskolin and 100 M IBMX (Fl), (F) 0.7 mM oleate supplemented with F], (G) 0.7 mM linoleate supplemented with FI and (H) 0.7 mM palmitate supplemented with F] for 24 h (n = 3). (I) Quantification of triglyceride levels by an assay for cells in control, 0.7 mM oleate and 0.7 mM palmitate without (w/o) or with (w/) F1 for 24 h (n = 4). (J) Apoptotic arnd necrotic labeling by P1 (propidium iodide) and Alexa F luor-488 conjugated annexin V for cells in 0.7 mM palmitate and 0.7 mM palmitate supplemented with F1 in the absence or presence of free fatty acid oxidation inhibitor etomoxir (n = 3). *: p<0.05. 100 Ag, 3 803.5 , 1 8 . cu §DCII 5:3 Dw/oFI 53-0‘antrl+Fl :2 ° Iw/FI :3 2.5 {TBP «22.5 H— IP+F1 31.5 a €10 : , . 30.5 . §00 ¥ 1 E palm ... ° ” Figure 4.6 Effect of FFAs and F1 on mitochondrial mass. (A) Mitochondrial mass fold change of cells in control, 0.7 mM oleate and 0.7 mM palmitate without (w/o) or with (w/) 10 uM forskolin and 100 uM IBMX (Fl) for 24 h (n = 3). (B) Mitochondrial mass fold change of cells in control and 0.7 mM palmitate without (w/o) or with (w/) F1 for 5 h, 12 h and 24 h (n = 3). *:p<0.05; **:p<0.01. 4.4.5 cAMP synergized with palmitate to alter mitochondrial morphology and integrity Mitochondria are not only primary sites for FFA B-oxidation, but also play an important role in cell death and survival [266]. In healthy cells, mitochondria exhibit elongated and connected morphology. When cells are subjected to apoptotic stimuli, mitochondria fragment into small and disconnected rrnitochondria [256, 257]. Control HepG2 cells exhibited long and connected thread-like mitochondria structure (Figure 4.7A). When the cells were subjected to 0.7 mM palmitate treatment, the mitochondrial morphology changed dramatically. The interconnected network was disrupted and collapsed into short disconnected morphology (Figure 4.7C). The elongated mitochondrial network also condensed and collapsed to the perinuclear locations (Figure 4.7C, arrow heads). The addition of F1 to the control cells did not alter the connectivity of the mitochondrial network (Figure 4.73, arrows), however supplementing the palmitate medium with Fl continued the disconnected and punctuate mitochondria structure (Figure 101 4.7D, arrow heads) in a higher population of cells (Figure 4.7E). No mitochondrial fragnentation was observed in the 0.2 mM palrrnitate condition (Appendix figure 4.23, arrows). When palmitate concentration was increased to 0.4 mM, mitochondrial fragnentation had begun to occur and a number of cells had disconnected and punctate mitochondrial structures (Appendix figure 4.2C, arrow heads), whereas the majority of cells still have elongated mitochondrial network as indicated by the arrow (Appendix figure 4.2C). As the palmitate concentration was further increased to 0.7 mM, more cells showed short and disconnected mitochondrial structures (Appendix figure 4.2D). Fragnentation of mitochondria is associated with cell death and the release of cytotoxic proteins that reside in the intermembrane space [256]. In accordance with the mitochondrial fragnentation observed in the high palmitate condition, we detected less Smac in the mitochondria and relatively more Smac in the cytosol (Appendix figure 4.3). Indeed, Smac colocalized in the mitochondria in the control (Figure 4.7F) and control supplemented with Fl conditions (Figure 4.7G), since almost no geen fluorescence was observed outside the mitochondria. However more cytosolic Smac (indicated by tlne geen fluorescence) was released into the cytosol in the palnnitate (Figure 4.7H) and palrrnitate supplemented with FI conditions (Figure 4.71), suggesting that the mitochondrial membrane integity was compromised. We also observed that the mitochondrial anti-apoptotic protein 3ch level was furtlner reduced by F1 supplementation (Appendix figure 4.3). 3ch is an anti-apoptotic protein that antagonizes pro-apoptotic proteins, such as Bax, thereby preventing the release of cytotoxic proteins from the nnitochondria [254]. The significant decrease in mitochondrial 3ch protein induced by the addition of F1 to palmitate treated cells may disrupt the outer 102 mitochondria membrane integity [267]. As a consequence, cytotoxic proteins, such as Smac which promotes caspase activation by inhibiting the inhibitor of apoptosis (lAP), are more likely to be released into the cytosolic compartment [254]. In support of this idea, caspase 3 activity was highest in the palmitate supplemented with Fl condition (Figure 4.8A), which had the highest level of mitochondrial fragmentation (Figure 4.7E) and lowest level of mitochondrial 3ch (Appendix figure 4.3). NN OUn *** So. % Cells with frag. mito. .l. .1. ctrl ctrl+Fl OUn Figure 4.7 Mitochondrial fragmentation and release of cytotoxic protein. (A—D) Mitochondrial morphology of cells in (A) control, (3) control supplemented with 10 uM forskolin and 100 M IBMX (Fl), (C) 0.7 mM palmitate and (D) 0.7 mM palmitate supplemented with F1 for 24h (n=3). Panels A—B exhibited elongated and tubular structure, while panels C—D displayed short and disconnected morphology. (E) Quantification of the percentage of cells with fragmented mitochondria as displayed in panels A—D (n = 3). *z p < 0.05; *"z p < 0.001. (F—l) Immunostaining of mitochondria (red), Smac (green) and nucleus (blue) for cells in (F) control, (G) control supplemented with Fl, (H) 0.7 mM palmitate and (l) 0.7 mM palmitate supplemented with F1 for 24 h (n = 3). 103 Since caspase 3 activity was activated by 0.7 mM palmitate and significantly increased by F1 supplementation (Figure 4.8A), we assessed whether caspase was involved in causing the cell death. When the pan caspase inhibitor Z-VAD-F MK was used to inhibit caspase activity, late apoptosis and necrosis were not reduced (Figure 4.83), suggesting that the cell death induced by palrrnitate and cAMP was caspase- independent. This is not too surprising given that the majority of the population under this condition is necrotic as opposed to apoptotic. 104 N O Dw/oFl if I 9"" lw/FI. ...-a U1 INN! l . 0 C- E. iii :- If.I £11 Casp-3 activity fold change 3 B ctrl 0.2-P 0.4-P 0.7-P 0.7-O 0.7-L 20 A Dctrl °\° lctrl+ZVAD 7,,“ Bctrl+Fl ”—5, '5 Inctrl+Fl+ZVAD 3 DP g IP+ZVAD E310 BP+F1 8 IP+FI+ZVAI e3 .9. *5 5 E < 0 E I... z: an: early apoptotic late apoptotic necrotic Figure 4.8 Effect of Caspase inhibition on apoptosis and necrosis. (A) Caspase 3 activity of cells in control (ctrl), 0.2 mM palrrnitate (0.2-P), 0.4 mM palmitate (0.4-P), 0.7 mM palnnitate (0.7-P), 0.7 mM oleate (0.7-O) and 0.7 mM linoleate (0.7-L) without (w/o) or with (w/) F I for 24 h (n = 4). (B) Apoptotic and necrotic labeling by P1 (propidium iodide) and Alexa Fluor-488 conjugated annexin V for cells in control (ctrl) and 0.7 mM palmitate (P) in the absence or presence of F1 and/or 10 uM pan-caspase inhibitor Z- VAD-FMK (Z-VAD) for 24 h. Early apoptotic cells: PI- annexin V+ cells; late apoptotic cells: PI+ annexin V+ cells; necrotic cells: PI+ annexin V. cells (n = 3). *: p<0.05; **: p<0.01; ***:p<0.001. 4.4.6 cAMP synergized with palmitate to enhance ROS generation in mitochondria Mitochondria are also the primary sites for ROS generation. Although palmitate B- oxidation was not the cause of cell death, generation of ROS at Complex I and Complex III during the process of oxidative phosphorylation through the electron transport chain in the mitochondria can induce cell death [260]. Excessive ROS generation can result in 105 cellular damage and cell death. Mitochondrial superoxide anion (02") was increased significantly in the palrrnitate condition after 24 hours (Figure 4.93) as compared with the control (Figure 4.9A). Moreover, the short, disconnected and perinuclear mitochondria appeared to have higher Oz" levels (Figure 4.93, arrow heads). Quantification of mitochondrial 02" levels indicated that palrrnitate did not induce an increase in mitochondrial 023 levels at 5 or 12 hour, but a significant increase was observed at 24 hours (Figure 4.9C). Elevating cAMP level by F1 synergistically increased the mitochondrial 02" levels at 24 hours (Figure 4.9C). 