Functions and mechanisms of action of CCN matricellular proteins

Abstract

Members of the CCN (CYR61/CTGF/NOV) family have emerged as dynamically expressed, extracellular matrix-associated proteins that play critical roles in cardiovascular and skeletal development, injury repair, fibrotic diseases and cancer. The synthesis of CCN proteins is highly inducible by serum growth factors, cytokines, and environmental stresses such as hypoxia, UV exposure, and mechanical stretch. Consisting of six secreted proteins in vertebrate species, CCNs are typically comprised of four conserved cysteine-rich modular domains. They function primarily through direct binding to specific integrin receptors and heparan sulfate proteoglycans, thereby triggering signal transduction events that culminate in the regulation of cell adhesion, migration, proliferation, gene expression, differentiation, and survival. CCN proteins can also modulate the activities of several growth factors and cytokines, including TGF-beta, TNFalpha, VEGF, BMPs, and Wnt proteins, and may thereby regulate a broad array of biological processes. Recent studies have uncovered novel CCN activities unexpected for matricellular proteins, including their ability to induce apoptosis as cell adhesion substrates, to dictate the cytotoxicity of inflammatory cytokines such as TNFalpha, and to promote hematopoietic stem cell self-renewal. As potent regulators of angiogenesis and chondrogenesis, CCNs are essential for successful cardiovascular and skeletal development during embryogenesis. In the adult, the expression of CCN proteins is associated with injury repair and inflammation, and has been proposed as diagnostic or prognostic markers for diabetic nephropathy, hepatic fibrosis, systemic sclerosis, and several types of cancer. Targeting CCN signaling pathways may hold promise as a strategy of rational therapeutic design.

Figure 1

Schematics of CCN protein structure and localization of their integrin binding sites. The six CCN proteins include CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1, ELM1), CCN5 (WISP-2, COP-1), and CCN6 (WISP-3). They share significant structural homology, including an N-terminal secretory signal peptide (SP), followed by modular domains (illustrated in different colors) with sequence homologies to insulin-like growth factor binding protein (IGFBP, module I), von Willebrand factor type C repeat (vWC, module II), thrombospondin type 1 repeat (TSP, module III), and a cysteine knot containing carboxyl domain (CT, module IV). Throughout the four modules are 38 cysteine residues that are highly conserved. CCN5 uniquely lacks the CT domain but conserves domains I–III. A protease-sensitive hinge region with no sequence homology among the CCN proteins separate domains II and III. Specific binding sites (black and hatched bars) for several integrins and HSPGs have been identified for CCN1 and CCN2 (Chen et al., 2000; Leu et al., 2003; Chen et al., 2004a; Leu et al., 2004; Gao and Brigstock, 2004; Gao and Brigstock, 2006).

Figure 2

CCN1 signaling and crosstalk with TNFα. Signal transduction initiated by CCN1, a prototypical member of the family, is mediated primarily through binding to α₆β₁ and syndecan-4 in fibroblasts to support activities including cell adhesion, although α_vβ₅ is also necessary for fibroblast migration and crosstalk with TNFα (Grzeszkiewicz et al., 2001; Chen et al., 2007). Cell adhesion to CCN1 activates FAK, paxillin, and Rac1, leading to actin cytoskeleton reorganization, cell spreading, and formation of filopodia and lamellipodia (Chen et al., 2001a). Adhesion to CCN1 also induces sustained ERK activation, an activity that is mediated through binding to α₆β₁-HSPG (Leu et al., 2004). CCN1 induces fibroblast apoptosis by activating p53 and Bax (Todorovic et al., 2005), and converts TNFα from a pro-mitogenic factor into a potent apoptotic molecule through the Rac1-dependent generation of ROS via 5-lipoxygenase and the mitochondria (Chen et al., 2007). CCN1/TNFα-induced apoptosis occurs rapidly (within 4 hours of treatment) without requiring de novo protein synthesis, indicating that CCN1 activates this pathway directly.

Figure 3

Interaction of CCN proteins with other molecules. CCN proteins interact with a variety of cell surface receptors and extracellular ligands, including various integrins, HSPGs, and LRPs (Lau and Lam, 2005; Gao and Brigstock, 2003; Mercurio et al., 2004). Receptors that interact with CCN proteins are shown schematically below the four conserved CCN domains and extracellular proteins that bind CCNs are shown above, aligned with the interacting CCN domains. Whereas CCN3 binds the receptor Notch (Sakamoto et al., 2002), CCN2 has been shown to bind BMPs and TGF-β through the vWC domain and VEGF through the TSP and CT domains (Abreu et al., 2002; Inoki et al., 2002). CCN2 also binds ECM proteins such as fibronectin and perlecan through the CT domain (Nishida et al., 2003; Chen et al., 2004b).