Related Experiment Videos
Down-regulation of the cyclin A promoter by transforming growth factor-beta1 is associated with a reduction in
M Yoshizumi1, H Wang, C M Hsieh
1Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Abstract:
Transforming growth factor (TGF)-beta1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-beta1 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-beta1. The TGF-beta1-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-beta1 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-beta1. TGF-beta1-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-beta1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.
Insights
Transforming growth factor-beta1 (TGF-β1) hinders cell cycle progression by reducing cyclin A gene expression. This occurs through decreased phosphorylation of activating transcription factor (ATF) and cyclic AMP-responsive binding protein (CREB).
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor-beta1 (TGF-β1) is a key regulator of cell proliferation and differentiation.
- Cyclin A is crucial for cell cycle progression, and its regulation is often disrupted in cancer.
- Understanding how TGF-β1 influences cyclin A expression is vital for comprehending cell cycle control.
Purpose of the Study:
- To elucidate the molecular mechanisms by which TGF-β1 down-regulates cyclin A gene expression.
- To identify the specific regulatory elements and signaling pathways involved in TGF-β1's effect on cyclin A promoter activity.
Main Methods:
- Reporter gene assays using cyclin A promoter constructs in mink lung epithelial cells.
- Site-directed mutagenesis of the activating transcription factor (ATF) binding site within the cyclin A promoter.
- Western blot analysis to assess protein levels and phosphorylation status of ATF-binding proteins.
- Treatment with phosphatase inhibitors (okadaic acid) and expression plasmids for protein kinase subunits.
Main Results:
- TGF-β1 significantly down-regulated cyclin A promoter activity, mediated by the ATF site.
- TGF-β1 treatment reduced the phosphorylation of cyclic AMP-responsive binding protein (CREB) and ATF-1, but not their overall abundance.
- Down-regulation of cyclin A promoter activity by TGF-β1 was reversed by phosphatase inhibition and by expressing cAMP-dependent protein kinase or PP2A inhibitors.
Conclusions:
- TGF-β1 down-regulates cyclin A gene expression by decreasing the phosphorylation of CREB and ATF-1.
- This mechanism involves the regulation of protein phosphorylation, likely through modulation of phosphatase activity.
- These findings provide insights into TGF-β1's role in cell cycle control and potential therapeutic targets.