02" is the precursor of stronger ROS, such as hydrogen peroxide (H202) and hydroxyl radical (HO°) [260]. When whole cell ROS levels were measured, higher ROS activity was detected in the palmitate condition 24 hours after treatment (Figure 4.9D). Sinnilar to the nnitochondrial superoxide levels, the cellular ROS level did not increase at 5 and 12 hours. However at 24 hours, FI induced a slight increase in ROS level in the palmitate condition albeit not significantly (Figure 4.9D). In order to evaluate whether ROS production contributes to the cell death induced by pahnitate and palrrnitate supplemented with FI, we used several ROS scavengers. The ROS scavengers employed were: DMU for hydroxyl radicals, catalase for hydrogen superoxide, Cu—DIPS and MnTBAP for superoxide. Employing DMU or catalase resulted in a decrease in both late apoptotic cells and necrotic cells caused by palrrnitate, however, the decrease was not significant (Figure 4.93). When DMU and catalase were used simultaneously, both late apoptosis and necrosis were reduced significantly in the palnnitate condition (Figure 4.9E). Similarly, DMU and catalase together significantly 106 reduced late apoptosis and necrosis in palmitate supplemented with F l. DMU itself also significantly reduced both late apoptosis and necrosis but catalase only significantly reduced necrosis (Figure 4.9E). The superoxide scavengers Cu-DIPS and MnTBAP did not decrease cell deatln (data not shown). This is likely due to the reduction of 023 by Cu-DIPS and MnTBAP to generate the stronger ROS, H202 and HO‘ [260] which would be expected to further continue the damage to the cells. Although the H202 and HO° scavengers reduced cell death, H202 and HO° are not likely the reason that cell death was irnitiated. At 5 and 12 hours of palrrnitate and palrrnitate supplemented with F I treatment, cell death had already been initiated (Appendix figure 4.4) but the ROS level did not increase until 24 hours (Figure 4.9D). However, ROS generation may be linked to mitochondrial fi'agnentation. At 12 hours, some cells already had fragnented mitochondria in the 0.7mM palmitate condition (Appendix figure 4.5A, arrow heads). This is especially the case for the cells with fiagnented nucleus (Appendix figure 4.53, rectangle), their nnitochondria were all short and disconnected (Appendix figure 4.5C, arrow heads). The short and disconnected mitochondria tend to have higher ROS levels (Figure 4.93, arrow heads). Therefore, mitochondria fragnentation precedes ROS generation and may have contributed to the ROS produced. 107 Ur DCUI nctrl+Fl n IP+F| NWA _l Mito. superoxide fold change 5h 12h D A , $.12 ... cnctrl g; *"‘ *H 303'0 uctrl+Fl "g '0 * "‘ 3%"! E 2.5 GP 8 8 UP+D o 2.0 IN" a I '0: '—] aP+C % 1.5 8 6 . :- IP+D+C w 1.0 I Q! 4 . .0: a o o a InPFl+D 0:05 I, '5 2 , :z aPFl+C 0.0 4 3 W [E , g IPFI+D+C 5h |2h 24h f; 0 , 4‘ .- . c 7 r ' early apovw“ We worm“ Figure 4.9 ROS and cell death. (A) Mitochondrial superoxide labeling for cells in control and (B) cells treated with 0.7 mM palmitate for 24 h. Arrowheads denote short and disconnected mitochondria which have higher superoxide levels (n = 3). (C) Mitochondrial superoxide levels fold change for cells in control and 0.7 mM palmitate without (w/o) or with (w/) 10 M forskolin and 100 pM IBMX (F1) for 5 h, 12 h and 24 h (n = 4). (D) Cellular ROS levels fold change for cells in control and 0.7 mM palmitate without (w/o) or with (w/) 10 M forskolin and 100 uM IBMX (F I) for 5 h, 12 h and 24 h (n = 3). (E) Apoptotic and necrotic labeling by P1 (propidium iodide) and Alexa Fluor- 488 conjugated annexin V for cells in control, 0.7 mM palmitate (P) and 0.7 mM palnrnitate supplemented with 10 M forskolin and 100 M IBMX (F I) in the presence of ROS inhibitors (n= 3). D: hydroxyl radical inhibitor DMU; CA: hydrogen peroxide inhibitor catalase. *: p<0.05; **: p<0.01; ***: p<0.001. 4.5 Discussion Lipotoxicity induced by palmitate has been demonstrated in a number of different cell types. In human HepG2 cells, both apoptotic behaviors, such as caspase 3 activation (Figure 4.8A) and annexin V labeling for phosphatidylserine (Figure 4.13), and necrotic behaviors, such as propidium iodide (PI) penetration (Figure 4.13), have been observed for palmitate-induced cell death. Many obese and diabetic patients have high plasma FFAs levels [250-252]. cAMP has been proposed as a potential drug target for type 2 108 diabetes [249] due to it ability to stimulate pancreatic B-cell insulin secretion. In light of the fact that elevated F FAs can induce hepatotoxicity, it warrants evaluating the effect of cAMP under the circumstance of high FFAs. In this study, we evaluated the effect of cAMP on hepatotoxicity and cell death under elevated FF As conditions. A high cAMP level achieved by adding 10 M forskolin and 100 pM IBMX (abbreviated as Fl) promoted palmitate-induced cell death in a synergistic manner (Figure 4.3 and 4.4). The increase in cell death caused by cAMP supplementation was mostly necrotic (Figure 4.3B), without an increase in the early apoptotic population (Appendix figure 4.4), and further appeared to be caspase-independent (Figure 4.88). Cell death caused by palmitate may be related to its lower ability to be stored in the form of triglyceride [264]. Reduced cytoxicity and cell death in HepGZ cells were achieved by inhibiting NADH dehydrogenase (complex I), which was accompanied by enhanced triglyceride storage [268]. While oleate and linoleate are more likely to be stored as triglyceride (Figure 4.5B-C), palmitate was less likely to be stored in the form of triglycerides (Figure 4.5D and 4.51). The lower ability of palmitate to be stored as triglyceride may contribute to its enhanced oxidation in mitochondria Indeed, our previous study showed that palmitate was oxidized more than oleate and linoleate by the HepGZ cells [163]. The total mitochondrial mass was increased significantly in the palmitate condition but not in the oleate condition (Figure 4.6A). Since mitochondria are the primary sites for FFA B-oxidation, we initially assumed that the induction of mitochondrial biogenesis would allow more palmitate to undergo B-oxidation and therefore have less triglyceride to be stored. cAMP can promote mitochondrial biogenesis by directly regulating mitochondrial related genes or indirectly by inducing the 109 expression of transcriptional coactivator, PGC-1a[269]. When F I was added to the different conditions, mitochondrial biogenesis was enhanced in all cases, but the extent was much higher in the palmitate condition (Figure 4.6A). However, the addition of F I did not cause a significant change in triglyceride levels (Figure 4.51). A time-dependent I study indicated that mitochondrial biogenesis occurred later than triglyceride storage (Figure 4.6B and Appendix figure 4.1), indicating that mitochondrial biogenesis would not likely contribute to reduced triglyceride storage in the palmitate condition. Therefore, even though Fl increased mitochondrial biogenesis (Figure 4.6A), triglyceride levels were not affected (Figure 4.51). Since the palmitate condition had higher [i-oxidation [163] and lower triglyceride storage (Fig. SI), we assessed whether palmitate B-oxidation is involved in palmitate-induced cell death. When B-oxidation was inhibited by etomoxir, no change in cell death was observed (Figure 4.51), suggesting that pahnitate B-oxidation was not the cause of cell death. Although B-oxidation was not the cause of cell death, generation of ROS by oxidative