Atypical antipsychotic drugs deregulate the cholesterol metabolism of macrophage-foam cells by activating NOX-ROS-PPARγ-CD36 signaling pathway.

BACKGROUND: Clinical reports indicate that schizophrenia patients taking atypical antipsychotic drugs suffer from metabolism diseases including atherosclerosis. However, the mechanisms underlying the detrimental effect of atypical antipsychotic drugs on atherosclerosis remain to be explored. METHODS: In this study, we used apolipoprotein E-deficient (apoe-/-) hyperlipidemic mice and apoe-/-cd36-/- mice to investigate the underlying mechanism of atypical antipsychotic drugs on atherosclerosis and macrophage-foam cells. RESULTS: In vivo studies showed that genetic deletion of cd36 gene ablated the pro-atherogenic effect of olanzapine in apoe-/- mice. Moreover, in vitro studies revealed that genetic deletion or siRNA-mediated knockdown of cd36 or pharmacological inhibition of CD36 prevented atypical antipsychotic drugs-induced oxLDL accumulation in macrophages. Additionally, olanzapine and clozapine activated NADPH oxidase (NOX) to generate reactive oxygen species (ROS) which upregulated the activity of peroxisome proliferator-activated receptor γ (PPARγ) and subsequently elevated CD36 expression. Inhibition of NOX activity, ROS production or PPARγ activity suppressed CD36 expression and abolished the detrimental effects of olanzapine and clozapine on oxLDL accumulation in macrophages. CONCLUSION: Collectively, our results suggest that atypical antipsychotic drugs exacerbate atherosclerosis and macrophage-foam cell formation by activating the NOX-ROS-PPARγ-CD36 pathway.

Structural and functional characterization of ß2 -glycoprotein I domain 1 in anti-melanoma cell migration.

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.

CCN family member 1 deregulates cholesterol metabolism and aggravates atherosclerosis.

AIM: CCN family member 1 (CCN1) is an extracellular matrix cytokine and appears in atherosclerotic lesions. However, we have no evidence to support the role of CCN1 in regulating cholesterol metabolism and atherosclerosis. METHODS: Apolipoprotein E-deficient (apoE-/- ) mice were used as in vivo model. Oxidized low-density lipoprotein (oxLDL)-induced macrophage-foam cells were used as in vitro model. RT-PCR and western blot analysis were used for evaluating gene and protein expression, respectively. Conventional assay kits were used for assessing the levels of cholesterol, triglycerides, and cytokines. RESULTS: We show predominant expression of CCN1 in foamy macrophages in atherosclerotic aortas of apoE-/- mice. In apoE-/- mice, CCN1 treatment worsened hyperlipidaemia, systemic inflammation, and the progression of atherosclerosis. In addition, CCN1 decreased the capacity of reverse cholesterol transport and downregulated the protein expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 in atherosclerotic aortas. Notably, CCN1 decreased the protein expression of cholesterol clearance-related proteins, including ABCG5, ABCG8, liver X receptor α (LXRα), cholesterol 7α-hydrolase and LDL receptor in liver, and exacerbated hepatic lipid accumulation. In macrophages, treatment with oxLDL increased CCN1 expression. Inhibition of CCN1 activity by neutralizing antibody or small interfering RNA attenuated the oxLDL-induced lipid accumulation. In contrast, cotreatment with CCN1 or overexpression of CCN1 augmented oxLDL-induced lipid accumulation by impairing apolipoprotein AI- and high-density lipoprotein-dependent cholesterol efflux, which was attributed to downregulation of LXRα-dependent expression of ABCA1 and ABCG1. CONCLUSION: Our findings suggest that CCN1 plays a pivotal role in regulating cholesterol metabolism and the development of atherosclerosis.

Novel effect of paeonol on the formation of foam cells: promotion of LXRα-ABCA1-dependent cholesterol efflux in macrophages.

Paeonol, a phenolic component purified from Paeonia suffruticosa (Cortex Moutan), is used in traditional Chinese medicine to treat inflammatory diseases. However, little is known about the effect of paeonol on cholesterol metabolism. We investigated the efficacy of paeonol on cholesterol metabolism and the underlying mechanism in macrophages and apolipoprotein E deficient (apoE(-/-)) mice. Treatment with paeonol markedly attenuated cholesterol accumulation induced by oxidized LDL in macrophages, which was due to increased cholesterol efflux. Additionally, paeonol enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Inhibition of ABCA1 activity with a pharmacological inhibitor, neutralizing antibody or small interfering RNA (siRNA), negated the effects of paeonol on cholesterol efflux and cholesterol accumulation. Furthermore, paeonol induced the nuclear translocation of liver X receptor α (LXRα) by increasing its activity. siRNA knockdown of LXRα abolished the paeonol-induced upregulation of ABCA1, promotion of cholesterol efflux and suppression of cholesterol accumulation. Moreover, atherosclerotic lesions, hyperlipidemia and systemic inflammation were reduced and the protein expression of ABCA1 was increased in aortas of paeonol-treated apoE(-/-) mice. Paeonol may alleviate the formation of foam cells by enhancing LXRα-ABCA1-dependent cholesterol efflux.

A novel anti-CCN1 monoclonal antibody suppresses Rac-dependent cytoskeletal reorganization and migratory activities in breast cancer cells.

CCN1, a secreted matrix-associated molecule, is involved in multiple cellular processes. Accumulating evidence supports that CCN1 plays an important role in tumorigenesis and progression of breast cancer. In this study, we have developed a novel CCN1 function-blocking monoclonal antibody (mAb), designated YM1B. YM1B binds to human CCN1 with high specificity, recognizing the native CCN1 structure with undisturbed disulfide linkages. Our analyses have mapped the YM1B recognition region to domain IV of CCN1, likely in proximity to the DM site. In breast cancer cells, CCN1 can induce actin reorganization, formation of lamellipodia, and cell migration/invasion through the αV integrins/Rac1/ERK signaling axis; these CCN1-dependent activities can be effectively suppressed by YM1B. Our results also suggest that YM1B may exert its CCN1-blocking effect by perturbing the interaction of CCN1 with vitronectin and fibronectin, which are ligands of αV integrins and instrumental for integrin activation. This CCN1-specific mAb may open a new potential avenue for therapeutic intervention of breast cancer progression.

The matricellular protein CCN1 suppresses lung cancer cell growth by inducing senescence via the p53/p21 pathway.

CCN1, a secreted matrix-associated molecule, is involved in multiple cellular processes. Previous studies have indicated that expression of CCN1 correlates inversely with the aggressiveness of non-small-cell lung carcinoma (NSCLC); however, the underlying mechanisms remain elusive. Using three NSCLC cell line systems, here we show that long-term treatment of cells with the recombinant CCN1 protein led to a permanent cell cycle arrest in G1 phase; cells remained viable as judged by apoptotic assays. CCN1-treated NSCLC cells acquired a phenotype characteristic of senescent cells, including an enlarged and flattened cell shape and expression of the senescence-associated ß-galactosidase. Immunoblot analysis showed that addition of CCN1 increased the abundance of hypo-phosphorylated Rb, as well as accumulation of p53 and p21. Silencing the expression of p53 or p21 by lentivirus-mediated shRNA production in cells blocked the CCN1-induced senescence. Furthermore, a CCN1 mutant defective for binding integrin α6ß1 and co-receptor heparan sulfate proteoglycans was incapable of senescence induction. Our finding that direct addition of CCN1 induces senescence in NSCLC cells provides a potential novel strategy for therapeutic intervention of lung cancers.

Latent membrane protein 1 of Epstein-Barr virus regulates death-associated protein kinase 1 in lymphoblastoid cell line.

The Epstein-Barr virus (EBV) infects and transforms primary B cells into lymphoblastoid cell lines (LCLs). We observed death-associated protein kinase 1 (DAPK1) upregulation in B cells following EBV infection and high DAPK1 levels in LCLs. DAPK1 participates in several apoptosis-inducing pathways, yet DAPK1 expression increased during B cell transformation. Data from LMP1 overexpression in LCLs and HeLa cells and from knocked down LMP1 in LCLs suggest LMP1 regulation of DAPK1 expression. We observed NF-κB signaling in DAPK1 upregulation by LMP1 with CTAR deletion mutants failing to induce DAPK1 expression and with Bay11 blocking DAPK1 expression. DAPK1 is inactive in LCLs due to insufficient stimuli, and is not regulated by Ser308 phosphorylation. However, DAPK1 in LCLs is functional, as evidenced by its quick mediation of cell death following UV or H(2)O(2) exposure, and increased survival among LCLs knocked down with DAPK. DAPK roles in EBV-infected B cells remain to be identified.

Nucleotide-binding domain of phosphoglycerate kinase 1 reduces tumor growth by suppressing COX-2 expression.

