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

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 molecul...

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Autores principales: Otterbein, Hannah, Mihara, Koichiro, Hollenberg, Morley D., Lehnert, Hendrik, Witte, David, Ungefroren, Hendrik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721813/
https://www.ncbi.nlm.nih.gov/pubmed/31434318
http://dx.doi.org/10.3390/cancers11081211
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author Otterbein, Hannah
Mihara, Koichiro
Hollenberg, Morley D.
Lehnert, Hendrik
Witte, David
Ungefroren, Hendrik
author_facet Otterbein, Hannah
Mihara, Koichiro
Hollenberg, Morley D.
Lehnert, Hendrik
Witte, David
Ungefroren, Hendrik
author_sort Otterbein, Hannah
collection PubMed
description 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.
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spelling pubmed-67218132019-09-10 RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5 Otterbein, Hannah Mihara, Koichiro Hollenberg, Morley D. Lehnert, Hendrik Witte, David Ungefroren, Hendrik Cancers (Basel) Article 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. MDPI 2019-08-20 /pmc/articles/PMC6721813/ /pubmed/31434318 http://dx.doi.org/10.3390/cancers11081211 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Otterbein, Hannah
Mihara, Koichiro
Hollenberg, Morley D.
Lehnert, Hendrik
Witte, David
Ungefroren, Hendrik
RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title_full RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title_fullStr RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title_full_unstemmed RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title_short RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
title_sort rac1b suppresses tgf-β-dependent chemokinesis and growth inhibition through an autoregulatory feed-forward loop involving par2 and alk5
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721813/
https://www.ncbi.nlm.nih.gov/pubmed/31434318
http://dx.doi.org/10.3390/cancers11081211
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