Effect of p38 kinase and cell cycle position on the expression of the pro-apoptotic Bcl2 family member PUMA in skeletal myoblasts

Skeletal muscle regeneration relies on myoblast stem cell differentiation and is a crucial response to muscle injury caused by trauma and numerous diseases. In skeletal myoblasts, cell death and differentiation are mutually exclusive biological endpoints that are both induced by culture in differentiation media. MyoD, the master muscle- specific transcription factor, is well-known to regulate the expression of muscle specific genes such as myogenin and the ensuing differentiation. However, we have previously reported that MyoD also plays a critical role in the expression of PUMA and apoptosis, rather than myogenin and differentiation, in a subset of myoblasts, thus diminishing the regeneration. It is, therefore, critical to understand the molecular events that distinguish between this coordinate regulation of differentiation and apoptosis by MyoD. p38 kinase is known to be required for the expression of myogenin. Herein, we report that pharmacological inhibition of p38, while diminishing the expression of myogenin, actually enhances the expression of PUMA. Since myoblast cell cultures are asynchronous, we hypothesized that cell cycle position may contribute to this molecular distinction. To investigate this possibility, we have successfully synchronized cultures and experiments are underway to determine the effect of cell cycle position on PUMA expression versus differentiation specific gene expression.https://engagedscholarship.csuohio.edu/u_poster_2016/1014/thumbnail.jp

Basal Signalling Through Death Receptor 5 and Caspase 3 Activates p38 Kinase To Regulate Serum Response Factor (SRF)-Mediated Myod Transcription

We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD