Abstract
We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and beta-catenin stabilization. Although it is well recognized that Wnt signaling leads to beta-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces beta-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-beta-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Galpha(12) and Galpha(13) compared with age-matched control counterparts. However, only Galpha(13) was found to be actively involved in PAR1-induced beta-catenin stabilization. Indeed, a dominant negative form of Galpha(13) inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Galpha(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 beta-catenin accumulation. It is of note that PAR1 also promotes the binding of beta-arrestin-2 to DVL, suggesting a role for beta-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 beta-catenin accumulation, no effect was observed on PAR1-induced beta-catenin stabilization. Collectively, our data show that PAR1 mediates beta-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Galpha(13)-DVL axis in cancer and beta-catenin stabilization.
Highlights
PAR1 is the first identified and prototype member of an established protease-activated receptor family
It was demonstrated that in both HCT116 colon cancer and HT-29 cell lines, the activation of PAR1 leads to marked nuclear -catenin localization [4]
We show in HT-29 cells that marked nuclear -catenin levels are observed following pretreatment (e.g. 2 h) with LiCl and activation of PAR1 (Fig. 1ai)
Summary
Plasmids and Reagents—The cDNA encoding G␣12, G␣13, and G␣q constructs, WT, and constitutively active and dominant negative forms were prepared as previously described [42]. cDNAs encoding mouse FLAG-Dvl, FLAG-⌬DIX-Dvl, FLAG-⌬PDX-Dvl, and FLAG-human -catenin were kindly provided by Dr Y. Western Blot Analysis—Cells were solubilized in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor mixture, including 5 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (Sigma), for 30 min at 4 °C. Nuclear Extract—Cells were solubilized in lysis buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, and 1 mM dithiothreitol), a protease inhibitor mixture (1:100), 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (Sigma) for 15 min at 4 °C. Membrane and Cytoplasmic Extracts—Cells were solubilized in lysis buffer containing 10 mM Tris-HCl, pH 8, 2.5 mM MgCl2, 5 mM KCl, 1 mM dithiothreitol, protease inhibitor mixture (1:100), 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (Sigma) for 30 min at 4 °C. Mouse anti-G␣12, antiG␣13, or anti-FLAG antibodies were added to the cell lysates.
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