The Roles of CCN1/CYR61 in Pulmonary Diseases.

CCN1 (cysteine-rich 61, connective tissue growth factor, and nephroblastoma-1), previously named CYR61 (cysteine-rich angiogenic inducer 61) belongs to the CCN family of matricellular proteins. CCN1 plays critical roles in the regulation of proliferation, differentiation, apoptosis, angiogenesis, and fibrosis. Recent studies have extensively characterized the important physiological and pathological roles of CCN1 in various tissues and organs. In this review, we summarize both basic and clinical aspects of CCN1 in pulmonary diseases, including acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), lung fibrosis, pulmonary arterial hypertension (PAH), lung infection, and lung cancer. We also emphasize the important challenges for future investigations to better understand the CCN1 and its role in physiology and pathology, as well as the questions that need to be addressed for the therapeutic development of CCN1 antagonists in various lung diseases.

CCN1/Cyr61 Is Required in Osteoblasts for Responsiveness to the Anabolic Activity of PTH.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvß3 and/or αvß5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvß3/ß5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvß3/ß5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

CCN1/CYR61-mediated meticulous patrolling by Ly6Clow monocytes fuels vascular inflammation.

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state, but their role in inflammation is still unclear. Here, using confocal intravital microscopy, we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins, platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets, Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level, we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation.

Evaluation of extracellular matrix protein CCN1 as a prognostic factor for glioblastoma.

Recently, research efforts in identifying prognostic molecular biomarkers for malignant glioma have intensified. Cysteine-rich protein 61 (CCN1) is one of the CCN family of matricellular proteins that promotes cell growth and angiogenesis in cancers through its interaction with several integrins. In this study, we investigated the relationships among CCN1, O(6)-methylguanine-DNA methyltransferase expression, the tumor removal rate, and prognosis in 46 glioblastoma patients treated at the Okayama University Hospital. CCN1 expression was high in 31 (67 %) of these patients. The median progression-free survival (PFS) and overall survival (OS) times of patients with high CCN1 expression was significantly shorter than those of patients with low CCN1 expression (p < 0.005). In a multivariate Cox analysis, CCN1 proved to be an independent prognostic factor for patient survival [PFS, hazard ratio (HR) = 3.53 (1.55-8.01), p = 0.003 and OS, HR = 3.05 (1.35-6.87), p = 0.007]. Moreover, in the 31 patients who underwent gross total resection, the PFS and OS times of those with high CCN1 expression were significantly shorter than those with low CCN1 expression. It was concluded that CCN1 might emerge as a significant prognostic factor regarding the prognosis of glioblastoma patients.

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.

The matricellular protein Cyr61 is a key mediator of platelet-derived growth factor-induced cell migration.

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6ß1 and αvß3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

CCN1 enables Fas ligand-induced apoptosis in cardiomyoblast H9c2 cells by disrupting caspase inhibitor XIAP.

Cell proliferation from pre-existing cardiomyocytes is a major source of cells for normal mammalian myocardial renewal or for regeneration after myocardial injury. These proliferative cardiomyocytes may act differently from the postmitotic cardiomyocytes in a stressed heart. Extracellular matrix molecule CCN1 is produced to promote Fas ligand (FasL)-induced cardiomyocyte apoptosis in mice with stress-induced cardiac injury. We aimed to investigate the effect of CCN1 on the proliferative cardiomyocytes. We used rat embryonic cardiomyoblast H9c2 cells to study the cardiotoxicity of CCN1. We found that FasL dose-dependently increased the X-linked inhibitor of apoptosis protein (XIAP) levels to prevent the progression of apoptosis in H9c2 cells. CCN1, though it did not induce apoptosis by itself, sensitized H9c2 cells to FasL-induced apoptosis. CCN1 functions by engaging its cell-surface receptor integrin α6ß1 and elevating reactive oxygen species levels, which leads to mitogen-activated protein kinase p38 activation, cytosolic Bax translocation to mitochondria, and the release of mitochondrial Smac and HtrA2 to cytosol. These elevated cytosolic Smac and HtrA2 dismantle the inhibition of XIAP, thereby facilitating the activation of caspase-3 and the apoptosis-induced by FasL. In summary, we demonstrated a novel mechanism underlying the resistance of cardiomyoblasts to FasL-induced apoptosis, and the pro-apoptotic function of CCN1 by disrupting this resistance.

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-ß signaling.

UNLABELLED: Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-ß signaling by scavenging TGF-ß thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. CONCLUSION: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

A novel p53/microRNA-22/Cyr61 axis in synovial cells regulates inflammation in rheumatoid arthritis.

