PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINE return on or before date due. MAY BE RECAILED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1M WM“ B ~ADRENERGIC STIMULATION INCREASES PROTEIN PHOSPHORYLATION AND SKELETAL MUSCLE a—ACTIN mRNA ABUNDANCE IN C2C12 MYOTUBES By Scott Allen Kramer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1998 ‘ e ABSTRACT B -ADRENERGIC STIMULATION INCREASES PROTEIN PHOSPHORYLATION AND SKELETAL MUSCLE or-ACTIN mRNA ABUNDANCE IN C2C12 MYOTUBES By Scott Allen Kramer B-adrenergic agonist (BAA) stimulation of skeletal muscle increases expression of muscle specific genes and muscle hypertrophy. The exact mechanism through which these actions occur remains unresolved. The objective of this study was to investigate the mechanism of BAA-stimulated muscle hypertrophy using the immortalized mouse hind limb skeletal muscle cell line, C2C12. The myogenic phenotype of C2C12 cells was characterized through assay of skeletal muscle specific gene expression (skeletal a-actin, SKA), protein (sarcomeric myosin, SM), and enzyme activity (muscle creatine kinase, MCK). Detection of SKA messenger ribonucleic acid (mRNA) abundance in C2C12 myotubes was accomplished using a SKA isoform specific, sequence verified, 105 bp digoxygenin labeled, polymerase chain reaction (PCR) generated probe homologous to 3591 bp-3696 bp of the 3’ untranslated region (UTR) region of the mouse SKA gene. Abundance of SKA mRN A was expressed per unit of 18S ribosomal ribonucleic acid (rRNA). The appearance of SKA mRN A occurred approximately 48 hours post confluence, increased as cells differentiated and fused to form myotubes, and paralleled the appearance of SM detected using an anti-SM antibody. A dose-response study suggested that, for the stimulation of SKA, the optimal concentration of the synthetic BAA, isoproterenol (ISO), was approximately 10'5 M. Further experiments revealed that the response to stimulation is maximal at 72 hours post stimulation and is depressed by the addition of the non-selective beta adrenergic receptor (BAR)-antagonist, propranolol (PRO), or protein kinase A (PKA) inhibitor, HA1004. The effects of BAA stimulation of PKA mediated phosphorylation (~P) of key proteins were determined using an anti-P- threonine antibody. Irnmunoblots of cell extracts revealed an ~90 kilo-Dalton (kDa) BAA-responsive, developmentally regulated, cytoplasmic protein in cell extracts of C2C 12 myotubes. The identity of the protein remains undetermined. ACKNOWLEDGEMENTS I would like to thank my committee members: Dr. M. Benson, Dr. W.G. Bergen, Dr. M.E. Doumit, Dr. J. Linz, Dr. H. Ritchie, and Dr. M. Vandehaar for their patience, understanding and input over the past six years. My special thanks go to Dr. ME. Doumit for setting an example to follow and all of his time and input over the past three years in wrapping up my dissertation research. I value his friendship and guidance. I would also like to thank Sharon Debar. Sharon has been my other two arms and legs over the past five years and I wouldn’t be as far as I am today if it weren’t for her. She has always been there for me and I really appreciate that. Dr. P.S. Weber has also had an impact on my graduate career. I wanted to thank her for setting an example and for her guidance when I was in need. I would also like to thank those people who I consider my personal support staff. These people have served as emotional, financial and technical advisors during my time spent at Michigan State University. I thank Rita House, Joanna Gruber, Ron Southwick and Betsy Booren for their friendship, advice, and assistance over the years. Not to discount the numerous friends and acquaintances I’ve made at Michigan State University who are too numerous thank in these acknowledgements. Finally, I’d like to acknowledge my family, Kelly Biersbach and Trevor Williams for being there, behind the scenes; who, when I felt I was walking a wire without a net, reassured me there was someone to catch me if I fell. That’s a great feeling and important to me; thank you. TABLE OF CONTENTS LIST OF TABLES ________________________________________________________________________________________________________________ ix LIST OF FIGURES ______________________________________________________________________________________________________________ x LIST OF ABBREVIATIONS ............................................................................................. xm INTRODUCTION ________________________________________________________________________________________________________________ 1 LITERATURE REVIEW ..................................................................................................... 9 Myogenesis ......................................................................................................... 9 Skeletal Muscle Structure .................................................................................. 10 MyofibrillarGene Expression ____________________________________________________________________________ l3 Actin .................................................................................................................... 14 Actin Isoform Switching ................................................................................... 17 Skeletal ot-actin .................................................................................................. l9 Transcriptional Regulation of Skeletal a-actin ................................................ 20 B—adrenergic Agonists ....................................................................................... 21 B-adrenergic Receptors ...................................................................................... 26 Factors Affecting Responsiveness to B-adrenergic Agonists .......................... 31 Manipulation of Animal Growth by B-adrenergic Agonists ........................... 36 Effect of B-adrenergic Agonists on Skeletal Muscle ....................................... 39 Signal Transduction Pathway Cross-Talk ........................................................ 4O Transcriptional Regulation by Extracellular Signals Through Phosphorylation .................................................................................. 46 F05 ....................................................................................................................... 43 Jun ....................................................................................................................... 49 Activator Protein-1 ............................................................................................. 50 Cyclic AMP Response Element Binding Protein ____________________________________________ 52 Influence of Phosphoproteins on Skeletal a-actin Transcription ____________________ 53 CHAPTER 1 - DEVELOPMENT AND CHARACTERIZATION OF A SKELETAL ot-ACTIN SPECIFIC PROBE Abstract 55 ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo vi Introduction 56 oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo Skeletal ot-Actin _________________________________________________________________________________________________ 58 Materials and Methods ______________________________________________________________________________________ 6O Plasmid ______________________________________________________________________________________________________ 60 Primers ....................................................................................................... 61 Polymerase Chain Reaction _____________________________________________________________________ 61 Sequencing ................................................................................................ 61 RNA Isolation ........................................................................................... 64 Northern Blot Analysis _____________________________________________________________________________ 68 Results and Discussion 70 oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo CHAPTER 2 - B-ADRENERGIC STIMULATION INCREASES SKELETAL a-ACTIN mRNA ABUNDANCE IN C2C12 MYOTUBES Abstract _______________________________________________________________________________________________________________ 80 Introduction ________________________________________________________________________________________________________ 82 Materials and Methods _______________________________________________________________________________________ 84 Cells ........................................................................................................... 84 Sample Preparation for Immunoblot Analysis ............................... _ ........ 84 SDS-PAGE and Western Blotting ___________________________________________________________ 85 Plasmids .................................................................................................... 86 Primers ....................................................................................................... 87 Polymerase Chain Reaction _____________________________________________________________________ 88 Sequencing ________________________________________________________________________________________________ 88 RNA Isolation ___________________________________________________________________________________________ 89 Northern Blot Analysis ............................................................................. 90 Muscle Creatine Kinase Activity .............................................................. 92 Developmental Time Course ................................................................... 92 Skeletal or-actin mRNA Abundance in Response to Increasing Duration of Isoproterenol Stimulation __________________________________ 93 B-Adrenergic Pathway Component Stimulation ..................................... 93 Statistical Analysis ___________________________________________________________________________________ 94 Results and Discussion 95 oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo CHAPTER 3 - B-ADRENERGIC STIMULATION INCREASES THE PHOSPHORYLATION OF A 90 kDa PROTEIN IN C2C12 MYOTUBES Abstract _______________________________________________________________________________________________________________ 125 Introduction ........................................................................................................ 127 Materials and Methods ....................................................................................... 131 Cells ........................................................................................................... 131 Sample Preparation ................................................................................... 131 SDS-PAGE and Western Blotting ___________________________________________________________ 132 Two-Dimensional Electrophoresis __________________________________________________________ 133 vii Duration of B-Adrenergic Stimulation on Protein Phosphorylation 133 Phosphodiesterase Inhibitor ..................................................................... 134 Cyclohexamide Treatment ....................................................................... 134 Cell Fractionation ..................................................................................... 135 Statistical Analysi _____________________________________________________________________________________ 135 Results and Discussion _____________________________________________________________________________ 139 CONCLUSION ..................................................................................................................... 1 74 IMPLICATIONS .................................................................................................................. 1 77 APPENDICES Appendix A “Touch-down” polymerase chain reaction protocol for the skeletal a—actin 105 bp probe ....................................................... 180 Appendix B Dyedeoxy chromatogram of the Barn HI/Pst-I 931 bp fragment ....................................................................................................... 181 Appendix C Dyedeoxy chromatogram of the Barn HI/Pst-I 451 bp fragment ...................................................................................................... 183 Appendix D “Touch-down” polymerase chain reaction protocol for the 18S rRNA 546 bp probe ______________________________________________________________________ 185 Appendix E Dyedeoxy chromatograms of the 18S rRNA 546 bp Fragment ..................................................................................................... 186 Appendix F Product index ................................................................... — ........................... 188 BIBLIOGRAPHY 190 ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo viii LIST OF TABLES Table 1 B-adrenergic activity in livestock ............................................................ 3 Table 2 Adrenergic receptor subtypes and post-receptor activity ....................... 32 Table 3 Adrenergic receptor subtype characterization _________________________________________ 35 ix LIST OF FIGURES Figure 1 Composite of signal transduction cascade interactions and skeletal a-actin mRN A abundance ooooooooooooooooooooooooooooooooooooooooooooooooooo Figure 2 Skeletal muscle structure .......................................................................... Figure 3 Catecholamine biosynthesis ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 4 Common B-adrenergic agonists ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 5 Adrenergic receptor activity ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 6 The B-adrenergic receptor ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 7 Schematic of signal transduction pathways and possible cross-talk interactions ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo Figure 8 Skeletal or-actin 105 bp PCR product ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 9 Sequence analysis of a PCR generated 105 bp fragment of mouse skeletal or-actin ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 10 Species and tissue Northern blot analysis using a 105 bp skeletal or-actin probe ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 11 Species skeletal muscle skeletal a-actin Northern bIOI ,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 12 105 bp probe competition experiment ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Figure 13 Skeletal a-actin mRNA/sarcomeric myosin developmental time course in C2C12 muscle cells Figure 14 Skeletal ot-actin mRN A abundance/muscle creatine kinase - activity developmental time course in C2C12 muscle cells ,,,,,,,,,,,,,,,,,,, Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Isoproterenol dose response on skeletal a-actin mRNA abundance in C2C 12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Isoproterenol dose response on skeletal a-actin mRNA and 188 rRNA abundance in C2C12 myotubes ............. Isoproterenol dose response on total protein abundance in C2C12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, F orskolin dose response on skeletal ot-actin mRNA abundance in C2C12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Forskolin dose response on skeletal a-actin mRNA and 18S rRNA abundance in C2C12 myotubes ............. Forskolin dose response on total protein abundance in C2C12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Duration of isoproterenol stimulation on skeletal a-actin mRNA abundance in C2C12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,,,, Duration of isoproterenol stimulation on skeletal or-actin mRNA and 18S rRNA abundance in C2C12 myotubes B-adrenergic pathway component modulation and skeletal a-actin mRNA abundance in C2C 12 myotubes ,,,,,,,,,,,,,,,,,,,,,,,, Summary figure-Chapter 2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Classical B-adrenergic signal transduction pathway ,,,,,,,,,,,,,,,,,, Anti-P-serine and anti-P-threonine immunoblot of C2C12 myotube cell extracts ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Identification of a ~90 kDa B-adrenergic responsive protein in C2C 12 myotube cell extracts ___________________________________________________ Anti-P-threonine immunoblot against B-adrenergic pathway component stimulated C2C 12 myotube cell ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo Anti-P-threonine immunoblot against isoproterenol stimulated C2C12 myotube cell extracts over time ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, xi oooooooooooo 101 103 105 108 110 112 115 117 119 123 128 137 139 142 144 Figure 30 Figure 31 Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Figure 37 Figure 38 Figure 39 Verification of a phosphorylation response to isoproterenol ,,,,,,,,,,,,,,,,, Initial fractionation of C2C12 myotube cell extracts ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Anti-P-threonine immunoblot against the sarcoplasmic fraction of C2C12 myotube cell extracts _______________________________________________________________ Anti-P-threonine developmental time course immunoblot against C2C12 myotube cell extracts ....................................................... Anti-P-threonine two-dimensional electrophoresis against the C2C12 myotube sarcoplasmic fraction .............................................. Coomassie Blue stained purified ~90 kDa protein transfer ,,,,,,,,,,,,,,,,,,, Identification of the subcellular location of ~90 kDa protein in C2C12 myotubes ...................................................................... VFl-C immunoblot against immunoprecipitated ~90 kDa protein of isoproterenol stimulated and control C2C 12 myotube microsomal fractionated cell extracts ...................................................... Summary figure- Chapter 3 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Dissertation summary figure .................................................................... xii 147 150 153 155 157 160 166 168 172 175 LIST OF ABBREVIATIONS oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooooooooooooooooooooooo ............................................................ oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooooooooooooooooooooo antibody adenylyl cyclase anti-digoxygenin Fab alkaline phosphatase activator protein-l aspartic acid Americain tissue culture collection beta-adrenergic agonist beta-adrenergic receptor beta-adrenergic receptor kinase basic logical alignment search tool base pair bovine serum albumen cardiac a-actin cyclic adenosine-monophosphate clenbuterol cAMP response element cAMP response element binding protein cholera toxin diacyl glycerol diethyl pyrocarbonate Dulbeccos modified eagles medium deoxyribonucleic acid epinephrine filamentous actin fetal bovine serum forskolin globular actin guanine diphosphate G-protein (inhibitory) glutamic acid G-protein (stimulatory) guanine triphosphate N-(2-guanidinoethyl)-5- isoquinolinesulfonamide xiii horseradish peroxidase H7 ................................................................ 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine IP3 ............................................................... inositol triphosphate ISO .............................................................. isoproterenol .1 SR ............................................................. junctional sarcoplasmic reticulum kb ................................................................ kilobase kDa .............................................................. kilo-Dalton Leu .............................................................. leucine MAPK ......................................................... mitogen-activated protein kinase MCK ........................................................... muscle creatine kinase Met .............................................................. methionine MCE ____________________________________________________________ mercaptoethanol mRNA ......................................................... messenger ribonucleic acid NFEP .......................................................... anti-phosphatase buffer NOREPI ...................................................... norepinephrine ~P ................................................................ phosphorylate/d/ion PAGE .......................................................... polyacrylamide gel electrophoresis PBS _____________________________________________________________ phosphate buffered saline PCR _____________________________________________________________ polymerase chain reaction PDE _____________________________________________________________ phosphodiesterase PDE-I _________________________________________________________ 4(3,butoxy,4-methoxybenzyl)imidizolidin-2-one PE ________________________________________________________________ phorbol ester pI _________________________________________________________________ isoelectric point P1P; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, phosphatidylinositol 4,5-bisphosphate PKA ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, cAMP dependent protein kinase PKC _____________________________________________________________ protein kinase C PRO ............................................................. propranolol RAC ____________________________________________________________ ractopamine RNA ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ribonucleic acid SDS ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, sodium dodecyl sulphate Ser ............................................................... serine SKA ____________________________________________________________ skeletal a-actin SM _______________________________________________________________ sarcomeric myosin SR ................................................................ sarcoplasmic reticulum SSC ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, sodium chloride/sodium citrate buffer ST ................................................................ somatotropin TBB ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, tris/EDTA buffer Th1 ............................................................... threonine TPA ............................................................. tissue plasminogen activator 2’D .............................................................. two-dimensional UTR ............................................................ untranslated region UV ............................................................... ultraviolet X-Gal .......................................................... 5-bromo-4-chloro-3-indoyl-B-galactosidase xiv INTRODUCTION Consumer demand for leaner food products and a recognition of this market by producers have fostered continued research on factors affecting lean and fat deposition in food- producing animals. Growth rate and body composition of livestock can be optimized to meet consumer needs for a leaner food product and to improve the efficiency of meat- animal production (Wray-Cahen et al., 1998). The field of study in animal science now referred to as growth biology has been concerned with the mechanisms of protein and fat deposition in food-producing animals (Bergen et al., 1996). Increasing the productivity of livestock species can be interpreted as increasing lean gain while depressing fat gain, or faster more efficient gain. Depending on the species, 50-70% of production costs are associated with feed. As a result, much research has been aimed at maximizing feed- conversion efficiency (Wray-Cahen et al., 1998). The feed-conversion ratio represents the interplay between the rate of body gain, the nature of the tissue deposited, and the inefficiencies of digestive processes (Wray-Cahen et al., 1998). Approximately 60% of total carcass weight is represented by skeletal muscle mass (Mulvaney, 1981). Skeletal muscle represents the primary economic product in a livestock production system; therefore, increasing productivity may increase economic gains. My long term goal is to investigate the mechanism by which growth-promoting hormones and other growth- promoting substances affect skeletal muscle protein accretion. This should lead to M‘a.',§..1'1" XuQL-b. ufl- r.. b ".fl‘v~r~- ‘0 O . tb‘..o\.uu\) \‘ “_ ' a urn... . 2.. 1' _ '- mun-“.5‘hku ;. ~44 ' 4 .... my“) xx‘ ‘\. ' I v . . I“""" '5. gen..- H Mut.\ .L d...“ u A (‘~ - ‘ I "xv-w. ch..‘,..;“5._'_~\‘v‘ J .._.. ‘ «q ”*2. . r . r “I...“ > I I :1- - A““ F H. u..- '5\\.‘\ ‘. 2‘1 2" 1' , .e um “It‘s C. {I ‘0 a r '1’ "Iu,.."‘ huk \ ’3‘. “I ~~Miu§“.~ um I 39..., _ ’ henyl¢.‘e r€\.:i: .. “U [it \ 3%..“ b q“ “ANALI 0c 9- n A 0“ strategies that improve the efficiency of lean tissue growth and would improve the economics of food animal production. Identification and interpretation of the responses to a variety of strategies utilized to modify body composition have been extensive. Regardless of the strategy applied, all attempt to shift the priority of nutrient allocation toward lean tissue deposition while simultaneously decreasing adipose tissue accretion (Hammond, 195 2, Wray-Cahen et al., 1998). Animal scientists have used a variety of strategies to increase animal productivity, from selective breedingto the use of exogenous agents. By the middle of the 1980's, availability of species-specific recombinantly produced somatotropin (ST), as well as orally active B-adrenergic agonists (BAA), encouraged a phenomenal amount of research on administration of ST and feeding BAA to animals (Bergen and Merkel, 1991). Extensive research has been conducted on the physiological effects and biochemical mode of action of repartitioning agents on protein and fat metabolism in meat-producing animals. Partitioning agents, such as ST or BAA, have increased rate of gain, improved efficiency of feed utilization, depressed fat deposition, and enhanced lean gain in livestock species (Asato et al., 1984, Baker and Kieman, 1983, Bergen and Merkel, 1991, Muir, 1988) (Table 1). Administration of exogenous agents to animals may improve the efficiency of lean tissue gain. A better understanding of the repartitioning mechanism is essential to (1) utilize nutrient repartitioning strategies to their potential and (2) address more specific questions regarding mechanism of action. Maximal lean gain translates into decreased feed/ gain, decreased waste, decreased time to market, and finally a greater Table l B-adrenergic activity in livestock Characteristic Poultry Ruminanu Swine Carcass protein (% increase) 6 10 4-8 Loineye area (% increase) - 15-20 9-15 Carcass fat (% decrease) 4-8 20-30 10-16 Back fat (% decrease) - 20-50 10-17 Abdominal fat (% decrease) 2-8 20-45 - ' Sheep and cattle data The table represents the effects of B-adrenergic activity in altering body composition across livestock species. The B-adrenergic agonists dramatically alter body composition toward that of a leaner animal (Muir, 1988). . no. '3' V- l ,. In-.. K h e... A‘,.- A” 1‘ as“: ;\ 035.. i o I. f.) .v .ZCIC‘ t". ~ I as. . I 4 I I“ M _ . i D a a w Les. 385.»... I. -9. 'fi.‘ \F o m‘cswgh p9“ h'.. l ”hula w-. ‘L LR‘.“ bk " a; . A" ‘i’ ‘3'” . Lab -\\IIS.A~:S L c W'.vr.'.\ T ~ - \\. M r my“. . ‘.‘ ’3 ‘9», ‘fi‘k 3‘ No \ ‘3 ‘ V: a ”‘3‘.- r» ”‘9- - . I tla‘QJ: return to the producer. Presently, the molecular mechanism by which these exogenous agents are acting is not completely understood. The focus of my dissertation research is to investigate the mechanism by which BAA affect skeletal muscle growth at the pretranslational level, as well as through signal transduction pathway mediated phosphorylation. SKA represents a myofibrillar protein and a unique candidate for studying skeletal muscle growth. Despite the high degree of homology across the nucleic acid and amino acid sequences of the six actin isoforrns, the 3’ UTR of these genes may confer specificity. The specificity in the 3’ UTR may allow for study of individual isoforrns. Unlike actin probes used previously, the isoform-specific probe will allow for a more precise measurement of a specific actin isoform. The initial objective was to isolate and characterize a SKA PCR generated probe homologous to a region of the 3’ UTR of the mouse SKA gene. The SKA specific probe will be useful for experimental analysis of the nuclear events preceding translation of SKA in C2C12 myotubes. Feeding of BAA to livestock species has been reported to increase muscle mass, total muscle protein, total RNA, fractional synthesis rates and the expression of myofibrillar protein genes (Bergen et al., 1989, Helferich et al., 1990, Smith et al., 1989, Koohmaraie et al., 1991). Helferich et al., (1990) report that feeding of the BAA, ractopamine (RAC), :r-aw :- ‘1‘: but. l " WIT"? “.I5 00.. a. b. 1 I ‘a‘fin‘ \- .\ ‘I ‘I‘ \\a‘. \'§ 5 \ ..". 't 5 A h _‘ “- .1 In“; .. .. . )II‘ 2 I"; 9“. ‘F. "mu. ,L._ ‘ ‘1 \ 'I 1“: ' *0. ' “3‘ an»...;\‘ §.\_ M _ i 3\"‘~.‘3 ~.‘ ‘ A ‘ ~ .15". ‘Iau-‘~ ‘ ‘ ‘juua .' h‘ "““S La. k. D ‘.II ._ a - ..' . “'*‘u..‘ :2: a by ' - v“Ly W- had increased SKA mRNA abundance in the longissimus dorsi muscle of finishing pigs. approximately two-fold above control animals. The second objective was to investigate the effect of BA stimulation of C2C12 myotubes on SKA mRNA abundance using a SKA isoform-specific probe. Furthermore, although the effects of feeding the BAA, RAC, to finishing pigs were clear, the in vivo study did not allow for an examination or verification of the signal transduction pathway/s involved in increased SKA mRNA abundance. The sub-objective of these sets of experiments was to examine and verify the mechanism of BAA stimulated increased SKA mRNA abundance in C2C12 myotubes. . Extracellular mediated events, such as BAA stimulation, may act through a complex interplay across signal transduction cascades. A major regulatory point in most signal transduction cascades is a specific kinase activated in response to stimulation at a particular membrane-bound receptor. Many kinases, across cascades, may converge on the same transcriptional regulator and may act in concert in activation of responsive genes (Figure 1). Many of these transcriptional regulators (trans-factors) are phosphoproteins. The phosphoproteins are ~P by the activated kinase in the appropriate cascade. Particular segments of DNA in the promoter region of the SKA gene suggest that several trans-factors (c-fos, c-jtm, AP-l , CREB) may bind the DNA and may initiate transcription. Several of 1 these known trans-factors have been reported to be responsive to stimulation by multiple signal transduction cascades. UvfiUToh m. Cr...» 4.. a dosage“ 2% 33:5 9 830825 onEoo a E @2562 on ~88 Amuoaoéécflc mEBEQ boss—awn: 3:23:85: _Eo>om .38an £895 328? 05 8 c3358 5 twang—new even 96: .323 E: .70 £86 .mmmov 268.5 EOE—smog 12833388“ .838 :o owuo>=oo .38 @8533 $8353 2:. (£8-38? BEE £26535 vomomoa :05 98 89830 .3353 cones—Smash Rama no 83388 a 3:323. Esme E toga—omen «5&ch of. 35353.. <73:— 5898 .523.» .3: 33328591339 Eco—.352. :3»: he 0:23:30 — 0.5»:— ‘b§§~ in. I Cut“ a, 1:13:13“ w - 5 i a... _ I: l. 1‘ Mar ,“‘~ . ruu' The final objective was to examine cell extracts of C2C12 myotubes to (1) identify a ~P response to BAA stimulation and (2) identify those responsive phosphoproteins. It is anticipated that the responsive proteins may be phosphoproteins involved in the BAA- stimulated muscle hypertrophic response in skeletal muscle. Investigation of BAA-stimulated SKA mRNA abundance and ~P response in C2C 12 myotubes may be critical in defining the mechanism and introducing new potentials related to BAA manipulation of animal growth. LITERATURE REVIEW Myogenesis Myogenesis is the formation of muscle fibers from mononucleated cells (Grant and Helferich, 1991). The development of skeletal muscle is a precisely orchestrated event occurring primarily by hyperplasia, prenatally. The number of muscle cells increases after birth. Postnatal growth occurs primarily through hypertrophy of existing fibers. The development of skeletal muscle begins soon after conception and can be divided into four stages (1) determination, (2) commitment, (3) differentiation and (4) innervation.The first stage is characterized by the irreversible transformation of the multipotential mesodermal precursor cells to the myogenic lineage, termed determination. Determination of precursor cells to myoblasts restricts the myoblasts to express muscle- specific genes upon differentiation (Grant and Helferich, 1991). Secondly, the committed cells proliferate until differentiation signals become present. Commitment to the myogenic lineage is accomplished through the coordinate expression of various myogenic regulatory factors. The myogenic regulatory factors act as transcription factors capable of initiating expression of various genes which are essential for differentiation to the myogenic phenotype. Konieczny and Emerson (1984) provided evidence for a family of regulatory genes that control the determination of precursor cells to muscle cell lineages. The third stage is differentiation. At differentiation, presumptive myoblasts withdraw from the cell cycle and begin to express the muscle-specific proteins and fusion to myotubes occurs. During differentiation and fusion of mononucleated myoblasts to multinucleated myotubes, environmental stimuli can alter the phenotypes that are expressed, resulting in fibers that are fast, slow, or mixed (fast/slow)(Grant and Helferich, 1991). Different isoforms of contractile proteins are synthesized as differentiation progresses and, in relation to myosin, may be related to fiber type (Grant and Helferich, 1991, Robbins et al., 1986). The last stage is recognized by innervation which is essential for proper functioning of the muscle as a tissue and growth. Skeletal Muscle Structure The highly ordered structure of skeletal muscle from the gross structure to the molecular level is depicted in Figure 2. The basic unit of the contractile process in striated muscle is the sarcomere (Squire, 1981, Richter et al., 1989) which is composed of thick and thin filaments tandemly arranged in the myofibril. Myofibrillar proteins, which represent more than 50% of the total protein content of skeletal muscle, include the structural Pr0t6ins of the thick and thin filaments, as well as those that regulate contraction (Grant and Helferich, 1991). The sarcomere is composed of 10-15 myofibrillar proteins. Of the l0 .. 030.31 .32 S eons—Baum . vim—83.x Fans: 25:0. .ocoEooBm 2: wee—a 98:82 .mcfiszwco. E :5. 0:88:80 .05 he 8288-380 0223:8an 98 Q 98 U m < S @2239 .ouoEooBm 05 2:: 2:08:80 o_w:_m a 5 5ng new 58a mo wcmcoEmoa 05 Serums—z 8 3208.5 fl £2..an 05 we .32 05 as 553:8ch .32... 8—329: 2333 05 8 $5. 3362058an macaw 05 Eat 832:3 282: 30.86 .«o 5:36.38 5388 05 3:82qu 23w: 2:. 9.32:: 9.92:: .3335 N «Laura 11 595m 3:233 0334 533a 12 I. 9. ”v -‘~RO- 29 R' m'»u~ouo_ p u . ' \ ? VIA '1‘va!) -- s. «it nun...» h .5. ‘0‘. 3:“; 1: . ‘ ‘ho um‘ F“ \ \t '1 m" 3 I s“ ' 1 me A 5‘ :41} ‘- A \ \IS. 0 3\ . ‘4‘: A ‘0‘“ .L H; myofibrillar proteins, only myosin, actin, troponin, and tropomyosin participate directly in the contractile event (Richter et al., 1989). Myosin is the predominant myofibrillar protein and accounts for 43% of the total myofibrillar protein. SKA, the second most abundant myofibrillar protein represents 22% of the total myofibrillar protein (Yates and Greaser, 1983, Grant and Helferich, 1991). Myofibrillar Gene Expression Regulation of myofibrillar gene expression seems to occur at the transcriptional level, with some of their RNA products exhibiting alternative exon splicing in the generation of multiple protein isoforrns (Richter et al., 1989). Some of these primary RNA transcripts are generated by differential initiation at alternate promoters (N abeshima et al., 1984, Periasamy et al., 1984, Robert et 31,. 1984, Richter et al., 1989). Muscle protein diversity can further be manifested by termination of primary transcripts at alternative 3’ untranslated sequences (Basi et al., 1984, Ruiz-Opazo et al., 1985, Bernstein et al., 1986, Rozek and Davidson, 1986, Richter et al., 1989 ). Two possible molecular mechanisms may explain the diversity in protein isoforrns: (1) the selective expression of one of the genes from a multigene family, dependent upon tissue and developmental stage-specific factors (Richter et al., 1989) , and (2) the generation of different protein isoforrns from a Single gene (Nadal-Ginard et al., 1987, Richter etal., 1989 ). Both mechanisms involve Specific cis- or trans— acting factors (Richter et al., 1989 ). A cis-acting factor refers to a DNA locus that affects the activity of DNA sequences on its own molecule of DNA. A traIlS-acting factor refers to a diffusable product able to act on all receptive sites in the 13 n 1 \a ”0".” 55“ JUUnA§QA I m w: «.g ‘5 ensues... . . .M A:‘~.. . A as'h‘.“ ‘ t...“ ‘ xa“ “- \HI ‘1‘. s -- L‘ ‘ 3‘ “fixe. . I u‘ Us; ‘: Q‘- ‘ . ~.. 5»... AJ‘Q “., \ r.‘. \“‘:g A c.‘ C ., B9,. ‘ s ‘g‘ u h ‘ “ “4;, ‘A it“- -* 9,5,. .. \i"- .0 cell (Richter et al., 1989 ). The components of the skeletal muscle contractile apparatus are products of gene families that each encode structurally and functionally related proteins (Cox et al., 1990) and include myosin heavy chain, myosin light chain, actin, tropomyosin, and troponin (Nadal-Ginard et al., 1982). The differential expression of these genes follows a complex program during muscle development (Cox et al., 1990). Myoblasts fi'om skeletal muscle can be cultured in vitro, either as primary cultures or muscle cell lines, and will fuse spontaneously to form myotubes (Cox et al., 1990, Buckingham, 1985). The fusion of proliferating myoblasts to form myofibers is accompanied by the developmental regulation of genes encoding a structurally diverse group of proteins that form the muscle sarcomere (Muscat et al., 1987). The change in contractile protein phenotype which accompanies the myoblast-to-myotube transition and subsequent modulation of transcript levels may be achieved by- regulating rates of transcription and/or by changes in export and stability of the RNA’s themselves (Cox et al., 1990). The morphological differentiation is accompanied by the accumulation of sarcomeric contractile protein mRNA’s, i.e. SKA, and the synthesis of corresponding protein, while non-muscle actin isoforms, [3 and y, are down-regulated (Alwine et al., 1979, Devlin et al., 1978, Gunning et al., 1987, Shani et al., 1981). Actin Actin is a globular-shaped molecule, approximately 5.5 nm in diameter, composed of a single polypeptide chain of 374 amino acids with a molecular weight of 42 kDa (Elizinga et al., 1973, Skjaerlund, 1993). The spherical molecule is termed G-actin (for globular l4 actin) and as such constitutes the monomeric (single molecule) form of actin. The fibrous nature of the actin filament occurs because the G-actin monomers link to form F-actin (fibrous actin) as shown in Figure 1. In F-actin, the G monomers are linked together in strands, much like beads on a string of pearls. Two strands of F-actin are spirally coiled around one another to form the “super-helix” that is characteristic of the actin filament. The actin filament in association with two other myofibrillar proteins (tropomyosin and troponin), form the thin filament involved in the contractile apparatus. Actins are highly conserved, ubiquitous proteins found in eukaryotes involved in cell motility and in the maintenance of cell structure (V andekerkhove et al., 1986, Mayer et al., 1984, Garner et al., 1989, Pollard and Weihing, 1974). In mammals, the actins are encoded by a multigene family giving rise to at least six different isoforms, each the product of a single gene, expressed in a tissue-specific manner or in the case of the cytoskeletal actins, ubiquitously (Barton et al., 1987, Vandekerkhove et al., 1986, Garner et al., 1989, Minty et al., 1981). The actin isoforms can be separated by isoelectric focusing. Separation of a, B, and y isoactins on a two-dimensional (2-D) gel is possible due to an alkaline stretch of amino acids in the B and y isoforms (Obinata et al., 1981). Skeletal and cardiac isofonns have very similar isoelectric points which is explained by their highly conserved amino acid sequence (V andekerckhove and Weber, 1987). Amino acid sequence analysis confirmed that the actins are greater than 90% homologous in amino acid sequence (Minty et al., 15 ~10~~ I w-‘§\ I‘. . L).......' a. u >. u | ‘1 --. u A. )1 Mrs. 3kg ..._,‘_ _‘. ; \L'bhbquk‘. -.. , 0“. Th.“ «Alf yaw 3“ 5“... 1“: ~. L“ "a. R H“ 'h . 'H. 5y. “¥‘: S 1981). Few differences have been reported in amino acid sequence for the same actin isoforrn across species (V andekerckhove et al., 1986). The six different isoactins can be classified according to the tissues in which they appear as the predominant isoforrns (V andekerckhove et al., 1986). Two isoforrns are present in the cytoplasmic microfilarnents of most or all cell types, while other actin isoforms are found in the contractile apparatus of skeletal, cardiac, and, smooth muscle (Mayer et a1. 1984, Minty et al., 1981). Non-muscle cells express two homologous isoactins generally referred to as B and y-cyotplasmic actins (V andekerckhove et al., 1986). Smooth muscle tissues express two closely related isoforms referred to as y smooth muscle and a smooth muscle actin (V andekerkhove etal., 1986). The 7 type is the major form in visceral tissue, while the a type appears as the major form in vascular smooth muscle (Vandekerkhove et al., 1986). In smooth muscle, the y and on types differ in about 20-23 residues from the non- muscle variants and are similar to, but distinct from, the major isoforrns expressed in striated muscle (V andekerkhove etal., 1986). Cardiac and skeletal striated muscles contain characteristic actin isoforrns which have identical molecular weights and isoelectric points, and only the complete amino acid sequence has revealed differences between the major isoforrn expressed in fast and slow twitch skeletal muscle and that in the rat heart ventricle (V andekerkhove et al., 1986). No fiber-type-specific or developmental isoforrns of skeletal a-actin have been found. The skeletal muscle type a- actin, differs from the cardiac variant by a Glu to Asp exchange at residues 2 and 3 and by exchanges of Met to Leu and Ser to Thr at positions 299 and 358, respectively. The 16 two latter exchanges are also typical of smooth and non-muscle actins (V andekerkhove et al., 1986). Pairs of these genes are differentially expressed throughout development and in adult tissues (Garner et al., 1989). Two sarcomeric actins, cardiac a-actin (CAA) and SKA have been defined (Garner et al., 1989). They are coexpressed at high levels during the development of striated muscle, but in the adult, the CAA and SKA isoforms predominate in cardiac and skeletal muscles, respectively (Garner et al., 1989). SKA and CAA isoforms migrate at 1.6 kb on an agarose gel while the non-muscle actin isoforms, [3 and y, migrate at approximately 2.1 kb on an agarose gel (Garfinklel et al., 1982, Mayer et al., 1984, Minty et al., 1981). Aetin Isoform Switching Regulation of a-actin expression occurs at the transcriptional level in a tissue specific mantler (V andekerckove et al., 1979, Richter et al., 1989). The actin genes are not linked in mammalian genomes (Czosnek et al., 1983, Gunning et al., 1984, Richter et al., 1989) and are unlinked to any other sarcomeric protein genes (Czosnek et al., 1983). Contrary to the non-muscle actin isoforms, CAA and SKA are synthesized in large amounts but with distinct tissue-specific and developmental patterns of expression (Bains et al., 1984, Devlim et al., 1979, Mohun et al., 1986, Nudel et al., 1986, Muscat et al., 1987, Minty et al., 1 982, Gunning et al., 1983, Singer et al., 1978). In replicating myogenic cells, B-actin is the predominant cytoplasmic isoform (Richter et a1, 1989). After fusion initiates, 17 "'- 3’“ *3,“ “2“:“0 ‘hu: ' b 4’" pg 'F‘ 5' ““3 u. .u . 1 (31:18:18. I “wen-«u. .3, {Nuanguz H W) .' . ‘1‘. hint; by \ ‘IIL - u.‘ “M80? m- 1 .‘ “~ '- .4 ‘5': ‘ “L“* “WNW .Ar ‘ ‘ ~ 3 ._| ‘ 11' "‘ ~3- ‘L..§“i": l-I‘.-.‘ ‘ I Any: III 3 F&‘* ' K he F's ‘g 711;“. ‘1 ., U i». 1. =1». \1312-13‘ \- synthesis of B-actin decreases and the sarcomeric a-actins begin to appear (Seiler-Tuyns et al., 1984). Older myotube cultures may undergo some of the changes in actin and myosin gene expression seen in vivo, and this situation can be manipulated to some extent in myogenic C2 cell line (Pinset et al., 1988, Weydert et al., 1987). Cox et al.(l990) reported that there is a sequential transition in transcription and RNA accumulation during myotube maturation which reflects the pattern of expression found during development in vivo from that of predominantly CAA to predominantly SKA. It has been suggested that one or the other isoform predominates in adult tissue to give rise to a selective functional advantage for that particular tissue. A precise fine tuning of gene expression may not be necessary or desirable at crucial stages of development when it is more important to generate large quantities of muscle proteins (Richter et al., 1989, Buckingham et al., 1985). During development, CAA appears to be the dominant iSot‘orm in both skeletal muscle and in heart (Minty et al., 1982). In adult human skeletal muscles, the SKA isoform predominates, but the CAA mRNA still represents 5% of the Sm‘corneric actin transcripts (Gunning et al., 1983). Along the same lines, in adult heart muScle, the CAA isoform predominates (Mayer et al., 1984, Waslyk et al., 1980). In pl’inlary chicken and human myoblasts differentiating in culture, similar patterns of on- actil'l isoform mRNA accumulation are observed (Hayward et al., 1982, Gunning et al., 198 7). CAA predominates, but SKA mRNA is transiently present at moderate levels and, subsfitquently, at 10 to 15% of adult muscle levels. In the frequently studied, imnlortalized, rodent myogenic cell lines, C2C12, L8, and L6, the patterns of sarcomeric a- 18 r" *9“. . mean-AN. ‘W‘wv... I“ :‘V‘ ‘|*\ u \| Skim (1..“ m 1 ‘ 5k: ‘39. "“1 (1‘32: 31);}: HI: {3 I): Sat. “2 1m .‘1 i'a"! H.“ “5154‘ mks actin mRN A differ substantially and are different from the patterns seen in primary cells (Muscat et al., 1987). CAA transcripts accumulate to extraordinary levels in C2C12 cells, but, are not detectable in L6 cells and only accumulate to 10 to 15% of adult levels in L8 cells. SKA transcripts in these cell types have similar patterns of accumulation and slowly rise to 10 to 15% of adult levels (Muscat et al., 1987, Bains et al., 1984, Hayward et al., 1982, Hickey et al., 1986, Minty et al., 1986). Functionally, the contractile apparatus in skeletal muscle and cardiac muscle appears to be the same regardless of the fact that different actin isoforms predominate. Skeletal a-actin Skeletal a-actin (SKA) represents a highly conserved muscle-specific protein in skeletal muscle. The fact that SKA is developmentally regulated makes SKA a unique candidate for studying muscle growth. Actin is the second most abundant contractile protein in skeletal muscle myofibrils and represents 22% of total myofibrillar protein (Skjaerlund et al., 1993). Even though myosin is more abundant, there have been over 12 myosin isoforms identified in skeletal muscle which presents a greater complication than just studying one actin isoform (Skjaerlund, 1993). The skeletal muscle isoform, SKA, represents greater than 95% of all actin present in mature skeletal muscle (Skjaerlund et al., 1993). The inherent problem with using SKA mRNA abundance as an index of myofibrillar gene expression lies in its highly conserved amino acid and nucleic acid sequence. The nucleic acid sequence across actins is? less than 2% divergent while the amino acid sequence has been shown to be less than 20% divergent, a sequence diversity 19 '1 |- 0‘ an. J12 OL 33: he ' ‘ t . S 1:" - ".1 tau 1th., 31"“: v» ‘ ”nu—1 u. "' .- 9“ 3.81105: : '.".'4 l 1.. .915.” 3,“ TnUSCflpllt T . _ .“x :83; . 2:2: C11 13 35.86. .V. ..'. _ 111:, ~. ..3.. I»:. due to the degeneracy of the genetic code (Buckingham et al., 1984, Buckingham, 1985. Skjaerlund, 1993). Nucleic acid probes for actin tend to cross-react with other actin isoforms, making data difficult to interpret. Nucleic acid homology comparisons across actin isoforms indicate that the 3' UTR may confer specificity across isoforms (Mayer et al., 1984, Minty et al., 1981, Wettenhall et al., 1982). Transcriptional Regulation of Skeletal a-actin Two upstream cis-acting elements that modulate the tissue specific transcription of the human CAA gene may represent similar sequences within the SKA promoter (Minty et al., 1986, Minty et al., 1986, Richter et al., 1989). These regions may be modulated by tissue-specific transcription factors (trans- factors) in skeletal muscle as they are in cardiac muscle. Muscat and Kedes (1987) revealed the complexity of the SKA promoter reporting specific domains which are responsible for gene transcription. The proximal domain (positions -153 to -87) is critical for tissue-specific expression and regulation in two myogenic cell systems (Muscat and Kedes, 1987). The cis-acting sequences 3’ of position -87 interact with factors present in both myogenic and fibroblastic cells and appear to define or act as the major component of the basal promoter domain. The distal 5’ domain from positions -1300 to ~626 and the proximal domain from positions ~153 to - 87 respond to muscle-specific factors and possibly modulate transcription in a synergistic fashion in C2C12 cells but not in L8 cells. 20 r». «aw—- « “A A 5‘...- & \— f‘ . . u. ‘ Cl». 531.). l — ‘. “'“ e... 1‘ ‘ \I-a.§‘.5.“..“ \ . ' . 3 ..‘~ " v a “‘5‘. O ‘- -1. ' . .I >fl 'FQ ~- “ d5 33¢.‘2 \k.h a... “*HLOIZ: 4.) "1 8‘s. ‘ .< 1. 1 . ‘a.\\ ‘;“K L“\“-~ 71‘), “'4 5‘. K‘K“ a ( J ,. 11 ‘ MO?» ‘ ‘h ‘15“; a: x U‘ ‘k ‘. The central domain between positions -626 and -153, although not required in either cell line, has a positive role in augmenting expression in L8 cells (two- to threefold) but not in C2C12 cells. Muscat and Kedes (1987) reported that these elements appear to be differentially utilized for maximal expression in different myogenic cells. Subsequently, the number and functional diversity of the regulatory domains and the distance they span raise the issue of their roles in the distinctive patterns of SKA mRN A accumulation during myogenesis in rodent cell lines and primary cell lines (Bains et al., 1984, Hickey et al., 1986, Minty et al., 1986, Hayward et al., 1982, Gunning et al., 1987). Their observations are compatible with the possibility that the particular combination of domains involved in vivo depends on the availability or relative levels of trans-acting factors in each cell type and the accessibility of the different domains to these factors. It is possible that the particular cell lines may differ in the kind and amount of trans-acting factors produced, emphasizing the complexity of the promoter'region and the variety of interactions which regulate its function. [3- Adrenergic Agonists The physiological BAA are represented by the catecholamines, epinephrine and norepinephrine. They are sequentially synthesized from the amino acid tyorsine in a series of hydroxylation, decarboxylation, and methylation reactions as (3,4)-dihydroxy derivatives of phenylethylamine (Figure 3) (Axelrod, 1971, Ungar et al., 1983). Synthetic BAA compounds exist which exhibit similar pharmacological and chemical properties to those of the endogenous catecholamines (Figure 4). These compounds can act through 21 .55.er coca—ESE use iota—$368“. deem—.9865 we moron a 5 0585: Bee 05:8 2: :55 2.5.05 3:83 05 5 3535:? Be 0553538: use. 655353 35550588 95:38 2.823: 2:. 2 2 585:»83 o5Ea-e58au m 0.53% 955353— mmeamxgeéafimfiéefiEeNeeefimeeeo‘ 6 055353an m5~b~e§§§ mSEemeQ a «55.2—3— emeiwefeomQ 3.6V eEEV a oemfiaieeaamueaezaa maeNAReaRAm mzmwebh a 0535:. 23 503008305 $50505 $5.83 5903533. 05055? .8030 can 055353 6555050030 35359??— 05 0.0505 005000.30 90503 0500350045 3:323 0:055? use 3032505; 30505 50:0 050 0550 wee 0.3.0:: 350030 05 3 5030 5 bra—:50 05 000505: 9 0050005. 08 35:03 0_w._0=0500-n .8050 .«0 00030.50 05. 005033 353—0005005— 335.“ 03% >05 .03 005520500000 38383 05 he 0030.50 35500 05 00050 3533 530350.35 050553 85.5”.“ 530:0.Gaé eeEEeU v 0.55..— 24 «308 muizmmoiw om 800385.00. m0 OS I _ Io IOIfolfo 1w l:2|£0l:.0l0!0: mz:2500 3% 50005 5805530 0 05 0005530 0050000 550505004“ 05 .3005: w5000 Emmcow< .0505 00—5—00 «0 0000000 0 95005550 5050000 n 50 d :0 50:50 .0 000 3050? 05500053 27 53500 003000.. 0350:0530 m 0.5»:— 055—00.. <05 0 AI 025 + 0,5, 28 50000: 00 000—03 00015—00 <0 0 ._0=m_0 0000:0500 0050w0 0300000000 05 t0 m555 00 00500050000000 00000000 05000000000 5 00335 000050— 050000 00.0 0000000500 000 0.023 0050 5000.500 >00— 550505 0000500 000 050500 0000:0005: .0596 0.0053055 .00 0000000 0 w500>000 050500 0010:8058 00 00000000 05000000000 05 00 0050 0050w0 0300000000 00; 0000300 b0005w00 50000000 050500 0000:3550 0:0 -005— .00300000 50500 50500000005505 -=0>00 0 0_ 00000000 590000000 00:. .802 .._0 00 00000005 00000000 0500000000 0005:: 05 0000000000 00000005: 003000000 00: 29 00000000 0_u00000.000-n 00:. 0 0.0:»:— three BAR subtypes, BAR-1, BAR-2, and BAR-3, each representing a separate molecular entity with distinct pharmacological properties (Mersmann, 1998). The BAR’s are present on most mammalian cells, but the distribution of subtypes and the proportion of each varies between and within tissues in a given species (Mersmann, 1998). Agonists that bind BAR’s activate the enzyme AC, whereas hormones that bind the a-adrenergic receptor inhibit this enzyme. Post-receptor signal transduction is thought to be the same for each of the BAR’s (Table 2). Factors Affecting Responsiveness to B-Adrenergic Agonists The response to BAA stimulation is dependent on several characteristics of the target tissue. Stimulation at the BAR with BAA results in a profound increase in the intracellular levels of cAMP. Three plasma membrane proteins are known to be required for this effect. The “target” must have functional BAR’s, (Moloney, 1991, Leflcowitz et al., 1983, Birnbaumer et al., 1985, Stiles et al., 1984), the stimulatory guanine nucleotide binding protein, and the enzyme AC. The interaction of the BAR complex with the G, catalyzes the release of GDP from the or subunit of the G protein ((1,), allowing the binding of GTP; this, in turn, leads to the direct activation of AC by a,-GTP (Levitski, 1988). Upon removal of the agonist, the activation persists until the intrinsic GTPase activity of the a, hydrolyzes the bound nucleotide (Hausdorff et al., 1990). In addition to the generation of cAMP from AC, two other factors, degradation of cAMP by phosphodiesterases (PDE) and the export of cAMP outside the cell, act to regulate cAMP generation. 31 l Table 2 3":'Jo~, .E F? .r’» A ......... w TU In ) . o0 Table 2 Adrenergic receptor subtypes and post-receptor activity a1 ............................................ Coupled to Protein kinase C, Calcium and 1P3 a2 ............................................ Coupled to Gi, inhibits adenylate cyclase B1 ............................................ Coupled to Gs, activates adenylate cyclase B2 ............................................ Coupled to Gs, activates adenylate cyclase B3 .................... ' ........................ Coupled to Gs, activates adenylate cyclase The subtypes of adrenergic receptors and post-receptor activities are presented. The subtypes are classified as either a or B and depending on the subtype have similar post- receptor activities. Alpha-adrenergic receptors act through a G inhibitory protein (G,) inhibiting the action of the enzyme adenylyl cyclase (AC). The B-adrenergic receptors act through a G stimulatory protein (G,) which activates the enzyme adenylyl cyclase and thereby elicits the characteristic cAMP mediated cascade of events. 32 .ma..‘~ I." ‘2... \r kal‘.k\v A. ‘Jfi‘flr' o m‘rwl: 30'. ‘Wfimaw ., '0 ‘uh ‘uh . s . Q--'..'...,, “M5‘\\'I | £‘919'1i0 p. ‘u’y. s“ 3‘ 1 s. “2‘ 3L0, , ”N‘g\" L h The relative distribution of BAR is another example of regulation of the response to BAA stimulation. There is a specific stimulatory G, protein by which BAR-1 and BAR-2 receptors are coupled to AC in the same manner. In contrast, activation of the a adrenergic receptor, specifically a—Z, inhibits AC by promoting the binding of a different G inhibitory protein (G,) (Birnbaumer etal., 1985). The relative distribution of these receptors may be a means of regulating the biochemical responsiveness of a tissue to adrenergic stimulation (Moloney, 1991). Regulation of receptor number is yet another factor affecting responsiveness to BAA stimulation (Moloney, 1991). The development of cell receptors and the intracellular mechanism for executing the response to receptor occupancy are a function of cell type and species (Mersmann, 1989). Another factor affecting receptor number which may represent a more acute control of receptor density is desensitization or down regulation (Stiles et al., 1984, Levitzki, 1986). Prolonged exposure to BAA’s results in a decline of receptor numbers at the cell surface and a reduced responsiveness to stimulation (Moloney, 1991 ). Desensitization may occur through uncoupling of receptor occupancy or through internalization of the receptor at the cell membrane for subsequent recycling and/or degradation (Hoffman, et al., 1979, Stiles et al., 1984, Su et al., 1980, Moloney, A., 1991). Hausdorff et a1. (1990) report that the m01¢cular mechanisms behind desensitization do not require internalization of the BAR, 33 p 1 . ”in arm”. 3, ~W-M5a .- . :1 , , i»'u‘\.1\~. 'i . “rat-xi - 0 , , J‘. ““h‘ikii'jlfl . 1 3: 3'0" Q t ”.5...“ 8)." réi’mm. u "~_L V .71 . V‘ ‘R )L‘Dn-p ih. '|_ ' .. a“ 3"“:er 1"“, l u. but rather an alteration through ~P by at least two kinases, PKA and BAR-kinase (BARK), which uncouples the receptors from the G, protein. Phosphorylation sites for each of these kinases are illustrated in Figure 6. It appears that both PKA and BARK are operative simultaneously, although a higher agonist concentration may be necessary to activate BARK (Hausdorfeta1., 1990). PKA may be elevated by agents other than BAA (heterologous desensitization) (Mills and Mersmann, 1995). Additional kinases also may be involved in BAR phosphorylation, including protein kinase C (PKC) and an unspecified tyrosine kinase (Leflrowitz etal., 1990, Mills and Mersmann, 1995). Different BAA have different affinities and, therefore, differing potencies relative to the responses observed (Table 3). Similar compounds may have differing effectiveness and, occasionally, opposite effects in biological systems (Moloney, 1991). Mills and Mersmann (1995) report that two points are important regarding specificity: 1) no agonist or antagonist is absolutely specific for a BAR-1 or BAR-2 because specificity resides in the relative concentration required to stimulate or inhibit the receptor, and 2) the classification system is circuitous in that tissues are classified as having specific receptor subtypes on the basis of their interaction with specific compounds, and compounds are classified as specific for receptors on the basis of their ability to bind with receptors on Specific tissues. Differences in the pharmacological properties of agonists and antagonists in different tissues have revealed that multiple tissues express more than one BAR subtype. The potency for activation by common agonists (isoproterenol, . epinephrine, and norepinephrine) has been delineated for each receptor subtype. 34 Table 3 Al .. O\ 0 0 a _ .0. a. 0. 0.. .m M V Vl‘l” T00 0: 15:1; To . h . .\ J fix “J. . - 4a. a 0.0.0 .00“ ...... A0 Table 3 Adrenergic receptor subtype characterization .nl ! . . NorepizEpi>lso ! . . (Subtype selective) Prazosin>>Yohimbine W Phentolamine, Phenoxybenzamine I' l' 'l . Vascular Smooth Muscle Hepatocytes Adrenergic Recenter Sum: 952 El 132 NorepizEpi>lso lso>EpizNorepi lso>Epi>>Norepi Yohimbine>>Prazosin Metoprolol>>Zinterol Zinterol>>Metoprolol Pbentolamine, Propranolol, Propranolol, Phenoxybenzamine Alprenolol Alprenolol Platelets Cardiac muscle Skeletal muscle White Adipocytes Vascular Smooth Muscle Hepatocytes The table represents adrenergic receptor subtypes and attributed characteristics. The various agonists and antagonists of each receptor subtype are presented relative to their potency. The respective tissues that the receptor sub-types are most prevalent also listed. 