1775132

pubmed:Citation

10

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.

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MEDLINE

0888-8809

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1488-97

pubmed-meshheading:1775132-Animals, pubmed-meshheading:1775132-Calcium, pubmed-meshheading:1775132-Cell Line, pubmed-meshheading:1775132-Chloramphenicol O-Acetyltransferase, pubmed-meshheading:1775132-Cobalt, pubmed-meshheading:1775132-DNA-Binding Proteins, pubmed-meshheading:1775132-Kinetics, pubmed-meshheading:1775132-Nimodipine, pubmed-meshheading:1775132-Plasmids, pubmed-meshheading:1775132-Prolactin, pubmed-meshheading:1775132-Promoter Regions, Genetic, pubmed-meshheading:1775132-Recombinant Fusion Proteins, pubmed-meshheading:1775132-Tetradecanoylphorbol Acetate, pubmed-meshheading:1775132-Tetrahydrofolate Dehydrogenase, pubmed-meshheading:1775132-Thyrotropin-Releasing Hormone, pubmed-meshheading:1775132-Transcription Factor Pit-1, pubmed-meshheading:1775132-Transcription Factors, pubmed-meshheading:1775132-Transfection

Mediation by calcium of thyrotropin--releasing hormone action on the prolactin promoter via transcription factor pit-1.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't