Abstract
In placental explants and cell lines, cAMP stimulates the biosynthesis of CG alpha- and beta-subunits and produces marked stimulation of CG secretion. We investigated the effect of a cell-permeable analog, 8-bromo-cAMP (8-Br-cAMP), on CG alpha- and beta-subunit gene expression in the JEG-3 placental cell line. RNA blot hybridization analysis demonstrated that 8-Br-cAMP increased CG alpha and beta mRNA levels 15- to 30-fold, consistent with 8-Br-cAMP regulation of CG biosynthesis at a pretranslational level. Transcription rates for CG alpha- and beta-subunit genes were determined in the absence and presence of 8-Br-cAMP using nuclear run-on assays. The basal transcription rates for the alpha- and beta-subunit genes were similar and increased 4- to 6-fold after treatment with 8-Br-cAMP. Expression of chimeric genes, consisting of alpha gene [1.5 kilobase (Kb)] or CG beta gene (0.3 Kb) 5'-flanking sequences ligated to the coding sequence of a reporter enzyme chloramphenicol acetyl transferase (CAT), was used to analyze the mechanism of 8-Br-cAMP stimulation of gene transcription. A 1.5-kb segment of the alpha- gene 5'-flanking sequence was expressed efficiently in JEG-3 cells and contains both cell-specific enhancers and cAMP response elements. Basal expression of the CG beta CAT reporter gene was not observed in either JEG-3 cells or HeLa cells, and expression was not stimulated by 8-Br-cAMP. The regulatory response elements of the CG beta gene must reside in sequences outside the 0.3-kb 5'-flanking region of the CG beta gene.
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