phosphorylation through the mitochondrial electron transport chain can damage cells [268]. Indeed, inhibiting NADH dehydrogenase (complex I), one of the primary sites for ROS generation, reduced the cytotoxicity induced by palmitate treatment [268]. Superoxide (023), which is the precursor of most ROS, was much higher in the palmitate condition and further increased upon Fl supplementation (Figure 4.9C). In addition to mitochondrial 02" levels, total cellular ROS levels were also much higher in the palmitate condition (Figure 4.9D). Mitochondrial 02" levels correlated with mitochondrial biogenesis (Figure 4.6B and 4.9C). Whether mitochondrial biogenesis promote mitochondrial Oz" generation or 110 mitochondrial 02" generation promote mitochondrial biogenesis still remains a question. From our results, it is unlikely that mitochondrial biogenesis itself promoted mitochondria] 02" generation, since Fl supplementation promoted mitochondrial biogenesis in the control and oleate conditions (Figure 4.6A) but no increase in mitochondrial 02" levels was observed in these two conditions (Appendix figure 4.6). It is possible that 02" generation may enhance mitochondrial biogenesis. Previous studies have suggested that ROS can enhance nuclear respiratory factor-l (N RF- 1) and mitochondrial transcription factor A (Tfam), which are involved in regulating mitochondrial biogenesis [270, 271]. Generation of ROS, however, may be linked to mitochondrial fiagmentation. A previous study has indicated that changes in mitochondrial morphology resulted in ROS overproduction [272]. We showed that palmitate induced morphology changes in the mitochondria, causing long interconnected networks (Figure 4.7A) to become short disconnected structures located in the perinuclear (Figure 4.7C). When HepGZ cells were subjected to palmitate and F1, mitochondrial fragmentation was further enhanced (Figure 4.7D), judging by the increase in the total number of cells with fragmented mitochondria (Figure 4.7E). Under the conditions of a high percent of mitochondrial fragmentation, mitochondrial Oz" and total cellular ROS levels were also high (Figure 4.9C-D). The occurrence of mitochondrial fragmentation precedes mitochondrial ROS generation. At 12 hours, mitochondrial fragmentation had already begun to occur in the 0.7 mM palmitate condition (Appendix figure 4.5), while ROS generation was not much higher than control until after 12 hours (Figure 4.9D). In addition, the fragmented mitochondria tend to have higher superoxide (02") levels as denoted by the arrow heads in Figure 4.9B. lll Thus it is likely that mitochondrial fragmentation contributed to ROS overproduction. ROS overproduction, in turn, further damages the mitochondria, and as a self defense mechanism, mitochondrial damage stimulates the synthesis of new mitochondria to ameliorate the damage [273]. However, what regulates mitochondrial fragmentation still remains an open question. Recent work in our lab indicated that endoplasmic reticulum (ER) stress was observed several hours after palmitate treatment (unpublished data). ER stress can induce the release of Ca2+ from the ER [274]. Release of Ca2+ can activate the Ca2+ and cahnodulin-dependent phosphatase calcineurin [275]. Calcineurin has been shown to dephosphorylate dynamin-related protein 1 (Drpl ), a key protein involved in mitochondria fragmentation, and therefore promote mitochondrial fiagmentation [27 6]. "Therefore, it is possible that pahnitate, which has been shown to induce ER [277], initiated Ca2+ release and activated calcineurin, which subsequently activates Drpl and leads to mitochondrial fragmentation. In summary, cAMP promoted cell death in the presence of high level of palmitate in a synergistic or additive manner in HepGZ cells. The many effects exerted by palmitate on the mitochondria, such as mitochondrial fragmentation, mitochondrial ROS generation, and mitochondrial biogenesis, were amplified by cAMP supplementation. Mitochondrial fragmentation appears to precede ROS generation and may have contributed to mitochondrial ROS overproduction, which in turn stimulated mitochondrial biogenesis. 112 CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS 5.1 Conclusions The capability of MSCS to differentiate into neural lineage cells, undergo rapid in vitro amplification, secrete grth factors, suppress immune cell proliferation as well as their ease of isolation, make them a promising source for treating neuronal diseases. Numerous in vitro studies have applied different methods to induce MSCS to differentiate into neural lineages, which include chemicals, growth factors, co-culture with neural lineage cells and transgene expression [4, 119, 138, 139, 144, 147-158, 278]. Various in vivo studies also indicate that MSCS can adopt neural lineage cell fates [131, 134, 195- 197]. Despite the ongoing research and application of MSCS for clinical applications, a clear understanding of whether MSCS can differentiate into truly functional neurons and the conditions required for generating ftmctional neurons to replace the damaged neurons is still lacking [6]. The ubiquitous second messenger cAMP, which has been shown to elicit neural differentiation in MSCS in 2001 [4] and since then been frequently employed in in vitro induction media [148, 153, 155, 156], has been indicated as an important regulator for regeneration of the central nervous system [36, 192, 193]. In particular, a prior report suggested that a combinatorial approach containing cAMP, neurotrophins and MSCS can enhance repair of spinal cord injury [279]. However, it remains unclear whether cAMP has adverse effect on MSCS and if cAMP can convert MSCS into functional neurons and thereby facilitate replacement of damaged neurons. The lack of understanding regarding these questions hinders the potential application of cAMP and MSCS in future therapeutic applications for treating neuronal diseases, such as spinal cord injury. Therefore, the aim 113 of this thesis work is to investigate the effect of cAMP on MSC differentiation to neural lineage and the potential mechanisms involved. The present study demonstrates that cAMP induced an early-phase neuron—like morphological changes and a late-phase neural marker expression and ftmction. While the early-phase morphological changes are likely due to cell shrinkage and cause subsequent apoptosis, the late-phase neuron marker expression occurrs gradually and does not rely on the appearance of the morphological changes. Both events are regulated by cAMP activated PKA, however, their regulation diverged downstream of PKA, since the morphological changes do not require transcription and translation while the expression of neural markers does. Moreover, cAMP also enabled MSCS to respond, with a rise in calcium signaling, to neuronal activators, namely, dopamine, glutamate and KCl. The strongest calcium response was observed with dopamine, followed by KCl and then glutamate. While the cells induced with cAMP showed a certain degree of neuronal function, they lacked the neuronal morphology and further, their responses to the three neuronal activators were quite heterogeneous, suggesting cAMP alone is not sufficient to differentiate all the MSCS into mature neurons. These results suggest that cAMP have both positive and negative effects on MSCS. The ability of cAMP to promote MSCs differentiate into neural lineages and obtain some neuronal function may facilitate the replacement of damaged neurons. However, cAMP also resulted in cell shrinkage and correlated apoptosis, which may reduce treatment efficacy and also possibly lead to inflammation. In light of the fact that activated CREB appears to co-localize with immature neurons and promote their maturation [206, 207], we hypothesized that it is involved in cAMP ll4 induced