Phosphoglycerate kinase 1 (PGK-1) is a multifunctional protein that is involved in the glycolytic pathway and the generation of the angiogenesis inhibitor angiostatin. In a previous study, we showed that the overexpression of full-length PGK-1 in Lewis lung carcinoma (LLC-1) can reduce tumor growth in vivo by downregulation of COX-2 expression. Phosphoglycerate kinase 1 has two functional domains: a catalytic domain (CD); and a nucleotide-binding domain (NBD). To identify the functional domain of PGK-1 responsible for its antitumor effects, we evaluated the tumorigenicity of LLC-1 cells overexpressing full-length PGK-1 (LLC-1/PGK), CD (LLC-1/CD), and NBD (LLC-1/NBD). Although no difference in tumor cell growth was observed in vitro, the tumor invasiveness was reduced in the LLC-1/PGK, LLC-1/CD, and LLC-1/NBD cells compared to parental LLC-1 cells in vivo. In addition, in vivo tumor growth retardation by LLC-1/CD and LLC-1/NBD cells was observed, similar to that by LLC-1/PGK cells. However, the reduced stability of COX-2 mRNA and downregulation of the COX-2 protein and its metabolite, prostaglandin E2, was only found in LLC-1/PGK and LLC-1/NBD cells. Low levels of COX-2 were also observed in the tumor mass formed by the modified cells when injected into mice. The results indicate that COX-2 suppression by PGK-1 is independent of its catalytic activity. COX-2 targeting by PGK-1 can be attributed to its NBD and is probably a result of the destabilization of COX-2 gene transcripts brought about by the mRNA-binding property of PGK-1.

IL-27 directly restrains lung tumorigenicity by suppressing cyclooxygenase-2-mediated activities.

Gene transfer of IL-27 to tumor cells has been proven to inhibit tumor growth in vivo by antiproliferation, antiangiogenesis, and stimulation of immunoprotection. To investigate the nonimmune mechanism of IL-27 that suppresses lung cancer growth, we have established a single-chain IL-27-transduced murine Lewis lung carcinoma (LLC-1) cell line (LLC-1/scIL-27) to evaluate its tumorigenic potential in vivo. Mice inoculated with LLC/scIL-27 displayed retardation of tumor growth. Production of IL-12, IFN-gamma, and cytotoxic T cell activity against LLC-1 was manifest in LLC/scIL-27-injected mice. Of note, LLC-1/scIL-27 exhibited decreased expression of cyclooxygenase-2 (COX-2) and PGE(2). On the cellular level, the LLC/scIL-27 transfectants had reduced malignancy, including down-regulation of vimentin expression and reduction of cellular migration and invasion. The suppression of tumorigenesis by IL-27 on lung cancer cells was further confirmed by the treatment with rIL-27 on the murine LLC-1 and human non-small cell lung carcinoma (NSCLC) cell lines. PGE(2)-induced vimentin expression, movement, and invasiveness were also suppressed by the treatment with rIL-27. Our data show that IL-27 not only suppresses expression of COX-2 and PGE(2) but also decreases the levels of vimentin and the abilities of cellular migration and invasion. Furthermore, inoculation of LLC/scIL-27 into immunodeficient NOD/SCID mice also exhibited reduced tumor growth. Our data indicate that IL-27-induced nonimmune responses can contribute to significant antitumor effects. Taken together, the results suggest that IL-27 may serve as an effective agent for lung cancer therapy in the future.

Anti-ribosomal phosphoprotein autoantibody triggers interleukin-10 overproduction via phosphatidylinositol 3-kinase-dependent signalling pathways in lipopolysaccharide-activated macrophages.

Anti-ribosomal phosphoprotein autoantibodies have been shown to be significantly associated with multiple manifestations of systemic lupus erythematosus (SLE). High levels of interleukin-10 (IL-10) have been demonstrated to contribute to lupus susceptibility and severity. In this study, we investigated the molecular mechanisms of anti-ribosomal phosphoprotein monoclonal antibody (anti-P mAb)-induced autoimmune responses. Anti-P mAb promoted IL-10 overproduction in a dose- and time-dependent manner in both lipopolysaccharide (LPS)-activated RAW 264.7 cells and primary human macrophages. Anti-P mAb enhanced phosphorylation of Akt (PKB; protein kinase B), extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 1/2 (JNK1/2), while phosphorylation of p38 remained unaltered. Furthermore, anti-P mAb decreased glycogen synthase kinase 3 (GSK3) activity and reduced the phosphorylation of I kappaB alpha in LPS-activated macrophages. The Syk, phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), JNK and ERK signalling pathways involved in anti-P mAb-triggered IL-10 secretion were also confirmed using various pharmacological inhibitors. In addition, nuclear factor (NF)-kappaB had negative regulatory effects on anti-P mAb-triggered IL-10 secretion. Using reporter plasmids containing the nuclear factor binding sites of NF-kappaB, cAMP-enhanced activation protein 1 (AP-1), serum response element (SRE) or cyclic AMP response element (CRE), treatment of anti-P mAb led to activation of the corresponding factors that bind to the AP-1 site, SRE and CRE in the LPS-activated macrophages. Furthermore, by transfection with reporter plasmids bearing various lengths of the IL-10 promoter, the AP-1 binding site, SRE and CRE were shown to be required for anti-P mAb-induced effects. Collectively, our results provide a molecular model for anti-P mAb-induced IL-10 overproduction in LPS-activated macrophages, which may play a role in the pathogenesis of SLE.

