Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61)

Abstract

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin ␣ 6 ␤ 1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel ␣ 6 ␤ 1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support ␣ 6 ␤ 1 -and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with ␣ 6 ␤ 1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished ␣ 6 ␤ 1 -HSPGmediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain ␣ v ␤ 3 -mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in ␣ 6 ␤ 1 -HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for ␣ 6 ␤ 1 -HSPG-dependent CCN1 activities and provide a strategy to investigate these ␣ 6 ␤ 1 -HSPG-specific activities in vivo.

αVβ3 integrin regulates heregulin (HRG)-induced cell proliferation and survival in breast cancer

“…In accordance with these results, we have previously reported that CYR61 can regulate the levels of a v b 3 expression as well as breast cancer cell sensitivity to taxol-induced cell damage in an HER2/neu-independent fashion (Menendez et al, 2004). CYR61 is also an important growth regulator in endothelial cells, mainly through its binding with the cell surface integrins a v b 3 and a 6 b 1 (Leu et al, 2002(Leu et al, , 2004. Since integrin ligation is required for endothelial cells responses to most proangiogenic factors (Stupack and Cheresh, 2003), it is reasonable to offer a model in which the HRG/CYR61/a v b 3 molecular connection may be orchestrating breast tumorigenesis and progression in at least two concerted modes: (1) by regulating endothelial cells survival and recruitment during tumor neovascularization in a paracrine fashion through an a v b 3 -dependent mechanism; and (2) by promoting breast cancer epithelial cell proliferation in an autocrine/paracrine manner either by increasing the activity of other growth factors (i.e.…”

The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing

“…3a), indicating that CCN1 can induce senescence as a cell adhesion substrate and this activity is unique among the ECM proteins tested. Consistent with impaired senescence in Ccn1 dm / dm mice, purified DM protein26 was completely unable to inhibit DNA synthesis, induce SA-β-gal expression, or trigger the SASP/SMS (Fig. 3b–d; Supplementary information, Fig.…”

“…Recombinant CCN1 and mutant proteins (D125A and DM) were produced using a baculovirus expression system in insect cells and purified by ion-exchange or immuno-affinity chromatography22, 26. Human FN, VN, mouse LN, rat tail Col.I were from BD Biosciences.…”

Inhibition of AP-1 by SARI negatively regulates transformation progression mediated by CCN1

“…CCN1 is a secretory integrin-binding protein that regulates angiogenesis, cell adhesion, migration, proliferation, survival and apoptosis (Chen et al, 2001;Leu et al, 2004). Alternatively, CCN1 has a complex role in tumorigenesis, showing divergent functions and expression profiles in cancers from different tissues (O'Kelly, 2005).…”