Promyogenic function of Integrin/FAK signaling is mediated by Cdo, Cdc42 and MyoD

The Integrin-mediated cell adhesion to the extracellular matrix is implicated in the control of proliferation, survival, migration and differentiation of myoblasts. Focal adhesion kinase (FAK) mediates signals from Integrins and plays an essential role in myotube formation. Cdo forms a multiprotein complex that includes other cell adhesion molecules like Cadherins and Boc. Multiple signals emanate from such complexes, including Cdc42 and p38MAPK pathways to activate MyoD. Here we show that C2C12 myoblasts cultured in suspension or on Poly-L-Lysine (PLL), a well known Integrin-independent substratum, failed to express Cdo and MyoD, while the expression of Cadherins and Boc was unchanged. In addition, the activation of Akt and p38MAPK as well as the expression of Cdc42 was affected in these cells. Overexpression of FAK rescued MyoD and Cdo expression as well as myotube formation of C2C12 cells on PLL. Furthermore, reintroduction of Cdo induced enhanced myotube formation on PLL and increased the expression of myogenic markers. Inhibition of ROCK or overexpression of Cdc42-V12 in C2C12 cells upregulated Cdc42 and MyoD expression and rescued defective myoblast differentiation. Taken together, these data indicate that the Integrin/FAK signaling pathway is required for myoblast differentiation by regulating the expression of the promyogenic factors, Cdo, MyoD and Cdc42.     

Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.     

CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis

Cyclin-dependent kinases (CDKs) restrict DNA replication origin firing to once per cell cycle by preventing the assembly of prereplicative complexes (pre-RCs; licensing) outside of G1 phase. Paradoxically, under certain circumstances, CDKs such as cyclin E-cdk2 are also required to promote licensing. Here, we show that CDK phosphorylation of the essential licensing factor Cdc6 stabilizes it by preventing its association with the anaphase promoting complex/cyclosome (APC/C). APC/C-dependent Cdc6 proteolysis prevents pre-RC assembly in quiescent cells and, when cells reenter the cell cycle from quiescence, CDK-dependent Cdc6 stabilization allows Cdc6 to accumulate before the licensing inhibitors geminin and cyclin A which are also APC/C substrates. This novel mechanism for regulating protein stability establishes a window of time prior to S phase when pre-RCs can assemble which we propose represents a critical function of cyclin E.     

Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation

Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.     

Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.     

Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein β (C/EBPβ) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPβ protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPβ protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPβ for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPβ levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPβ, suggesting that the regulation of C/EBPβ turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPβ for degradation by the 26 S proteasome.     

Transcriptional analysis of the titin cap gene

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.     

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1^KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1^KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1^KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1^KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1^KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP^KO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1^KD cells restored myotube formation of Toca-1^KD cells. Thus, our results suggest that Toca-1^KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.     

Differential effects of Hsp90 inhibition on protein kinases regulating signal transduction pathways required for myoblast differentiation

As derivatives of the Hsp90-inhibitor and tumoricidal agent geldanamycin move into phase II clinical trials, its potential for triggering adverse effects in non-tumor cell populations requires closer examination. In this report, the effect of geldanamycin on the differentiation and survival of C2C12 myoblasts was investigated. Treatment of differentiating C2C12 myoblasts with geldanamycin blocked myogenin expression, inhibited myotubule formation, and led to the depletion of three Hsp90-dependent protein kinases, ErbB2, Fyn, and Akt, and induction of apoptosis. ErbB2 levels declined rapidly, while Fyn and Akt levels decreased at a slower rate. Geldanamycin blocked the interaction of Hsp90 and its "kinase-specific" co-chaperone Cdc37 with Fyn, indicating that Fyn is an Hsp90-dependent kinase. Pulse-chase experiments indicated that geldanamycin caused newly synthesized Akt and Fyn to be degraded rapidly, but geldanamycin had little effect on the turnover rate of mature Fyn and Akt. Curiously, total cellular Src (c-Src) protein levels and the turnover rate of newly synthesized c-Src were unaffected by geldanamycin. While, geldanamycin had no effect on the levels of the putative Hsp90 client protein MyoD expressed in C2C12 cells, geldanamycin disrupted the interaction of Cdc37 with MyoD. Thus, inhibition of Hsp90 caused C2C12 cells to become depleted of multiple signal transduction proteins whose functions are essential for myoblast differentiation, and muscle cell survival, suggesting that geldanamycin derivatives may have the prospective of adversely affecting the physiology of certain sensitive muscle cell populations in vivo.     

Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers

The satellite cell compartment provides skeletal muscle with a remarkable capacity for regeneration. Here, we have used isolated myofibers to investigate the activation and proliferative potential of satellite cells. We have previously shown that satellite cells are heterogeneous: the majority express Myf5 and M-cadherin protein, presumably reflecting commitment to myogenesis, while a minority is negative for both. Although MyoD is rarely detected in quiescent satellite cells, over 98% of satellite cells contain MyoD within 24 h of stimulation. Significantly, MyoD is only observed in cells that are already expressing Myf5. In contrast, a minority population does not activate by the criteria of Myf5 or MyoD expression. Following the synchronous activation of the myogenic regulatory factor+ve satellite cells, their daughter myoblasts proliferate with a doubling time of approximately 17 h, irrespective of the fiber type (type I, IIa, or IIb) from which they originate. Although fast myofibers have fewer associated satellite cells than slow, and accordingly produce fewer myoblasts, each myofiber phenotype is associated with a complement of satellite cells that has sufficient proliferative potential to fully regenerate the parent myofiber within 4 days. This time course is similar to that observed in vivo following acute injury and indicates that cells other than satellite cells are not required for complete myofiber regeneration.