Altered Transforming Growth Factor β Signaling in Epithelial Cells when Ras Activation Is Blocked

We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor β1 (TGFβ) and TGFβ2 was associated with a rapid activation of both Ras and extracellular signal-regulated kinase 1 (Erk1) (Mulder, K. M., and Morris, S. L. (1992) J. Biol. Chem. 267, 5029-5031; Hartsough, M. T., and Mulder, K. M. (1995) J. Biol. Chem. 270, 7117-7124). In order to determine whether Ras was required for TGFβ regulation of both Erk1 and downstream components associated with TGFβ-mediated growth inhibition, the intestinal epithelial cell (IEC) line IEC 4-1 was transfected with a vector containing a dominant-negative mutant of Ras (RasN17) under the control of an inducible metallothionein promoter. Using two different RasN17-transfected clones treated with ZnCl2, we demonstrate here that induction of Ras expression by at least 4-fold completely abrogated the TGFβ-mediated activation of Erk1. Moreover, the RasN17-mediated reversal of the TGFβ effect on Erk1 was dependent upon the level of expression of the dominant-negative protein. ZnCl2 treatment of control cells transfected with the empty vector did not alter Ras expression or the activation of Erk1 by TGFβ. In order to determine whether the activation of Ras by TGFβ was required for the growth inhibitory effect of TGFβ, we examined TGFβ2 effects on Cdk2-associated histone H1 kinase activity, cyclin A protein expression levels, and DNA synthesis in two intestinal epithelial cell clones transfected with RasN17. In cells expressing RasN17, we observed a 50% reversal of the inhibition of Cdk2 activity, a 78% reversal of the down-regulation of cyclin A protein expression, and a 21% reversal of the inhibition of DNA synthesis by TGFβ. Collectively, these results indicate that Ras activation is obligatory for TGFβ-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGFβ. We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor β1 (TGFβ) and TGFβ2 was associated with a rapid activation of both Ras and extracellular signal-regulated kinase 1 (Erk1) (Mulder, K. M., and Morris, S. L. (1992) J. Biol. Chem. 267, 5029-5031; Hartsough, M. T., and Mulder, K. M. (1995) J. Biol. Chem. 270, 7117-7124). In order to determine whether Ras was required for TGFβ regulation of both Erk1 and downstream components associated with TGFβ-mediated growth inhibition, the intestinal epithelial cell (IEC) line IEC 4-1 was transfected with a vector containing a dominant-negative mutant of Ras (RasN17) under the control of an inducible metallothionein promoter. Using two different RasN17-transfected clones treated with ZnCl2, we demonstrate here that induction of Ras expression by at least 4-fold completely abrogated the TGFβ-mediated activation of Erk1. Moreover, the RasN17-mediated reversal of the TGFβ effect on Erk1 was dependent upon the level of expression of the dominant-negative protein. ZnCl2 treatment of control cells transfected with the empty vector did not alter Ras expression or the activation of Erk1 by TGFβ. In order to determine whether the activation of Ras by TGFβ was required for the growth inhibitory effect of TGFβ, we examined TGFβ2 effects on Cdk2-associated histone H1 kinase activity, cyclin A protein expression levels, and DNA synthesis in two intestinal epithelial cell clones transfected with RasN17. In cells expressing RasN17, we observed a 50% reversal of the inhibition of Cdk2 activity, a 78% reversal of the down-regulation of cyclin A protein expression, and a 21% reversal of the inhibition of DNA synthesis by TGFβ. Collectively, these results indicate that Ras activation is obligatory for TGFβ-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGFβ.