Blockade of TCFβ₃ up-regulation of p27^Kip1 and p21(^ip1 by expression of RasN17 in epithelial cells

Our previous data demonstrated that Ras activation is necessary and sufficient for transforming growth factor β (TGFβ)-mediated Erk1 activation, and is partially required for the inhibition of cyclin-dependent kinase 2 (Cdk2) activity, cyclin A expression and DNA synthesis by TGFβ (KM Mulder and SL Morris, J. Biol. Chem., 267: 5029 ± 5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270: 7117 ± 7124, 1995; and MT Hartsough et al., J. Biol. Chem., 271: 22368 ± 22375, 1996). Here, we examined the kinetics and role of Ras in TGFβ₃-mediated effects on specific G₁ cell cycle components in TGFβ-sensitive (4-1) acid TGFβ-resistant (4-6) intestinal epithelial cells (IEC's). Our results indicate that inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) completely abrogated the ability of TGFβ₃ to up-regulate both CKI's. In contrast, the ability of TGFβ₃ to up-regulate p27^Kip1 and p21^Cip1 was maintained in ZnCl₂-treated control cells. Inactivation of Ras also completely blocked the rapid TGFβ-mediated increase in new synthesis of p27^Kip1 protein. Moreover, up-regulation of p21^Cip1 protein levels and new synthesis of p27^Kip1, as well as the association of these CKI's with Cdk2, preceded the decrease in Cdk2 activity by TGFβ. Collectively, our results suggest that p21^Cip1 and p27^Kip1 are upstream effecters of the TGFβ-mediated inhibition of Cdk2 activity in IEC 4-1 cells, and demonstrate that Ras activation is obligatory for TGFβ-mediated up-regulation of these CKIs in untransformed epithelial cells.