NF-κB independent Cell Survival Regulation in Differentiated Skeletal Muscle
Creators:Hill, Eric M.
Advisor:Guttridge, Denis C.
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. School of Biomedical Science Honors Theses; 2010
The transcription factor NF-κB has been shown to inhibit the differentiation of skeletal muscle cells via numerous molecular mechanisms and is believed to play an integral role in several skeletal myopathies. NF-κB has also been shown to play a vital role in inhibiting programmed cell death by up-regulating specific anti-apoptotic genes following a stressful stimulus. Interestingly, it has been reported that terminally differentiated skeletal muscle cells devoid of NF-κB activity do not undergo apoptosis when treated with the inflammatory cytokines interferon-γ and tumor necrosis factor alpha (TNFα). In this study, we examine the reported phenomenon using TNFα, an extrinsic signal for apoptosis as well as a potent activator of NF-kappaB, to better determine the role of NF-kappaB in the survival of skeletal muscle cells at different stages of the differentiation program. Using a C2C12 cell line stably expressing a plasmid coding for a dominant negative form of IκBα (IκBα-SR), it was shown that myoblasts lacking competent NF-κB activity were sensitized to TNFα-induced apoptosis, while identical IκBα-SR myotubes were not. To determine if C2C12 myotubes possess an intact TNFα signaling pathway, Western blotting for phosphorylated forms of p38 and JNK as well as reverse transcriptase-PCR for TNFα-activated genes were performed. It has recently been claimed that skeletal muscle differentiation evokes a natural reduction of apoptotic peptidase activating factor-1 (Apaf-1), a vital pro-apoptotic protein. Western blot analysis of IκBα-SR myoblasts and myotubes confirm that the natural reduction of Apaf-1 is present in C2C12 cultures, as well as indicate that the reduction is not dependent on NF-κB. To determine if this reduction in Apaf-1 could be the mechanism responsible for the NF-κB independent inhibition of apoptosis seen in skeletal myotubes, C2C12-IκBα-SR myoblasts were transiently transfected with an Apaf-1 expression plasmid and differentiated before being treated with TNFα. The over-expression of Apaf-1 in IκBα-SR myotubes did not rescue sensitivity to TNFα-induced apoptosis, as measured by levels of cleaved caspase-3 following treatment. Collectively, these findings indicate that the transcription factor NF-κB is vital to the survival of skeletal myoblasts, but not required to inhibit TNFα-induced apoptosis in skeletal myotubes. Additionally, we show that the reduction of Apaf-1 along the myogenic program is not required for the survival of terminally differentiated skeletal myotubes treated with TNFα. Future elucidation of the role of NF-κB and important anti-apoptotic mechanisms in mature skeletal muscle could lead to a better understanding of skeletal muscle differentiation as well as novel insight into numerous skeletal muscle diseases, including the pediatric muscle cancer rhabdomyosarcoma.
Research on Research Fellowship, Digital Union, The Ohio State University
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