Regulation of the c-fos protooncogene by growth hormone.

Growth hormone (GH) is a major regulator of normal cell growth and differentiation. Interaction of GH with its cell surface receptor initiates a signal transduction cascade leading to changes in gene expression. This dissertation analyzed GH-mediated gene expression using the c-fos protooncogene as a model to gain insight into the mechanisms used by GH to regulate gene expression. GH induces c-fos, a gene involved in growth and differentiation, in a rapid and transient manner. Northern blot analysis was used to compare GH-regulated c-fos expression with the expression of the early growth response gene-1 (egr-1), another early response gene with regulatory elements and induction kinetics similar to that of c-fos. GH-induced egr-1 expression followed a time course and dose dependence similar to those of c-fos. The region of the GH receptor (GHR) required for GH mediated expression of egr-1 and c-fos was localized to the N-terminal half of the cytoplasmic domain by analysis of Chinese hamster ovary cells expressing truncated GHRs. Within this region of the GHR the proline-rich Box 1 region was found to be essential for the expression of these genes. To identify c-fos promoter sequences targeted by GH, promoter-reporter plasmids were transiently transfected into GH-responsive cells and analyzed for GH inducibility. The c-fos serum response element (SRE) alone was able to mediate gene expression in response to GH. Electrophoretic mobility shift assays (EMSA) of nuclear proteins from GH-treated cells revealed the binding of the serum response factor (SRF) and an additional factor likely to be a member of the ternary complex factor (TCF) family. The TCF family member Elk-1 was found to be phosphorylated on serine 383 following GH stimulation, an event likely to lead to transcriptional activation via the SRE. Additional evaluation of the c-fos promoter revealed another GH sensitive regulatory element, the sis-inducible element (SIE). EMSA demonstrated that the SIE bound a nuclear complex following GH stimulation. Using specific antibodies in EMSA, the GH induced complex was found to include members of the STAT (signal transducers and activators of transcription) family. The STAT proteins are widely used by members of the cytokine receptor family, including GH, which signal via the JAK tyrosine kinases. The present findings identify a region of the GHR required for c-fos and egr-1 expression. Two sequences in the c-fos promoter and transcription factors that interact with them are identified as targets for regulation by GH. Based on our understanding of the signaling events initiated by binding of GH to its receptor and analysis of the elements of the c-fos promoter regulated by GH, this thesis links several important signaling pathways initiated at GHR with the regulation of nuclear events.Ph.D.Animal PhysiologyBiological SciencesCellular biologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130068/2/9712037.pd