The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility

TY - JOUR

T1 - The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility

AU - Ungefroren, Hendrik

AU - Witte, David

AU - Fiedler, Christian

AU - Gädeken, Thomas

AU - Kaufmann, Roland

AU - Lehnert, Hendrik

AU - Gieseler, Frank

AU - Rauch, Bernhard H

PY - 2017/12/20

Y1 - 2017/12/20

N2 - BACKGROUND: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2.METHODS: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2-ALK5 physical interaction.RESULTS: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2-AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2-AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5.

AB - BACKGROUND: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2.METHODS: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2-ALK5 physical interaction.RESULTS: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2-AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2-AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5.

KW - Cell Line, Tumor

KW - Cell Movement

KW - Humans

KW - MAP Kinase Signaling System

KW - Protein Binding

KW - Protein Serine-Threonine Kinases/genetics

KW - Receptor, PAR-2

KW - Receptor, Transforming Growth Factor-beta Type I

KW - Receptors, G-Protein-Coupled/antagonists & inhibitors

KW - Receptors, Transforming Growth Factor beta/genetics

KW - Transforming Growth Factor beta1/metabolism

U2 - 10.3390/ijms18122776

DO - 10.3390/ijms18122776

M3 - SCORING: Journal article

C2 - 29261154

VL - 18

SP - 2776

JO - INT J MOL SCI

JF - INT J MOL SCI

SN - 1661-6596

IS - 12

ER -