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.(ABSTRACT TRUNCATED AT 250 WORDS)
Carr
F E
FE
Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C. 20307.
Galloway
R J
RJ
Reid
A H
AH
Kaseem
L L
LL
Dhillon
G
G
Fein
H G
HG
Smallridge
R C
RC
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D015759:Ionomycin / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013972:Thyrotropin / Q000096:biosynthesis / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D012333:RNA, Messenger; D015759:Ionomycin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D013973:Thyrotropin-Releasing Hormone; D005707:Gallic Acid; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D013972:Thyrotropin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium