Inhibition of Skeletal Myogenesis by Activated Raf-kinase

Open Access
Thomson, Season R.
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
October 10, 2003
Committee Members:
  • Sally E Johnson, Committee Chair
  • Avery August, Committee Member
  • Andrew Thomas Henderson, Committee Member
  • Roland M Leach, Committee Member
  • Lorraine M Sordillo, Committee Member
  • skeletal myogenesis
  • Raf-kinase
  • TGF-beta
  • AP-1
  • transformation
  • MAPK
The mobility of the human body is dependent upon the contractile function of skeletal muscle. Proper formation of skeletal muscle is a complex process involving specification of mesodermal progenitors to a muscle lineage, proliferation of myoblasts, and subsequent differentiation of the myoblasts into myofibers that produce the proteins required for muscle contraction. Initiation of intracellular signaling cascades by growth factor binding to cell surface receptors precisely controls myogenesis. Many factors are capable of disrupting muscle formation by interfering with various components involved in the process. Ras is a membrane localized GTPase activated by many growth factors that is capable of inducing multiple signaling pathways, one of which is the conventional Raf/MEK/MAPK pathway. Constitutively activated forms of Raf-kinase induce morphological transformation and are inhibitory to terminal differentiation of skeletal muscle cells in vitro. The activation of the transcription factor activator protein-1 (AP-1) by this kinase appears to be responsible for the cellular changes in phenotype. However, the mechanism behind the block to differentiation is independent of this phenomenon and remains unidentified. Raf-kinase exerts its effects by altering the expression of genes that may positively or negatively affect muscle fiber formation. Several putative Raf-regulated genes were identified in a subtractive library screen of Raf-transduced myoblasts. One of these genes was slightly downregulated in Raf-transformed avian myoblasts and was found to be highly homologous to a human Tax Responsive Element Binding Protein 107 (TaxREB107). We pursued the possibility that chick TaxREB107 (cTaxREB107) may be involved in transcriptional regulation during myogenesis. Our findings suggest a positive role for cTaxREB107 in the regulation of muscle gene expression during development of skeletal muscle. Therefore, expression of cTaxREB107 may be impeded by Raf-kinase in order to negatively affect myogenesis. To further extend our understanding of the block to myogenesis that is imposed by activated Raf-kinase, a stable myogenic cell line expressing an estrogen receptor-Raf chimeric protein was created. Induction of high levels of Raf-activity was found to prevent fiber formation. Interestingly, conditioned media from the Raf-inducible cell line inhibited fiber formation in the parental cell line, which suggests the possibility that a secreted factor is responsible for the block to differentiation of skeletal muscle cells. Gene expression and activity of transforming growth factor b1 (TGFb1), which is a well-known inhibitor of the myogenic program, was found to be elevated in cells expressing high levels of activated Raf-kinase. However, addition of a TGFb1 inhibitor did not restore muscle fiber formation in these cells. These results suggest that although TGFb1 expression and activity is increased, the induction of this growth factor is not solely responsible for the block to myogenesis that is imposed by activated Raf-kinase. Although cellular secretion of TGFb1 is not necessarily the cause of the Raf-imposed block to muscle formation, a TGFb-like factor is probably responsible for these effects. Treatment of myoblasts with exogenous TGFb has been shown to suppress the accumulation of MEF2 to the nucleus. MEF2 retention in the cytoplasm also occurs in muscle cells overexpressing activated Raf-kinase, thereby providing a link between TGFb-induced and Raf-induced myogenic inhibition. Therefore, we chose to study the interaction of the TGFb and Raf-kinase pathways during myogenesis. Our results indicate that Raf-kinase and TGFb similarly induce transcriptional activity of activator protein (AP-1). Fra-2 and c-Jun were found to be the primary AP-1 DNA binding components in Raf-expressing myoblasts as well as those treated with TGFb. However, Western blot analysis of both TGFb-treated and Raf-expressing myoblasts revealed that Fra-1 protein levels were elevated. AP-1 tether proteins were used in order to analyze the effects of specific subunit composition of AP-1 on myogenesis. c-Jun~Fra-1 and c-Jun~Fra-2 both suppressed muscle-specific reporter gene activity and were inhibitory to myofiber formation. Furthermore, c-Jun~Fra~1 was found to directly affect MEF2 function, while c-Jun~Fra-2 was shown to be inhibitory to MRF activity. This data provides evidence for specificity of AP-1 components in the regulation of subsets of genes during skeletal myogenesis. In conclusion, the data provided here suggests that Raf-kinase controls a complex pattern of gene expression. While the exact mechanism behind the Raf-imposed block to myogenesis has not been completely elucidated, the results of this study indicate that Raf regulates expression of cTaxREB107, members of the TGFb superfamily and specific AP-1 subunits.