OBJECTIVE: We previously showed that Cyr61 acts to promote fibroblast-like synoviocyte (FLS) proliferation and Th17 cell differentiation, suggesting that Cyr61 plays an important role in mediating the joint inflammation and damage in rheumatoid arthritis (RA). The aim of this study was to investigate whether Cyr61 expression is regulated at the posttranscription level, and if so, how this regulation connects to other etiologic factors in RA. METHODS: Expression of microRNA-22 (miR-22) in synovial tissue was detected by real-time polymerase chain reaction (PCR) using miRNA-specific TaqMan MGB probes. MicroRNA-22 promoter activity was analyzed using a Dual-Luciferase Reporter Assay. Cytokine expression was measured by enzyme-linked immunosorbent assay, and the expression of other factors was measured by real-time PCR or Western blotting. RESULTS: MicroRNA-22 directly targeted the 3'-untranslated region of Cyr61 messenger RNA and inhibited Cyr61 expression. Expression of miR-22 was down-regulated and was negatively correlated with Cyr61 expression in RA synovial tissue. Furthermore, wild-type p53 activated miR-22 transcription by binding to the promoter region of the miR-22 gene, while the mutant forms of p53 frequently found in RA synovial tissue were shown to have lost the ability to activate miR-22 expression. As a result, miR-22 was down-regulated, contributing to the overexpression of Cyr61 in RA FLS. CONCLUSION: Our results not only reveal a novel mechanism whereby p53 is involved in the posttranscriptional regulation of Cyr61 expression via miRNA-22, but also provide a molecular explanation for the role of somatic mutations of p53, which are frequently observed in RA synovial tissue, in the etiology of this autoimmune disease.

Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1ß in chronically sun-exposed human skin.

Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1ß (IL-1ß). CCN1 and IL-1ß are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1ß, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1ß actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1ß by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1ß and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.

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.

Role of ß-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

Extracellular matrix protein CCN1 regulates cardiomyocyte apoptosis in mice with stress-induced cardiac injury.

AIMS: Expression of extracellular matrix protein CCN1 is induced in end-stage ischaemic cardiomyopathy in humans, and after cardiac ischaemia and reperfusion in experimental animal models. Despite its well-documented angiogenic activities, CCN1 increases the cytotoxicities of the tumour necrosis factor family cytokines, which promotes apoptosis in fibroblasts. We aimed to determine the physiological function of CCN1 in an injured heart. METHODS AND RESULTS: To assess the function of CCN1 in vivo, knock-in mice carrying the apoptosis-defective mutant allele Ccn1-dm were tested in an isoproterenol (ISO)-induced myocardial injury model (100 mg/kg/day of sc injected ISO for 5 days). Compared with wild-type mice, Ccn1(dm/dm) mice were remarkably resistant to ISO-induced cardiac injury; they showed no post-treatment cardiomyocyte apoptosis or myocardial tissue damage. ISO cardiotoxicity was dependent on Fas ligand (FasL) and its downstream signalling. Using primary cultures of cardiomyocytes isolated from rats, we demonstrated that CCN1 sensitized FasL-mediated apoptosis by engaging its cell-surface receptor integrin α6ß1 and up-regulating intracellular reactive oxygen species (ROS), which activated mitogen-activated protein kinase p38, and increased cell-surface Fas expression. CONCLUSION: CCN1 is a critical pathophysiological regulator that mediates cardiomyocyte apoptosis during work-overload-induced cardiac injury. CCN1 increases cellular susceptibility to Fas-induced apoptosis by increasing ROS and cell-surface Fas expression.

Cyr61, a matricellular protein, is needed for dendritic arborization of hippocampal neurons.

The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a ß1-integrin-dependent manner.

The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis.

CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvß3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance.

Inverse expression of cystein-rich 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) in borderline tumors and carcinomas of the ovary.

Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.

NUR77 inhibits the expression of TIMP2 and increases the migration and invasion of HTR-8/SVneo cells induced by CYR61.

OBJECTIVE: To investigate the function and mechanism of CYR61 on the migration and invasion of the trophoblast cell line, HTR-8/SVneo cells. STUDY DESIGN: The mRNA and protein levels of NUR77 in the placentas of normal and preeclampsia (PE) women were evaluated using real-time PCR and Western blot, respectively. Paraffin-embedded tissues were processed for localization of NUR77 protein in placental villus by immunohistochemistry. HTR-8/SVneo cells were cultured in the presence of CYR61, Ad-NUR77 or a small interfering RNA for NUR77 (Ad-sinur77). The expression of NUR77 in the HTR-8/SVneo cells was detected and the effects of CYR61 on the migration and invasion of HTR-8/SVneo cells were assessed in wound-healing and transwell experiments, respectively. Gelatin zymography was used to measure the MMP2 release in HTR-8/SVneo cells. RESULTS: NUR77 is significantly decreased in the placenta of women with PE compared with the levels during a normal pregnancy. CYR61 can significantly increase the expression of NUR77 in HTR-8/SVneo cells. CYR61, as well as NUR77, can promote HTR-8/SVneo cells migration and invasion, which can be blocked by Ad-sinur77. Both CYR61 and Ad-nur77 reduced the mRNA expression of TIMP2 in HTR-8/SVneo cells. CONCLUSIONS: CYR61 may promote HTR-8/SVneo cells migration and invasion through the upregulation of NUR77, leading to the increase of MMP2 release and the downregulation of TIMP2 expression.

Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis.

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvß5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.