Down-regulation of the cyclin A promoter by transforming growth factor-beta1 is associated with a reduction in phosphorylated activating transcription factor-1 and cyclic AMP-responsive element-binding protein

Transforming growth factor (TGF)-β1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-β1 down-regulates cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A promoter into mink lung epithelial cells in the absence and presence of TGF-β1. The TGF-β1-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-β1 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-β1. TGF-β1-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-β1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.