Thyrotropin-Releasing Hormone Regulation of Thyrotropin β-Subunit Gene Expression Involves Intracellular Calcium and Protein Kinase C

Our previous studies demonstrated TRH stimulation of TSHB 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 TSHβ gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca²⁺]i, or activate protein kinase C on TSHβ promoter activity in transfected GH₃ cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSHB 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 TSHB 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 TSHB gene expression until 48 h (5-fold). Similar effects on prolac-tin/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 l-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity. However, TRH stimulation of TSHβ 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 TSHβ subunit gene. These results strongly suggest that both protein kinase C and [Ca²⁺] mobilization are important factors in mediating TRH-induced TSHβ gene expression.