Down-regulation of the cyclin a promoter by transforming growth factor-beta 1 is associated with a reduction in phosphorylated activating transcription factor-1 and cyclic AMP-responsive element-binding protein

Transforming growth factor (TGF)-beta 1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-beta 1 down-regulates cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A prometer into mink lung epithelial cells in the absence and presence of TGF-beta 1, The TGF-beta 1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolished down-regulation, Surprisingly, although TGF-beta 1 treatment for 24 h markedly decreased cyclin A promoter activity, it did not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic AMP-responsive binding protein (CREB), However, we detected 90 and 78% reductions (by Western analysis) in phosphorylated CREB and ATF-1, respectively, in mink lung epithelial cells treated with TGF-beta 1, TGF-beta 1-induced down-regulation of cyclin A promoter activity was reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection with plasmids expressing the cAMP dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A), These data indicate that TGF-beta 1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.