35 7 Has a :5; “is“. p, this.“ \- ‘HI'F ‘V'F W Iv‘p 5050...; u. t I ' ' "30 40.. “0““ “,4 a m aims: ' . . fin“, “LAN-OI: & IF “~ ”W" . 5‘- ya‘ “k ' § ”.9 '- _ mm 30.2: ‘m 0“ . N0hsagz‘AS‘ ’ V :2 ,, - ' 3430", . 1x‘ Tn. ‘ 1'” 1"; .. h‘ .f,. a How a cell with multiple BAR subtypes integrates the function of the receptor subtypes remains unclear (Mills and Mersmann, 1995). Despite the apparent similarities in agonist binding in different tissues and activation of AC, other differences have been reported when addressing BAR activation. Whereas PRO completely blocks the activation of AC in adipose tissue, it did not block ISO-stimulated AC in muscle. That PRO did not function as an antagonist in muscle is explained by the fact that this ligand stimulated AC in pig muscle membranes. PRO alone did not stimulate AC activity in adipose tissue membranes, but was as efficacious as ISO in pig skeletal muscle (Spurlock et al., unpublished results). PRO is a potent BAR-1 and BAR-2 antagonist and has been shown to be a partial agonist in the cloned BAR-3 from humans, but not from mice (Blin et al., 1994). The strong agonist response to PRO in pig skeletal muscle clearly distinguishes the BAR in this tissue from those in pig adipose tissue or from tissues in other species examined. Other factors which may influence BAA action include absorption, degradation, excretion, and counter activities by other systems (Mills and Mersmann, 1995). Manipulation of Animal Growth by B-Adrenergic Agonists Synthetic BAA are the most potent agents that can promote skeletal muscle growth in many species of animals (Deschaies et al., 1980, Beennann et al., 1986, Wallace et al., 1987, Yang et al., 1989, Sainz et al., 1990, Bergen et al., 1989, Hanrahan et a1. 1986). The main advantage with these compounds is that they are orally active and can be incorporated into the diet of food-producing animals. Protein deposition in muscle cells 36 f. a:.‘o~..a . 0. ha..-» ». il"‘l9.u‘,’ . . hafiicxi. . . . E Q I. I‘ 404' ‘ .U m... ..., “11:13. ‘ e ,‘L\-i1. COW” h.~,‘ I-) H, 4 ‘77 [I II J. J in culture can also be enhanced by BAA and this effect can be blocked by B-antagonists (Anderson et al., 1990, Bergen etal., 1991). The fact that the BAA effect can be blocked by a B-antagonist implies that the eflect is mediated via the BAA pathway; however, a non- BAR stimulation in skeletal muscle has also been proposed (Bergen et al., 1989, 1991). The mechanism by which BAA influence protein accretion in muscle remains unresolved. The effects may be mediated at a pretranslational level. Skeletal muscle total RNA content, a-actin, myosin light chains 1 and 3 mRN A abundance are increased in animals fed BAA (Smith et al., 1989, Helferich et al., 1990). The proposed mechanism for BAA-induced muscle protein accretion is through a G,- protein linked protein kinase cascade (Bergen etal., 1991 , Bowman et al., 1969). Most aspects of adrenergic stimulation do not ordinarily require an increase in the synthesis of new proteins, and subsequent events may involve a complex interplay among various proteins by covalent modification through ~ P/dephosphorylation (Iwaki et al., 1990). Protein ~P regulates a diverse range of cellular responses (Meek et al., 1992). Although many of these regulated proteins have been identified and characterized in other systems, many newly discovered or yet undiscovered proteins may play a pivotal role in understanding the biochemical mechanism behind BAA-induced muscle protein accretion (Iwaki et al., 1990, Meek et al., 1992). Oral administration of synthetic BAA to food-producing or laboratory animals has been shown to increase muscle growth and alter carcass composition to that of a leaner animal 37 I". X ‘..".C.". u ‘lqr u 005 5...“, . a" at V0" - A5.au.. «- -. §~0.- . wan-0t . . -~a..-.-. (Hanrahan et al., 1986, Beermann, 1989, Anderson et al., 1992). Growth rate may or may not be increased, but efficiency is generally improved 10-20% and is primarily the result of the change in composition of gain to a greater percentage of protein and less fat (Mills and Mersmann, 1995). Another characteristic of BAA is increased dressing percentage, indicating carcass tissues are the primary target for BAA action (Mills and Mersmann, 1995). The first reports of BAA manipulation of animal growth centered on the effects of BAA stimulation in lambs (Baker et al., 1984), poultry (Dalrymple et al., 1984), cattle (Ricks et al., 1984), and swine (Dalrymple et al., 1984). The agonists for which the most data are available include ractopamine (RAC), clenbuterol (CLEN), cimaterol, L-644- 969, and salbutarnol (Mills and Mersmann, 1995). With the exception of RAC, the agonists are reported to have BAR-2 selectivity. Skeletal muscle BAR’s have been identified and have been shown to be predominantly BAR-2 (Sainz et al., 1990). Each agonist is not equally effective in each species, and species differences exist in their magnitude of response to the agent tested (Mills and Mersmann, 1995). Considering postnatal growth of skeletal muscle is primarily the result of hypertrophy and that skeletal muscle DNA is not affected in BAA-treated animals suggests increased protein synthesis. The increase in muscle mass attributed to BAA stimulation may be attributed to an increase in muscle protein synthesis, a decrease in muscle protein degradation, or both (Mersmann, 1998, Beennann et al., 1985, Koomahraie et al., 1991). Increased rates of protein synthesis have been reported for pigs treated with RAC (Bergen et al., 1989, Helferich et al.,l990) as well as an increase in the amount of RNA transcript 38 i. .4. ,. ‘msuq..g\ -. l r.‘|.‘fll\. WV-K... - 0 ‘ ‘fP‘ ‘ a . .. I. ‘ "‘ Al I ‘ !.'\.' 13‘ I v.‘,‘ . ‘ ll". Kn“: and \ '0’». . ‘- ' v 5i“ ‘ R?) :4. n 'l‘ .0 A s. 4.“ . “-4.8: 3'?- “ ‘ I l‘ for several muscle proteins including myosin light chain, SKA (Helferich et al., 1990, Grant et al., 1993, Killefer et al., 1994, Smith et al., 1989). Decreased rates of protein degradation upon BAA treatment are reflected by a decrease in the activity and mRN A abundance for the cathepsins and soluble proteases (calpains) while that of an endogenous protease inhibitor (calpastatin) increased (Mersmann, 1998, Koohmaraie et al., 1991, Higgins et al., 1988, Wang and Beennann, 1988, Bardsley et al., 1992, Killefer et al., 1994). Mills and Mersman (1995) indicate that considerable interest has focused on the BAR subtypes that mediate increased protein accretion in skeletal muscle. Mills and Mersmann (1995) emphasize that the BAR’s that mediate skeletal muscle growth may differ from those that mediate adipose tissue metabolism and this may alter the relative effectiveness of a select BAA to alter body composition. Effect of B-Adrenergic Agonists on Skeletal Muscle BAA stimulation via G, protein results in the elevation of intracellular cAMP levels and subsequent activation of the cAMP-dependent protein kinase (PKA). PKA, a serine/threonine kinase, is capable of ~P a wide array of proteins including nuclear phosphoproteins involved in cell growth. Although the biochemical mechanisms that mediate adrenergic effects are not completely understood, the effects are usually rapid (Iwaki et al., 1990). The effects do not usually require an increase in the synthesis of new proteins. BAA stimulation cascade effects are transmitted through cyclic nucleotide 39 i l “I“ h n. 31:11:11” " . I. I .‘. - .> '1 A“. ~Q‘\: ~ ‘ \W‘: .‘I‘ q . "““‘t-.‘ . 0 ~ . “in. ‘fi’ 15% , 0'. 3‘ I - r~a‘ k,.“ ‘:§\ "Ai‘ V :09)... “M a \. ““‘w-s, |‘ VA IEJFJ‘Y (Stull et al., 1977, Tsien, 1977, Jones et al., 1986), lipid-derived signaling molecules (Iwaki et al., 1990), and the ~P of critical cell substrates and enzymes (Brostrum et al.. 1970) Signal Transduction Pathway Cross-Talk The basic structure of many of the major signaling pathways is well established. Signaling pathways may be composed of a variety of proteins including specific receptors, GTP-binding proteins, second messenger generating enzymes, protein kinases, target functional proteins, and regulatory proteins (Nishizuka, 1992, Barnard, 1992). Interactions across signaling pathways are diverse and include potentiation, cooperation, synergism, antagonism, and co-transmission (Iwaki et al., 1990). The secondary or tertiary messengers that translate the signals into molecular responses at the level of gene expression are not understood and are presumably multifactorial (Iwaki et al., 1990) (Figure 7). The BAR is coupled to at least two intracellular signal pathways, including cAMP generation (Bishopric et al., 1992, Citri et a1.) and Ca“ entry (Yatani et al., 1989). In practice, Ca“ and cAMP-dependent signaling pathways are closely intertwined, in part because PKA is involved in the regulation of Ca” homeostasis at many intracellular sites, and both pathways may converge on the same transcriptional activator (Bishopric et al., 1992, Sheng et al., 1990). Car and cAMP have been implicated in eukaryotic gene- regulatory pathways (Bishopric et al., 1992, Roesler et al., 1988). The cell surface 40 5500000000 050w 550.55 000.000-5000 00.50000 .0 00000.5 .05 00000000 0000.... 0050000000 00 000005005 50:00500 003500 505 0:0 00000000 55000500. w5_0:w.0 00v. 50.00.0050: 0:0w 5000-0 5.0.00.0 00:03.5 >05 00050050005 b05052 >00. 0:0 0003500 00050000500. 5:50 5050 0.0.0005. 000.0500 .0. 55000005. 500000.00 0.50000 0:0 0.330.000 000.000.050.00 .0030 .00 0.005055 0 0005.0 41 2% E3 32% 4 g muzoxwm HQO—O\ om 0005000505 + \st 055-0 2 <1: 0092052 4/ a + “.00: 5000050090100 max/20 U< / .\ 350050 00 09$ 4 [I\\ 0000—00 $00 + . 00 J 0%. «+0 "a”. «0.- Ab‘bp 0U. . mines. Y ~;‘|:.. \ 0.0.0...- fin-0F“. .h\- 5‘, . 1 - ._' N” 1 ‘5‘I-‘sys. 0 0“ Ki‘bLD {CE IM“ . ‘ t~\::ii ~ ‘ 1*» “al.. ‘99 K 1 v3. the : trf’ ‘ . 09 ‘w t lq.Ur,\ u receptor-mediated generation of second messenger molecules, such as cAMP and Ca". and the subsequent activation of protein kinases and phosphatases is a widespread mechanism of signal transduction in mammalian cells (Rozengurt, 1986). Many transcription factors are phosphoproteins and their functions could, therefore, be regulated by phosphorylation/dephosphorylation events (Mitchell et al., 1989). The pathways leading from cAMP and PKA are incompletely understood. Phosphorylation of 408 ribosomal protein S6 is elicited by agents, including cAMP and PKA, in a wide variety of tissues which correlates with increased rates of translation (Wettenhall et al., 1982, 1984, Gressner et al., 1980, Stefanovic et al., 1986). Although PKA can directly ~P S6, there are also two kinase families: the 70 kDa and 90 kDa kinase families (Sturgill et al., 1991, Erikson, 1991). The signaling cascade regulating the 70 kDa family is poorly understood but it can be inhibited by rapamycin and stimulated by cAMP (Chung ct al., 1992, Kahan et al., 1992), In contrast, the 90 kDa family is ~P and activated by mitogen-activated protein kinase, MAP kinase (Sturgill et al., 1990, Chung et al., 1991). Furthermore, upon stimulation, MAP kinase has been shown to translocate from the cytoplasm to the nucleus and influence transcriptional events through regulation of factors such as c-myc, c-fos, c-jun and ATP-2 (Davis, 1993). In L6 cells, COS-7 cells and PC12 cells, cAMP has been shown to activate MAP kinase (Davis, 1993, Faure et al., 1994, Frodin et al., 1994, Thompson et a1. 1996). Both PKA and elevated levels of cAMP have been shown to stimulate ribosomal protein S6 43 . ~ H.- v. a” . §h\ . p u\ o l a a I fit! .‘~3§ 1.‘.su6\. - “"ua '> 305).... .A. A uqo - h‘"\ -‘5-‘n'b& § ‘P ‘0”. A ‘ i u “M‘HA-h A; . ‘00,. up A- (b ... I/I T""1r 4.:...-.“~" '0‘ i I \ ~.. . "l- "m‘h ' 4.. “‘37 v; phosphorylation which may promote the recruitment of mRNA to increase the number of polysomes (Wettenhall et al., 1982,1984, Gressner et al., 1980, Chung et al., 1992). Is it possible that cAMP may alter translation and/or transcription rates through a mechanism involving MAP kinase in differentiated C2C12 cells? In mammalian cells, the ability to activate the mitogen-activated protein (MAP) kinase cascade is a feature common to many extracellular stimuli including growth factors, hormones, and neurotransmitters (Pelech et al., 1992, Crews et al., 1992). The various stimuli which can activate the MAP kinase cascade employ distinct initial signaling pathways (F rodin et al., 1994). In the myocyte, the BA, ISO, stimulates protein synthesis and mimics other hypertrophic agents in activating MAPK (Bogoyevitch et al., 1996, Mills, 1998). Activation of MAPK is Ca++ dependent but not cAMP dependent, suggesting the involvement of multiple G proteins in BAR signaling (Mills, 1998). In I-IEK293 cells, isoproterenol activates MAPK via the By subunit of Gi . Only the BARK ~P receptor activates MAPK suggesting desensitization of the G, pathway is the signal to activate the Gi pathway (Daaka et al., 1997, Mills, 1998) however, at the same time, MAPK activation in the myocytes is pertussis toxin insensitive, indicating multiple pathways to MAPK activation are possible (Mills, 1998). Some stimuli activate receptor tyrosine kinases or non-receptor tyrosine kinases (Klausner et al., 1991, Gupta et al., 1993, Gardner et al., 1993, Ahn et al., 1991, Sturgill et al., 1988, Boulton et al., 1991). Other stimuli activate G protein-coupled receptors generating second messengers, including diacylglycerol (DAG) or Ca", or activating ion channels (Bading et al., 1991, 0‘. l’ . > ‘ ‘ »;“‘ than 0 3" W- . I . ”Cut ' b ..,_ _. ‘5".Q 3s- ‘fi;...’ L an“, .. I W, \’ ‘H ‘ I‘Lb-H ‘£‘ '3‘ P3350» ‘ "“JIUOS Ct Mixi‘ I 'l‘w. . Nbi, :‘lr’hl u\ “A“. '32:». Ely et al., 1990, Vournet-Craviari et al., 1993, Winitz et al., 1993). Frodin et a1. (1993) report that cAMP activates the MAPK cascade in PC12 cells; the significance here lies in the fact that the data indicates a cAMP/MAPK cascade link (Frodin et al., 1994). Frodin and co-workers (1994) show a cAMP effect and a synergistic effect between cAMP and phorbol ester (PE), which activates PKC. Such an interaction may be used by different ligands that stimulate cAMP generation or phosphatidylinositol breakdown, respectively, or by ligands that stimulate both pathways. Various studies have suggested a requirement for intracellular Ca” in BAR-mediated actions (Horn et al., 1988, Putney, 1978, Argent et al., 1985 ). BAR activation has also been reported to directly alter Ca“ flux mechanisms in parotid acinar cells (Horn et al., 1988, Kanagasuntheram et al., 1976, Butcher, 1980, Scott et al., 1985, Takemura, 1985, Dreux et al., 1986, Helman et al., 1986, Nauntofte et al., 1987); however, these intracellular events have not been characterized (Horn et al., 1988). Horn et a1. (1988) report that in rat parotid cells, there is an interaction between the cAMP and phosphoinositide intracellular signaling systems. Horn et al. (1988) report that ISO stimulates PIP-2 turnover and mobilizes Ca“ from an intracellular, carbachol-sensitive Ca“ pool via a mechanism involving BAR and cAMP. BAR’s activate the G protein, G,, which stimulates Ca" currents by both cytoplasmic, indirect, and membrane-delimited direct pathways (Yatani et al., 1989, Gilman , I987, Yatani et al., 1987, Brown et al., 1988). The BAA, ISO, increases Ca“ currents through a cAMP pathway (Yatani et al., 1989, Trautwein, et al., 1987). It has been shown in single-channel patch clamp studies 45 0’ p 5 i ' Gt: 1‘15 BAR an: 0.... 3.2.; - E‘E-l‘h meflafiflnli§r [ti-1:2: b‘ 3:“ 5.11112: m Pk marine SAP Ssh-23:1: :1 a], Li: 5M CXpregg a... cam mm 0’ J that the BAR and G, are directly coupled to the L-channel (Yatani et al., 1989). In theory. signal mechanisms involving Ca“ entry through the myocardial L channel should be activated by any agent that increases intracellular cAMP, since the channel is ~P and activated by PKA (Reuter et al., 1982), and this activation accounts for the bulk of measurable BAR stimulated Ca“ entry (Yatani et al., 1988, Hartzell et al., 1991). Bishopric et al. (1992) report that cAMP and forskolin (FOR) had relatively minor effects on SKA expression as compared to ISO and cholera toxin (CTOX). Bishopric (1992) suggests that BAA induction of SKA may require participation of a-CTOX-sensitive G, protein which would be bypassed by FOR and cAMP. They have also shown that inactivating PKA has no effect on the upregulation of SKA by ISO. They report that in cardiac myocytes, downstream elements of the cAMP/PKA pathway are not required. Transcriptional Regulation by Extracellular Signals Through Phosphorylation Changes in cellular gene transcription patterns induced by extracellular signals are thought to be important for many biological processes (Bohmann, 1990). The transmission of gene regulatory signals through the cytoplasm is mediated by signaling pathways, of which protein kinases are important components. Recent evidence suggests that communication between the cytoplasm and the nucleus relies on signal-dependent ~P/dephosphorylation of transcription factors (Bohmann, 1990). To activate or repress transcription, transcription factors must be located in the nucleus, bind DNA, and interact with the basal transcription apparatus. Accordingly, extracellular signals that regulate transcription factor activity may affect one or more of these processes. Most commonly, 46 regulation is achieved by reversible ~P. Phosphorylation of a transcription factor by several different kinases, or a kinase linked to more than one pathway, is a simple mechanism that allows different signals to converge at the same factor. Many growth- factor induced genes themselves encode transcription factors, and it is presumably the interaction between these factors and pre-existing factors, including those activated by the stimulus itself, that ultimately determines the response of the cell to the stimulus (Hill et al., 1995). Given the potential complexity of such interactions, it is conceivable that even if two different stimuli induce the same set of genes, small differences in relative expression might result in qualitatively different patterns of subsequent transcription. The growing list of such nuclear effector proteins include the transcription factors, CREB, Jun, Fos, NFKB, Myc and Myb. Four of which (Jun, F os, Myc, and Myb) are products of proto-oncogenes, and two tumor suppressor proteins, p53 and the product of the retinoblastoma susceptibility gene (pRB), and the simian virus 40 (SV 40) large tumor antigen (T antigen) which is a multifunctional viral protein involved in DNA replication, transcription and cellular transformation (Meek et al., 1992). Several of these proteins are ~P by the same protein kinases, suggesting that signals may coordinately target these key proteins (Meek et al., 1992). Although it is clear that BAR stimulation must generate intracellular mediators that reach the nucleus and consequently activate skeletal muscle-specific gene expression, the precise signaling mechanisms that link the occupancy of the receptor with gene induction remain unknown (Iwaki et al., 1990). 47 1 ll -.. v‘o‘ F05 Lo . ..‘. 7‘5. ‘F U». xv“ “.5. ‘0‘ k.~ . “. ~44 Expression of the proto-oncogenes c-fos and c-jun is induced by a wide variety of agents. such as mitogens, differentiation factors, specific pharmacological agents, stress, and heat shock (Greenberg et al., 1984, Kruijer et al., 1984, Muller et al., 1984, Lamph et al., 1988, Ryder et al., 1988, Ransone et al., 1990). Induction is rapid and transient and occurs at the level of transcription (Ransone et al., 1990) and the regulation of c-fos and c-jun expression is via not only their gene products, but also by related transcription factors (Rasone et al., 1990). F03 According to its putative role as a master switch in cell proliferation and differentiation, c-fos transcription is increased in response to various stimuli. Depending on the cell line, these stimuli include growth factors and cytokines like PDGF, FGF, EGF, NGF, TNFa, TGF B, thyrotropic hormone, PE, Ca" ionophore, metal ions, neurotransmitters, heat shock, cAMP, and UV irradiation. Expression of the fos gene appears within minutes of induction, reaches maximal levels by 30-60 minutes and is essentially undetectable by 120 minutes (Ransone et al., 1990). The rapid induction is likely to involve post- translational modifications of the fos protein (Ransone et al., 1990). In the presence of inhibitors of protein synthesis, the c-fos mRN A is induced at the normal rate, but remains detectable for up to 4-6 hours (Ransone et al., 1990). This suggests that, following mitogen treatment, pre-existing factors are utilized for fos transcriptional activation (Treisman, 1986, Sassone-Corsi et al., 1987). The c-fos protein can repress the 48 . ‘ o "‘ 0 a,” 1038. W550!“ i: mentioned ex." on 1“. ‘0‘ ‘- ' 0:".qu Oil Ol ads:- pazuays u: the c: ‘ . 11:17:; me c-fos pr 233cm 0.01;: al..1991.\'erma e transcription of the c-fos gene (Lucibello et al., 1989, Sassone-Corsi 1988c, Schonthal et al., 1988, Wilson et al., 1988). As mentioned earlier, the induction of the fos gene may be carried out through the mediation of adenylate cyclase and PKC pathways, two major signal transduction pathways in the cell (Ransone et al., 1990). Although multiple protein-binding sites within the c-fos promoter were identified, for most of the inducers listed above the respective cis-acting element that mediates the effect has yet to be determined (Angel et al., 1991, Verma et al., 1987). Inspection of the c-fos promoter reveals the presence of a 20 bp region required for responsiveness to serum and growth factors and is identified as a palindromic sequence known as the dyad symmetry element at approximately -308 bp (Angel et al., 1991). Further investigation reveals the presence of two cAMP-dependent response elements at positions -60 and -350 required for induction by agonists of the AC pathway (Sassone-Corsi et al., 1988, Gilman et al., 1986). Induction of the c-fos gene requires either activation of the serum response factor or ~P of CREB protein by a catalytic subunit of PKA (Ransone et al., 1990). The c-fos protein represents an excellent example of regulation across signal transduction pathways. Jun The prom-oncogene, c-jun, is a component of the AP-l transcription factor family involved in the mediation of nuclear events elicited by extracellular stimuli (Bohman et al., 1987, Angel et al., 1988, Woodgett, 1990, Pulverer et al., 1991). In contrast to the 49 , , , ‘ ”I 3 3 '"9 b. :Jiix amul» 9‘ ‘1‘ Q a P.‘l\' 0'. a. \\luu W'Qflfwr } U. “v 01.1. I nul‘I-[jni H‘ lb‘kb“.‘ ‘ ' ' I”: 1" .,,H EM A 1‘2...‘ IKi. ‘ V H, ‘_ 0. ,. .301“: ‘ 9.: u . W , Lhufle'fit '3‘ ‘ . ir";?"‘i' ”“Mn . I a s“ PKC at," M ma. extensive delineation of the regulatory elements in the c-fos promoter, little is known about the c-jun gene. Agents which induce the c-fos gene generally induce c-jun transcription (Ransone et al., 1990). The c-jun promoter has an AP-l binding site that is recognized by the fos/jun complex (Ransone et al., 1990). Unlike the fos gene, the c-jun gene is positively regulated by its own gene product (Ransone et al., 1990, Angel et al., 1988, Lamph et al., 1990). The c-jun protein is negatively regulated by ~P of residues at the carboxy terminus which are de~P in response to PE (Binetruy et al., 1991, Pulverer et al., 1991) and CREB protein (Lamph et al., 1990). Suppression of c-jun by CREB can be overcome by ~P of CREB with the catalytic subunit of PKA (Lamph et al., 1990). Interestingly, purified c-jun is not a substrate for either PKC or PKA, indicating that PKC activation does not directly lead to modification of the c-jun protein (Hai et al., 1988, Angel et al., 1991). A further explanation of the regulation of the c-jun gene extends beyond the scope of this literature review. Suffice to say that c-jun is regulated by the same elements which regulate c-fos expression with the main difference being that the c-jun protein elicits positive feedback on its own expression. Activator Protein-l Activator protein-1, AP-l, is the collective name for a class of transcription factors that is ftmctionally characterized by the ability to bind to promoter or enhancer elements containing TGACTCA or related sequences (Bohmann, 1990). The members of the AP-l family of transcription factors include c-jun, jun-B, jun-D, c-fos, F 05 B, Fra-l, and Fra-2 (Bohmann, 1990). Structurally, all these proteins share a conserved region consisting of 50 I I 9' a or“! b l‘bEu. Miser 113.9th ..”. ’V . m ‘1 ‘2 I.. b‘ the leucine repeat dimerization domain (leucine zipper) and an adjacent basic DNA binding domain (Bohmann, 1990). The functional form of AP-l is a dimer that is composed of either two jun monomers, or of one jun and one fos or fra monomer (Bohmann, 1990). AP-l was first identified as a transcriptional factor that binds to an essential cis-element of the human metallothionein 11 promoter and has also been recognized as a TPA response element of several genes whose transcription is induced in response to treatment of the cells with a PE tumor promoter (Karin et al., 1991, Huang et al 1991). Evidence suggesting that protein ~P is involved in the regulation of AP-l activity came from the observation that the positive effect of AP-I binding sites on the transcription of linked genes could be increased by exposure of cells to TPA (Bohmann, 1990, Angel et al., 1987, Lee et al., 1987). TPA is a potent activator of PKC (Nishizuka, 1984). Other agents which lead to PKC activation, such as serum, and growth factors, also induce expression of these genes (Imbra et al., 1986, Imbra et al., 1987 Angel et al., 1987, Brenner et al., 1989, Matn'sian et al., 1986). Inhibitors of PKC block those responses (Imbra et al., 1987 Angel et al., 1987, Brenner et al., 1989). Comparison of several transcription response elements led to the derivation of a palindromic consensus sequence that is recognized by AP-l : 5’-TGA G/C TCA-3’ (Angel et al., 1987, Lee et al., 1987). Insertion of synthetic oligodeoxynucleotides that form efficient AP-l binding sites in front of the thyrnidine kinase promoter renders it TPA inducible (Karin et al., 1991). Sequences that deviate from the consensus in positions essential for AP-l binding do not confer such a response (Angel et al., 1987). Hence, AP-l binding is sufficient for conferring a transcriptional response to PKC activation (Karin et al., 1991). 51 Cyclic AMP Response Element Binding Protein Cyclic AMP response element binding protein (CREB) was initially identified as an activity that could bind to cAMP-responsive promoter elements (CRE’s) (Bohmann, 1990). The situation with CREB is different than that of AP-l in that a clear connection has been discovered between an inducer (cAMP) , a kinase (protein kinase A), a ~P site (set-133) and a transcriptional effect (Bohmann, 1990). cAMP activates PKA, which is capable of entering the nucleus and ~P CREB, the cAMP response element binding protein, on ser 133 (Gonzalez et al., 1989). The CREB~P is then capable of binding to the CRE, cAMP response element, and enhances transcription of linked promoters (Boularand et al., 1995, Kinane et al., 1993, Rangan et al., 1996). Iwaki and coworkers first demonstrated that the or or B adrenergic stimulation increased induction of the immediate early genes c-fos and c-jun. Since these genes encode either known or putative transcriptional factors, their induction has been proposed to regulate gene transcription during growth factor stimulation, thereby influencing cellular growth and/or differentiation (Iwaki et al., 1990). Previous research by Bishopric and coworkers (Bishopric et al., 1992 a,b) has indicated a direct relationship between specific transcription factor activation (c-fos, c-jun) and signal transduction pathway cross-talk in the regulation of SKA gene expression in cardiac myocytes. The regulation of SKA gene expression in skeletal muscle may very well involve such a network of communication. Figure 1 represents a diagram compiled from literature in different cell types across 52 signaling pathways. The diagram represents the possible complexity that may exist in trans-factor regulation of gene expression across signal transduction pathways. Influence of Phosphoproteins on Skeletal or-actin Transcription Early work by Bishopric et al.(1992) indicated that norepinephrine (N E) induces both hypertrophy and SKA gene expression in cultured neonatal rat myocytes (Bishopric et al., 1992, Bishopric et al., 1992). The response of cardiac myocytes to NE involves one or both or and B-adrenergic signal pathways (Bishopric et al., 1992, Simpson et al., 1982, 1985). In confluent myocytes, SKA induction required only the BAA component and could be reproduced by the BAA, ISO, instead of NE (Bishopric et al., 1992). Bishopric et al. (1992) reported that expression of the proto-oncogenes c-fos and c-jun is activated very early in the hypertrophy response of myocytes to ISO, preceding SKA expression by several hours (Bishopric et al., 1992). Overexpression of transfected c-fos and c-jun in cardiac myocytes and in P19 teratocarcinoma cells strongly and selectively activated the SKA promoter in the absence of serum or other growth stimuli (Bishopric et al., 1992). The data presented by Bishopric et al. (1992 ) support a possible functional relationship between early proto-oncogene expression and SKA induction during signal-mediated hypertrophy of rat cardiac myocytes (Bishopric et al., 1992). Analysis of the SKA promoter did not reveal a canonical AP-l consensus element (Bishopric et al., 1992); however, there is evidence for binding of AP-l to non-consensus sequences (Owen et al., 1990, Takimoto et al., 1989, Gaub et al., 1990, Bishopric et al., 53 1992) and there are several elements in the proximal promoter region with partial homology to the AP-I consensus (TCACTCA) (Taylor et al., 1988, Bishopric et al. 1992). One of the sequences overlaps with the first CArG box, which has been implicated in muscle-specific expression of the a-actins (Minty et al., 1987, Miwa et al., 1987 a,b), and is a binding site for myocardiocyte nuclear proteins within the -153 to -36 region of the SKA promoter. My dissertation research focuses on the mechanism regulating the BAA-stimulated increase in SKA mRNA abundance in skeletal muscle. I will focus on the signal transduction pathway components and their involvement in transcriptional regulation of SKA using a SKA isoform specific DNA probe. Further insight on the regulation of SKA mRNA abundance following BAA stimulation will be obtained by examining the ~P status of proteins in cell extracts. Phosphorylation regulates a wide array of processes in the cell including trans-factor activation. Trans-factor activation is directly involved in the regulation of gene transcription. Information regarding the mechanism behind BAA stimulation of SKA mRNA abundance obtained through examination of pathway modulation may be beneficial in the optimization of this particular repartitioning strategy. 54 EVELOI CHAPTER 1 DEVELOPMENT AND CHARACTERIZATION OF A SKELETAL a-ACT IN SPECIFIC DNA PROBE Abstract A DNA probe homologous to 105 bp of the 3’ untranslated region of the mouse SKA gene corresponding to bp 3591 to 3696 was prepared by the PCR using a “touch-down” procedure. The PCR fragment was sequenced on a 6% acrylamide gel using the USB Sequenase kit as per manufacturer’s instructions. The sequence was verified by comparison to the catalogued sequence of mouse SKA and corresponds to 3591 bp to 3696 bp. The amplified fragment was purified and size verified on a 1.2% agarose gel. Purification of the fragment from the agarose gel was accomplished using the Bio-Rad Prep-A-Gene kit as per manufacturer’s instructions. The isolated and purified fragment was resuspended and stored in sterile milli-Q water. In order to determine the usefulness of the probe in agriculturally important species, the probe was tested against total RNA across various livestock species and isoform specificity across various tissue types. Species and tissues tested included mouse, rat, chicken, porcine, ovine, and bovine brain, heart, skeletal muscle, stomach, and liver. Results indicate specificity to the SKA isoform in mammalian species. No activity was detected in any chicken tissues or non- skeletal muscle tissues in the mammalian species tested. 55 \i “‘1 lllei ' - ..“JD°‘ N.§l “ a “'3‘; fi‘b'rgll A Introduction Skeletal muscle is comprised of a variety of proteins. The contractile apparatus is composed of two of the more predominant proteins in muscle, actin and myosin. Actin is unique in that it is a member of a large group of structurally related isoproteins which are also among the most abundant proteins in eukaryotic cells. In addition to their major role in muscle contraction, the actins are involved in maintenance of cell structure and organelle motility, including cytoplasmic streaming, phagocytosis, and cell division (V andekerckhove cta1., 1986, Mayer et al., 1984, Garner et al., 1989). At least six different vertebrate actin isoforms have been identified, each a product of a different gene expressed in different cells or even in the same cell at different stages of development (V andekerckhove et al., 1986, Garner et al., 1989, Minty et al., 1981). The six actins identified include SKA, CAA, aorta smooth muscle a-actin, smooth muscle a-actin, and two cytoplasmic actins B— and y-actin. These isoforms can be separated by isoelectric focusing, although they are greater than 90% homologous in amino acid sequence (Minty et al., 1981). Amino acid sequence analyses, confirmed and extended by the DNA sequences of different actin genes have failed to reveal amino acid differences between the same isoactins in different species (V andekerckhove et al., 1986). The six different isoactins can be classified according to the tissues in which they appear as the major 56 forms (Vandal. lady-Qtopjl‘ “.3730 no :1. mil? mm 1\ ii"! f z 1 “we- “fill? if. ' n' '. ”a" - A be“: “Cit restate: from forms (V andekerckhove et al., 1986). Two isoforms are present in the cytoplasmic microfilaments of most or all cell types, while other actin isoforms are found in the contractile apparatus of skeletal, cardiac, and smooth muscle (Mayer et al., 1984, Minty et al., 1981). Non-muscle cells express two homologous isoactins generally referred to as B and y-cytoplasmic actins (V andekerckhove et al., 1986). Smooth muscle tissues express two closely related isoforms, referred to as y smooth muscle and or smooth muscle actin (V andekerckhove et al., 1986). The 7 type is the major form in visceral tissue, while the or type appears as the major form in vascular smooth muscle (V andekerckhove et al., 1986). In smooth muscle, the y and or types differ in about 20-23 residues from the non-muscle variants and are similar to, but distinct from, the major isoforms expressed in striated muscle (V andekerkhove et al., 1986). Cardiac and skeletal striated muscles contain characteristic actin isoforms which have identical molecular weights and isoelectric points, and only the complete amino acid sequence has revealed difi’erences, specifically in the rat heart ventricle (Vandekerkhove et al., 1986). No fiber- type-specific isoforms of actin have been found. The skeletal muscle type a skeletal actin differs from the cardiac variant by a Glu to Asp exchange at residues 2 and 3 and by exchanges of Met to Leu and Ser to Thr at positions 299 and 358, respectively. The two latter exchanges are also typical of smooth and non- muscle actins (V andekerkhove et al., 1986). Pairs of these genes are differentially expressed throughout development and in adult tissues (Garner et al., 1989). Two sarcomeric actins, CAA and SKA have been defined (Garner et al., 1989). They are 57 :03);de 3? 7 CIA and Ski l 262.116: :1 al.. 1 Skeletal u-tctir Skim; u-actir. Tn: fact ha: SK at 5'0. type is lid: is the sec: 0... . , a “5.3335 -26 0 l melt isoform. mam: skeletal . are high] }' conse.‘ Considerably in I H “”0"is fMm t 1984). The 3’ L“ 984-931va et af coexpressed at high levels during the development of striated muscle but in the adult. the CAA and SKA isoforms predominate in cardiac and skeletal muscles, respectively (Garner et al., 1989). Both actin isoforms migrate at 1.6 kb on an agarose gel. Skeletal a-actin Skeletal a-actin represents a highly conserved muscle-specific protein in skeletal muscle. The fact that SKA is (1) muscle specific and (2) is not characterized by developmental and fiber type isoforms, make SKA a unique candidate for studying muscle growth. Actin is the second most abundant contractile protein in skeletal muscle myofibrils and represents 22% of total myofibrillar protein (Yates and Greaser, I983). The skeletal muscle isoform, skeletal a-actin, represents greater than 95% of all actin present in mature skeletal muscle. The inherent problem with using skeletal or-actin as an index of myofibrillar gene expression lies in the highly conserved nucleic acid sequence among the actin family of genes. Nucleic acid probes for actin tend to cross react among actin isoforms, making data difficult to interpret. Segments of the 3’UTR are similar across species and other sequences may be not only isoform specific but also species specific (Gunning et al., 1984, Skjaerlund, 1993). The amino acid sequences of the different actins are highly conserved whereas the 3’ UTR of some actin mRN As have diverged considerably in their nucleotide sequence. The 3’UTR shows the greatest diversity across isoforms, even though select sequences are conserved across species (Gunning et al., 1984). The 3' UTR of many genes may confer specificity across isoforms (Mayer et al., 1984, Minty et al., 1981, Wettenhall et al., 1982). 58 fat objective 'I ‘v‘v ' Lt .. mblalb DAOL ‘ I ‘ . ”W‘s" Pffl-t K‘sgbn‘auyh Di; \ ~ O major. ofS The objective of the initial study was to isolate and characterize a SKA-specific PCR- generated probe homologous to a region of the 3’ UTR of the SKA gene. The isoform- specific probe will be useful for experimental analysis of the nuclear events preceding translation of SKA mRN A. 59 PH 2 1‘ £9.57, fl‘t“~é "lb ,~ Materials and Methods Plasmid Inconsistencies in the pMACT-a (Hu et al., 1986) plasmid map required verification of sequences before initiation of experiments. A 931 bp Bam HI fragment corresponding to bp 3071-4002 of the mouse SKA gene in pMACT-or was subcloned into the plasmid pcDNA3 (Stratagene, Inc., La Jolla, CA) for sequence verification. Following the sequence verification of the 931 bp fragment, a 437 bp Pst-I/BarnHI fiagment corresponding to bp 3565-4002 of the mouse SKA gene was subcloned into pBluescript II SK+/- (Stratagene, Inc., La Jolla, CA). The intentions were to allow for maximal replication by PCR of the 437 bp fragment to be collected by plasmid preparation for use as a PCR template. Bacteria were transformed and screened on X-gal plates as per established protocols (Maniatis et al., 1989). Plasmid preps were performed on selected colonies. BamHI/Pst-I digests of the p451 were run on a 1% agarose gel, size verified by comparison to a 100 bp ladder (Gibco, Grand Island, NY), excised and purified using the Prep-A-Gene kit (Bio-Rad, Hercules, CA). A set of appropriate primers was chosen for PCR amplification of a 105 bp region within the 3’ UTR of the SKA gene using a “touch- down” procedure. Initially, the 437 bp Pst-I/Bam HI fragment was used as template for amplification of a 105 bp region of the 3’UTR. Subseqently; the 105 bp amplified and purified fragment was used as template for PCR in further amplification reactions. 60 “‘Iu. MW“. 