METABOLIC EFFECTS OF THYROID HORMONE IN CELL CULTURE

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Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.