Programmed cell death (apoptosis) is induced concomitant with differentiation in skeletal myoblasts and other systems. In skeletal myoblasts, these two processes result in mutually exclusive physiologically important endpoints. However, the coordinate regulation of these two processes is not understood. Dissecting this coordinate regulation could enable selective manipulation relevant to the amelioration of disease states associated with muscle degeneration and to the effectiveness of any treatment utilizing skeletal myoblast transfer. The long-term goal of this laboratory, therefore, is to elucidate the molecular mechanism(s) responsible for this coordinate regulation. Guided by our preliminary data, the short-term goal of the funded proposal is to test the hypothesis that MyoD is both sufficient and necessary to induce mitochondrial disruption and apoptosis by affecting one or more of the three pro-apoptotic pathways previously identified by this laboratory to play a role in the apoptotic process in skeletal myoblasts. By performing experiments outlined in Aim 1 of the funded proposal, we have determined that ectopic expression and activation of MyoD in fibroblasts is sufficient to induce apoptosis and increase the expression of PUMA. We have also determined that MyoD is necessary for the induction of apoptosis and the increased expression of PUMA in that 23A2 and C2C12 skeletal myoblasts silenced for MyoD expression were defective in their ability to undergo apoptosis and to increase their expression of PUMA when compared to respective parental controls myoblasts. Guided by this new data, we propose to test the hypothesis that PUMA expression is directly regulated by MyoD by pursuing a new Aim (AIM 3) to determine if MyoD controls the expression of PUMA via binding to regulatory elements within the extended promoter. We propose to identify, through the creation of pGL3 basic luciferase reporter constructs and analysis under conditions with and without active MyoD in skeletal myoblasts and fibroblasts, the minimal cis-acting MyoD responsive element in the PUMA gene. We will then assess binding of MyoD to this element by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP).
These studies will dissect a new biological role for MyoD. Furthermore, these findings could be significant in the effort to ameliorate the myoblast apoptosis that contributes pathologically to a variety of diseases and in the study of myoblast transfer as a therapeutic approach. Funding this revision will create a new job, accelerate the tempo of the research and facilitate the training of more undergraduate students.
| Harford, Terri J; Kliment, Greg; Shukla, Girish C et al. (2017) The muscle regulatory transcription factor MyoD participates with p53 to directly increase the expression of the pro-apoptotic Bcl2 family member PUMA. Apoptosis 22:1532-1542 |
| Ebron, Jey Sabith; Weyman, Crystal M; Shukla, Girish C (2013) Targeting of Androgen Receptor Expression by Andro-miRs as Novel Adjunctive Therapeutics in Prostate Cancer. J Cancer Ther 4:47-58 |
| Harford, Terri J; Shaltouki, Atossa; Weyman, Crystal M (2010) Increased expression of the pro-apoptotic Bcl2 family member PUMA and apoptosis by the muscle regulatory transcription factor MyoD in response to a variety of stimuli. Apoptosis 15:71-82 |
| Freer-Prokop, Margot; O'Flaherty, John; Ross, Jason A et al. (2009) Non-canonical role for the TRAIL receptor DR5/FADD/caspase pathway in the regulation of MyoD expression and skeletal myoblast differentiation. Differentiation 78:205-12 |
