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.(ABSTRACT TRUNCATED AT 250 WORDS)