Suppression of protein kinase C theta contributes to enhanced myogenesis in vitro via IRS1 and ERK1/2 phosphorylation.
BACKGROUND
Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.


RESULTS
In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.


CONCLUSIONS
Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.
Center for Diabetes and Endocrine Research, Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, OH 43614, USA. JenniferW.Hill@utoledo.edu.
Joseph S
JS
Marino
Terry D
TD
Hinds
Rachael A
RA
Potter
Eric
E
Ondrus
Jeremy L
JL
Onion
Abigail
A
Dowling
Thomas J
TJ
McLoughlin
Edwin R
ER
Sanchez
Jennifer W
JW
Hill
D000818:Animals; D002454:Cell Differentiation; D002459:Cell Fusion; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D024510:Muscle Development / Q000235:genetics*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000074121:Protein Kinase C-theta; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482127:Map2k1 protein, mouse; C482129:Map2k2 protein, mouse