6 it Primers Primers were chosen according to accepted protocol (Maniatis et al., 1982). The primers were prepared at the Michigan State University Macromolecular Structure Facility. The oligonucleotides: WGB] (5’ TTG GAG CAA AAC AGA ATG GCT G 3’) and W082 (S’ATA GAT TGA CTC GTT TTA CCT CA 3’) were chosen as primers for the polymerase chain reaction and represented sequences complementary to bp 3674-3696 and 3591-3612 bp of the mouse SKA gene, respectively. Polymerase Chain Reaction A digoxygenin-labeled probe was created using the Boehringer Marmheim Biochemicals- Digoxygenin PCR Kit (BMB, Indianapolis, IN) using the Barn HI/Pst-I 437 bp fragment as the initial template and the oligonucleotide primers WGB 1/2 in a “touch-down” procedure. The amplified fragment was size verified using a 100 bp DNA ladder (Gibco, Grand Island, NY) at 105 bp (Figure 8) and purified from an agarose gel using the Prep- A-Gene kit from Bio-Rad. The “touch-down” procedure allows for efficient amplification of templates which have higher calculated annealing temperatures (>50) (see Appendix A). Sequencing A 931 bp Barn HI fragment subcloned in the initial plasmid clone pS3’0t of pcDNA3 was sequenced at the Michigan State University Plant Research Laboratory using the 61 500—505 2E0? 50300—05 as oo. 0 0. 500500500 .3 an we 50 00553 003 050 0_ 505005 05... .050w 5000-0 50200—0 00505 05 .50 50305 0050—05055: .m .50 50305 as mo. 0 05000505 0050050 «Um 00:. 500000.. :530050505: MUm 5000-0 030.80 05 .50 005005 05 050 .0» 035050005 00050w0 fan.— 0 050500 05mm 05,—. .2625 505 5.. 0.: .538 30.3.0 0 2:0: 62 .; , 4!. _. .a 5.5.4. .ULYHK... s , B n‘ firs. .3 .50 §\-.s..i.._..h1.v. 63 v- - ., 4; til-utOX‘ l :4 if Mist 5‘. 1‘, .'.‘ A 9 Unfit "Q \Ohh; “AAl\ W3. { 1,531 men 103", R11 Told: i Dyedeoxy procedure from the T7 dye primer or Sp6 dye primer, a system designed by Applied Biosystems Inc. The Dyedeoxy method of sequencing is similar to the Sanger dideoxy method which represents a controlled interruption of enzymatic replication. The Dyedeoxy chromatogram is pictured in Appendix B. A 437 bp Barn HI/Pst-l fragment was subcloned into pBluescript for further verification of the proper or-actin sequence and is intended to be the initial template for the PCR of the 105 bp fiagment. The 437 bp fiagment was sequenced using the Sequenase protocol (United States Biochemical (U SB), Cleveland, OH) from the T7 and T3 promoters and verified by comparison to the published sequence of mouse SKA on GenBank (Name: MUSACASA, Accession: M12347) to be homologous to 3565 bp through 4002 bp of the mouse SKA gene (see Appendix C). The 105 bp fi'agment obtained from the PCR reaction using the Barn HI/Pst-I 451 bp fragment of the pBluescript plasmid clone and WGBI/2 primers was sequenced using the Sequenase protocol (U SB) and verified by comparison to the published sequence of mouse SKA on GenBank (Name: MUSACASA, Accession: M12347) to be homologous to 3591 bp through 3696 bp of the mouse SKA gene (Figure 9). RNA Isolation Total RNA was obtained from distinct tissues across species: bovine, ovine, porcine, rat, mouse, and chicken. Tissues were chosen to represent those tissues predominantly .NmOB 50 563 505.0 00 005055000 050 005.5005: 050 050.5000. 505.50. 0.000000 0.5050w 0 50 050m 5000 -0 50.8.0 00:05 05 .0 00505000 0050.32. 0... 0. 50.05 30.0505 0 5505.0 00502550000 03 50..00.-..50> do 009-. 0. 00 .0mm 50¢ 50.w05 080.0555: .m 5000-0 5.0.00.0 00505 0... 05 00.500. 00 0. 00500:. mun. a... no. 05 «0 00505000 05 0055-500 0.0».050 00505.00m 50505 080.0555: .m 050w 5000-0 5.0.00.0 00505 05 .0 50505 5. ma. 0 .0 00505000 00:00.05: 05 0050005005 505 05.. 0500 5300-0 3.0.8.... 00505 0... 00 50.00.. 0000—0503:: .M 0 .0 505?...— E. m..— 00005050u 100. 0 .0 0.0.0.050 00505.00m 0 0.50.0. 65 0000 0000 .0 30: .0 <0>.65 0505500050 .5000. 05. 5. 50. 0.. .0 02.500. 003 3.3.000. 005 50350000.. .55005050 050 0.0005 0.03 00.0000 505.55.. 050 58.0.50 5. ..50050.0: 05. 5.500500. 00.0500 05 .. .95. 5.0.5 050 AM... 50>: .Ahm. 50050.0 .50. .5005 A35. 0.0055 5.0.00.0 005.05. 005000.00. 00500.. 05 .- 05.0.00. 5.00 0.0055 -505 05. 5.000.008 00500.. .05.0 0 5. 5.055 .0 0050050 05. 05 0055-500 0. 5.0.00. 5.00-0 5.0.00.0 05. .0. 050.0 05. .0 5.05.0000 05% .00050 050 6.500 .w.0 58.0.50 ..0. .00505 0005.05. 00.0000 05... 00.0000 50505505 0.00.003. 050 5.0.0.050. .5050 000.00 550.00. 5.00-0 .0.0.00.0 05. .0. 0....0000 0. 050.0 5.000 5.0.00.0 05 no. 05. .05. 0.00.0. 05500.. 05... 050.0 5050. 030.05.05.55 .m 05 no. 00505 05. .05.0w0 000.0...505 00.0000 050 00500.. .00.00 50.. <21 .05. m: o. .0 0.0.0.050 .0... 5.05502 .0 05500.. 05. 0.0 020.003 .0... 5.05.507. 5.50-0 5.0.00.0 0500.. 050 00.025 5— 0...»..— 71 IIJIIIFIJIIITL mm M1. Fm DU 2m mAHH0 05. 5. w5055 .0 0050050 0.0.0500 05. 050 00.0500 <20. .05. 0.0055 .0.0.00.0 50505505 5. 5.. 5.. .0 05.055 .0 0050020 05. 05 030550500 0. 5.00-0 .0.0.00.0 .0 5.0.00. 50505505 05. 0. 050.0 5.000 50.0.8.0 05 3.05. .0 5.0...0000 05 ... 00.0000 .0.0>00 000.00 <20. 0.0055 .0.0.00.0 .0.0.w1 o. .0 0.00.050 .0.5 50.0.02 05. 05000.00. 0.50.. 05 H .0... 5.05.52 5.00-0 5.0.00.0 0.0055 5.0.00.0 00.000m 2 0.5»... 74 Al 5.. 5.. It is unlikely that the band of cross-reactivity at 1.8 kb represents either the B or y isoforms. These isoforms are typically detected at 2.1 kb, and hybridization with a transcript, at that particular molecular weight, was not apparent in the skeletal muscle sample or other tissues which would predominantly express those particular isoforms as well as in skeletal muscle. A competition experiment was performed with 1,000 fold excess of unlabeled 105 bp probe in a standard hybridization to examine the specificity of the binding at the 1.8 kb. The competition experiment suggested that the binding at 1.8 kb was specific as the intensity at 1.6 kb and 1.8 kb diminished in parallel to that of the 1.6 kb band (Figure 12). A BLAST homology search was performed on the 105 bp fragment and did not suggest any homologous base pairings with non-skeletal muscle a-actin isoforms. The same RNA blot was incubated without cDNA probe and exposed only to the anti-Dig Fab fragment to rule out the possibility that the antibody was leading to the detection of the cross-reactivity. Results with the antibody alone suggest that the anti-Dig Fab fragment is not influencing the cross-reactivity at 1.8 kb (Figure 12). The band of cross-reactivity appears to be present only in the rodent species and remains unidentified. The PCR generated DNA probe homologous to a 105 bp region within the 3' UTR of the mouse SKA gene is SKA isoform specific across several mammalian livestock species. The results are consistent with the observation that the 3’UTR of the actins show great diversity across isoforms and species (Shani et al., 1981, Gunning etal., 1984) and that a 76 5.. w. .0 0505 05. .0 005000000 05.00500 0. 0.0....55 003 005.05 55.00.00 05. 5. 5< 05. .05. 0.00000 .. .6. 0. 05.0.0”. .50. w. .0 w5.055 0....0000 m5..00wm50 Am. 050 A<. 5003.05 b.0505. 0500 05. .0 50.5.5.0 55 w. 050 5.. .0 00505 05. 55.00.00 3.30:0“. 55.000 0005.05 5. 00.00.05. 00 0.500080 55.00.00 050 55.00.0055 5. 050.0 05 .< 00 0500 .0. 050.0 05050.55 .0 0008.0 0.0. 5...... 5...: < 00 0500 8. .0.0055 .0.0.0..0 00505 .50) (see Appendix A). 188 rRNA A digoxygenin labeled probe was created using the Boehringer Mannheim Biochemicals- Digoxygenin PCR Kit (BMB, Indianapolis, IN) using the Barn HI/SphI 750 bp fragment as the initial template and the oligonucleotide primers MED 1/2 in a “touch-down” procedure. The amplified fragment was size verified using a 100 bp DNA ladder (Gibco, Grand Island, NY) at 546 bp and purified from an agarose gel using the Prep-A-Gene kit from Bio-Rad. The “touch-down” procedure allows for efficient amplification of templates which have calculated higher annealing temperatures (>50) (see Appendix D). Sequencing Skeletal a-actin Fragments A 931 bp Barn HI fragment subcloned in the initial plasmid clone pS3’a of pcDNA3 was sequenced at the Michigan State University Plant Research Laboratory using the 88 Dyedeoxy procedure from the T7 dye primer or Sp6 dye primer, a system designed by Applied Biosystems Inc. The Dyedeoxy method of sequencing is similar to the Sanger dideoxy method which represents a controlled interruption of enzymatic replication. The Dyedeoxy chromatogram is pictured in Appendix B. The 105 bp fragment obtained fi'om the PCR reaction using the Barn HI/Pst-l 451 bp fragment of the pBluescript plasmid clone and WGBl/2 primers was sequenced using the Sequenase protocol (United States Biochemical (U SB), Cleveland, OH). The sequence of the 105 bp fragment was verified by comparison to the published sequence of mouse SKA on GenBank (Name: MUSACASA, Accession: M12347) to be homologous to 3591 bp through 3696 bp of the 3’ UTR of the mouse SKA gene. l8S rRNA 546 bp Fragment The 546 bp fragment representing the 18S rRNA was sequenced using a dye-deoxy procedure with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Norwalk, CT). The chromatogram is presented in Appendix E. The 546 bp fragment is homologous to 1211 bp to 1757 bp of the rat 188 rRNA. RNA Isolation Total RNA was isolated from C2C12 myotube cultures in 60 mm diameter dishes using 1 ml Trizol reagent (GIBCO BRL. Co., Grand Island, NY). Myotubes were scraped from the 60 mm dishes with a rubber policeman then allowed to sit at room temperature for 89 fifteen minutes to allow for the complete dissociation of nucleoprotein complexes before an initial pre-spin at 12,000 x g for 5 minutes at 4° C. The supernatant was then extracted against chloroform (.2 ml chloroform per 1 ml Trizol reagent), and phase separation achieved by centrifugation at 12,000 x g for 15 minutes at 4° C. The RNA was precipitated from the extract with isopropanol (.5 ml per 1 ml Trizol reagent) stored on ice for 10 minutes, then centrifuged at 12,000 x g for 20 minutes at 4° C. The RNA pellet was then washed by vortexing with 75% ethanol (1 ml per 1 ml Trizol reagent) and centrifuged at 7,500 x g for 5 minutes at 4° C. The RNA pellet was resuspended in sterile milli-Q diethylpyrocarbonate (DEPC) treated RNase free water and stored at -80° C until analysis. RNA solutions were scanned from 220 nm - 320 nm, the A260/A280 ratio was determined, and RNA concentration calculated from the A260. Typical total RNA yield from a 60 mm dish containing mature myotubes was approximately 100 pg. Northern Blot Analysis Ten micrograms total RNA extracted from myotube samples was electrophoretically separated on a denaturing 1.2% agarose, 2.2 M formaldehyde gel at ~60 V for 3 hours. A control RNA, isolated from mouse hind-limb muscle, was used in all studies to aid in the normalization of hybridization data across studies. RNA was transferred to positively charged nylon membranes (Boehringer Mannheim Biochemicals, Indianapolis, IN) overnight with a 10x SSC solution. Following Northern transfer, the membranes were allowed to dry slightly and were then UV crosslinked using a Spectroline transilluminator (model # 302) for 3 minutes before prehybridization in a standard 90 hybridization buffer (5X SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, and 1% blocking reagent) for 8 hours. Blots were hybridized with a digoxygenin labeled 105 bp PCR generated SKA probe at 42° C for 8 hours. After hybridization, membranes were washed (2 x 5 minutes) in a 2x SSC/ .1% SDS solution at 42° and then (2 x 15 minutes) in a .1x SSC/ .1% SDS solution at 42° C. Membranes were blocked for 30 minutes and then exposed to anti-Dig Fab fragments (l : 15,000) (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 30 minutes. Membranes were washed (2 x 15 minutes) in a .3% Tween-20 Maleic acid buffer (100 mM maleic acid, 150 mM NaCl; pH 7.5), rinsed in a Tris buffer (100 mM Tris-HCl, 100 mM NaCl; pH 9.5) for one minute and exposed to the chemilluminescent AP detection substrate, CDP-Star (Boehringer Mannheim Biochemicals, Indianapolis, IN), for five minutes. Membranes were then sealed in plastic bags and exposed to x-ray film. Hybridization of RNA to the 105 bp SKA probe was quantified by videodensitometry. Size was verified at 1.6 kb using an RNA marker (Promega Co., Madison, WI). Following the SKA hybridization, the blots were stripped by a 5-minute wash in DEPC water followed by a boiling DEPC water/ 0.1% SDS (w/v) wash for 10 minutes. The blots were then placed in the prehybridization buffer for 8 hours then incubated with an 188 rRNA probe. Detection of the 18S rRNA was performed as previously mentioned for detection of SKA mRN A. The level of expression of the SKA mRNA was calculated as the ratio of the intensity of the SKA band to the intensity of the 188 rRNA band. This presents the level of specific mRNA per unit rRNA and minimizes any variation in loading of the gel. The ratios are expressed as percentages of the mean value for control tissue cultures on each 91 blot to enable direct comparison between blots. RNA data were expressed per 185 rRN A abundance and relative to 2% FBS control treatment. The 18S rRNA band was utilized as a constituitively expressed, non-fluctuating marker. The rRNA represents approximately 85% of all total RNA in the cell and codes for the proteins involved in the ribosomal translational machinery. Muscle Creatine Kinase Activity Cells at each time point of the developmental time course, from rapidly dividing myoblasts to mature myotubes, were washed 3 times in PBS, overlaid with 200 ul .05 M glycylglycine buffer, pH 6.75, and stored at -80° C until assay. The cells were scraped from plates using a rubber policeman, sonicated, and a 10 ul sample assayed for creatine kinase activity using the Sigma (47-UV) kit and manufacturer’s instructions (Sigma Chemical Co., St. Louis, MO.) (Szasz et al., 1976). DNA was quantified spectrophotometrically using the Hoescht 33258 reagent as described by West et al. (1985) using calf thymus DNA as the standard. Creatine kinase activity was expressed per unit DNA. Developmental Time Course RNA was isolated from C2C12 cells at various stages of development, from rapidly dividing myoblasts to mature myotubes. Ten micrograms total RNA were loaded per lane and electrophoretically separated as described previously. The RNA was transferred to a positively charged nylon membrane and subjected to Northern blot analysis with the 92 SKA and 188 rRNA probes. Bands were quantified using videodensitometry using the Bio-Rad MultiAnalyst System (Bio-Rad, Hercules, CA). Skeletal a-actin mRNA Abundance in Response to Increasing Duration of Isoproterenol Stimulation Maximal response of SKA mRN A abundance as a result of BAA stimulation was determined over a series of time points post differentiation. Cells were maintained as previously described. Fresh treatment media (10'5 M ISO in DMEM/2% F BS) were applied at 24 hour intervals beginning at 6 days after addtion of fusion media to cells. Time points included a 72, 48, 24, 8, 4, and 1 hour. Treatments 8, 4, and 1 hour were created in present media and returned to plates for the assigned time interval to minimize the response of the cells to fresh media and also keep treatments consistent across time points. B-adrenergic Pathway Component Stimulation Stimulation of various components of the BAA stimulatory pathway may indicate the role of each component in eliciting the BAA stimulated increase in SKA mRN A abundance. Key points in the BAA pathway include the G, protein, AC and PKA, stimulated by CTOX (Sigma Chemical Co., St. Louis, MO.), FOR (Sigma Chemical Co., St. Louis, MO), and cAMP (Sigma Chemical Co., St. Louis, MO.), respectively. The fact that several protein kinases may converge on the same transcriptional activator suggests that more than a single pathway may be involved in BAA stimulation of SKA transcription. 93 Involvement of PKA and PKC pathways was addressed by including PE (Sigma Chemical Co., St. Louis, MO.), a potent activator of PKC. Other pathway component modulators included the potent inhibitors of PKA and PKC; HA1004 (ICN, Costa Mesa, CA), and staurosporine (Sigma Chemical Co., St. Louis, MO.) respectively, and H7 (Sigma Chemical Co., St. Louis, MO.), a potent inhibitor of both PKA and PKC. Inhibitors were preincubated for one hour before the addition of specified agonist. Statistical Analysis Data were analyzed through analysis of variance by using the program of SAS (SAS, 1996). Differences among means were tested for significance (P<.05) using the method of Tukey or comparison across treatments by contrast. 94 Results and Discussion Skeletal a-actin mRNA/Sarcomeric Myosin [Muscle Creatine Kinase Activtiy Developmental Time Course The SKA mRNA band at 1.6 kb progressively increased in intensity during differentiation of C2C12 muscle cells. Initial appearance of the 1.6 kb band was approximately 48 hours post confluence and paralleled SM protein accumulation (Figure 13). The developmental pattern of expression of SKA mRNA and the SM is consistent with the established expression of muscle-specific expression of key contractile proteins. Caravatti et al., (1982) determined that the corresponding mRNAs coding for myosin heavy chain and for a-actin are detectable immediately before the initiation of myofibrillar protein synthesis. Devlin and Emerson (1978) reported that myofibrillar proteins accumulate at the same time, have similar synthetic rates, and reach steady state levels at the same time. The results presented by Caravatti et al. (1982) demonstrated a close temporal correlation between muscle mRN A accumulation and protein synthesis during myogenesis. Muscle creatine kinase activity, which was measured over a similar developmental time course, followed a similar pattern to that of the developmentally expressed SKA mRNA and SM, reaching peak activity at differentiation (Figure 14). An RNA sample was obtained at each time point parallel to the sample obtained for MCK and exhibited expression consistent with the pattern observed for the previous SKA 95 000.05050 0.50w005 05. ..5.5x0 0:00 0005. .05. 5.00.05. 0:00 m. 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The developmental data represents the characteristic expression of key components in skeletal muscle differentiation and suggests that the C2C 12 system is an appropriate in vitro system in which to study muscle-specific gene/protein expression. Isoproterenol Dose Response A BAA dose response study was undertaken utilizing the non-selective BAR agonist ISO. Myotubes were exposed to ISO at 10" M to 10‘5 M for 48 hours, then harvested for RNA isolation. Isoproterenol treatment resulted in a dose-dependent increase in SKA mRNA abundance with maximal expression at either 10‘5 M (Figure 15). Surprisingly, SKA mRN A abundance was lower (p<.05) than the 2% FBS control at low concentrations of ISO (10'9-10‘6 M). This response appears to be due to a decrease in SKA mRNA and not a result of expressing the data per 18S rRNA. Separate SKA mRNA and 188 rRNA values are plotted tandemly in Figure 16. No differences in 185 rRNA were detected. Presently, no explanation is available for the decrease in SKA mRN A abundance at lower ISO concentrations. Further, no change in total protein was observed across the dose- response study (Figure 17). 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UR. 005.0000 00 535w 0.03 0:00 ..vn5. 0055.005 0.000 5. 005005550 <2y.5 5.00-0 .0.0.00.0 .05.x05 0.0.5550 0. ..050.0.0.000. ..0.50w0 0.305500-.. 05.00.00-505 05.0 50505500500 .05..00 05 055.500 0. 500.0..0055 003 005.0 0050000. 0000 .0.50w0 55050.00-.. < 0055.005 «.000 5. 005005550 5.0.0.0 .0.0. 50 0050000.. 