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Altered transforming growth factor signaling in epithelial cells when ras activation is blocked
M T Hartsough1, R S Frey, P A Zipfel
1Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Abstract:
We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor beta1 (TGFbeta) and TGFbeta2 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 TGFbeta regulation of both Erk1 and downstream components associated with TGFbeta-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 TGFbeta-mediated activation of Erk1. Moreover, the RasN17-mediated reversal of the TGFbeta 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 TGFbeta. In order to determine whether the activation of Ras by TGFbeta was required for the growth inhibitory effect of TGFbeta, we examined TGFbeta2 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 TGFbeta. Collectively, these results indicate that Ras activation is obligatory for TGFbeta-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGFbeta.
Insights
Transforming growth factor beta (TGFbeta) requires Ras activation for extracellular signal-regulated kinase 1 (Erk1) activation in intestinal cells. Ras is also partially required for TGFbeta-induced growth inhibition.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor beta (TGFbeta) inhibits intestinal epithelial cell growth.
- TGFbeta signaling involves Ras and extracellular signal-regulated kinase 1 (Erk1) activation.
- The precise role of Ras in TGFbeta-mediated growth inhibition is not fully understood.
Purpose of the Study:
- To investigate if Ras is essential for TGFbeta-induced Erk1 activation.
- To determine if Ras is required for TGFbeta-mediated growth inhibition in intestinal epithelial cells.
- To elucidate the downstream effects of Ras in TGFbeta signaling.
Main Methods:
- Transfection of intestinal epithelial cells (IEC 4-1) with a dominant-negative Ras mutant (RasN17) under an inducible promoter.
- Treatment with ZnCl2 to induce RasN17 expression.
- Analysis of Erk1 activation, Cdk2 activity, cyclin A protein levels, and DNA synthesis following TGFbeta treatment.
Main Results:
- Induction of RasN17 expression abrogated TGFbeta-mediated Erk1 activation in a dose-dependent manner.
- RasN17 expression partially reversed TGFbeta-induced inhibition of Cdk2 activity (50%), cyclin A down-regulation (78%), and DNA synthesis (21%).
- Control cells without RasN17 showed normal TGFbeta responses.
Conclusions:
- Ras activation is obligatory for TGFbeta-mediated Erk1 activation in intestinal epithelial cells.
- Ras plays a partial role in the growth inhibitory effects of TGFbeta.
- These findings clarify the role of Ras in TGFbeta signaling pathways relevant to intestinal cell growth and potentially cancer.