Ningyu Chen

Adhesion of Human Skin Fibroblasts to Cyr61 Is Mediated through Integrin α6β1 and Cell Surface Heparan Sulfate Proteoglycans

The angiogenic inducer Cyr61 is an extracellular matrix-associated heparin-binding protein that can mediate cell adhesion, stimulate cell migration, and enhance growth factor-stimulated DNA synthesis in both fibroblasts and endothelial cells in culture. In vivo, Cyr61 induces neovascularization and promotes tumor growth. Cyr61 is a prototypic member of a highly conserved family of secreted proteins that includes connective tissue growth factor, nephroblastoma overexpressed, Elm-1/WISP-1, Cop-1/WISP-2, and WISP-3. Encoded by an immediate early gene, Cyr61 synthesis is induced by serum growth factors in cultured fibroblasts and in dermal fibroblasts during cutaneous wound healing. We previously demonstrated that Cyr61 mediates adhesion of vascular endothelial cells and activation-dependent adhesion of blood platelets through direct interaction with integrins αVβ3 and αIIbβ3, respectively. In this study, we show that the adhesion of primary human skin fibroblasts to Cyr61 is mediated through integrin α6β1 and cell surface heparan sulfate proteoglycans (HSPGs), which most likely serve as co-receptors. Either destruction of cell surface HSPGs or prior occupancy of the Cyr61 heparin-binding site completely blocked cell adhesion to Cyr61. A heparin-binding defective mutant of Cyr61 was unable to mediate fibroblast adhesion through integrin α6β1 but still mediated endothelial cell adhesion through integrin αVβ3, indicating that endothelial cell adhesion through integrin αVβ3 is independent of the heparin-binding activity of Cyr61. These results identify Cyr61 as a novel adhesive substrate for integrin α6β1 and provide the first demonstration of the requirement for HSPGs in integrin-mediated cell attachment. In addition, these findings suggest that Cyr61 might elicit disparate biological effects in different cell types through interaction with distinct integrin receptors.

CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family

CCN3 (NOV) is a matricellular protein of the CCN family, which also includes CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). During development, CCN3 is expressed widely in derivatives of all three germ layers, and high levels of expression are observed in smooth muscle cells of the arterial vessel wall. Altered expression of CCN3 has been observed in a variety of tumors, including hepatocellular carcinomas, Wilms tumors, Ewings sarcomas, gliomas, rhabdomyosarcomas, and adrenocortical carcinomas. To understand its biological functions, we have investigated the activities of purified recombinant CCN3. We show that in endothelial cells, CCN3 supports cell adhesion, induces directed cell migration (chemotaxis), and promotes cell survival. Mechanistically, CCN3 supports human umbilical vein endothelial cell adhesion through multiple cell surface receptors, including integrins αvβ3, α5β1, α6β1, and heparan sulfate proteoglycans. In contrast, CCN3-induced cell migration is dependent on integrins αvβ3 and α5β1, whereas α6β1 does not play a role in this process. Although CCN3 does not contain a RGD sequence, it binds directly to immobilized integrins αvβ3 and α5β1, with half-maximal binding occurring at 10 nm and 50 nm CCN3, respectively. Furthermore, CCN3 induces neovascularization when implanted in rat cornea, demonstrating that it is a novel angiogenic inducer. Together, these findings show that CCN3 is a ligand of integrins αvβ3 and α5β1, acts directly upon endothelial cells to stimulate pro-angiogenic activities, and induces angiogenesis in vivo.

The Angiogenic Factor Cysteine-Rich 61 (CYR61, CCN1) Supports Vascular Smooth Muscle Cell Adhesion and Stimulates Chemotaxis through Integrin α6β1 and Cell Surface Heparan Sulfate Proteoglycans

Cysteine-rich 61 (CYR61, CCN1) is a heparin-binding, extracellular, matrix-associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. CYR61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor-stimulated mitogenesis in fibroblasts and endothelial cells. Although the expression of CYR61 has been observed in arterial walls, its function in vascular smooth muscle cells (VSMCs) has not been examined to date. Here we show that purified CYR61 supports VSMC adhesion in a dose-dependent, saturable manner through integrin α6β1 with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin α6β1 and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still ...

Identification of a Novel Integrin α6β1 Binding Site in the Angiogenic Inducer CCN1 (CYR61)

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including α6β1. Previous studies have shown that CCN1 interaction with integrin α6β1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin α6β1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-α6 and anti-β1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating α6β1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other α6β1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits α6β1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin α6β1 directly, we perform affinity chromatography and show that integrin α6β1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin α6β1, providing the basis for the development of peptide mimetics to examine the functional role of α6β1 in angiogenesis.

Cytotoxicity of TNFα is regulated by integrin‐mediated matrix signaling

Cytokines of the tumor necrosis factor (TNF) family regulate inflammation and immunity, and a subset of this family can also induce cell death in a context‐dependent manner. Although TNFα is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFκB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFα without perturbation of NFκB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins αvβ5, α6β1, and syndecan‐4, triggering the generation of reactive oxygen species (ROS) through a Rac1‐dependent mechanism via 5‐lipoxygenase and the mitochondria, leading to the biphasic activation of JNK necessary for apoptosis. Mice with the genomic Ccn1 locus replaced with an apoptosis‐defective Ccn1 allele are substantially resistant to TNFα‐induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFα cytotoxicity, providing the contextual cues from the extracellular matrix for TNFα‐mediated cell death.

Identification of a Novel Integrin αvβ3 Binding Site in CCN1 (CYR61) Critical for Pro-angiogenic Activities in Vascular Endothelial Cells

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin αvβ3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin αvβ3. Immobilized synthetic V2 peptide supports αvβ3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin αvβ3 and abolished αvβ3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified αvβ3 and impaired in αvβ3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds αvβ3 and supports αvβ3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary αvβ3 binding site in soluble CCN1, whereas additional cryptic αvβ3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin αvβ3 and provide a new approach for dissecting αvβ3-specific CCN1 functions both in cultured cells and in the organism.

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61) SELECTIVE INACTIVATION OF INTEGRIN α6β1-HEPARAN SULFATE PROTEOGLYCAN CORECEPTOR-MEDIATED CELLULAR FUNCTIONS

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 α6β1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel α6β1 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 α6β1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with α6β1 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 α6β1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain αvβ3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in α6β1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for α6β1-HSPG-dependent CCN1 activities and provide a strategy to investigate these α6β1-HSPG-specific activities in vivo.