0000 .050.0.0.000. h. 0.5»... VIN “fl [HI/8n uopenueouoo uya101d 106 10'8 M 10'7 M 10‘6 M 10'5 M 10" M [SO [SO ISO ISO ISO F o rskolin Dose Response Forskolin is a potent stimulator of AC which increases levels of intracellular cAMP. Stimulation of AC by FOR should result in an increased level of SKA mRNA abundance as observed with BAA treatment, since AC is a key component in the BAA signaling pathway. Myotubes were exposed to FOR at 10'8 M to 10“ M for 48 hours, then harvested for RNA isolation. FOR treatment resulted in a dose-dependent increase in SKA mRN A abundance with maximal expression at 104 M (Figure 18). As observed in the ISO dose response, SKA mRNA abundance was lower (p<.05) than 2% FBS control SKA mRNA abundance at low FOR concentrations (10"-10" M). In the case of FOR: DMSO was used as a carrier, however, the effect of the carrier on decreased SKA mRN A abundance must be considered irrelevant considering higher concentrations of FOR contained greater amounts of DMSO and the response was incremental with increasing FOR concentration. DMSO volume never exceeded 8 ul in the 2 ml media/dish. The SKA mRNA and 188 rRNA abundance values are tandemly plotted in Figure 19. No differences in 188 rRNA across treatments were observed. The data suggest that, as in the ISO dose response, SKA mRNA abundance decreases at the lower concentrations of FOR. No explanation is available at this time to describe the decrease in SKA mRNA abundance at the lower concentrations of either ISO or FOR. Consistent with findings in the ISO dose response experiment, no significant increase in total protein was observed in response to FOR (Figure 20)- 107 .Amo.V0. .05500 mm". gm 05. 0. 0>..0.0. 005005550 <2~.5 5500-0 .0.0.050 000.000 055005.550 0. 00.00000 5.00.0.0. .0 050505500500 .030. 05. .0.505.0.0x055 0..5000.0 050 0.w5.0..0.5m ..moV0. .05500 mm... fim 0. 050.0. 5.00.0.0. S. we. .0 005005550 <2M.5 5500-0 550.050 .0 50.000.0x0 .05.x05 0.00.05. 0.0m. <20. 0... .0 ..55 .00 00000.0..0 0.0 0.00 5500.0 .0.0.050 05 H .05050..0500000.> w5.05 0000.050 0.03 0.0.5 05. 0005.05 50500.00 0>..000.00.-505 w5.30..00 050.0 <20. mm. 50 5.3 005055050. 050 0000.50 0.03 0.0.5 0500 05... 050.0 5500.0 550.050 05 mo. 0 .05.0w0 0050.505 050 050.5505 50.05 0. 08.0.0505 ..0w w5..5.0500 000.0w0 5%.. 0 50 55. 0.03 0055.005 0. 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Abundance of SKA mRNA increased over 1, 4, 8, 24 , and 48 hours exposure to ISO reaching maximal levels at 72 hours post treatment. The response at 72 hows was significantly greater than the 2% FBS control (p<.05) (Figure 21-22). B-adrenergic Pathway Component Modulation Skeletal a-actin mRN A abundance was increased in response to stimulation of C2C 12 myotubes with ISO (10'5 M). The response to ISO stimulation was abolished by addition of propranolol (PRO) (104 M) (a non-selective BAR antagonist) in combination with ISO. The reduction in SKA mRN A abudance in response to PRO suggests that the ISO stimulated increase is occurring through the BAR mediated signal transduction cascade. In contrast to the previously reported dose response study, no increase in SKA mRNA abundance was detected in response to FOR (10‘ M). In the same study, however, the addition of cAMP (1 mM) increased levels of SKA mRNA abundance relative to 2% FBS control (p<.05)(Figure 23). The lack of a FOR effect may be attributable to inactive FOR in the series of experiments on pathway component modulation. Inhibition of kinases BAA stimulated signaling pathways would be expected to depress the SKA mRNA response to BAA stimulation, if the increased mRNA abundance is due 114 ..moVE .05500 mm. {cm 0... 50... .0.00.w 0..5005.5w.0 003 050 0.50.. 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E 25 0. .302: 05 2 L: 08232.8. .0. .2 L: 009.08. .0. .25 _ .025 0.. .2 L: 02.05.02. 05 .2 1: 0822208. .0. .2 1: 0820.208. .0 .208 00. $0 5. 0.5.502. 05323. 0... 08000.00. 80.w0.0 00 .. 0.50.. 5. .0. 0.0.0.5008 .0 00..00 0 5.3 808.00.. m5.30=0. 00..0. <2)... mm . \<2~.8 5..00-0 .0.0.000 0... 0.0 00.0.00n. 0030.00 50..0500500 .05w.0 08.05200-.. 0... .0 0.50500800 00.. .0 0.0.0.5008 0..3 00.00.. 0.03 0005.008 .0.—ONO 0005.008 0.000 5. 005005500 <2~.8 5..00-0 8.0.8.0 050 50..-0500.5 .50500800 0030.00 0.3053000 mm 0.5..— 119 01.0m— modvn. NECEAOm. modv... Ax0NA...=.._.<0 0 0w50.... M5...5000 0. 0005.008 0.000 .0 50..0.58..0 0.9052004. 5005 0020000 005005500 <2~.8 5..00-0 .0.0.000 00000.05. 0... .0... 0.00.05. 0:500”. A28 m0. vac—<2 .0 C). so: .0.050.00.0 .0 50...000 00.00 00000000 003 .050.0.0.000. 00 50..0.58..0 0.905200-.. 80.. w5.._500. 005005500 <2~.8 5..00-0 .0.0.000 5. 0000.05. 0.... ..0..500 mm. {cm 0... 0>000 005005500 ..0.0. 005005500 <2~.8 5..00-0 .0.0.000 00000.05. 5. 000500. C2 0.... .050.0.0.000. 00 .0.0000. 03.05200-.. 0... .0 50..0.58..0 ”30.>0. 5. 0005.008 .0.. 0.00 5. 005005500 m .003: Z 50......0058. + 5: 0.0000 g 2.02: 43:29:.- + luv X /. “Y: 02. .- 0.0.-0 a! I 4/ 02 «G... + 2500.000. 0005.0. v5.0.2 5._=00.5_0U\~+0U / 4/.=>_<0 III 0.0 .0550..0 00 0%. [K 0: 0000.0. N+00+ fl\q.r. 02.00.00.550 2.0.0... 5.30:0 ./ =5. 9 <0 124 Abstract B-adrenergic agonist stimulation via G, protein results in the elevation of intracellular cAMP and subsequent activation of cAMP-dependent protein kinase, PKA, a serine/threonine kinase which is capable of phosphorylating a wide array of proteins. The objective was to determine if the response of differentiated C2C12 muscle cells to BAA stimulation was manifested by an increase in protein phosphorylation. An apparent increase in phosphorylation of an ~ 90 kDa protein was observed in response to increasing concentrations of isoproterenol (ISO). Exposure of C2C12 myotubes to a combination of ISO and a phosphodiesterase inhibitor (PDE-I) (10 uM) enhanced the phosphorylation of the ~ 90 kDa protein. Further investigation indicated an increased phosphorylation of the ~90 kDa protein in response to dbt-cAMP (1 mM), FOR (10“ M) and CTOX (500 ng/ml). Responses observed appear to be mediated by an increase in protein phosphorylation rather than an increase in protein synthesis, since cyclohexamide (CI-IX) (10 ug/ml ) did not alter the response observed in treated cells. Anti-P-threonine. immunoblots of extracts of cells at progressive stages of development revealed that appearance of the ~90 kDa phosphoprotein followed a developmental pattern of expression which paralleled that of sarcomeric myosin (SM). Further characterization of the protein via cell fractionation revealed the protein to be cytoplasmic rather than nuclear or myofibrillar. These results indicate the presence of a cytoplasmic, ~90 kDa 125 phosphoprotein that is phosphorylated in response to BAA stimulation in C2C12 myotubes. 126 Introduction Skeletal muscle is the primary target tissue for enhanced protein accretion in animals fed BAA (Bergen et al., 1996, Skjaerlund et al., 1993, Paterson et al., 1984, Gordon et al., 1984). BAA’s also stimulate protein synthesis and accretion in cultured muscle cells (Bergen et al., 1996, Shani et al., 1981, Anderson et al., 1990). The mechanism by which BAA influence protein accretion in muscle remains unresolved. Extracellular signals regulate gene expression by triggering signal transduction cascades that result in the modulation of transcription factor activity. Signal transduction cascade gene activation is most commonly achieved by changes in the phosphorylated state of nuclear proteins. Phosphorylation affects transcription factor activity at several distinct levels. It can modulate their intracellular localization by controlling the association with other proteins, have both negative and positive effects on their binding activity, and modulate the activity of their transcriptional activation domains. In addition to phosphorylation, protein-protein interactions also have an important role in mediating a cross-talk at the nuclear level between different signaling pathways (Karin, 1991) (Figure 1). The proposed mechanism for BAA induced muscle protein accretion is through a Gs-protein linked cAMP-dependent protein kinase cascade (Bergen et al., 1991, Bowman et al., 1969) (Figure 25). Most aspects of adrenergic stimulation do not ordinarily require an increase in the synthesis of new proteins. Post-receptor events may involve a complex 127 .0050000. 00.0.58..0 0.w.050.00-n 00. w5...0..0 ..00 00. 5.0..3 05.0.0.0 .0 >050 00.3 0 w5..0.>.0000000 .0 0.00000 500. 0.0 0..55050 0030.00 00 .. .0..55050 0.3.0.00 0>..00 050 0.0.0.53. 0 0.5. 00.0.0000.0 < 0005... 5.0.0.0 00.0>..00 00 .- .< 00050. 5.0.0.0 .0005... 5.0.0.0 .50050000 03.0.0 00. w5..0>..00 .0 0.00000 500. 0. 02.00 .0206 ..0w500005. 050000 00. 0. 0..-< .0.5..000..5. .0 50.0.0>500 00. 00.00.00 000.000 0.00.5000. 000.98 0.00.5000 0.50050 00. 00.0>..00 00.03 055050.... 5.0.0.0 0.0.0.585 -0 00. .0 50..0.0000.0 000500 .0.0000. 00. .0 w5.05.m. 50.0000. 0.m.050.00-.. 05500-050580... 00. .0 .0.50w0 0.905200-.. 0 .0 m5.05.0 >0 00.0...5. 0. 0000000 w5..05w.0 00.. .0 50..0.58..m $030.00 5000500505 .05w.0 0.w.050.00-n .00.000.0 00. 0.0.000 50..0..05... 00... 0030.00 50.350058. .0530 0.305.004. .00.000.0 mm 0.5»... 128 0050000. in. 0 AI 05.40 + 5: 000.000 0.00.5000 5.0.0.0 mU / <0. 5.0 129 interplay among various proteins by covalent modification through phosphorylation /dephosphorylation (Iwaki et al., 1990). Protein phosphorylation regulates a diverse range of cellular responses (Meek et al., 1992). Although many of these regulated proteins have been identified and characterized in other systems, many newly discovered or yet undiscovered proteins may play a pivotal role in the biochemical mechanism behind BAA induced muscle protein accretion (Iwaki et al., 1990, Meek et al., 1992). Identification of skeletal muscle proteins phosphorylated in response to BAA is essential for understanding the biochemical mechanism behind BAA-induced muscle protein accretion (Beermann et al., 1986). The objective is to identify proteins phosphorylated in response to BAA stimulation in C2C 12 myotubes. It is anticipated that the proteins identified represent a phospho-protein involved in the BAA hypertrophic response. 130 Materials and Methods Cells C2C12 muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Approximately 50,000 cells/dish were seeded into 60 mm diameter dishes in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO Inc., Grand Island, NY) containing 10% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO). Cells were grown to confluence in DMEM containing 10% FBS in a humidified atmosphere of 5% CO2 and 95% air at 37° C. Upon confluence, growth media was replaced with differentiation media (DMEM, 2% FBS) to stimulate myotube formation. Medium was replaced every 48 hours unless otherwise indicated during the experiments. Sample Preparation Myotube cultures were rinsed 3 times in phosphate buffered saline, harvested using a low salt NFEP homogenization buffer (.05 M NaF, .01 M EDTA, .075 M NaCl, .015 M NazHPO” pH 7.0), and sonicated using a Branson Sonic Power Co., Sonifier-cell disrupter 350 at setting #4 for approximately 5 seconds. Aliquots of each sample were saved for protein and DNA quantification, and for SDS-PAGE. Protein was determined using the method of Bradford (Bradford, 1976) using commercial reagents (Bio-Rad, 131 Hercules, CA). DNA was determined fluorometrically using the Hoescht 33258 reagent as described by West (1985). Sample aliquots for SDS-PAGE were prepared by mixing equal portions of the aliquot and hot 2x treatment buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% MCE, pH 6.8). Samples not used immediately were stored at -80°C and heated for 5 minutes at 95°C before loading onto gels. SDS-PAGE and Western Blotting Samples were loaded on an equal protein basis on an 8% acrylamide (37.5:1) separating gel with a 4% acrylamide stacking gel and fiactionated at 165 volts for approximately 1 hour. Proteins were electrOphoretically transferred to Immobilon P membrane (Millipore, Inc., Bedford, MA) at 100 V for 1 hour. Membranes were allowed to air dry and stored until blotting. Initially, membranes were blocked for 1 hour in blocking buffer (1% BSA, 05% Tween 20 in Tris-buffered saline). Following blocking, blots were incubated with either a rabbit anti-P-thr. (#61 -8200) (.5 ug/ml) or anti-P-ser. (#61-8100) primary antibody (Zymed Technologies, Inc., San Francisco, CA). Antibodies were titrated for optimal concentration for use in C2C 12 myotube extracts. Following incubation with primary Ab, the blots were incubated with an anti-rabbit secondary antibody conjugated to either alkaline phosphatase (AP) or horseradish peroxidase (HRP) (120,000) (Sigma Immunochemicals, St. Louis, MO). Membranes were washed three times (5 minutes each) in blocking buffer following each Ab incubation. Antibody binding was visualized by exposure of membranes to colorimetric AP reagent (BCIP/NBT; Bio-Rad, Hercules, 132 CA) or chemilluminescent HRP substrate (Super-Signal Chemilluminescent Substrate for Western Blotting; Pierce, Rockford, IL). Two-Dimensional (2-D) Gel Electrophoresis 2-D gel electrophoresis was utilized to further identify the responsive protein relying on separation by the protein’s isoelectric point (pl) and molecular weight. 20 ul aliquots of the cytoplasmic fraction of cell extracts were applied to 3.5% tube gels cross-linked with piperazine diacrylamide containing 9M urea and 2% Bio-Lyte ampholytes (1 part 3/10 and 2 parts 5/7) according to manufacturers instructions (Bio-Rad, Hercules, CA). The tube gel was run on a 8% acrylamide (37.5: 1) slab gel for further fractionation by molecular weight. Estimation of the unknown proteins pI was determined through comparison to 2-D standards (Bio-Rad, Hercules, CA) run in parallel to the cytoplasmic fraction sample. Duration of B-adrenergic Stimulation on Protein Phosphorylation C2C12 myotubes were stimulated with the BAA, ISO (105 M) for various lengths of time to determine the time of maximal phosphorylation in response to ISO stimulation. Cell maintenance was performed as described earlier. Media were replaced every 24 hours during treatment. Treatments were initiated 48 hours prior to harvest and continued through 30 minutes prior to harvest. Treatments were initiated in this fashion to maintain all cells in culture for the same amount of time and to minimize the confounding effects of fresh media at later time points. Treatment at time points 8, 4, 1 hour and 30 minutes 133 were prepared by removing media from cells, adding ISO to the media, and redistributing this media to plates so new media was not introduced at those time points. The control samples were maintained in DMEM + 2% FBS for the 48 hour duration of the study. Phosphodiesterase Inhibitor B-adrenergic stimulation increases intracellular cAMP. The enzyme phosphodiesterase (PDE) breaks down cAMP, thereby eliminating the BAA induced response. Treatment with a phosphodiesterase inhibitor (PDE-I) (4-(3-butoxy-4-methoxybenzy1)-imidizolidin- 2-one) (Sigma Chemical Co., St. Louis, MO) in a BAA-responsive system should result in increased levels of intracellular cAMP and a potentiated/enhanced response to BAA stimulation. C2C12 myotubes were pre-incubated with a PDE—I (1 ug/ml) for 1 hour prior to and during stimulation with ISO for either 30 minutes or 1 hour before harvesting as described above. Cyclohexamide Treatment The effects of BAA stimulation are usually rapid in onset, often occurring within minutes of agonist binding, and do not ordinarily require an increase in the synthesis of new proteins. Treatment of C2C12 myotubes with cyclohexamide (CHX) (Sigma Chemical Co., St. Louis, MO) would indicate if the response to ISO stimulation was due to the synthesis of new proteins or an increased phosphorylation of the protein of interest. C2C12 myotubes were pre-incubated with CHX (1 ug/ml) for one hour prior to and during stimulation with ISO for either 30 minutes or 1 hour before harvesting as 134 described above. The time points chosen, 1 hour or 30 minutes, represent time points that are rather early after stimulation to see a phosphorylation response. Therefore, an increased phosphorylation response observed at either time point would strongly indicate that the response observed was due to phosphorylation rather than increased protein synthesis. Cell Fractionation Cells were rinsed 3x in NFEP buffer, scraped with a rubber policeman, and Dounce homogenized. Samples were centrifuged at 1,000 x g for 15 minutes. The supernatant was saved and represented the cytoplasmic fraction while the pellet was resuspended in NFEP buffer and represented the myofibrillar and nuclear fractions. Further fractionation of the sarcoplasmic fraction was achieved by centrifugation of the sarcoplasmic fraction at 100,000 x g for 1 hour to pellet the membrane fraction. The sample was washed once with NFEP buffer and recentrifuged at 100,000 x g for an additional hour. Aliquots of each sample were analyzed by protein and DNA assay and by SDS-PAGE as described above. Statistical Analysis Data were analyzed through analysis of variance by using the program of SAS (SAS, 1996). Differences among means were tested for significance (P<.05) using the method of Tukey or by comparison across treatments by contrast. 135 Results and Discussion Phospho-Amino Acid Profiles Initially, cell extracts of untreated mytotubes were prepared as described earlier and immunoblotted using either an anti-P-thr or anti-P-ser primary Ab in an effort to determine what the phosphoprotein profile for each antibody looked like. The anti-P-thr Ab labeled a greater number and overall intensity of bands relative to the anti-P-ser Ab (Figure 26) which revealed only light labeling of a few phospho-proteins (Figure 26). We then chose to examine the effect of BAA stimulation on C2C12 myotube cell extracts in comparison to control cell extracts in order to identify any potential phosphorylation responses in the protein profile. Cell extracts were prepared and blotted against the anti- P-thr Ab in an attempt to identify any responsive protein/s. Identification of a B-adrenergic Responsive Protein Initially, myotubes were stimulated with two concentrations of ISO (10" M and 10" M) as well as two concentrations of PDE-I (10 uM or 100 uM). An ~90 kDa protein appeared to be phosphorylated in response to BAA stimulation by ISO (10" M) (Figure 27) as determined by an increased band intensity relative to the 2% FBS control. Although other bands appeared that may have shown an increased phosphorylation response to BAA stimulation, the intensity at ~90 kDa suggested that the ~90 kDa protein may be 136 aoBoasfifim camcoobuifiew 05 E 353 8:25 we oceafifiovoa a 83:2: «:5 .05—cocoa oEoEteBo a smack: eon—£3888 3.5 5:889 .omfianamonn 0:28.? o. teaming 09 “398-55.. .3 83 n< $9.88 2:. .3335 SEE. GER: mdv assign—adieu .5 2.5m: m3 octomdéca an $56 mama: vote—noeseg 28 0:83.58 :2? 2 35.33: .m0 97kD —>_ 66kD —” 45kD ——> 31kD H... . 21kD —> .0850 boa—awe .3 93.53:. 0.... .3 .00....m:0.:_ bfiwzm 0:0 .0.:ow0 0_w.0:0.€0-n 0 96 82.000 06 .3 0026:. 0a. 2 0.80%.: 0.32.00: gusto—Ewen: 0:... 00:29:: N. UmU :. :o..0_bosmmo:a 60.033. 0300:0023. 0.0w.a0>:_ 9 0.02050 300... 0 m. 50.8: 000. O02 05 :0... 0.8me 5505:0050 005.0 .0 9:05.00: 8.32:. 00007.0.vennmosazofiheoaom. Am. 0:0 3:288:00. 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III. .-p. ill-III I... ‘1 .0. 1 a. .— ' ..D‘ ..i T TTI T T' a “N a “M :0— me A: 3 G0— 5: G0— a: A: 2K more abundant and therefore an easier candidate to study than proteins at lower band intensities. An apparent increase in phosphorylation of a ~ 90 kDa protein in response to either concentration of ISO and a slight increase in phosphorylation response to either concentration of PDE-I was detected. The response observed indicates that BAA stimulation through the BAR by ISO can be potentiated by the addition of a PDE-I. The data suggest that ISO stimulated phosphorylation is the result of a cAMP linked cascade in C2C12 myotubes. The phosphorylation of the ~90 kDa protein appears to be dependent on a cAMP regulated cascade mechanism most likely through the activation of protein kinase A (PKA). Pathway Component Modulation Further investigation into the mechanism of the phosphorylation of the ~90 kDa protein was accomplished by targeting direct components within the proposed BAA pathway. FOR (10‘ M), ISO (10'5 M), RAC (10* M), cAMP (lmM), PE (2 uM), and CTOX (500 ng/ml) were administered to C2C12 myotubesifor 48 hours. At 48 hours, the cells were harvested in NFEP buffer and cell extracts prepared as described above. Based on densitometric scans, an approximate three-fold increase in intensity of an anti-P-thr labeled band above 2% FBS control myotube extracts was observed through stimulation by components of the BAA pathway. 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A... 0800.8 cm .0 0881 . 