title: Thyrotropin-releasing hormone regulation of thyrotropin beta-subunit gene expression involves intracellular calcium and protein kinase C.
abstract: 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)
authors.affiliation: Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C. 20307.
authors.forename: F E
authors.initials: FE
authors.lastname: Carr
authors.forename: R J
authors.initials: RJ
authors.lastname: Galloway
authors.forename: A H
authors.initials: AH
authors.lastname: Reid
authors.forename: L L
authors.initials: LL
authors.lastname: Kaseem
authors.forename: G
authors.initials: G
authors.lastname: Dhillon
authors.forename: H G
authors.initials: HG
authors.lastname: Fein
authors.forename: R C
authors.initials: RC
authors.lastname: Smallridge
mesh_terms: 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*
chemical_list: 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