Transforming Growth Factor- β 1/Activin Receptor-like Kinase 5-Mediated Cell Migration is Dependent on the Protein Proteinase-Activated Receptor 2 but not on Proteinase-Activated Receptor 2-Stimulated Gq-Calcium Signaling

Mol Pharmacol. 2017 Nov;92(5):519-532. doi: 10.1124/mol.117.109017. Epub 2017 Aug 25.

Abstract

Transforming growth factor-β (TGF-β), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-β1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-β receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-β signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-β1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-β1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-β1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-β1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-β1 synergy may involve TGF-β1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-β's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.

MeSH terms

  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology*
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Cell Movement / physiology*
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism*
  • HEK293 Cells
  • Humans
  • Oligopeptides / pharmacology
  • Protein Serine-Threonine Kinases / metabolism*
  • Receptor, PAR-2
  • Receptor, Transforming Growth Factor-beta Type I
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, Transforming Growth Factor beta / metabolism*
  • Transforming Growth Factor beta1 / metabolism*

Substances

  • F2RL1 protein, human
  • GB88 compound
  • Oligopeptides
  • Receptor, PAR-2
  • Receptors, G-Protein-Coupled
  • Receptors, Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • Protein Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type I
  • TGFBR1 protein, human
  • GTP-Binding Protein alpha Subunits, Gq-G11