Cyr61 Protein Research Articles

Expression of Cyr61, CTGF, and WISP-1 Correlates with Clinical Features of Lung Cancer

BackgroundCCN family, comprising six members (Cyr61, CTGF, Nov, WISP-1, WISP-2, WISP-3), is involved in the stimulation of cell proliferation, migration, adhesion, angiogenesis, and tumorigenesis. Several studies have shown that expression of Cyr61, CTGF, and WISP-1 affects the tumorigenic potential of lung cancer cells in vitro. However, the correlation of expression of CCN family proteins and clinical features of lung cancer remains unknown.Methodology and Principal FindingsIn the present work, we quantified the mRNA levels of Cyr61, CTGF, and WISP-1 in samples from 60 primary lung cancers and their matched normal lung tissues by quantitative real-time PCR assay. Downregulation of the Cyr61 and CTGF genes and upregulation of the WISP-1 gene were found in primary lung cancers compared to the paired normal lung tissues. Immunohistochemistry analysis also disclosed a similar expression pattern of Cyr61, CTGF, and WISP-1 protein in paired lung cancer tissues. Statistical analysis revealed significant associations between expression of either Cyr61 or CTGF with tumor stage, tumor histology, metastasis, smoking, and family history at diagnosis. A significant correlation also existed between WISP-1 expression with tumor histology, and patient age. Moreover, expression levels of Cyr61 and CTGF correlated with survival of the lung-cancer patients.ConclusionsOur results suggest that Cyr61, CTGF, and WISP-1 might be implicated in the development and progression of primary lung cancers, and their levels might serve as valuable prognostic markers, as well as potential targets for therapeutic intervention.

Cyr61 is upregulated in pre‐osteoclastic cells by low level cadmium exposure

Low-level cadmium exposure causes bone loss in vitro and in vivo as a result of increased osteoclastic activity. A microarray analysis of bone cell RNA indicated that the CCN protein, Cyr61, was the most upregulated gene 2 and 4 hours after cadmium exposure in vivo. Upregulation of Cyr61 in osteoblasts was hypothesized to signal osteoclast aggregation and fusion mediated by the αVβ3 integrin. Cadmium-induced Cyr61 expression was investigated in the osteoblastic cell line, 7F2 and a pre-osteoclastic cell line, RAW264.7. RNA and protein were isolated from cells after exposure to 100 nM cadmium. Beta-estradiol and 1,25 vitamin D were used as positive controls for Cyr61 expression in osteoblastic cells. RT-PCR and semi-quantitative PCR was performed using Cyr61 and GAPDH primers. Preliminary results of PCR and Western blotting show distinct Cyr61 RNA and protein expression in RAW 264.7 cells after cadmium exposure. Little cadmium-induced Cyr61 expression was observed in 7F2 cells. These data confirm immunohistochemical staining data from our lab that pre-osteoclasts expressed Cyr61 10 hrs after cadmium exposure whereas cadmium did not induce Cyr61 protein expression in another osteoblastic cell line. These data suggest an independent function for Cyr61 in the aggregation of osteoclasts without the contribution of osteoblastic signaling. Supported by the APS Undergraduate Summer Research Fellowship.

IGFBP-3 and IGFBP-10 (CYR61) up-regulation during the development of Barrett's oesophagus and associated oesophageal adenocarcinoma: potential biomarkers of disease risk

Dys-regulation of the insulin-like growth factor (IGF) system increases the risk of a number of malignancies. The aim of this study was to investigate the role of members of the IGF binding protein (IGFBP) superfamily in the development of oesophageal adenocarcinoma (EAC) and their possible use as markers of disease risk. Expression of IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 was assessed using Real-Time-polymerase chain reaction (PCR) and immunohistochemistry in oesophageal tissues from Barrett's oesophagus (BE) patients with and without associated EAC, and in control subjects. IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 mRNA levels were up-regulated in Barrett's (n=17) and tumour tissue of EAC patients (n=18) compared with normal tissue of control subjects without BE or EAC (n=18) (p<0.001). Over-expression of IGFBP-3 and IGFBP-10/CYR61 proteins was observed in Barrett's, dysplastic and tumour tissue of EAC cases (n=47 for IGFBP-10; n=39 for IGFBP-3) compared with adjacent normal epithelium (p<0.050). Notably, IGFBP-3, IGFBP-4, and IGFBP-10/CYR61 expression in Barrett's tissue of EAC cases (n=17) was significantly (p<0.001) higher than in Barrett's tissue of BE patients with no sign of progression to cancer (n=15). Overall, the results suggest that members of the IGFBP superfamily are up-regulated during oesophageal carcinogenesis and merit further investigation as markers of EAC risk.

CYR61 (CCN1) Protein Expression during Fracture Healing in an Ovine Tibial Model and Its Relation to the Mechanical Fixation Stability

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

Cyr61, a Member of the CCN Family, is Up-Regulated in Endometrium From Women With Polycystic Ovarian Syndrome Compared to Normal Controls

ObjectiveTo evaluate the temporal and spatial distibution of Cyr61 in normal endometrium throughout the menstrual cycle and compare its expression in endometrium from women with polycystic ovarian syndrome (PCOS). We also studied the regulation of Cyr61 in the well-differentiated, hormone-responsive endometrial cell line, ECC1.DesignThis is a retrospective study on 21 endometrial samples from the proliferative and secretory phase of the menstrual cycle in normal women and women with hyperandrogenism and PCOS. Regulation of Cyr61 was performed in vitro using the well-differentiated endometrial cell line, ECC1.Materials and methodsWestern blot analysis of Cyr61 was performed on protein lysates of untreated ECC1 cells and cells treated for 6 and 12 hours with diethylstilbestrol (DES; 10-8M) in the presence or absence of the anti-estrogen ICI 182,780. Immunohistochemistry on endometrial samples and western blot analysis was performed using the specific Cyr61 polyclonal antibody (generously provided by Dr. Lester Lau, UIC, Chicago, IL). Additional staining was performed for the estrogen receptor alpha (ERa) and a marker of cell proliferation, Ki67. Twenty-one endometrial biopsies were obtained from both fertile and PCOS women during the menstrual cycle. Statistical analysis was performed using ANOVA with Bonferroni correction with significance at the 95% confidence interval (p < 0.05).ResultsConclusionThe Cyr61 protein is a sensitive marker of estrogen activity and is significantly increased in the endometrium of women with PCOS in both the proliferative and secretory phase. Further studies will be required to investigate the mechanism for this over-expression of Cyr61, but evidence suggests that failure of down-regulation of estrogen receptors in the secretory phase may play a role. ObjectiveTo evaluate the temporal and spatial distibution of Cyr61 in normal endometrium throughout the menstrual cycle and compare its expression in endometrium from women with polycystic ovarian syndrome (PCOS). We also studied the regulation of Cyr61 in the well-differentiated, hormone-responsive endometrial cell line, ECC1. To evaluate the temporal and spatial distibution of Cyr61 in normal endometrium throughout the menstrual cycle and compare its expression in endometrium from women with polycystic ovarian syndrome (PCOS). We also studied the regulation of Cyr61 in the well-differentiated, hormone-responsive endometrial cell line, ECC1. DesignThis is a retrospective study on 21 endometrial samples from the proliferative and secretory phase of the menstrual cycle in normal women and women with hyperandrogenism and PCOS. Regulation of Cyr61 was performed in vitro using the well-differentiated endometrial cell line, ECC1. This is a retrospective study on 21 endometrial samples from the proliferative and secretory phase of the menstrual cycle in normal women and women with hyperandrogenism and PCOS. Regulation of Cyr61 was performed in vitro using the well-differentiated endometrial cell line, ECC1. Materials and methodsWestern blot analysis of Cyr61 was performed on protein lysates of untreated ECC1 cells and cells treated for 6 and 12 hours with diethylstilbestrol (DES; 10-8M) in the presence or absence of the anti-estrogen ICI 182,780. Immunohistochemistry on endometrial samples and western blot analysis was performed using the specific Cyr61 polyclonal antibody (generously provided by Dr. Lester Lau, UIC, Chicago, IL). Additional staining was performed for the estrogen receptor alpha (ERa) and a marker of cell proliferation, Ki67. Twenty-one endometrial biopsies were obtained from both fertile and PCOS women during the menstrual cycle. Statistical analysis was performed using ANOVA with Bonferroni correction with significance at the 95% confidence interval (p < 0.05). Western blot analysis of Cyr61 was performed on protein lysates of untreated ECC1 cells and cells treated for 6 and 12 hours with diethylstilbestrol (DES; 10-8M) in the presence or absence of the anti-estrogen ICI 182,780. Immunohistochemistry on endometrial samples and western blot analysis was performed using the specific Cyr61 polyclonal antibody (generously provided by Dr. Lester Lau, UIC, Chicago, IL). Additional staining was performed for the estrogen receptor alpha (ERa) and a marker of cell proliferation, Ki67. Twenty-one endometrial biopsies were obtained from both fertile and PCOS women during the menstrual cycle. Statistical analysis was performed using ANOVA with Bonferroni correction with significance at the 95% confidence interval (p < 0.05). Results ConclusionThe Cyr61 protein is a sensitive marker of estrogen activity and is significantly increased in the endometrium of women with PCOS in both the proliferative and secretory phase. Further studies will be required to investigate the mechanism for this over-expression of Cyr61, but evidence suggests that failure of down-regulation of estrogen receptors in the secretory phase may play a role. The Cyr61 protein is a sensitive marker of estrogen activity and is significantly increased in the endometrium of women with PCOS in both the proliferative and secretory phase. Further studies will be required to investigate the mechanism for this over-expression of Cyr61, but evidence suggests that failure of down-regulation of estrogen receptors in the secretory phase may play a role.

