title: Mediation by calcium of thyrotropin--releasing hormone action on the prolactin promoter via transcription factor pit-1.
abstract: Mediation by Ca2+ of TRH action on the PRL promoter was investigated by both additivity and pharmacological studies and by techniques that probe more gene-proximal events. TRH required the presence of Ca2+ in the medium for stimulation of transient expression in GH3 cells of a PRL-chloramphenicol acetyltransferase (PRL-CAT) construct containing proximal PRL promoter sequences [(-187)PRL-CAT]. Chronic 12-O-tetradecanoyl phorbol-13-acetate down-regulation of cellular protein kinase C did not block induction of expression of (-187)PRL-CAT by either Ca2+ or TRH. In studies with Ca2+ blockers, the Ca2+ flux inhibitors cobalt ion and nimodipine blocked induction of (-187)PRL-CAT expression by either Ca2+ or TRH. On the other hand, the Ca2+ immobilizers 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyltetraester and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate blocked induction of expression of this construct by Ca2+ but not by TRH, suggesting that TRH regulation of the PRL promoter may be dependent on Ca2+ fluxes but insensitive to Ca2+ immobilization. We have shown previously that the PRL promoter pit-1 binding site 1P is a TRH response element. In the present studies, Ca2+ regulation studies with 5'-deletion mutants of (-204)PRL-CAT showed that (-75)PRL-CAT, containing the single pit-1 binding site 1P, also contains a Ca2+ response element. The observation that two copies of a site 1P oligomer transferred a Ca2+ response to either of the two minimal constructs (-39)PRL-CAT or (-39)mouse metallothionein-CAT showed that site 1P is an independent Ca2+ response element. Analysis of site 1P mutants yielded a strong correlation between the ability to bind pit-1 and to transfer a Ca2+ response. In addition, coexpression of a mutant pit-1 possessing reduced trans-activational activity strongly inhibited TRH regulation of (-187)PRL-CAT and partially blocked Ca2+ regulation of this construct. We conclude that Ca2+ mediates TRH action on the PRL promoter, and that pit-1 represents a gene-proximal mediator in this signalling pathway.
authors.affiliation: Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York 10029.
authors.forename: G Z
authors.initials: GZ
authors.lastname: Yan
authors.forename: C
authors.initials: C
authors.lastname: Bancroft
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003035:Cobalt / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D009553:Nimodipine / Q000494:pharmacology; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D003035:Cobalt; D009553:Nimodipine; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium