Protease-activated Receptor-1 (PAR1) Acts via a Novel Gα13-Dishevelled Axis to Stabilize β-Catenin Levels

Abstract

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and ␤-catenin stabilization. Although it is well recognized that Wnt signaling leads to ␤-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces ␤-catenin stabilization independent of Wnt, Fz (Frizzled), and the coreceptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-␤-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both G␣ 12 and G␣ 13 compared with age-matched control counterparts. However, only G␣ 13 was found to be actively involved in PAR1-induced ␤-catenin stabilization. Indeed, a dominant negative form of G␣ 13 inhibited both PAR1-induced Matrigel invasion and Lef/ Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-G␣ 13 association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased ␤-catenin accumulation. It is of note that PAR1 also promotes the binding of ␤-arrestin-2 to DVL, suggesting a role for ␤-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated ␤-catenin accumulation, no effect was observed on PAR1-induced ␤-catenin stabilization. Collectively, our data show that PAR1 mediates ␤-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced G␣ 13 -DVL axis in cancer and ␤-catenin stabilization.PAR1 (protease-activated receptor-1) is the first identified and prototype member of an established protease-activated receptor family. In addition to the traditional role of PAR1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1-3). We have established a novel link between hPar1 and ␤-catenin stabilization, both in transgenic mouse mammary glands and in a wide spectrum of tumor cell lines (4). Protease-activated receptors are part of a large seven-transmembrane-spanning G protein-coupled receptor (GPCR) 2 family (5, 6), shown to couple to G␣ i/o , G␣ q , or G␣ 12/13 within the same cell type (7). Of the 16 G␣ genes found in the mammalian genome, the G␣ 12 subfamily is of particular interest to cancer biologists. G␣ 12 and its sister family member, G␣ 13 , are the only heterotrimeric G proteins that are capable of transforming fibroblasts when overexpressed in their wild-type (WT) form (8 -10). Recent studies have demonstrated that G␣ 12 is markedly up-regulated in adenocarcinoma of the breast and have identified G␣ 12 protein as an important regulator of breast and prostate cancer invasion (11,12). The G␣ 12 protein subunit also plays a role in disrupting cadherin-␤-catenin i...

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure

“…Critical downstream effectors of PI3-kinase signaling include the serine/threonine kinases Akt and mTorc1. 36 Additional potential pathway was indicated recently by Bar-Shavit et al 37 demonstrating an intracellular link between PAR1 and b-catenin stabilization independent of Wnt, but through the PAR1-induced Ga 13 -disheveled axis.…”

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

“…Thrombin activation of PAR1 has been shown to promote β-catenin stabilization in endothelial cells and colorectal cancer cells (35,36). In cancer cells, thrombin-induced stabilization of β-catenin occurs through Gα 13 -mediated recruitment of Dvl-1 (35). However, Gα 12/13 proteins also are known to stabilize β-catenin directly by interacting with the cytoplasmic domain of cadherins, causing β-catenin dissociation and translocation to the nucleus (37).…”

“…In addition to its function in β-catenin stabilization induced by Wnt/Fz, Dvl appears to mediate β-catenin stabilization stimulated by classic GPCRs. Thrombin activation of PAR1 has been shown to promote β-catenin stabilization in endothelial cells and colorectal cancer cells (35,36). In cancer cells, thrombin-induced stabilization of β-catenin occurs through Gα 13 -mediated recruitment of Dvl-1 (35).…”

PAR1 plays a role in epithelial malignancies: Transcriptional regulation and novel signaling pathway

“…Both G a12 and G a13 showed a marked induction in the expression when analyzed by immunohistological staining on hPar 1 -transgenic (tg) mice mammary tissues as compared with agematched control counterparts. However, only G a13 was actively involved in PAR 1 -induced b-catenin stabilization (30). Indeed, a dominant negative form of G a13 potently inhibited both PAR 1 -induced b-catenin Lef/Tcf transcriptional activity as well as Matrigel invasion.…”