Proteomic identification of differentially‐expressed genes in human gastric carcinomas

Although genetic alterations in proto-oncogenes, tumor-suppressor genes, cell cycle regulators, and cell growth factors have been implicated in the process of human gastric carcinogenesis, the principle carcinogenic mechanisms are not fully understood. In this study, we used a proteomic approach to search for genes that may be involved in gastric carcinogenesis and that might serve as diagnostic markers. We identified nine proteins with increased expression and 13 proteins with decreased expression in gastric carcinomas. The two most notable groups included proteins involved in mitotic checkpoint (MAD1L1 and EB1) and mitochondrial functions (CLPP, COX5A, and ECH1). This suggested that there are links between dysfunctions in these processes and gastric carcinogenesis. We also observed the differential expression of HSP27 and CYR61 proteins in gastric carcinoma, whose expression is known to be altered in other types of tumors. Furthermore, the study identified proteins whose function in gastric carcinomas was previously unsuspected and that may serve as new molecular markers for gastric carcinomas. Importantly, immunohistochemical analyses confirmed that the levels of expression of MAD1L1, HSP27, and CYR61 were altered in gastric carcinoma tissues. Therefore, our study suggested not only that the proteins identified in this study can be useful diagnostic markers but also that a proteomics-based approach is useful for developing a more complete picture of the pathogenesis and function of gastric carcinomas.

Increased expression of Cyr61 (CCN1) identified in peritoneal metastases from human pancreatic cancer

