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

Frances E. Carr, Richard J. Galloway, Ann H. Reid, Laura L. Kaseem, Gurpreet Dhillon, Henry G. Fein, Robert C. Smallridge

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3721-3728
Number of pages8
JournalBiochemistry
Volume30
Issue number15
DOIs
StatePublished - Apr 1 1991

ASJC Scopus subject areas

  • Biochemistry

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    Carr, F. E., Galloway, R. J., Reid, A. H., Kaseem, L. L., Dhillon, G., Fein, H. G., & Smallridge, R. C. (1991). Thyrotropin-Releasing Hormone Regulation of Thyrotropin β-Subunit Gene Expression Involves Intracellular Calcium and Protein Kinase C. Biochemistry, 30(15), 3721-3728. https://doi.org/10.1021/bi00229a019