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

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.     

Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene

Previous investigations have shown that Ca2+ strongly and specifically stimulates endogenous PRL gene expression by GH3 cells. In this study, addition of Ca2+ to Ca2+-deprived GH3 cells yielded a large (ca. 8-fold) stimulation of transient expression of a transfected PRL-chloramphenical acetyltransferase (CAT) construct containing ca. 1 kilo-base-pair of the PRL promoter region, but only a slight (less than or equal to 2-fold) nonspecific stimulation of CAT activity directed by any of three control promoters: dihydrofolate reductase, Rous sarcoma virus, or thymidine kinase. In GH3 cells never deprived of Ca2+, expression of a PRL-CAT construct was specifically stimulated and inhibited, respectively, by the dihydropyridine voltage-dependent Ca2+ channel modulators Bay K8644 and nimodipine; Ca2+ can thus regulate expression of an exogenous PRL promoter in cells incubated under physiological Ca2+ conditions. By employing a combined protocol, in which Ca2+-deprived cells are exposed to Ca2+ in the presence of Bay K8644, a very large (greater than 35-fold) but still promoter-specific induction of expression of a PRL-CAT construct was obtained. Analysis of 5'-deleted PRL-CAT constructs implied that the PRL gene Ca2+ response element is contained entirely within the first 174 base pairs of upstream flanking DNA sequence.     

Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)