neural differentiation of MSCs. However, whether CREB can regulate calcium signaling in response to neuronal activators was not known until this current study. To investigate the role of CREB in calcium signaling in MSCS, a stable cell line expressing the dominant-negative CREB (M 1 -CREB) was established. Introducing the dominant negative CREB greatly decreased the calcium signaling elicited by the neuronal activators but not by the positive control, ATP. Further analysis indicated that downregulating CREB activity reduced the expression of membrane receptors, such as dopamine receptor 1, which may contribute to the reduction in calcium response. Surprisingly, while down-regulating CREB reduced the calcium response, it facilitated the adoption of a neuron-like morphology. It appears that CREB has contrasting effects, encouraging neuronal function while inhibiting neuron-like morphology. It is unclear how CREB manages this; perhaps the timing of CREB activation may be important in achieving both morphological and functional differentiation. Contrasting effects on differentiation were reported for the Wnt/B-catenin signaling pathway. Early activation of Wnt/B-catenin signaling promoted cardiac lineage of embryonic stem cells (ESCs), while late activation of Wnt/B-catenin signaling inhibited cardiac lineage differentiation of ESCs [23 8, 239]. Therefore, the design of the induction media may need to be optimized in such a way that CREB activity is tuned to allow both functional and morphological neural lineage differentiation of MSCS. Apart from its role in differentiation, cAMP also initiated apoptosis in MSCS and enhanced palmitate-induced cell death in HepGZ cells. While cAMP itself is not harmful to HepG2 cells, it promoted cell death in the presence of high levels of palmitate in a synergistic manner. The many detrimental effects exerted by palmitate, such as 115 mitochondrial fragmentation and mitochondrial ROS generation, were significantly amplified by cAMP. Mitochondrial fiagmentation appears to precede ROS generation and may have contributed to the mitochondrial ROS overproduction, which is partially responsible for palmitate and cAMP induced cell death. cAMP has been proposed as a potential drug target for type 2 diabetes [249], since it appears to enhance insulin secretion. In light of the fact that palmitate is elevated in obesity and diabetes [250-252] and can induce hepatotoxicity, administering cAMP to type 2 diabetic patients may further the adverse effects on the liver, given that our results suggest that cAMP promotes cell death with palmitate in a synergistic manner in HepGZ cells. In summary, the current study revealed that cAMP is able to induce MSCs to neural lineage cells with some neuronal function in vitro. Its downstream signaling component, CREB, plays a role in the acquisition of neuronal function. The findings fi'om the MSC study shed some light on the application of cAMP and MSCS for treating neuronal diseases which could help better design future in vitro differentiation strategies. In addition, the pro-apoptotic affects of cAMP observed in HepGZ cells, i.e. promoting cell death and mitochondrial dysfunction, provides some insights , into the potential application of cAMP for treating type II diabetes. 5.2 Future directions 5.2.1 To enhance neural lineage differentiation of MSCS MSCS are naturally heterogeneous, with varying morphologies and sizes, different growth rates and differentiation abilities [108, 113, 115, 280, 281]. Such heterogeneity introduces uncertainty in cell fate determination and also compromises the differentiation 116 efficiency. To better control and increase differentiation efficiency, enrichment of a sub- p0pulation of cells with high neural differentiation potential is desired. The most abundant subpopulations in rat MSCS are the rapidly growing spindle-like cells, star-like cells and small round cells. Another subpopulation of cells with flat morphology also appeared frequently in culture. Although these cells are morphologically heterogeneous, they all express the same known markers for MSCs [108]. Currently it is not possible to separate these subpopulations of MSCS with known surface markers. A potential way to enrich MSCS with higher neural lineage differentiation is to select out a sub-population of cells that express certain neural lineage markers, since uninduced MSCS already express several neuronal/glial markers and these cells may have higher ability to differentiate into neural lineage cells [282]. However, all the examined neural lineage markers in the aforementioned study, such as nestin and GFAP [282], are intracellular proteins and cannot be used for live cell sorting. During our study, around 12% of MSCS was observed to express dopamine receptor 1 (D1), which is a membrane bound protein and therefore can be used for live cell sorting. These D1- positive cells are likely to have a higher potential to differentiate into neurons since they already possess some neuronal features. In addition, to obtain a more amenable sub-population of cells for differentiation, the differentiation induction media needs to be further improved. A good induction protocol may require different components at different stages. For example, development of midbrain dopaminergic neurons includes four stages and different factors are required at the different stages, i.e., Shh and FGF 8 for specification of precursors to the dopaminergic fate, and Wnt5a and F GF20 for development of postrnitotic dopaminergic 117 neurons [283]. Therefore, the composition of the induction media may need to be optimized for better differentiation. 5.2.2 Using polymer surfaces and modeling to study the relationship between cell morphology and differentiation capability While it is unlikely that current known cell surface markers can be used to separate the subpopulations of MSCS with different morphologies, we observed that cell morphology can be tuned by the substrates, with some surfaces favoring a particular morphology, which could affect the selection of a subpopulation of cells [284]. Moreover, it is likely that addition of cAMP may favor further selection of the subpopulation of cells with higher neural lineage differentiation potential. Therefore, another future work is to apply polymer surfaces to enrich cells for neural lineage differentiation. To test if indeed certain subpopulations can be enriched by different culture surfaces and whether cAMP addition can facilitate the selection of cells with higher neural lineage differentiation potential, the following approaches may be taken. 1) Single cell-derived cells can be obtained by cloning cylinders. The subpopulations arising fiom different single cells can be labeled with cell tracer beads (which comes in different colors and can be retained in cells for several weeks). 2) Different culture substrates, such as agarose, chitosan and polyelectrolyte multilayers (PEMs), can be prepared and tested for the cell cultures. The labeled subpopulations of MSCS can be combined and cultured on the different substrates to evaluate whether a certain population has a better ability to survive. cAMP can then be supplemented to the media and immunostained using neuronal markers to assess which subpopulation have a higher potential to differentiate into neural lineage cells. 