Negative regulation of the myogenic pathway in the neuronal cell line RT4-B8
Auvenshine, Ronald Christopher
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Neurons and skeletal muscle cells may share a common lineage, although once thought to arise from distinct precursor pools. While the majority of skeletal muscle is derived from mesoderm, a small percentage arises from the ectodermally derived neural crest, which also gives rise to the entire peripheral nervous system. In addition, cells which express the skeletal muscle-specific marker myf-5 have been identified in the mouse central nervous system early in development. Finally, certain types of neural tumors give rise to myoblasts when cultured. Studies in our laboratory indicate that based on the endogenous expression of MyoD, the peripheral neurotumor derived neuronal cell line, RT4-B8, may provide a unique model to study this lineage relationship. In vivo, the MyoD family of basic helix-loop-helix transcription factors has been shown to play a pivotal role in the regulation of skeletal muscle myogenesis. Significantly, the ectopic expression of MyoD in many non-muscle cell types can activate the entire myogenic program. The focus of this study was to gain an understanding of the regulation of MyoD function in a neuronal cell type. Our results demonstrate that despite the expression of both MyoD mRNA and protein, the myogenic pathway was not activated in RT4-B8. This finding is unique in that, to our knowledge, no other cell has been reported to express endogenous MyoD and lack the ability to activate the myogenic pathway. The inability of RT4-B8 cells to activate the myogenic pathway was demonstrated by immunoblot analyses, which revealed that the muscle-specific myogenin, desmin, or a-sarcomeric actin genes were not expressed in RT4-B8. In addition, myotubes did not form when RT4-B8 cells were grown in culture conditions that induced the differentiation of C2C12 mouse myoblasts. The MyoD expressed by RT4-B8 was wild type with respect to the size of the protein and length of the mRNA. Finally, MyoD protein was present in the nuclei of RT4- B8 cells, and capable of binding to DNA in vitro. The results of the experiments conducted in this study suggest that the endogenous expression of MyoD is not sufficient to activate the myogenic pathway in RT4-B8. We therefore conclude that the block to myogenesis in RT4-B8 may lie downstream of the expression of MyoD.