A functional analysis of Wnt inducible signalling pathway protein −1 (WISP-1/CCN4)

Abstract

Wnt-1 inducible signalling pathway protein 1 (WISP-1/CCN4) is an extracellular matrix protein that belongs to the Cyr61 (cysteine-rich protein 61), CTGF (connective tissue growth factor) and NOV (CCN) family and plays a role in multiple cellular processes. No specific WISP-1 receptors have been identified but emerging evidence suggests WISP-1 mediates its downstream effects by binding to integrins. Here we describe a functional analysis of integrin receptor usage by WISP-1. Truncated WISP-1 proteins were produced using a baculovirus expression system. Full length WISP-1 and truncated proteins were evaluated for their ability to induce adhesion in A549 epithelial cells and β-catenin activation and CXCL3 secretion in fibroblasts (NRK49-F cells). Subsequent inhibition of these responses by neutralising integrin antibodies was evaluated. A549 cells demonstrated adhesion to full-length WISP-1 whilst truncated proteins containing VWC, TSP or CT domains also induced adhesion, with highest activity observed with proteins containing the C-terminal TSP and CT domains. Likewise the ability to induce β-catenin activation and CXCL3 secretion was retained in truncations containing C-terminal domains. Pre-treatment of A549s with either integrin αVβ5, αVβ3 or β1 neutralising antibodies partially inhibited full length WISP-1 induced adhesion whilst combining integrin αVβ5 and β1 antibodies increased the potency of this effect. Incubation of NRK49-F cells with integrin neutralising antibodies failed to effect β-catenin translocation or CXCL3 secretion. Analysis of natural WISP-1 derived from human lung tissue showed the native protein is a high order oligomer. Our data suggest that WISP-1 mediated adhesion of A549 cells is an integrin-driven event regulated by the C-terminal domains of the protein. Activation of β-catenin signalling and CXCL3 secretion also resides within the C-terminal domains of WISP-1 but are not regulated by integrins. The oligomeric nature of native WISP-1 may drive a high avidity interaction with these receptors in vivo.

The Matrix Revolution: Matricellular Proteins and Restructuring of the Cancer Microenvironment

“…CCNs act through multiple mechanisms to regulate a plethora of dynamic cellular processes (11,101,116). In particular, these proteins activate ILK/Akt, MAPK, and associated (11,101,123,124), for example syndecan-4 and Notch in the case of CCN1/2 and CCN3, respectively. Interestingly, CCN proteins may be activated by proteolytic cleavage (125,126).…”

WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway

“…Wnt1 inducible signaling pathway protein 1 (WISP1), namely CCN4, belongs to the CCN (Cysteine-rich protein 61, Connective tissue growth factor, and Nephroblastoma overexpressed gene) family (Stephens et al, 2015). CCN family proteins are ubiquitously expressed in mammalian tissues and excert their biological functions including extracellular matrix (ECM) synthesis, cell proliferation and vascular remodeling (Chong et al, 2011;Maiese et al, 2008;Stephens et al, 2015).…”

“…CCN family proteins are ubiquitously expressed in mammalian tissues and excert their biological functions including extracellular matrix (ECM) synthesis, cell proliferation and vascular remodeling (Chong et al, 2011;Maiese et al, 2008;Stephens et al, 2015). Accumulating studies have shown that WISP1 inhibits cell apoptosis and promotes cell survival through activation of protein kinase B (Akt) signaling pathway (Hou et al, 2013;Su et al, 2002;Venkatesan et al, 2010;Wang et al, 2012).…”

WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

“…Depending on context, WISP1 activates a variety of downstream signaling, including focal adhesion kinase, RAS/RAF/ MEK/ERK, NF-B, TGF-␤, and PI3K/AKT pathways (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). Functionally, WISP1-initiated signals regulate various biological processes, including cell adhesion, proliferation, differentiation, survival, motility, and wound healing/tissue repair (38,39).…”

“…Compared with CCN1-3, the components and steps of WISP1 signaling are less characterized, but putative integrin recognition sites exist within VWC, TSP, and CT domains (22). In vitro binding assays and functional assays with integrinblocking antibodies implicated that ␣5␤1, ␣v␤3, and ␣v␤5 were involved in WISP1 signaling, and these integrins were essential for WISP1-induced activation of focal adhesion kinase, Rac, RAS/RAF/MEK/ERK, JNK, or NF-B pathways in epithelial cells, fibroblasts, bone marrow stromal cells, or cancer cells (26,30,31,(33)(34)(35)37).…”