3) Finally, 118 a modeling approach as described in [284] can be used to predict the range of surface stiffness that favors the selection of a certain subpopulation of MSCS with the highest neural lineage differentiation. This study is not limited to neural lineage differentiation, but can also be extended to differentiation of MSCS into other lineages such as osteoblasts, through modulating surface characteristics and induction media. It addition to MSC, the approach could be applied to ESCs and induced pluripotency stem (iPS) cells, which are often highly heterogeneous [285-288]. ESCs and iPS cells have great potential for future cell-based therapies since they can differentiate into all cell types, however, the intrinsic heterogeneity may lead to uncontrolled differentiation and cause teratoma formation. It is likely modulation of culture substrate mechanics may help to reduce heterogeneity in ESCs and iPS cells and thereby restrict their differentiation into specific lineages. 5.2.3 Determine how CREB regulates D1 mRNA and protein levels It is intriguing that knocking down CREB activity reduced D1 protein level but increased D1 mRNA levels. There are two possible explanations for why knock-down of CREB could result in upregulation of D1 mRNA. First, Dl promoter contains a putative CRE sites and can be potentially regulated by the CREB/ATP family transcription factors. While CREB is an activator for cAMP-responsive genes, it can induced the expression of a transcriptional inhibitor, inducible cAMP early repressor (ICER), which can compete with CREB for binding to the CRE sites and thereby repress transcription of cAMP- responsive genes [34]. Promoter analysis reveals that the D1 promoter contains a half CRE sequence (Appendix figure 3.5A). We have observed that cAMP treatment caused 119 constant upregulation of ICER (Appendix figure 3.58). It is possible for ICER to bind to the CRE site and inhibit D1 transcription. When CREB activity is knocked down, expression of ICER would be inhibited, thereby relieving the transcriptional inhibition of D1. Alternatively, CREB may modulate D1 transcription indirectly through regulating other transcription factors. Transcription of D1 is known to be regulated by the Sp family proteins [289, 290] and the D1 promoter contains several Sp binding sites (Appendix figure 3.5A). While Spl activates D1 transcription, the other Sp family protein Sp3 inhibits Dl transcription [290]. Transcription of Sp3 was shown to be upregulated by c- fos [291], a transcription factor that is activated by CREB [34], c-fos was observed to be transiently upregulated by cAMP in our study (Appendix Figure 3.5C). Therefore, it could be possible that CREB indirectly inhibits D1 transcription by upregulating c-fos expression, which increases Sp3 expression and thereby represses Dl transcription. While such transcriptional regulation could contribute to the upregulation of the D1 mRNA levels, it does not explain why the D1 protein level decreases. One possible explanation could be posttranscriptional regulation of the D1 protein. microRNAs (miRNAs), which mainly regulates mRNA by binding to the 3’-untranslated (3’-UTR) regions of the mRNA to suppress translation [292], are potential candidates for the reduced D1 protein level. A recent study suggested that expression of D1 can be modulated by the miRNA miR-504 [293]. It is not known how miR-504 expression is regulated. A possible scenario is that knock-down of the CREB activity is due to an upregulation of the miR-504 levels by a yet unknown mechanism, and thereby leading to down-regulation of the D1 protein level. 120 5.2.4 Combining experiments and modeling to delineate signaling pathways involved in regulating CREB and ATF4 activation of HepG2 cells Previously, we showed that palmitate down-regulated cAMP [163, 177]. In contrast to the down-regulation of cAMP by palmitate, we observed that its downstream signaling component, CREB, was activated by palmitate. Similarly, the activating transcription factor 4 (ATF4), which also belongs to the CREB/ATP farme transcription factors, was elevated by palmitate. While CREB is predominantly anti-apoptotic, regulating the transcription of anti-apoptotic genes such as 3ch [294, 295] and Bcl-xL [296], prolonged activation of ATF4 is considered pro-apoptotic, inducing C/EBP homologous protein (CHOP, also known as GADD153), which is a major component of the endoplasmic reticulum (ER) stress-mediated apoptotic pathway [297]. Therefore, the coordinated regulation of CREB and ATF4 may exert an important role in cell fate determination. Accordingly, an approach that integrates both experimental and modeling data may help to delineate the signaling events involved and enhance our understanding of how palmitate reduces cAMP levels while enhancing CREB activation and further explain how cAMP synergizes with pahnitate to enhance cell death. While little is known about the transcriptional regulation of ATF4, our preliminary results suggest that ATF4 may be transcriptionally regulated by CREB and vice versa. A Boolean network model is currently being developed to delineate the upstream pathways that lead to CREB and ATF4 activation. Delineation of the upstream signaling network initiated by palmitate that led to CREB and ATF 4 activation may facilitate our understanding of how palmitate activates both anti- and pro-apoptotic pathways to elicit a pro-apoptotic cell fate. 121 5.3 Overall conclusions and impact Taken together, 'the work in this thesis suggests that a combinatorial approach of cAMP and MSCS for treating neuronal diseases, such as spinal cord injury, may be appropriate since cAMP is able to guide MSCs to differentiate into neural lineage cells. However, careful attention should also be paid to the fact that cAMP may also result in undesirable effects, such as apoptosis of MSCS. In general, this study advances our understanding regarding the potential of MSCS to differentiate into neural lineage cells and obtain some neuronal function upon stimulation by cAMP. Although the neural lineage differentiation ability of MSCS is not the same as neural stem cells (N SCs), ESCs or iPS cells, MSCs have their own advantages with respect to therapeutic applications. For example, MSCS can be obtained more easily than NSCs and do not have the moral or political issues associated with ESCs. They are also much safer for clinical applications than iPS cells, which are more likely to form tumor and are quite different from ESCs in many ways [298]. Therefore, a better understanding of MSCS is still necessary and could potentially greatly advance the application of stem cells for clinical applications. Although current studies indicate that the ability of MSCS to repair neuronal damage is mainly related to their ability to secrete growth factors, the ability of MSCS to differentiate into neural lineage cells cannot be denied and would be beneficial to promoting neuronal repair. As such, future work aimed at purifying subpopulations of MSCS to obtain ones with higher ability to differentiate into neural lineages is desirable. Therefore, our ongoing research is to use a multi-prong approach, including optimization of induction media, modification of culture substrate and construction of models, to aid the selection of a subpopulation of MSCS that may have higher neural induction 122 efficiency. Obtaining an enriched cell source may advance the research regarding neural lineage differentiation of MSCS and facilitate the therapeutic application of MSCS for treating neuronal diseases. 123 APPENDICES APPENDIX 1. ISOLATION AND ENRICHMENT OF MSCS AND SEPARATION OF SINGLE-COLONY DERIVED MSCS This work has been published in Journal of Visualized Experiments Zhang, L. and Chan, C. Isolation and enrichment of rat mesenchymal stem cells (MSCS) and separation of single-colony derived MSCS. J Vis Exp. 2010 Mar 22;(37). pii: 1852. doi: 10.3791/1852. Abstract MSCS are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCS from rat femurs and tibias. The isolated cells were further enriched against two MSCS surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCS were depleted of HSCs. MSCS are naturally quite heterogenous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCS markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately. Protocol 1. Isolation of rat MSCS 124 Mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rat as previously described [152, 299]. Isolated MSCS can adhere to plastic surface and easily expand during in vitro culture. 