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

• Main Authors: Hannah Otterbein, Koichiro Mihara, Morley D. Hollenberg, Hendrik Lehnert, David Witte, Hendrik Ungefroren
• Format: Article
• Language: English
• Published: MDPI AG 2019-08-01
• Series: Cancers
• Subjects: RAC1B; ALK5; PAR2; cell migration; pancreatic carcinoma; RNA interference; TGF-β
• Online Access: https://www.mdpi.com/2072-6694/11/8/1211

The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-&#946;1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-&#946; 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 <i>F2RL1</i> that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-&#946;1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-&#946;1-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-&#946;1-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-&#946;1 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&#8217;s ability to suppress ALK5 abundance and TGF-&#946;1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-&#946;1-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 <i>F2RL1</i>/PAR2 expression. Since <i>F2RL1</i> itself is a TGF-&#946; target gene and its upregulation by TGF-&#946;1 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-&#946;-driven cell motility and growth inhibition.