Combination of Fasl and GM-CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma.

Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte-macrophage colony stimulating factor (GM-CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor-specific response. To investigate whether the combination of FasL and GM-CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC-1) cells that are transduced with GM-CSF (LLC/GM-CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM-CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM-CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/FasL or LLC/FasL/GM-CSF develop tumors. Specific memory immune response and delayed LLC-1 tumor growth are found in mice immunized with LLC-1/FasL or LLC-1/FasL/GM-CSF. Furthermore, therapeutic effects are observed only when LLC-1/FasL/GM-CSF tumor vaccine is employed to retard growth of preexisting LLC-1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC-1 and LLC-1/FasL/GM-CSF. In addition, IL-12 production, cytotoxic T-cell activity and IgG against LLC-1 are manifested in mice injected with LLC/FasL/GM-CSF. Our data show that FasL-induced pathway triggers expression of proinflammatory cytokines, including IL-1 beta, IL-6, MIP-2 and MCP-1, while GM-CSF-dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene-based delivery with FasL and GM-CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo.

Integrin-dependent functions of the angiogenic inducer NOV (CCN3): implication in wound healing.

The novel angiogenic inducer CCN3 (NOV, nephroblastoma overexpressed) 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). CCN3 is broadly expressed in derivatives of all three germ layers during mammalian development, and its deranged expression is associated with vascular injury and a broad range of tumors. We have shown that CCN3 promotes proangiogenic activities in vascular endothelial cells through integrin receptors and induces neovascularization in vivo (Lin, C. G., Leu, S. J., Chen, N., Tebeau, C. M., Lin, S. X., Yeung, C. Y., and Lau, L. F. (2003) J. Biol. Chem. 278, 24200-24208). In this study, we show that CCN3 is highly expressed in granulation tissue of cutaneous wounds 5-7 days after injury and is capable of inducing responses in primary fibroblasts consistent with wound healing. Purified CCN3 supports primary skin fibroblast adhesion through integrins alpha(5)beta(1) and alpha(6)beta(1) and induces fibroblast chemotaxis through integrin alpha(v)beta(5). We show that CCN3 is a novel ligand of alpha(v)beta(5) in a solid phase binding assay. Although not mitogenic on its own, CCN3 also enhances basic fibroblast growth factor-induced DNA synthesis. Furthermore, CCN3 up-regulates MMP-1 and PAI-1 expression but interacts with TGF-beta1 in an antagonistic or synergistic manner to regulate the expression of specific genes. These findings, together with its angiogenic activity, support a role for CCN3 in cutaneous wound healing in skin fibroblasts and establish its matricellular mode of action through integrin receptors.

Targeted mutagenesis of the angiogenic protein CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-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 alpha6beta1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha6beta1 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 alpha6beta1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha6beta1 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 alpha6beta1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alphavbeta3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha6beta1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha6beta1-HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha6beta1-HSPG-specific activities in vivo.

Identification of a novel integrin alphavbeta3 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 alphavbeta3 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 alphavbeta3. Immobilized synthetic V2 peptide supports alphavbeta3-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 alphavbeta3 and abolished alphavbeta3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alphavbeta3 and impaired in alphavbeta3-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 alphavbeta3 and supports alphavbeta3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alphavbeta3 binding site in soluble CCN1, whereas additional cryptic alphavbeta3 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 alphavbeta3 and provide a new approach for dissecting alphavbeta3-specific CCN1 functions both in cultured cells and in the organism.

Identification of a novel integrin alpha 6 beta 1 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 alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 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 alpha 6 beta 1. 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-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 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 alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.

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, Wilm's tumors, Ewing's 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 alphavbeta3, alpha5beta1, alpha6beta1, and heparan sulfate proteoglycans. In contrast, CCN3-induced cell migration is dependent on integrins alphavbeta3 and alpha5beta1, whereas alpha6beta1 does not play a role in this process. Although CCN3 does not contain a RGD sequence, it binds directly to immobilized integrins alphavbeta3 and alpha5beta1, 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 alphavbeta3 and alpha5beta1, acts directly upon endothelial cells to stimulate pro-angiogenic activities, and induces angiogenesis in vivo.

Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells.

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.