Identification of extracellular matrix proteins (ECM) associated with tumor cell metastasis may generate targets for future therapy against pancreatic cancer metastases. We hypothesized that comparison of ECM-associated gene expression in primary and metastatic pancreatic tumors would identify ECM proteins associated with pancreatic metastasis. A clinically relevant model of pancreatic cancer was used to generate RNA from primary and metastatic tumors; it was evaluated by microarray analysis with subsequent cluster analysis. Target genes (Cyr61 and integrins alpha(v) and beta(3)) identified by microarray analysis were confirmed by reverse transcription polymerase chain reaction and immunohistochemistry analysis. Peritoneal metastases at sites distant from the primary tumor were present in all animals bearing orthotopic tumors. High-density microarray comparison of gene expression in metastases versus primary pancreatic tumors identified a greater than twofold increase in the expression of Cyr61, a secreted matricellular protein that binds to integrins. Reverse transcription polymerase chain reaction confirmed the microarray results, and immunohistochemistry analysis demonstrated increased Cyr61 protein and persistent alpha(v)beta(3) expression in peritioneal metastases. Additionally, immunohistochemistry demonstrated increased collocalization of Cyr61 and alpha(v) in metastases relative to primary tumor. The ECM protein Cyr61 shows increased expression in metastatic lesions in a clinically relevant model of pancreatic adenocarcinoma. Protein analysis confirms the microarray results and collocalization of Cyr61, and alpha(v) suggests that interaction between Cyr61 and alpha(v)beta(3) promotes formation of peritoneal metastases.

Coxsackievirus B3 Infection Induces cyr61 Activation via JNK To Mediate Cell Death

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.

A Novel Mechanism of Plasmin-Induced Mitogenesis in Fibroblasts.

The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased 3H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.

Induction and function of CYR61 (CCN1) in prostatic stromal and epithelial cells: CYR61 is required for prostatic cell proliferation.

CYR61 is an extracellular matrix-associated protein that promotes adhesion, migration, and proliferation of endothelial cells and fibroblasts. Prostate enlargement, which frequently causes the urethral compression, is often histologically observed as stromal and epithelial hyperplasia in an enlarged gland. To determine whether or not CYR61 has relevance to the progression of benign prostatic hyperplasia (BPH), we investigated the induction of CYR61, and also examined its function in both prostatic stromal and epithelial cells. Recombinant CYR61 protein was used for the examination of the activity of CYR61 as to cell adhesion and proliferation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was utilized to screen for inducers of the CYR61 gene in prostatic cells. Finally, the effects of an anti-sense oligonucleotide, which could reduce the production of CYR61, on the morphology and growth of prostatic cells were also examined. Recombinant CYR61 protein promotes prostatic cell adhesion and proliferation. The mRNA for CYR61, a growth factor-inducible immediate early gene, was markedly induced by fetal bovine serum (FBS) within 1 hr, and strongly induced by transforming growth factor-beta1 (TGF-beta) for at least 19 hr following stimulation. The suppression of CYR61 production with an anti-sense oligonucleotide causes obvious morphological changes of prostatic cells. Furthermore, we have shown that CYR61 is necessary, at least in part, for FBS-induced prostatic cell proliferation, because dramatic inhibition of cellular growth was caused by the suppression of CYR61 production with the addition of the anti-sense oligonucleotide before FBS stimulation. In this study, we demonstrate that serum growth factors induce the CYR61 gene in both stromal and epithelial cells, and that CYR61 plays functional roles in cell adhesion, morphology, and proliferation, supporting its involvement in benign prostatic enlargement. These results strongly suggest that CYR61 is a key molecule, and therefore could be a potential therapeutic target in prostatic hyperplastic growth.

Increased expression of CYR61, an extracellular matrix signaling protein, in human benign prostatic hyperplasia and its regulation by lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous lipid growth factor that is thought to play important roles in cell proliferation and antiapoptosis and therefore may have roles in the development and progression of benign prostatic hyperplasia (BPH). CYR61 (CCN1), on the other hand, is a growth factor-inducible immediate early gene that functions in cell proliferation, differentiation, and extracellular matrix synthesis. Here we show the close relationship between LPA-induced expression of CYR61 and prostate enlargement. CYR61 mRNA and protein were dramatically up-regulated by 18:1 LPA (oleoyl-LPA) within 1 and 2 h, respectively, in both stromal and epithelial prostatic cells. G protein-coupled receptors, i.e. Edg-2, Edg-4, and Edg-7, for LPA were also expressed in both stromal and epithelial prostatic cells. Furthermore, on DNA microarray analysis for normal and BPH patients, CYR61 was found to be related to the development and progression of BPH, regardless of symptoms. Although CYR61 mRNA was synthesized in hyperplastic epithelial cells, in many cases of BPH, CYR61 protein was detected in both the epithelial and stromal regions of BPH patient tissues. The functional contribution of CYR61 to prostatic cell growth was demonstrated by recombinant CYR61 protein and anti-CYR61 neutralizing antibodies, which inhibited CYR61-dependent cell spreading and significantly diminished cell proliferation, respectively. In conclusion, these data support the hypothesis that LPAs induce the expression of CYR61 by activating G proteincoupled receptors and that CYR61 acts as a secreted autocrine and/or paracrine mediator in stromal and epithelial hyperplasia, demonstrating the potential importance of this signaling mechanism in the disease.

