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RhoB, not RhoA, represses the transcription of the transforming growth factor beta type II receptor by a mechanism
Jalila Adnane1, Edward Seijo, Zhi Chen
1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Oncology, University of South Florida, Tampa, Florida 33612, USA.
Abstract:
The transforming growth factor-beta (TGF-beta) type I (T beta R-I) and type II (T beta R-II) receptors are responsible for transducing TGF-beta signals. We have previously shown that inhibition of farnesyltransferase activity results in an increase in T beta R-II expression, leading to enhanced TGF-beta binding, signaling, and inhibition of tumor cell growth, suggesting that a farnesylated protein(s) exerts a repressive effect on T beta R-II expression. Likely candidates are farnesylated proteins such as Ras and RhoB, which are both farnesylated and involved in cell growth control. Neither a dominant negative Ha-Ras, constitutively activated Ha-Ras, or a pharmacological inhibitor of MEK1 affected T beta R-II transcription. However, ectopic expression of RhoB, but not the closely related family member RhoA, resulted in a 5-fold decrease of T beta R-II promoter activity. Furthermore, ectopic expression of RhoB, but not RhoA, resulted in a significant decrease of T beta R-II protein expression and resistance of tumor cells to TGF-beta-mediated cell growth inhibition. Deletion analysis of the T beta R-II promoter identified a RhoB-responsive region, and mutational analysis of this region revealed that a site for the transcription factor activator protein 1 (AP1) is critical for RhoB-mediated repression of T beta R-II transcription. Electrophoretic mobility shift assays clearly showed that the binding of AP1 to its DNA-binding site is strongly inhibited by RhoB. Consequently, transcription assays using an AP1 reporter showed that AP1-mediated transcription is down-regulated by RhoB. Altogether, these results identify a mechanism by which RhoB antagonizes TGF-beta action through transcriptional down-regulation of AP1 in T beta R-II promoter.
Insights
RhoB protein represses transforming growth factor-beta (TGF-beta) signaling by down-regulating T beta R-II expression. This repression occurs via inhibition of the transcription factor AP1, impacting tumor cell growth and TGF-beta response.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- Transforming growth factor-beta (TGF-beta) signaling is mediated by T beta R-I and T beta R-II receptors.
- Farnesyltransferase inhibition increases T beta R-II expression, enhancing TGF-beta signaling and tumor cell growth inhibition.
- This suggests a farnesylated protein negatively regulates T beta R-II expression.
Purpose of the Study:
- To identify the farnesylated protein responsible for repressing T beta R-II expression.
- To elucidate the mechanism by which this protein affects TGF-beta signaling and tumor cell growth.
Main Methods:
- Ectopic expression of Ras and Rho family proteins (RhoA, RhoB).
- Analysis of T beta R-II promoter activity and transcription.
- Deletion and mutational analysis of the T beta R-II promoter.
- Electrophoretic mobility shift assays (EMSA) and reporter gene assays for AP1 activity.
Main Results:
- Ectopic RhoB, but not RhoA, significantly decreased T beta R-II promoter activity and protein expression.
- RhoB expression led to resistance to TGF-beta-mediated growth inhibition.
- A RhoB-responsive region in the T beta R-II promoter was identified, containing an AP1 binding site critical for repression.
- RhoB inhibited AP1 binding to DNA and down-regulated AP1-mediated transcription.
Conclusions:
- RhoB antagonizes TGF-beta signaling by transcriptionally down-regulating T beta R-II expression.
- This antagonism is mediated through RhoB's inhibition of the transcription factor AP1 at the T beta R-II promoter.
- RhoB plays a role in regulating TGF-beta pathway activity and tumor cell response to TGF-beta.