CCN1 induces hepatic ductular reaction through integrin αvß5-mediated activation of NF-κB.

Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvß5/αvß3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvß5/αvß3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvß5/αvß3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvß5/NF-κB/JAG1 as a critical axis for biliary injury repair.

Matricellular protein CCN1 promotes regression of liver fibrosis through induction of cellular senescence in hepatic myofibroblasts.

Liver fibrosis occurs as a wound-healing response to chronic hepatic injuries irrespective of the underlying etiology and may progress to life-threatening cirrhosis. Here we show that CCN1, a matricellular protein of the CCN (CYR61/CTGF/NOV) family, is accumulated in hepatocytes of human cirrhotic livers. CCN1 is not required for liver development or regeneration, since these processes are normal in mice with hepatocyte-specific Ccn1 deletion. However, Ccn1 expression is upregulated upon liver injuries and functions to inhibit liver fibrogenesis induced by either carbon tetrachloride intoxication or bile duct ligation and promote fibrosis regression. CCN1 acts by triggering cellular senescence in activated hepatic stellate cells and portal fibroblasts by engaging integrin α6ß1 to induce reactive oxygen species accumulation through the RAC1-NADPH oxidase 1 enzyme complex, whereupon the senescent cells express an antifibrosis genetic program. Mice with hepatocyte-specific Ccn1 deletion suffer exacerbated fibrosis with a concomitant deficit in cellular senescence, whereas overexpression of hepatic Ccn1 reduces liver fibrosis with enhanced senescence. Furthermore, tail vein delivery of purified CCN1 protein accelerates fibrosis regression in mice with established fibrosis. These findings reveal a novel integrin-dependent mechanism of fibrosis resolution in chronic liver injury and identify the CCN1 signaling pathway as a potential target for therapeutic intervention.

TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release.

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)ß(5), α(6)ß(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.

Matricellular protein CCN1 activates a proinflammatory genetic program in murine macrophages.

CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NF-kappaB activity and enhancing the apoptotic activity of Fas ligand and TRAIL. In this paper, we show that CCN1 supports the adhesion of macrophages through integrin alpha(M)beta(2) and syndecan-4, activates NFkappaB-mediated transcription, and induces a proinflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-12b), chemokines (MIP-1alpha; MCP-3; growth-related oncogenes 1 and 2; and inflammatory protein 10), and regulators of oxidative stress and complement (inducible NO synthase and C3) and downregulation of specific receptors (TLR4 and IL-10Rbeta) and anti-inflammatory factors (TGF-beta1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NF-kappaB by CCN1, leading to the synthesis of cytokines including TNF-alpha and inflammatory protein 10; and a delayed response resulting from CCN1-induced TNF-alpha, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1beta and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates proinflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation.

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines.

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFalpha is an apoptotic inducer in some cancer cells, it activates NFkappaB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFkappaB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFalpha, thus converting TNFalpha from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTalpha, FasL, and TRAIL. Mechanistically, CCNs function through integrin alpha(6)beta(1) and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding alpha(6)beta(1)-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an alpha(6)beta(1)-HSPG-binding defective CCN1 are blunted in TNFalpha- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFalpha and related cytokines.

Matrix protein CCN1 is critical for prostate carcinoma cell proliferation and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins alphavbeta3 and alpha6beta4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Calpha-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy.

Fas-mediated apoptosis is regulated by the extracellular matrix protein CCN1 (CYR61) in vitro and in vivo.

Although Fas ligand (FasL) is primarily expressed by lymphoid cells, its receptor Fas (CD95/Apo-1) is broadly expressed in numerous nonlymphoid tissues and can mediate apoptosis of parenchymal cells upon injury and infiltration of inflammatory cells. Here we show that CCN1 (CYR61) and CCN2 (CTGF), matricellular proteins upregulated at sites of inflammation and wound repair, synergize with FasL to induce apoptosis by elevating cellular levels of reactive oxygen species (ROS). CCN1 acts through engagement of integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, leading to ROS-dependent hyperactivation of p38 mitogen-activated protein kinase in the presence of FasL to enhance mitochondrial cytochrome c release. We show that CCN1 activates neutral sphingomyelinase, which functions as a key source of CCN1-induced ROS critical for synergism with FasL. Furthermore, Fas-dependent hepatic apoptosis induced by an agonistic monoclonal anti-Fas antibody or intragastric administration of alcohol is severely blunted in knock-in mice expressing an apoptosis-defective Ccn1 allele. These results demonstrate that CCN1 is a physiologic regulator of Fas-mediated apoptosis and that the extracellular matrix microenvironment can modulate Fas-dependent apoptosis through CCN1 expression.

Functions and mechanisms of action of CCN matricellular proteins.

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.

Cytotoxicity of TNFalpha 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 TNFalpha is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFkappaB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFalpha without perturbation of NFkappaB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins alpha(v)beta(5), alpha(6)beta(1), 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 TNFalpha-induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFalpha cytotoxicity, providing the contextual cues from the extracellular matrix for TNFalpha-mediated cell death.

The matrix protein CCN1 (CYR61) induces apoptosis in fibroblasts.

Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin alpha6beta1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type-specific manner and that integrin alpha6beta1-HSPGs can function as receptors to induce p53-dependent apoptosis.

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 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.

CYR61 (CCN1) is essential for placental development and vascular integrity.

CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.