v-Src inhibits myogenic differentiation by interfering with the regulatory network of muscle-specific transcriptional activators at multiple levels

Abstract

The conversion of skeletal myoblasts to terminally differentiated myocytes is negatively controlled by several growth factors and oncoproteins. In this study, we have investigated the molecular mechanisms by which v-Src, a prototypic tyrosine kinase, perturbs myogenesis in primary avian myoblasts and in established murine C2C12 satellite cells. We determined the expression levels of the cell cycle regulators pRb, cyclin D1 and D3 and cyclindependent kinase inhibitors p21 and p27 in v-Srctransformed myoblasts and found that, in contrast to myogenin, they are normally modulated by differentiative cues, implying that v-Src affects myogenesis independent of cell proliferation. We then examined the levels of expression, DNA-binding ability and transcription-activation potentials of myogenic regulatory factors in transformed myoblasts and in myotubes after reactivation of a temperature-sensitive allele of v-Src. Our results reveal two distinct potential modes of repression targeted to myogenic factors. On the one hand, we show that v-Src reversibly inhibits the expression of MyoD and myogenin in C2C12 cells and of myogenin in quail myoblasts. Remarkably, these loci become resistant to activation of the kinase in the postmitotic compartment. On the other hand, we demonstrate that v-Src efficiently inhibits muscle gene expression by repressing the transcriptional activity of myogenic factors without affecting MyoD DNAbinding activity. Indeed, forced expression of MyoD and myogenin allows terminal differentiation of transformed myoblasts. Finally, we found that ectopic expression of the coactivator p300 restores transcription from extrachromosomal muscle-specific promoters.

Delineating v-Src downstream effector pathways in transformed myoblasts

“…Consistently, accumulation of musclespecific proteins was also increased (Figure 2d), clearly Signalling pathways in v-Src myoblasts L Ciuffini et al indicating that inhibition of MEK activity promoted differentiation of v-Src-transformed myoblasts. We had previously shown that forced expression of the cdk inhibitor p27 causes QMb-LA29 to exit from the cell cycle, yet does not promote myogenic differentiation (Falcone et al, 2003). To determine whether the MEK pathway is directly involved in the v-Src block of differentiation or acts by keeping cells proliferating (Figure 1b), we infected QMb-LA29 with adenoviruses encoding for p27 and b-gal and, after 24 h, added PD98059 or U0126 to the culture medium for 2 days.…”

“…Although exit from the cell cycle and commitment to differentiation are necessarily linked processes, inhibition of myogenesis by v-Src in both avian and murine myoblasts can occur independently of cell proliferation (Falcone et al, 2003). In myocytes, v-Src negatively affects transcription of muscle-specific genes by targeting the function of MRFs, in part by interfering with their interaction with co-regulators that control chromatin remodelling (Falcone et al, 2003). However, the cytoplasmic pathways activated by v-Src that convey the inhibitory signals from the cell periphery to the nucleus, eventually leading to block of musclespecific transcription, have not yet been defined.…”

“…For example, myogenic cells, a prototypic model cell system for in vitro differentiation, are prevented to undergo terminal differentiation when transformed by oncoproteins including v-Src (Olson, 1992;Alema`and Tato`, 1994) that interfere with the expression and/or the activity of myogenic regulatory factors (MRFs) (Lassar et al, 1989;Russo et al, 1997;Perry et al, 2001;Falcone et al, 2003). However, the signalling pathways that lead to inhibition of MRFs function have remained largely obscure.…”

“…Earlier work showed that primary avian myoblasts transformed by a conditional v-Src kinase are extremely well suited to study all facets of the transformed phenotype, such as control of proliferation (Falcone et al, 2003), block of differentiation (Falcone et al, 2003) and alterations of myofibrillar assembly in myocytes (Castellani et al, 1995;Gallo et al, 1999). We have previously established that v-Src affects the expression of muscle-specific genes in quail myoblasts independently of the effects on the cell cycle (Falcone et al, 2003). Thus, the necessity arises of identifying the signalling pathways involved in transformation and/or in block of myogenesis.…”

Células satélites musculares

“…2o) p27 kip -há uma relação inversa entre a capacidade proliferativa da CS e a abundância da proteína p27 Kip, podendo estar envolvida no crescimento e na hipertrofia musculares (27) . 2p) v-Src -é uma típica tirosina-quinase, inibindo a diferenciação miogênica por meio da inibição da expressão de MyoD e miogenina (28) . Outros candidatos a marcadores das CS encontram-se em investigação: o antígeno de superfície celular Sca-1 (29)(30) , a glicoproteína Leu-19, o fator anti-apoptótico Bcl-2 (31)(32) , entre outros.…”