RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5.

Hannah Otterbein,Morley D Hollenberg,Hendrik Ungefroren,David Witte,Hendrik Lehnert,Koichiro Mihara

doi:10.3390/cancers11081211

Abstract

The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by F2RL1 that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-β1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-β1-induced expression of PAR2. Further, the simultaneous knockdown of PAR2 and RAC1B rescued Panc1 cells from a RAC1B knockdown-induced increase in ALK5 abundance and the ALK5-mediated increase in TGF-β1-induced migratory activity. Conversely, Panc1 cells with stable ectopic expression of RAC1B displayed reduced ALK5 expression, an impaired upregulation of PAR2, and a reduced migratory responsiveness to TGF-β1 stimulation. However, these effects could be reversed by ectopic overexpression of PAR2. Moreover, the knockdown of PAR2 alone in Panc1 cells and HaCaT keratinocytes phenocopied RAC1B’s ability to suppress ALK5 abundance and TGF-β1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-β1-induced PAR2 mRNA expression was sensitive to pharmacological inhibition of MEK-ERK signaling. Our data show that in pancreatic and skin epithelial cells, downregulation of ALK5 activity by RAC1B is secondary to suppression of F2RL1/PAR2 expression. Since F2RL1 itself is a TGF-β target gene and its upregulation by TGF-β1 is mediated by ALK5 and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and PAR2 that is efficiently suppressed by RAC1B to restrict TGF-β-driven cell motility and growth inhibition.

Highlights

RAC1B is an alternative splice product derived from the human RAC1 gene
Since F2RL1 itself is a transforming growth factor (TGF)-β target gene and its upregulation by TGF-β1 is mediated by activin receptor-like kinase 5 (ALK5) and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and proteinase-activated receptor 2 (PAR2) that is efficiently suppressed by RAC1B to restrict TGF-β-driven cell motility and growth inhibition
TGF-β-Dependent PAR2 Expression Is Negatively Regulated by RAC1B in an ALK5-Dependent Manner

Summary

Introduction

RAC1B is an alternative splice product derived from the human RAC1 gene. This isoform differs from RAC1 by an in-frame insertion of 57 nucleotides, comprising an additional exon (exon 3b) [1].Cancers 2019, 11, 1211; doi:10.3390/cancers11081211 www.mdpi.com/journal/cancersRAC1B can promote cell cycle progression and apoptosis resistance, its role in other processes involved in driving tumor progression, like epithelial–mesenchymal transition (EMT), cell motility, and metastasis is less well known. That RAC1B suppresses EMT, random cell migration, and growth inhibition induced by TGF-β1, while RAC1 promotes both processes [2,3,4,5]. Previous data from our group indicated that RAC1B suppresses expression and TGF-β1-mediated upregulation of activin receptor-like kinase 5 (ALK5), the prototype type I receptor for TGF-β [8]. This suppression was associated with decreased activation of Smad [2], MKK3/6-p38, and MEK-ERK signaling [3]. The molecular mechanism(s) underlying RAC1B-mediated inhibition of ALK5 expression and function remains obscure, i.e., whether regulation is executed at the level of the TGFBR1 promoter to prevent de novo transcription, or whether the RAC1B effect on ALK5 is indirect via other mechanisms

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Conclusion