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Ets-2 has an important role in controlling the differentiation of the placenta. Here we show by truncation and mutational analysis that two closely spaced Ets-2 binding sites in the proximal promoter of the human chorionic gonadotropin beta5 (hCGbeta5) gene constitute a major enhancer for hCGbeta gene expression in JAr and JEG-3 human choriocarcinoma cells and in mouse NIH3T3 cells. Contrary to a previous report, we also demonstrate that the ability of Ets-2 to enhance transcription is subject to control by the Ras/MAPK pathway, although this relationship is less easily demonstrable in JAr and JEG-3 choriocarcinoma cells than in the 3T3 cells because the former already possess a fully activated MAPK pathway and contain Ets-2 phosphorylated at threonine residue at T72. Coexpression of Ets-2 and activated Ras in 3T3 cells led to activation of MAPK/ERK kinase 1/2, phosphorylation of Ets-2 at T72, and an approximately 120-fold up-regulation of reporter gene expression from a short (-175) hCGbeta promoter. Fold activation in JAr and JEG-3 cells was rather less (20- to 30-fold), but basal activity was much higher. These effects on promoter activity were largely reversed in presence of the MAPK inhibitor PD98059, which prevents ERK1/2 activation, and partially reversed by mutating T72 on Ets-2. We finally show that the ability of 8-bromoadenosine-cAMP to stimulate hCGbeta promoter activity in JAr and JEG-3 cells occurs with a short promoter lacking the upstream elements previously considered to be essential for cAMP activation of the gene and, through mutational analysis, confirm that the major cAMP effects on the hCGbeta promoter are mediated through the proximal Ets-2 enhancer. The data are consistent with the hypothesis that Ets-2 has a general and possibly essential role in controlling the activity of genes associated with trophectoderm differentiation.

molecular endocrinology

A Central Role for Ets-2 in the Transcriptional Regulation and Cyclic Adenosine 5′-Monophosphate Responsiveness of the Human Chorionic Gonadotropin-β Subunit Gene