1.1 The animal is put into an anaesthesia chamber and anaesthetized for around five minutes. During the anaesthesia, the rate of blinking, breathing and motor activity of the aminal is observed. Immediately after the animal stops motor activity and its blinking rate became infrequent, remove the animal from the anaesthesia chamber. 1.2 The animal is layed down on an operation station and killed by cervical dislocation. Femurs and tibias are cut off from the back limbs and the attached skin and muscles are removed. 1.3 The dissected femurs and tibias are put in 70% isopropanol for a few seconds and transferred to 1X D-PBS. 1.4 In a biosafety cabinet, the femurs and tibias are transferred to a 10 cm dish containing DMEM. Each bone is then held with tweezers and the two ends are cut open with a scissor. A 22G needle is attached to a 31111 syringe and filled with DMEM, then the marrow is flushed into a 50ml tube by inserting the needle to one open end of the bone. This is repeated 2~3 times for each bone. When all the marrows are obtained, cells are resuspended and the cell suspension is passed through a 70 um cell strainer to remove the bone debris and blood aggregates. 1.5 Cells are centrifuged at 200g, 4 °C for 5 minutes and the supernatant is removed by aspiration. After that, cells are resuspended in 25 ml MSC medium (DMEM containing 10% F BS and 1% Pen-Strep). 10 ml cell suspension is seeded into each 10 125 cm culture dish for a total of two dishes. The culture dishes are kept in a 37 0C and 5% C02 incubator for l~2 weeks. Medium is changed every 2~3 days. . Enrichment of rat MSCS A number of surface proteins have been used to enrich MSCS, including CD54, CD90, CD73, CD105 and CD271 [107-109]. In this study, we used CD54 and CD90 as markers to enrich MSCs by magnetic cell sorting. 2.1 When cells reached around 80% confluency, the medium is aspirated and 4~5ml trypsin-EDTA is added to each dish. The dishes are put back to the incubator for around 5 minutes to allow cell detachment. Once cells are detached, equal amount of culture medium is added to inactivate trypsin. Cell suspension is collected into a 15 ml tube and centrifuged at 200g, 4 °C for 5 minutes. 2.2 The next steps describe how to enrich MSCS by two surface markers CD54 and CD90 according to the manual for cell separation using BD lMagnet. Cell pellet is resuspended in cell staining buffer (3% heat inactivated FBS in 1X D-PBS) at 20 million cells/ml. Biotinylated CD54 antibody (0.25 pg per million cells) and biotinylated CD90 antibody (0.15 pg per million cells) is added and mixed gently with the cell suspension. After incubation on ice for 15 minutes, labeled cells are washed with an excess volume of 1X BD lMag buffer. The labeled cells are spinned down at 200g, 4 0C for 5 minutes. 2.3 The BD lMag streptavidin particles are vortexed thoroughly, and 40 pl particles are added for every 10 million cells. The labled cells are mixed with the particles 126 thoroughly and incubated at 6~12 0C for 30 minutes. This allows the streptavidin particles to bind to the biotinylated anti-CD54 and anti-CD90, which is bound to the surface proteins CD54 and CD90 respectively. 2.4 During the incubation time, a round-bottom test tube is labled to collect the positive fraction. After incubation, the labeling volume is brought to 20 million cells/ml with 1X BD lMag buffer and the labeled cells are transferred to the positive-fiaction collection tube. The positive-fraction tube is placed onto the BD IMagnet for 6 minutes. Afier that, the supernatant is removed with a glass Pasteur pipette with the positive-fiaction tube still on the BD lMagnet. 2.5 The positive-fraction tube is removed from the BD IMagnet and placed on ice. lml ice cold 1X BD lMag buffer is added to the tube and cells are resuspended by gentle mixing. The tube is placed back onto the BD IMagnet for 24 minutes. The supemant is removed with a new glass Pasteur pipette. This step is repeated one more time. 2.6 The tube is removed from the BD IMagnet and cells are resuspended in culture medium. seed one 75cm2 flask for maintaining the cells and a 10 cm dish for flow cytometry. 3. Verification of surface marker expression by flow cytometry Flow cytometry analysis is performed to verify the cells we obtained express CD54 and CD90. The HSC marker CD45 is used to confirm that the MSCs are depleted of HSCs. 127 3.1 Cells are trypsinized with trypsin-EDTA when they become ~80% confluent.,Trypsinized cells are collected into a 15 ml tube and centrifuged at 200g, 4 0C for 5 minutes to collect cells. 3.2 After aspirating the supernatant, cells are washed with 1X D-PBS once. Cells are then resuspended in cell staining buffer (1X D-PBS containing 2% FBS and 0.05% sodium azide) to a final concentration of 5~10 million cells/ml and kept on ice. Cell suspension are aliquoted into six labeled tubes (1 . cells only; 2. isotype control IgGZa; 3. Isotype control IgG]; 4. CD45; 5. CD54; 6. CD90), with 100 pl each tube. 3.3 Isotype controls and primary antibodies at appropriate concentrations (lgG2a and IgG]: 20 pl per million cells; CD45: 0.5 pg per million cells; CD54: 0.25 pg per million cells; CD90: 0.15 pg per million cells) are added to the cell suspension and incubated at 4 0C for 30 minutes. 3.4 After washing cells with 1X D-PBS twice, they are resuspended in 100 pl cell staining buffer. SA-PE (streptavidin- phycoerythrin, use 0.15 pg per million cells) is added to every tube and the cell suspensions are incubated at 4 0C for 30 minutes in dark. 3.5 Labeled cells are washed with 1X D-PBS twice and resuspended in 400 pl cell staining buffer, and then transferred to a falcon tube for flow cytometry analysis. 4. Separation of single-colony derived MSCs 128 MSCS is a heterogeneous population composed of different subpopulations with different cell shape, growth rate as well as differentiation ability [281]. However, all the subpopulations express the known MSC markers and therefore it is not feasible to use markers to separate out these subpopulations. Therefore, we applied cloning cylinders to separate out the different subpopulations, which are colonies formed by single cells. 4.1 Cells are plated at about 50~100 cells per 10 cm dish and incubated in a 37 °C and 5% C02 incubator for l~2 weeks. During this period, colonies are examined with an inverted microscope. Once the colonies have reached big enough size (better more than 100 cells in each colony), the well isolated (no surrounding colonies) colonies are marked with a sharpie at the bottom of the dish. 4.2 After aspirating medium and washing with 1X D-PBS, sterile cloning cylinders are gently placed around the marked colonies. The picked colonies should be far away from each other such that every cloning cylinder only contains one colony. 