Pituitary Calcium Channel Modulation and Regulation of Prolactin Gene Expression

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Abstract
The dihydropyridine Ca2+ channel modulators (-) Bay K 8644 (R5417) and nimodipine were used to study the role of voltage-gated Ca2+ channels in the regulation of PRL gene transcription in GH3 cells. Fusion constructs containing 5''-flanking sequences from the rat PRL gene linked to either the bacterial chloramphenicol acetyltransferase (CAT) gene or the firefly luciferase gene were transiently expressed in GH3 cells and the transcriptional response to Ca2+ channel modulators was assessed. The Ca2+ channel agonist R5417 enhanced the transcriptional of a PRL-CAT fusion gene containing 2.5 kilobase (kb) pairs of the 5''-flanking sequence. This response was completely blocked by Ca2+ channel blocker nimodipine demonstrating that sequences in the PRL 5''-flanking region confer response to Ca2+. Transfection with PRL-CAT constructs containing 2.5 kb to 0.6 kb pairs of 5''-flanking sequence were responsive to Ca2+, although those which contained the distal enhancer region (positions-1765 to -1495) had much higher basal expression. The possibility that the distal enhancer might contain Ca2+-responsive elements was tested by comparing the response to R5417 and TRH for both the proximal enhancer region (.apprx. first 300 base pair of the 5''-flanking sequence) and distal enhancer regions linked to the thymidine kinase promoter and CAT. The results demonstrate that these two regions contribute to the overall transcriptional response to Ca2+ and TRH. The distal region does not confer a response to phorbol ester, while the proximal region is responsive to that treatment. Linking the distal enhancer to the proximal enhancer region (-306 to + 34) or the distal region and the PRL promoter (-39 to +34) demonstrated that response to R5417 and TRH is conferred by both the distal and proximal regions, but that only the proximal region can confer response to phorbol ester. The response of the proximal and distal regions to a combination of treatment with the Ca2+ channel agonist and TRH was no greater than that of TRH alone, suggesting overlapping mechanisms mediate the responses. To further analyze the sequence of the distal region required for response to Ca2+ and TRH, deletions and mutations were made in that region. Deletions in the distal enhancer that progressively remove the binding sites for the pituitary specific protein Pit-1 reduced basal enhancer activity and response to R5417 and TRH. These results show that movement of Ca2+ through "L" type channels can regulate the synthesis of PRL at the level of transcription and that both the distal and proximal regulatory regions are involved in conferring response to Ca2+ and TRH, while only the proximal region mediates the response to phorbol ester.