Activin induces hepatocyte cell growth arrest through induction of the cyclin-dependent kinase inhibitor p15INK4B and Sp1.

In this report, we examined the role of activin in the regulation of cell growth inhibition of human hepatocarcinoma cells. Using RNase protection assay for various cell cycle regulators and Western blotting experiments, we show that activin treatment of HepG2 cells leads to increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p15INK4B. Furthermore, transient co-transfection studies of the p15INK4B promoter/luciferase construct performed in HepG2 cells demonstrates that activin induction of the p15INK4B promoter is mediated through the Smad pathway. p15INK4B gene promoter mapping analysis revealed a 66-bp region within the proximal domain of the promoter, which contains a consensus site for the transcription factor Sp1, as critical for mediating the activin effect on p15INK4B gene expression. Finally, gel mobility shift experiments, using the Sp1 consensus site, revealed increased DNA binding of Sp1 in response to activin treatment of HepG2 cells, further confirming the involvement of Sp1 in activin-mediated p15INK4B gene promoter activation. Together, our data indicates an important role for the cyclin-dependent kinase inhibitor p15INK4B in activin-induced cell cycle arrest in liver cells.