4.3 A volume of 100 pl trypsin-EDTA is added to each cloning cylinder and the dish is put back to the incubator for ~5 minutes. After 5 minutes, cells are checkecd under the microscope to see whether they are rounding up. When the cells have lifted up, an equal amount of culture medium is added to inactivate trypsin. The cell suspension was then transferred to a 60 mm dish containing 3ml prewarrned culture medium. Morphology can be trackedover the next few days. Representative Results 129 According to the protocol described in the part for rat MSCS isolation, plastic adherent MSCs should be visible the next day after plating. As cells continue to proliferate, the confluent cells should look like the cells shown in Appendix figure 1.1A. When cells reach ~80% confluency (Appendix figure 1.lB), subculturing can be carried out. During subculture, trypsin-EDTA was used to detach cells and lifted cells are small and round as shown in Appendix figure 1.1C. Appendix figure 1.1 Phase contrast images of rat MSCS. (A) Confluent MSCS. The majority of cells are spindle-like or star-like. (B) MSCS around 80% confluency. (C) Lifted cells after trypsinization are small and round. Once MSCs are enriched by magnetic cell sorting, flow cytometry analysis is performed to verify the surface marker expressions. If the enrichment is good, the cells should show positive staining against MSC markers CD54 and CD90 but negative against the HSC marker CD45 (Appendix figure 1.2). Isotype controls IgG2a and IgG] are used as negative controls. When cells are seeded at proper clonal density, colonies should rise from single cells. Appendix figure 1.3A represents a colony formed by a single cell. Cloning cylinders can then be used to separate the colonies and cells derived from the colonies can be cultured separately. Appendix figure 1.3B and 1.3C represents cells derived from two individual colonies. The cells derived from colony l are spindle like (Appendix figure 1.38) whereas the cells derived from colony 2 are round (Appendix figure 1.3C). 130 isotype ctrl-l — isotype ctrl-2 — CD45 — CD54 3 — CD90 C D O U 1 1’! 1“ j, l f 01'". . rvarn‘ . "’4" . . ....-n . . ..4.... 100 101 102 103 104 Fluorescence intensity Appendix figure 1.2 Flow cytometry analysis of MSCs for surface markers. MSCs were labeled with antibodies against IgG2a (isotype control 1), IgG] (isotype control 2), CD45, CD54 and CD90. MSCS expressed CD54 and CD90 but not CD45. Appendix figure 1.3 Colony formation by MSCS and single-colony derived cells. MSCs cultured at clonal density form individual colonies. These colonies can be separated by cloning cylinders and cells from different colonies can be cultured separately. (A) A representative colony formed by MSCS when plated at clonal density. (B) Spindle-like cells derived from one colony. (C) Round cells derived fiom another colony. Discussion This protocol describes how to isolate and enrich MSCS. A method to separate the single-colony derived cells is also incorporated. There are several steps that are important for a successful isolation, enrichment and colony separation. While doing cell isolation 131 from rat, it is recommended to filter through a cell strainer or a sterile nylon mesh of similar size to get rid of the blood clots and bone debris. After plating the cells overnight, many dead cells will be floating in the medium and the dead cells are removed by replacing with fiesh medium which should help the growth of the attached cells. The magnetic cell sorting in this protocol describes how to perform a positive selection, and similar procedures can be used to perform a negative selection. The amount of antibodies to be added may differ and optimization is required to achieve better sorting. This is also true for labeling the cells for flow cytometry analysis. If not running flow cytometry analysis for the labeled samples immediately, samples can be fixed with 2% formaldehyde and run later. However, long-term storage is not recommended since this tends to increase the auto-fluorescence and sacrifice sample quality. The key part for the colony separation is seeding at the right cell density (which should be optimized experimentally) and locating single clones that are not surrounded by other clones. If there are other clones nearby, the cloning cylinder may encompass the nearby clones and the cells obtained will no longer be from one clone. When placing the cloning cylinder over the clone, also be careful not to slide it over the dish surface as this will cause the silicon grease at the bottom of the cloning cylinder to cover the cells and prevent the trypsin from reaching the cells to detach them. 132 Appendix Table 1.1 Specific reagents and equipment Name of the reagent Company Catalogue number Comments (optionaD 1X D-PBS Invitrogn 14040-133 DMEM Invitrogen 1 1885-084 Cell strainer BD Bioscience 352350 F BS Invitrogen 26140-079 Pen-Strep Invitro gen 1 5 140 Trypsin-EDTA Invitrogen 25200-056 BD Imagnet BD Bioscience 552311 Biotin mouse IgGZa BD Bioscience 555572 Biotin mouse IgGl BD Bioscience 555747 Biotin anti-rat CD54 Cedarlane Labs CL054B Biotin anti-rat CD90 Cedarlane Labs CL005B Biotin anti-rat CD45 Cedarlane Labs CL001B BD Imag buffer BD Bioscience 552362 Round-bottom tube BD Bioscience 352063 Streptavidin particles BD Bioscience 557812 SA-PE R&D systems F0040 Cloning cylinder Sigma C2059 133 APPENDIX 2. SUPPLEMENTARY METHODS AND FIGURES FOR CHAPTER 2 Supplementary methods Quantitative real time polymerase chain reaction (RT-PCR) Cells were treated as indicated and mRNA was extracted using the RNA extraction kit from Qiagen according to the manufacture’s instruction. mRN A was then reverse transcribed into cDNA using the cDNA synthesis kit from Bio-Rad. The following primer sets (Eurofins MWG Operon) were used for PCR: actin (5’- CCCTAGACT'I‘CGAGCAAGAGA-3’, 5’- AGGAAGGAAGGCTGG AAGAG—3’), NSE (5’-ACCACATCAACAGCACCATC-3’, 5’-T'I‘GTTCTCAGTCCCATCCAA-3’), Tujl (5 ’-TAGTGGAGAACACGGATGAGA-3 ’, 5 ’-GCAGACACAAGGTGGTTGAG-3 ’), GFAP (5’-GCTCCAAGATGAAACCAACC-3 ’, 5’-AACCTTCCTCTCCAGATCCA-3 ’). Amplification of the cDNA templates were detected by SYBR Green Supermix (Bio-Rad) using Real-Time PCR Detection System (Bio-Rad). The cycle threshold (CT) values for each condition were determined by the Mle software. Colony-forming units (CFU) assay CFU assay was used to assess the self-renewal capability of MSCS [300]. In brief, 50~100 cells were plated in a 10 cm culture dish and the dish is incubated in a 5% C02 incubator at 37°C for 10~14 days. Colonies were stained with 0.5% crystal violet (Sigma) in methanol for 5~10 minutes at room temperature and the number of colonies were scored with a scoring grid. 134 Adipogenesis induction Adipogenesis induction was carried out according to the protocol provided on the Millipore website. Cells were induced with adipogenesis induction media (cell culture media supplemented with 10 mM dexamethasone (Sigma), 0.5 M IBMX (Sigma), 10 mg/mL insulin (Sigma) and 20 mM indomethacin (Si gma)) after reaching confluency. This corresponds to differentiation day l. The induction media was changed on day 3 and day 5. On day 7, the media was changed to adipogenesis maintenance media (cell culture media containing 10 mg/mL insulin) for two days. On days 9, 11 and 13, the media was replaced with fiesh adipogenesis induction media. On day 15, the media was replaced with fi'esh adipogenesis maintenance media. On days 17 and 19, the media was replaced with fresh adipogenesis induction media. On day 21, the cells were fixed and lipid droplets were stained using Oil Red 0 solution (Sigma). Osteogenesis induction Osteogenesis induction was carried out according to the protocol provided on the Millipore website. Confluent cells were induced with osteogenesis induction media (cell culture media supplemented with 1 mM dexamethasone (Sigma), 0.1 M ascorbic acid 2- phosphate (Sigma), 1 M glycerol 2-phosphate (Calbiochem) and 200 mM L-glutamine (Sigma)). Media was changed every two days for a total of 14 days. Calcium deposition can then be visualized by Alizarin Red (Sigma) staining. Oil Red 0 staining for lipid droplets 135 Cells were fixed with 4% formaldehyde for 30 minutes at room temperature. After carefully removing the fixative, the cells were rinsed three times with PBS, and twice with DI water. The cells were then covered with enough Oil Red 0 solution and incubated for 50 minutes at room temperature. Excess dye was removed and the cells were rinsed three times with DI water. The cells stained with Oil Red 0 can be visualized using phase contrast imaging (with a color camera). Alizarin Red staining for calcium deposits Cells were fixed with 70% ice cold ethanol for 1 hour. After carefully removing the fixative, the cells were washed twice with DI water for 5~10 minutes each. Alizarin Red solution was added to stain the cells for 30 minutes after removing the water. The cells were rinsed four times with DI water before phase contrast imaging (with a color camera). Supplementary Figures Appendix figure 2.1 Self-renewal and multi-lineage differentiation ability of MSCS. (A) Colony-forming units (CF U) assay indicates that MSCS can form colonies and have self- renewal property. (B) Oil Red 0 staining for oil droplets indicates MSCS can differentiate into adiocytes. (C) Alizarin Red staining for calcium deposit indicates MSCS can differentiate into osteoblasts. 