title: Protein kinase D2 is an essential regulator of murine myoblast differentiation.
abstract: Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.
authors.affiliation: Department of Internal Medicine I, University of Ulm, Ulm, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kleger
authors.forename: Christiane
authors.initials: C
authors.lastname: Loebnitz
authors.forename: Ganesh V
authors.initials: GV
authors.lastname: Pusapati
authors.forename: Milena
authors.initials: M
authors.lastname: Armacki
authors.forename: Martin
authors.initials: M
authors.lastname: Müller
authors.forename: Stefan
authors.initials: S
authors.lastname: Tümpel
authors.forename: Anett
authors.initials: A
authors.lastname: Illing
authors.forename: Daniel
authors.initials: D
authors.lastname: Hartmann
authors.forename: Cornelia
authors.initials: C
authors.lastname: Brunner
authors.forename: Stefan
authors.initials: S
authors.lastname: Liebau
authors.forename: Karl L
authors.initials: KL
authors.lastname: Rudolph
authors.forename: Guido
authors.initials: G
authors.lastname: Adler
authors.forename: Thomas
authors.initials: T
authors.lastname: Seufferlein
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D018485:Muscle Fibers, Skeletal; D009132:Muscles / Q000166:cytology / Q000502:physiology; D032446:Myoblasts / Q000166:cytology*; D010766:Phosphorylation; D020033:Protein Isoforms; D000091283:Protein Kinase D2; D011494:Protein Kinases / Q000502:physiology*; D012038:Regeneration; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology
chemical_list: D020033:Protein Isoforms; D000091283:Protein Kinase D2; D011494:Protein Kinases