Thyrotropin releasing hormone (TRH) plays a pivotal role in the hypothalamic regulation of thyroid stimulating hormone (TSH) secretion from the anterior pituitary and is widely distributed in extrahypothalamic neuroendocrine tissues. Although this neuropeptide plays a key role in the transduction of information between neuronal and endocrine systems, little is known about the factors that regulate TRH gene expression. Identification of the DNA sequence and cloning the trans-acting factor cDNA that regulates TRH gene expression would provide a great deal of insight into the mechanism of neuropeptide hormone expression in normal and ectopic hormone secreting neuroendocrine cells. The major aims of this proposal are to 1) identify the cis-acting DNA sequences that determine tissue-specific expression of the TRH gene, 2) characterize the trans-acting factor(s) that bind to the TRH sequences important for gene expression, 3) isolate and sequence a cDNA encoding the TRH trans-acting factor. My preliminary results demonstrate that the TRH fragment between -47 and -113 bp is important for TRH gene expression. Within this fragment are two sequences that are homologous to the tissue- specific/cAMP enhancer (TSE/CRE) of the somatostatin (SS) gene. Studies of the elements with its surrounding nucleotides suggest they can function as enhancer elements and are protected by factors in nuclear extracts in DNase I footprinting assays. Footprinting assays with competition studies demonstrate that the TRH-protein complex can be dissociated equally with either the TRH of the SS promoter element. The sequence homologies shared by the neuropeptide genes, TRH and SS, suggest that these homologous elements might be binding sites for trans-acting factors shared by different neuroendocrine cells and ectopic neuropeptide secreting tumors. The TRH and SS secreting medullary thyroid carcinoma cell line, CA77, provides and ideal model to test the theory that a single trans-acting factor can regulate the expression of both neuropeptide genes. Elucidation of the cellular and molecular mechanisms underlying the expression of the TRH gene will increase our understanding of the control of the pituitary-thyroid axis by the brain and the molecular basis of peptide hormone expression in neuroendocrine cells.