136 Appendix figure 2.2 Cytoskeleton staining for actin filaments and microtubules. MSCs were treated with 10 pM forskolin and 100 pM IBMX (F I) for 1 hr. Green: microtubules; red: actin filaments; blue: nucleus. Appendix figure 2.3 Morphology of MSCS treated with staurosporine (1 pM) 1 hour. (A) Microtubules, (B) Actin filaments, (C) Nucleus and (D) Overlaid image of (A-C). 137 Appendix figure 2.4 Morphology of MSCS treated with 10 pM forskolin and 100 pM IBMX (F1) for 12 hours. The cell denoted by the arrow head has changed morphologically and lifted up from the surface. The cells denoted by the arrows are not as anchored to the surface and eventually may lift up. 6 Q) * ‘t DCIrl g5 l ' lFl1d ‘54. 2 .9 E3 9 _a_>2 E m1 E 0 NSE Tuj1 GFAP Appendix figure 2.5 mRNA levels of neural markers NSE, Tujl and GFAP. MSCS were in control media or induced with 10 pM forskolin and 100 pM IBMX (F1) for one day. (n=3 for NSE and Tuj 1, n=2 for GFAP). *: p<0.05. 138 C 1 2 3 4 Fl - - + + ‘2 Rp—cAMP - + - + NSE -~ w W 3:15. . GFAP a I: I m GAPDH in w w w Appendix figure 2.6 PKA regulates morphology and marker expression. (A) MSCS induce with 10 pM forskolin and 100 pM IBMX (F1) for one hour. (B) MSCs pretreated with 10 pM Rp—cAMPS for 30 minutes and induced with Fl supplemented with 10 pM Rp-cAMPS for 1 hour. (C) Expression of neural markers NSE, Tujl and GFAP. MSCs Were treated with F I in the absence or presence of 10 pM Rp-cAMPS for 24 hours. Appendix figure 2.7 Morphology of MSCs treated with FI and paclitaxel. (A) Morphology of MSCS treated with 10 pM forskolin and 100 pM IBMX (F1) for 3 hours followed by fresh Fl medium for 1 hour. (B) Morphology of MSCs treated with F1 in the presence of 0.4 pM paclitaxel for 3 hours followed by fresh F I medium for 1 hour. 139 600 500 400 - 300 200 100 cAMP levels (10"—3 nmol/ug) O 3h 0.511 54:: .::..c:, 5.24:: .—.mm ~01: v—Nm 0h 0.5h .S ‘0. o _ ctrl F I pulse 1 F I pulse 2 F 1 pulse 3 2.0 .. l N NSA Les—1 51 l \ A a ' A 6 - c“ gtv‘ r» we)" “09% ‘ fixes ‘ 1.5 1.0 Cells (million) 0.5 0.0 1 Appendix figure 2.8 cAMP levels and live cells in different treatments (A) Intracellular cAMP levels for untreated cells, or cells treated with 10 pM forskolin and 100 pM IBMX (F1) for 0.5, 1, 2 and 3 hours (F I pulse 1), or after the first pulse treated again with F l for 0.5, 1, 2 and 3 hours (FI pulse 2), or after the second pulse treated again with F1 for 0.5, l, 2 and 3 hours. (B) Number of viable cells in control and F1 treated culture, in the absence or presence of 1 pg/ml actinomycin D (ActD), 10 pg/ml cycloheximide (CHX) or 0.4 pM paclitaxel (Ptx) for 3 hours followed by fresh F I medium for another 21 hours (n=3). *: p<0.05, NS: not significant. 140 APPENDIX 3. SUPPLEMENTARY FIGURES FOR CHAPTER 3 A \ W 3,2020 o’ o’ 8 *MSC >§ 2v °J £0.15 Ml-MSC cg) \’ 0%)“? E é é és 50.10 CREB ' ‘V-d €0.05 U o 1 2 3 4 5 6 Days in culture Appendix figure 3.1 Evaluation of MSCs expressing Ml-CREB. (A) Expression of CREB for MSCS expressing the control vector and MSCS expressing the dominant negative Ml-CREB (Ml-MSCS). (B) Proliferation curves for MSCs and Ml-MSCs during culture. (C) Colony formation of Ml-MSCs (expressing Ml-CREB). 503m 20]: m Appendix figure 3.2 Time-dependent morphological changes upon 10 pM forskolin and 100 pM IBMX (Fl) induction of MSCS (expressing control vector) and Ml-MSCs (expressing Ml-CREB). (A-B) MSCS and Ml-MSCs in control medium (left: MSCs; right: Ml-MSCs). (C-D) MSCS and Ml-MSCs induced with F I for 2 days. (E-F) MSCS and Ml-MSCs induced with F l for 4 days. (G-H) MSCS and Ml-MSCs induced with F1 for 6 days. (I-J) MSCS and Ml-MSCs induced with F l for 8 days. (K) Magnified view of Ml-MSCs induced with F1 for 8 days. Arrows denote a typical cell (Ml-MSCS) that adopted a distinct morphology. 141 U.) C iii NN OM LDH release (%) G p—o O LII 0 dfl Y 5‘5 Y \QX\00Y10X100? “$0.00 Appendix figure 3.3 LDH release as indication of toxicity of treatment. Cells were incubated in control media, media containing 5 pM forskolin and 50 pM IBMX (F5150), 10 pM forskolin and 100 pM IBMX (F101100), 20 pM forskolin and 200 pM IBMX (FZOIZOO) and 40 pM forskolin and 400 pM IBMX (F4OI400) for 24 hours. NS: not significant; **:p<0.01; ***: p<0.001. O O A O\ 00 O O O GO/Gl phase cells (%) N O ‘ $1 9.0.; 9.; $1 9.0.; 9.," Q(11‘, 9‘33” so 083’ QC «1? 993 so 093» ctrl Fl Appendix figure 3.4 CREB and GO/Gl phase cells. MSCs were transfected with the control vector pCMV, the constitutively active CREB (VP16-CREB), the negative control scramble siRN A and CREB siRN A. Transfected cells were incubated in medium in the absence (ctrl) or presence of 10 pM forskolin and 100 pM IBMX (F I) for 20 hours and cell cycle distribution was assessed after that (n=3). *: p<0.05; **: p<0.01. 142 Spd'l Sp J—E -1 943 -934 -538 -430 B1:1 ->0h 1h 3h 6h 12h 24h C lCER “can..-“ 1:1—>0h .5h 1h 2h 3h 6h 12h actin ~~* 8”“ c-fos — a F‘- i F1 ->0d 1d 2d 4d 6d GAPDH-— ......____..——-----. ICER mflwfi. actin ~“fi‘” Appendix figure 3.5 D] promoter and expression of ICER and c-fos. (A) D1 promoter contains a CRE site and several Sp sites. (B) ICER expression upon 10 pM forskolin and 100 pM IBMX (F I) treatment for the indicated time. (C) c-fos expression upon FI treatment for the indicated time. ' 143 APPENDIX 4. SUPPLEMENTARY FIGURES FOR CHAPTER 4 Appendix figure 4.1 Oil Red 0 staining for triglyceride. (A) cells treated with 0.7 mM oleate for 5 h, (B) cells treated with 0.7 mM palmitate for 5 h, (C) cells treated with 0.7 mM oleate for 12 h, and (D) cells treated with 0.7 mM palmitate for 12 h. 144 Appendix figure 4.2 Mitochondrial morphology. (A) control cells in medium/BSA, (B) cells treated with 0.2 mM palmitate for 24 h, (C) cells treated with 0.4 mM palmitate for 24 h, (D) and cells treated with 0.7 mM palmitate for 24 h. mitochondrial cytosolic ctrl ole palm ctrl ole palm F|-+-+-+-+-+-+ Bcl2 -" "" "" ‘“ ~~ Smac _ — - .... COXIV ° 1'” GAPDH Appendix figure 4.3 Western blot of mitochondrial and cytosolic fractions. Cells were in control, 0.7 mM oleate and 0.7 mM palmitate in the absence or presence of 10 pM forskolin and 100 pM IBMX (F I) for 24 h (n = 3). 145 ‘JF 10 A Dctrl ‘3) 8 Dctrl+Fl 2 #:1- :3 [2P .3 6 IP+F1 ** 8 l 8 C. 08 4 .2 7 i E 2 g , re 0 4 12:1?- early late necrotic early late necrotic apoptotic apoptotic apoptotic apoptotic 5h 12h Appendix figure 4.4 Apoptotic and necrotic labeling by P1 (propidium iodide) and Alexa Fluor-488 conjugated annexin V. Cells were in control (ctrl) and 0.7 mM palmitate (P) without (w/o) or with (w/) 10 pM forskolin and 100 pM IBMX (F1) for 5 h and 12 h. Early apoptotic cells: PI_ annexin V+ cells; late apoptotic cells: PIJr annexin V+ cells; necrotic cells: PI+ annexin V— cells (n = 3).*: p < 0.05; "z p < 0.01. \ 10.0pm Appendix figure 4.5 Mitochondrial morphology of cells treated with 0.7 mM palmitate for 12 h. (A) Some cells already had fragmented mitochondria, as indicated by the arrowheads. 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