Angiogenic protein Cyr61 is expressed by podocytes in anti-Thy-1 glomerulonephritis.

Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.

Expression of Angiogenic Factor Cyr61 during Neuronal Cell Death via the Activation of c-Jun N-terminal Kinase and Serum Response Factor

The immediate early gene, cyr61, is transcriptionally activated within minutes by serum and serum growth factors. The encoded Cyr61 protein is secreted into the extracellular matrix and promotes cell adhesion and migration. In this study, we sought to examine the expression profile of cyr61 gene during neuronal cell death induced by various toxic stimuli and the mechanisms involved. Our data show that toxic stimuli, such as etoposide, significantly increased cyr61 mRNA levels in immortalized hippocampal progenitor (H19-7) cells. Cyr61 transcriptional activation was corroborated at the protein level as well. To identify the upstream signaling cascades involved in cyr61 gene induction, the blocking effect of either JNK or p38 kinase-signaling pathway on cyr61 induction in response to etoposide was tested. Transfection of the cells with a kinase-deficient mutant MEKK, an upstream activator of JNK, significantly decreased the cyr61 expression induced by etoposide. In contrast, cyr61 mRNA levels did not change after pretreatment with SB203580, the p38 kinase inhibitor. When the induction of cyr61 was tested by using several of its deleted promoters driving the expression of reporter gene, the promoter activation occurred primarily within the region containing an SRE-like CArG box. In addition, the SRF, which binds to the CArG site, was directly phosphorylated by active JNK. Furthermore, the blockade of cyr61 gene expression using an antisense encoding cyr61 sequence significantly inhibited the cell death induced by etoposide. Overall, these results suggest that the induction of the immediate early gene, cyr61, is important for neuronal cell death in the central nervous system hippocampal progenitor cells, and JNK activation, but not of p38, as well as the subsequent SRF phosphorylation are involved in cyr61 gene induction.

Early detection of cysteine rich protein 61 (CYR61, CCN1) in urine following renal ischemic reperfusion injury

Acute renal failure (ARF) has a high morbidity and mortality. Many therapies have worked in animals but were unsuccessful in clinical trials. The inability to diagnose ARF early may have impaired the success of these trials. We screened a subtraction library to search for potential disease markers that would be induced rapidly after renal injury. Mice and rats were subjected to 30 to 40 minutes of bilateral ischemia. mRNA for Cyr61, a secreted growth factor-inducible immediate early gene, was markedly up-regulated at two hours in the kidney but not other organs following renal ischemia. In situ hybridization studies suggested Cyr61 was synthesized in the proximal straight tubule. Cyr61 protein was analyzed by capture with heparin beads followed by Western blotting. Induction of Cyr61 protein could be detected in the kidney within one hour, peaked at four to eight hours, and remained elevated for at least 24 hours following ischemia. Cyr61 protein was detected in urine at three to six hours and peaked at six to nine hours after renal injury. Cyr61 was not detected after volume depletion, which is often difficult to differentiate from ARF. The secreted, cysteine-rich, heparin binding protein Cyr61 is rapidly induced in proximal straight tubules following renal ischemia, and excreted in the urine where it might serve as an early biomarker of renal injury.

Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction.

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.

The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) 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 alpha(6)beta(1) with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin alpha(6)beta(1) and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.

The Angiogenic Factor Cysteine-Rich 61 (CYR61, CCN1) Supports Vascular Smooth Muscle Cell Adhesion and Stimulates Chemotaxis through Integrin 6 1 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 induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.

Localization of Cyr61 mRNA and Protein in Prostate Cancer

Localization of Cyr61 mRNA and Protein in Prostate Cancer

The angiogenic factor Cyr61 is induced by the progestin R5020 and is necessary for mammary adenocarcinoma cell growth.

Cyr61 is a secreted pro-angiogenic factor that belongs to an emerging family of growth regulators classified as CCN (CTGF/Cyr61/NOV). Work in our laboratory has focused on sex steroid regulation of Cyr61 and its role in hormonal carcinogenesis. In this study, both Cyr61 mRNA and protein were induced by the progestin, R5020, in T47D mammary adenocarcinoma cells in a dose- and time-dependent fashion. Cyr61 gene induction by R5020 was transcriptionally regulated by progesterone receptor (PR) as the antiprogestin, RU486, and actinomycin D blocked induction completely. Moreover, Cyr61 was upregulated by epidermal growth factor (EGF) but not by R5020 in the PR-MDA-MB-431 mammary adenocarcinoma cell line, underscoring the necessity of PR. The functional significance of progestin induction of Cyr61 in breast cancer cell growth was demonstrated by anti-Cyr61 neutralizing antibodies, which diminished R5020 and EGF-dependent DNA synthesis by 30%. Moreover, anti-Cyr61 neutralizing antibodies reduced the synergistic effects of R5020 and EGF on T47D cell growth by 30%. Accordingly, protein lysates generated from stage II invasive ductal carcinomas (n = 20) were analyzed in order to determine the relevance of Cyr61 expression in the context of breast tumorigenesis. Remarkably, increased Cyr61 protein expression was observed in greater than 50% of primary breast tumor lysates that were progesterone receptor (PR)+ but estrogen receptor negative. Taken together, our data suggest that in addition to its proangiogenic activity, Cyr61 may be a novel mediator of progesterone activity in enhancing growth-factor-driven tumor growth in breast cancer.

Promoter function of the angiogenic inducer Cyr61gene in transgenic mice: tissue specificity, inducibility during wound healing, and role of the serum response element.

The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, stimulates cell migration, and enhances growth factor-induced cell proliferation. Cyr61 also promotes chondrogenic differentiation and induces neovascularization. In this study, we show that a 2-kb fragment of the Cyr61 promoter, which confers growth factor-inducible expression in cultured fibroblasts, is able to drive accurate expression of the reporter gene lacZ in transgenic mice. Thus, transgene expression was observed in the developing placenta and embryonic cardiovascular, skeletal, and central and peripheral nervous systems. The sites of transgene expression are consistent with those observed of the endogenous Cyr61 gene as determined by in situ hybridization and immunohistochemistry. The transgene expression in the cardiovascular system does not require the serum response element, a promoter sequence essential for transcriptional activation of Cyr61 by serum growth factors in cultured fibroblasts. Because the serum response element contains the CArG box, a sequence element implicated in cardiovascular-specific gene expression, the nonessential nature of this sequence for cardiovascular expression of Cyr61 is unexpected. Furthermore, the Cyr61 promoter-driven lacZ expression is inducible in granulation tissue during wound healing, as is synthesis of the endogenous Cyr61 protein, suggesting a role for Cyr61 in wound healing. Consistent with this finding, purified Cyr61 protein promotes the healing of a wounded fibroblast monolayer in culture. In addition, we mapped the mouse Cyr61 gene to the distal region of chromosome 3. Together, these results define the functional Cyr61 promoter in vivo, and suggest a role of Cyr61 in wound healing through its demonstrated angiogenic activities upon endothelial cells and its chemotactic and growth promoting activities upon fibroblasts.