60.800.000.08 + 3. n.o. ..0:80.088. 5. 0080.8 cm .0 .3. .-o. ..0:80.088. A.,... .800 . .0 .3... c. 02.0.00. 80.800.02.808 + .3. ..o. ..0:80.0800. Am. .80.. 000.0 .3. ..o. ..0:80.088. 5. 0080.8 cm .0 .3... c. ..0..0...:. 000.800.02.800: + .3. 0.0. ..0:80.088. .0. .0888 cm .0 3. .-o. ..0:80.088. 3. ..0.800 mm... {an .5 ”00800088 0000.. $000.80 808.8 080000040000 :0 8.00 00..0.000088. 000 000.0808 00...: 0. 080.000.. .900... $0 :0 :0. 0.03 0.00.80 ..00 000.an 2000 .8800 0:0 00.00.. 80... 0.0.08 .0.0. 08080.08 :0... 0.00.80 :00 0008.8 «.000 0. 088.08.... 800.0888. 0. 0002.00. 00..0.80000000 0 ..0 00..00....0> cm 0.0»... 147 .. . . .. , .Ili.‘ i r n‘.‘ .0: .EGme0m< TTTT TTT 90.: Aux—m amv 90.00 5.8 :10: A... :3 148 synthesis (Figure 30). Considering a phosphorylation response is observable within 1 h suggests that the response is due to protein phosphorylation rather than an increase in newly synthesized protein. Cell Fractionation Initially, we were hoping to identify a phospho-protein involved in myofibrillar gene expression. Determining the cellular fraction in which the ~90 kDa protein was most predominant would aid in further identification of the protein. Cellular fractionation was accomplished as described above. We were unable to successfully isolate a pure nuclear fiaction most likely due to rupture of nuclear membranes during Dounce homogenization We had, however, two distinct fractions: a myofibrillar/nuclear fraction and a sarcoplasmic fraction. Characterization of fractions included assaying each fraction for DNA as described previously. While the sarcoplasmic fraction had virtually no DNA, the presumptive nuclear fraction remained devoid of DNA also; most of the DNA resided in the myofibrillar fraction. Initially, further purification of the fraction was not required. It was apparent through comparison of immunoblots of the fractions, using either the anti- P-thr Ab or the anti-sarcomeric myosin Ab (NA-4), that the ~90 kDa protein was predominant in the sarcoplasmic fraction (Figure 31). The search for the identity of the ~90 kDa protein revealed several candidates at approximately 90 kDa in either the microsomal or cytoplasmic fractions. 149 0000.008 0.000 ..0 :0..00... 880080800 0... 8 000.00. 80.08 0m... 002 0... .0... 0:0 88.00.. .0.0..00 .08.0.0 03. ..0 800.088 0... 0.00.08 0:003. 88.00... 880080800 0... 8 80.08 00.. 002 .0 0:00 0.8.0 0 ..0 08088008.. 0... 0.00.08 0< 0:.:08...-..-..:0 0... 8.00 0.8000088. 88.00... 0.8008088 0... 8 00>.0000 003 8.080 0: 0......» 80008 8.080800 8.00800. :0..00... .00.00: .0....00008 0... 8 0:00 00.0. 00m 0.080 0 0. 0800 .0< 0.0.7. 0... .00. 0.00.08 0.800888. 08:00....-..-..:0 .0 800.08 8.080800 .00..0 8880 000200888. 0:0 000.0808 ....Q>.. 0 0. 08.0.88. 80>» 0.00 0.00.800 8 .004... $0 .00 00.0.0800 0:0 0.000 80.08 .0000 :0 :0 00000. 0.03 008.00.... .00..00... 0.8008088 0:0 .00.00:\.0....0...0>8 0 008.00... .0880 03. 08.80.. 00.088 0. .0. w x 000.. .0 0000....800 003 080008000 ..00 00... 08800080.. 0080A. 0:0 .0....00 8.087. .1 00m 8 0000.0 88 00 80... 0080.00 0.03 0000.008 ~— 08 .:0..0:0..00.. ..00 0000.... 02.0.8888 003 80.08 0m... 002 0... ..0 08.000. .0.0..00000 0... ..0 :0..00.....:00. 0.0.2.00 :00 000.008 SUN“. .0 08.80.80...— .0...:. in 080.8 150 G:00.|Wv 9:05N.IIV a“ .Ohflm .0:: .38 00.08.000-02 0:02. 0.85.08 151 The search results suggested that further fractionation was required to identify which fraction, within the sarcoplasmic fraction, contained the ~90 kDa phospho-protein. Fractionation by centrifugation at 100,000 x g revealed that ~90 kDa phospho-proteins exist in both the microsomal and cytoplasmic fraction (Figure 32). The ~90 kDa phospho-protein band in the microsomal fraction declined with BAA treatment, while the ~90 kDa band of the cytoplasmic fraction increased with BAA stimulation. Developmental Time Course Identification of the ~90 kDa protein was further investigated through examination of ~P over a developmental time course. Cell extracts were prepared from untreated C2C12 at various stages of development: from rapidly dividing myoblasts to fully differentiated myotubes. Cell extracts from those time points were then immunoblotted against the anti-P-thr Ab. The same extracts were run in parallel to NA—4 (sarcomeric myosin) for comparison to a well defined muscle-specific protein. The same cell extracts were run on an 8% PAGE and the gel stained to visualize the protein profile of the cell extracts across the development time course. Results indicate the ~90 kDa protein represents a protein which is present from early development through differentiation. Phosphorylation- of the ~90 kDa protein is developmentally regulated and is further confirmed by its paralleled expression to a well defined muscle specific protein, SM (Figure 33). 152 00..00... .08000.0.8 00. 8 380880008 000.00. 0.0.0.8 0A... 002 0... .0... 00.000000 0< 00.000.078-000 00 00.00 0808000088. 000.0808 80 >8 0 0. 00008000.. 000 0..0> no. .0 .000... $0 00 00.0.0800 003 08.00... 0000 088000.80. 0.0.0.8 .0.0. ..8 0. 08.00... 0.800.800? RU 000 88.00... .08000.0.8 C. 88.00... 880080800 5. .00..00... 8.0.0.838 A...0...0000 03. 0.. 0.0. 08.00... 0.800.800.00 0... ..0 08.000000... .00..0. 0 0.000080. 00.00008 .0.000088. 0..... 0.00..00 :00 000.088 «.000 ..0 08.00... 80.00.800.00 00. .00.000 .0.000088. 00.000.588.00. NM 0..—0.8 153 G800 Ilv T T T T T T T :8: 90:0 Emu 90.00 G0. 00 a... 0: Q.— 00m 154 .0.0088 000800.00 ..0 08000800 0... 0.8.0008 000 00.0.000. 8..0.00880.0>00 0. 0.0.0.8 0A... 082 00. ..0 0000.8.0080008 00. .00. 0.000000 .0.000088. 00.000.58.00 00 8. .0.0.08 00000.8..0 8..0.00880.0>00 00.0080 0.0008 000000 ..03 0 0.000080. 0803 .0.000088. Y.0000 0.008 00 .. .0< 00.000.88-000 00 .0 20-0.2. 0< 0.0088 0..0800.00-..00 00 .00..0 0800 00000880000 003 0808000088. 000.0808 8Q>8 0. 00008000.. 000 m0<8 08.0 00 00. 0.03 0.0>.0.8 08.. .00880.0>00 00. 000.00 0.00.0.0 ..00 000.088 2000 80... 0.0.0.8 .0.0. 01 0. 000000000 .008 0.000 3.. 8... .000000000 .008 0.000 00 .0. .000000000 .008 0.000 0.. P... 600000000 .008 0.000 ..N .m. 0.00.0088 8000000 5. 0.00.0088 080.>.0 8.0.80. 610.. ”00.000080. 00000 0000.088 0.0.08 0. 0.00.0088 00.0.80 8.0.80. 80... ..00880.0>00 0000.0. 0..00 2000 80... 00.0808 0.03 0.00.0.0 :00 .8808 0A... 002 00. 0.. 08.000.00.000 8.0000. .0008 0. 0008.008 003 800.0 00.000 08.. .0.00880.0>00 < 0.00.2.0 :00 000.088 0.000 .00.000 .0.000088. 00.000 08.. .0.00880.0>00 00.000.58-000. mm 0..—0...... 155 my... 3 11.. III. 0000i \I-lullll... 0.0088 IV ..,. . H TTTT 90:0 amv 90.00 G000 90.0: Q0. can G0. cam .0.m 30.08.08.880 ..0 .8 0 .0 0.0.0.8 0. 0.0.0.8 00.0. 002 00. 00.00>0. 00.00000 D-N 0. .0.000088. 00.000.078-000 00. 00 000008800 000.0808 80. >8 0. 00000000.. 000 > m0. .0 mO<8 08.0 00 00.0.0800 003 00.0008.0 000000 00 8- ..00 000. 00.80.8.00 .8. £00 0..08 N 000 0...“ ..08 .v 00.8.00880 0.80-0.0 gm 000 00.0 3.0 00.0.0.000 00.80.8.00... 0800.088 0..? 000.00-000.0 .00 000. 00.80.8.00 08.0.0 0 00 00.0.0800 003 0.0.0.8 000000 0.800.800.00 .0.0. 0.8 o. 86.08.08.880 00..0000.80. 0.8800 .1 mm 000000 0.800.800.00 00. .00 0.08.000 Q-N 0000.0. 00.88000 003 0.0.0.8 0m... 002 00. ..0 0000000000. .00..0... 00..00... 0.800.800.00 000.088 «.000 00. .00.000 0.00.0080..00.0 .0.—0.058.003. 00.000.58-00... 00 0.00.8 157 £0.00 V H T TT a: a...” 39. 90.00 90.00 G0. 0: A: can 158 .oh bofigxoaam .3 E a «a 2838 2 £2th «Ox col 2: 3392 mcSucflm D..N 8 BESSEE oE:025d-::m 05 .3 :omtmmEoU 65582: mo>m 2 Bumbag: can > m2 3 m0 Bx:__-mmo.u Em 2.3 025258 $m.m a co 3:238 33 £28m cocoa.“ o_Emm_moo8m .83 w: o. bofigxoaam mas—8852 29:9. 3 mm documfi 883—383 2: mo max—mg Q-m .3325 3.9883 33 £085 «Qu— oo: 2: mo cosmocccuE 55.5.» 523....— 3833353. 2.59:: SUNHV 2: 3:3»: 50.8.1358.» _a:c_n:o=._e-c5 onmaoohfidéuay em unsur— 157 3.5 .V H T TT a: gun name 0&9» aha Gu— e: A: can 158 Two-Dimensional (2-D) Electrophoresis Positive identification of the protein remained elusive and more information was essential for data-base searching. Two-dimensional electrophoresis was undertaken using 20 ul representing 10 pg of the sarcoplasmic fraction to approximate the ~90 kDa protein’s pI. Two-dimensional electrophoresis revealed a protein/s at ~90 kDa and by comparison to two-dimensional standards (Bio-Rad, Hecules CA) revealed the isoelectric point (p1) of the responsive protein to be approximately 5.6 (Figure 34). Protein Purification and N-terminal Sequencing Following two-dimensional gel electrophoretic analysis and unsuccessful data-base searching, N-terminal sequencing was attempted at the Michigan State University Macromolecular Structure Facility. Sequencing required the acquisition of pure 90 kDa sample. Cell extracts were prepared from C2C12 myotubes and run on an 8% preparative gel. The ~90 kDa band of interest was dissected out of the gel and subjected to elution in 1x TBE. The slurry was then centrifuged at 3,000 x g for 30 minutes and supernatant saved for analysis. The supernatant was then dialyzed against .lx TBE for 2 hours followed by water overnight. The dialate was removed from tubing and concentrated using a Centricon device; pore size 30 kDa. The sample 'was run on an 8% PAGE, transferred to PVDF membrane, and stained with amido black stain; thus revealing a prominent band at ~90 kDa and two other less intense bands at ~60 and 30 kDa. The latter bands may represent degradation products of the ~90 kDa band, as it is highly improbable that these bands were excised with the ~90 kDa band initially (Figure 35). 159 .23 «9. col 2: .23 vomfixo b.8528“ 9:805 55 558 £805 «Dv— oo2 05 go $2605 :ozmwfiwoc 0352. “5353 3:. 3:3 82: 35 “mowwsm 22.89:. 832 a :25 3 $55 8:8 95 05 .«o 3:39:— of. $3.605 :ocmcflwov 383588 an? on can cc boa—«meoaam 8 32:0 03. can max 002 8 2m :5on $53 2: .mwSB 856% :5 Em: 025 go 3:085 05 "”3835 28582: 3:35 2; any. cat “a v53 29% a .3 855:8 05 big o. :36 0:3 232280 5:3 3:33 .83 28582: 2; 65582: m0>m m 9 watchman: EH .m0..00 0: .05 50.0000 2 ._ .20.. 03.530.005.55. 00.0358 3:80.058. 05 0:0 :0. 03.530.005.55 3.500 05 .0550 33055.5. n< 05:00.5-0-_50 05 0580.00. 3:00 0:008 0..... 00:80.0. .0.. 6. 55.00... 5500—0050 0:0 92. 358055 5500500 05 0. Am. 5500... 0.5.00—000.00 05.3 50.55.00... 359.0 05 m. .3355 00.0.0.0 .5.. 05:00.5-0-.50 05.0 3. 55:050. 5500—000.8 3:555... 09— 00550 05 .053 5.3 00.330555 0:0 6:05.505 .350 0 0. 0050050.. .m0<0 {on :0 00.0308 d< 55:050. 5500—000.8 35555. 0D.— 00550 :0 53 020.330.005.55 0.03 0.30 00.0358 3:20.508. 0:0 3.500 mm”. .xa 05.3 :0500... 3580.55 2:. 450500... 0.5003050 0:0 3580.55 05 0. 00.55.00... .23.. 0:0 0.00; v .00 32 .-o C 3:20.508. 53 00.0358 0.03 805.005 N. US 03.530.005.55 :0.00_0:00-.m00: 05 0. 00. 0.5.00—00.00 .0 3580.05 05 .053 5 8.00.0:00 3.0>8 .3 00:25.0 05 ”50.0.0 000. oaz 05.3 55005505 05 .0.. £0.08 055050 :0 5.3030 023.005 «:30 5 50.0.0 0:0— 00... :0 3 55000. 53:02.8 0:. .3 5.50.5500— 3. 0.5»...— 166 UH DU Si. EU uh EU 0 2 m Alla—0.: Alla—«Zn Tame Alla—0.00 Alla—0.00 Alla—0.0: All 90.25 167 .528: 83830.. umEmsEOoEm 388:3. we. on 2: 6: m_ £28: 85:58 $9828-83 «Ox col 2: 85 :28: 05 wfitoqgm 82:3 588: 838:2 2:82:85». .a:o:o::.n «9. co a: 8:28 05 E 822:8? >8 momma 8: 88 83058:: 05 .8228 5 485882: on 8 8:82 m_ z 233 888.: 38883:: 05 :_ 828: :53 we. ca 2: 88.83 :ousamooaogfig U; n; 05 35 2865 83o::EE_ 05 :_ 8.88:: Sam .89:an 83:88 _o:888:8_ no 838$ 388385 F: won—Bob: .0::8 mmm e\om no 888.“ 358388 Am: .888: 3:88:28 8.22:? .o:22oa8_ vogéooaogfiafi RC .888.“ 3:58:28 .0::8 mm: o\om vegfiooaogfiem ADV £3292: 85:85 388.89% be 858...: 2883823 :38 Amy don—doze .0::8 mmn— {am no 888.: 2883028 .82 A3 688882 855 888$ :8 2.392: 38883:: «58 88385 3:228:8m 8 35:8 mm”— $~ an £82: 52852 SEQ—:88 388:3 we. ca 8§_q_ooao:::::_ 65am: BBQSEEm u; m> 2: 3:828: 8:888 83 2:. 8255 :8 83:88.5 38889:: 2.5:»:— 2Unu .838 6:: 8.25:8 _::o:o.:..._cm_ a: £89:— aA—u— cal 8.a._&uo..ao:::_:: 85:»: 83:555: 0.3?» pm 8:»:— 168 AB CD Elf 169 intensity of a band at ~90 kDa was misinterpreted. In reality, the ~90 kDa band in the cytoplasmic fraction was as intense as the ~90 kDa band in the initial sarc0plasmic fraction. The observation that the ~90 kDa protein was as intense in the, diluted, cytoplasmic fraction should have suggested the predominance of the protein in that fraction. The fractionation data indicate not only that several ~90 kDa proteins may be present in the sarcoplasmic fraction but that the BAA stimulated phosphorylation response may have been less obvious in earlier non-fractionated samples due to the apparent decrease in phosphorylation of the ~90 kDa protein of the microsomal fraction. The presence of multiple ~90 kDa proteins may also explain the difficulties encountered upon purification of the ~90 kDa band from cell extract preparative gels used for initial sequencing procedures where sequence analysis appeared indeterminable. The identity of the ~90 kDa cytoplasmic protein remains undetermined. Another candidate for the ~90 kDa BAA responsive cytoplasmic protein may be phosphorylase. Phosphorylase represents a cyotplasmic protein which is ~P in response to adrenergic stimulation and appears to migrate at approximately at the same MW on an acrylamide gel (Alvarez et al., 1992). The only problem lies in the fact that the ~P site on phosphorylase is at a serine residue (Rawn, 1989). I am using an anti-P-thr Ab. Zymed laboratories (San Francisco, CA) assures that the anti-P-thr Ab does not cross-react with ser~P or tyr~P. It may be that the Ab is cross-reacting with the ser~P or perhaps with the pyridoxal-phosphate present in phosphorylase. The same cell extracts will have to be immunoprecipitated with an anti-phosphorylase Ab and examined as were the previous 170 -l‘ Jul . ‘5 J SR 90 kDa protein immunoprecipitates to be certain if the phosphorylated protein is glycogen phosphorylase. BAA stimulation of C2C12 myotubes increaes the ~P of an ~90 kDa cytoplasmic protein. Stimulation of various BAA signal transduction pathway components results in a similar increase to that observed upon stimulation at the BAR (Figure 38). The identity of the if ~90 kDa cytoplasmic protein and its role in BAA-induced events in skeletal muscle remain undetermined. l7l 8088888: 8880.. 808:: £88828 we. ca... 05 8 .0802 05:. .8358: 08:2 808:: 80:88.: :E .8 88888 68885.8: 88:8 mm: o\o~ 08 8 0288: 888:8 BOND 8 8088 288888 «D0. cm... 8 .8 88—88888 88088 8 8:80: A: vo: 888888: 3 88000.. 290880-: 05 8 88888 ”3030: E 883:8: 8888 888 ..58888: 8 83—588: =80>o 05 $8880: 8888 05. m 83:85 .0...—we ~8.8.8.6 an 0..—.3..— 172 m ham-0:2 A, X, 88:088. + e C + a @ ~.m,_.mm + Jon—:92.- />Uv_._ : 058-0 a! 4/ :2 38:3— 1 VE<~2 5385.856 / .0880 «U 0 a l \mvnx + 2.80.89. J25 TI 06 b:(\\. v «322%? TI ,0 2:80 <~_8o:0\< 4/ :8. + E w a: 173 .vofigofiufi. 252:2 £22m 283323 «Du. col 2: be 3:52 2: $5558 03:2 5205 «co—Evan“. m2m awn—o: Z 5:988“: + a @ 59me + 4255.... />uv... ..z .. "$5-0 ..2 <2 .— 326. ES). 5.255.656 J: 88.2 ~+ a I \\\\\\\~ :.w. A&.( + 5.9.2.9.. S .I am / .0555 «0 0a. {\\\ v «0+4! 9 238+ $315.6 \« j :5. + «a 173 CONCLUSION The data presented indicate that BAA stimulation of C2C 12 myotubes increases mRN A abundance for SKA and increases the phosphorylation of an ~90 kDa cytoplasmic protein. The pathway modulators and results are depicted in the summary figure (Figure 39). The significance lies in the observation that BAA stimulation of muscle-specific gene expression and ~P in the immortalized skeletal muscle cell line, C2C12, are occurring through a cAMP-dependent cascade mechansim. Interpretation of data in the literature suggests an inconsistent response to BAA stimulation. Inconsistencies may be interpreted as inherent differences in regard to species and tissue type expression of BAR subtypes and differing pharmacological properties at receptors of various agonists. Data presented support BAA stimulation of skeletal muscle specific events through a BAR mediated cAMP-dependent cascade mechanism. 174 80580.08: 8.082 8.088 09. cat 0... .8 3.80... 2: $0350.. 000:... £088 80.82.084.242“. 0 .38.... 8.8.08 0. £08... an... ca: 0... .8 confibofimonq .00.000.0... 2.. .0... 0.008... 0.0.. 2: $8089.80 x0358 0.m.0..0...0-n 08...? .8 8.8.2.5... .380... 80228 8.0 00>» .5.. col .0 8.338888 800020... 0..... .5088 0.800828 0%. col :0 .8 8.3.0.8888 2.. 80020:. 0.088»... ~.U~U .8 8.3.58.6 0.92.080-.. 688058... 0.000000 03:... E088 .=0.0..0..0.Yn.2<0 a :82... 8.238 m. 00:88.... m 80.2.2 cm 9 88.88.... + h: . 253.: Zak-ww— +e I6 M. 88.8.... / I1 .- 3 .5 9.8-0 UV... iv... \mz. 886. 0:82 + 2.80.8.9. 5.258926 V25 I ca .0888 so 0%.. (\ 2. 080.0. 1.0.. . fizz—0.50 AI 7 8.5.0 z_xc..<._._.o__u\q J .3. + s. 176 IMPLICATIONS The magnitude of response relative to altered body composition of livestock species treated with a BAA is dramatic and reproducible across species (Muir, 1988). To date, BAA are not federally approved for use as a growth promotants/metabolism modifiers in livestock species. Government approval of BAA usage is centered on the issue of BAA residues in meat. The fact that BAA are not approved for use suggests that further investigation into their mechanism of action is required in order to optimize this particular strategy. Further complications relative to BAA usage include the issue of meat tenderness. Considering the dramatic effects on body composition, the use of BAA in a livestock production setting merits strong attention. A major focus in United States meat-animal production settings has been the meat tenderness issue. Unfortunately, use of BAA to has been shown to not only increase lean tissue gain, but decrease meat tenderness across species (\Vheeler et al., 1992, Pringle et al., 1993, Gwartney et al., 1991). Research regarding the correlation between BAA treatment and meat tenderness has received considerable attention. BAA treatment has been reported to increase mRN A 177 abundance for the protease inhibitor calpastatin (Koohmaraie et al., 1991). Furthermore. it has recently been reported that the calpastatin promoter region contains domains responsive to cAMP-dependent protein kinase (PKA) stimulation (Cong et al., 1998). These findings indicate a direct relationship between BAR stimulation and decreased meat tenderness. These findings are not very promising for strong advocates of BAA’s. If the product is unacceptable to the consumer there is no benefit to the producer regardless of the advantages in muscle deposition and fat reduction. Further investigation of the BAA signal transduction pathway is essential if that production strategy is to be utilized. An investigation into the interaction across signaling pathways as expressed earlier may address previous questions or expose new options to modify the BAA response to optimize product approval by the government and the consumer. In relation to further examination of the mechanism of action of BAA; it may very well be that one particular strategy may not be sufficient to meet producer and consumer demands. Information regarding mechanism of action may indicate a synergy between growth promotants or related strategies. Furthermore, genetic manipulation of livestock species can not be left unmentioned. A strategy which optimizes muscle growth and fat reduction yet also meets producer/consumer demands may be represented by a 178 commercially available transgenic animal fed a BAA acting in synergy to create the desired product. 179 APPENDICES "1 ~a..—= Appendix A Appendix A Polymerase chain reaction cycle set-up for the 105 bp skeletal a-actin digoxygenin labeled probe Annealing temperature formula: 4 (#GC) + 2 (#AT) = +/- 5° :C. 94°C 94 60 72 94 58 72 94 55 72 94 50 72 4 time 4 minutes 1 minute, 10 seconds “ ‘6 ‘6 6‘ 1 minute, 10 seconds “ ‘5 6‘ ‘6 1 minute, 10 seconds 6‘ 66 6‘ ‘6 1 minute, 10 seconds ‘6 ‘6 £6 ‘6 hold 3 cycles 2 cycles 2 cycles 30 cycles 180 Appendix B Appendix B Dyedeoxy chromatogram of the Barn HIlPst-I 931 bp fragment 181 1...1t11<1.1.11114_4111.1_111114:111. .1411.1114101444)41.14411131114114114.41‘ at 41111411111111.4144L11111141 14.1.1 1.. 1 411.1111. {411141.11 1.111% (1414.114... 41.14.41 1111. 1&1.11411’11 11111411. 1.111111411411114413’11141. 1....z...4..1:.<.112