THYROTROPIN-RELEASING-HORMONE REGULATION OF THYROTROPIN BETA-SUBUNIT GENE-EXPRESSION INVOLVES INTRACELLULAR CALCIUM AND PROTEIN-KINASE-C

Our previous studies demonstrated TRH stimulation of TSH-beta-gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH-beta-gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH-beta-promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH-beta-promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH-beta-promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH-beta-gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity. However, TRH stimulation of TSH-beta-gene expression was inhibited by decreasing both calcium mobilization and protein kinase C activity. These effects were mediated through the same 180 bp DNA sequence in the 5'-flanking region of the rat TSH-beta-subunit gene. These results strongly suggest that both protein kinase C and [Ca2+] mobilization are important factors in mediating TRH-induced TSH-beta-gene expression.