Involvement of hypoxia-inducing factor-1alpha-dependent plasminogen activator inhibitor-1 up-regulation in Cyr61/CCN1-induced gastric cancer cell invasion

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.     

CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.     

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.     

Cyr61/CCN1 is a tumor suppressor in human hepatocellular carcinoma and involved in DNA damage response

Cyr61/CCN1 is a secreted extracellular matrix associated protein involved in diverse biological functions and plays multiple roles in tumorigenesis. Cyr61 was down-regulated in HCC tumor tissues as observed in our previous cDNA microarray study, but its potential role in hepatocarcinogenesis is still unclear. To explore the biological significance of Cyr61 in HCC development, over-expression of this gene was established in HCC cell lines and its effects on cell proliferation, adhesion, migration and invasion were analyzed in this study. Cyr61 expression was down-regulated in HCC tumors as measured by quantitative real-time PCR and its protein level was decreased in most HCC cell lines as detected by Western blot. Over-expression of Cyr61 in HCC cell lines suppressed cell proliferation in monolayer and anchorage-independent growth in soft agar, whereas down-regulation of Cyr61 by siRNA increased cell proliferation rate. Over-expression of Cyr61 also significantly enhanced adhesion activities of HepG2 cells to various ECM proteins. Moreover, stably transfected HepG2-Cyr61 cells showed inhibited cell mobility (40-45%) and reduced invasiveness (30-40%) compared to HepG2-Neo controls. Furthermore, upon exposure to 5-Fluorouracil and UV irradiation, Cyr61 was rapidly induced in both p53(+/+) HepG2 and p53(-/-) Hep3B cells. However, only HepG2 cells showed increased G2/M phase arrest with concomitant up-regulation in p53 and p21 levels, suggesting that Cyr61 may play an active role in regulating HCC cell growth involving p53 as well as alternative pathways. In conclusion, we demonstrated that Cyr61 is a tumor suppressor in hepatocarcinogenesis and is involved in DNA damage response.     

Cyr61 suppresses growth of human endometrial cancer cells

Cyr61 (CCN1) is a member of the CCN protein family; these secreted proteins are involved in diverse biological processes such as cell adhesion, angiogenesis, apoptosis, and either growth arrest or growth stimulation depending on the cellular context. We studied the role of Cyr61 in endometrial tumorigenesis. Levels of Cyr61 were decreased in endometrial tumors compared with normal endometrium. Knockdown of Cyr61 expression by RNA interference in a well differentiated endometrial adenocarcinoma cell line (Ishikawa) stimulated its cellular growth. Conversely, overexpression of the protein in the undifferentiated AN3CA endometrial cancer cell line decreased their growth concurrently with increased apoptosis in liquid culture. These same cells had decreased clonogenic capacity and a nearly complete loss of tumorigenicity in vivo. Furthermore, partially purified Cyr61 suppressed growth of endometrial cancer cells. The increased apoptosis in these endometrial cancer cells with forced overexpression of Cyr61 was associated with elevated expression of the pro-apoptotic proteins Bax, Bad, and TRAIL (tumor necrosis factor receptor-associated ligand). Cyr61-induced caspase-3 activation and depolarization of mitochondrial membrane. In summary, endometrial cancer cells have decreased expression of Cyr61 compared with normal endometrium, and this lowered expression may provide the transformed cells a growth advantage over their normal counterpart.     

CCN3 and calcium signaling

The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.     

Temporal and spatial expression of CCN3 during retina development

NOV/CCN3 is one of the founding members of the CCN (Cyr61 CTGF NOV) family. In the avian retina, CCN3 expression is mostly located within the central region of the inner nuclear layer. As retinal development progresses and this retinal layer differentiates and matures, CCN3 expression forms a dorsal–ventral and a central–peripheral gradient. CCN3 is produced by two glial cell types, peripapillary cells and Müller cells, as well as by horizontal, amacrine, and bipolar interneurons. In retinal neurons and Müller cell cultures, CCN3 expression is induced by activated BMP signaling, whereas Notch signaling decreases CCN3 mRNA and protein levels in Müller cells and has no effect in retinal neurons. In Müller cells, the CCN3 expression detected may thus result from a balance between the Notch and BMP signaling pathways. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

The CCN family of genes: a perspective on CCN biology and therapeutic potential

The CCN family of genes currently comprises six secreted proteins (designated CCN1-6 after Cyr61/CCN1; ctgf/CCN2; Nov/CCN3; WISP1/CCN4; WISP2/CCN5, WISP3/CCN6) with a similar mosaic primary structure. It is now well accepted that CCN proteins are not growth factors but matricellular proteins that modify signaling of other molecules, in particular those associated with the extracellular matrix. CCN proteins are involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Since their first identification as matricellular factors, the CCN proteins now figure prominently in a variety of major diseases and are now considered valid candidates for therapeutic targeting. Dissection of the molecular mechanisms governing the biological properties of these proteins is being actively pursued by an expanding network of scientists around the globe who will meet this year at the 5th International Workshop on the CCN family of Genes, organized by the International CCN Society ( http://ccnsociety.com ), home for an international cadre of collaborators working in the CCN field.     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP up-regulation

The aggressiveness of a tumor is partly attributed to its resistance to chemotherapeutic agent-induced apoptosis. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. Here we established a cell model system to examine whether stable expression of Cyr61 in MCF-7 cells can confer resistance to apoptosis and identify possible participating mechanisms. We showed that stable cell lines overexpressing Cyr61 had acquired a remarkable resistance to apoptosis induced by paclitaxel, adriamycin, and beta-lapachone. Most interesting, gel shift and reporter assays showed that the Cyr61-overexpressing cells had significantly increased NF-kappaB activity compared with neo control cells. Blockage of NF-kappaB activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-IkappaB or with an NF-kappaB decoy rendered them more susceptible to anti-cancer drugs-induced apoptosis. In addition, several NF-kappaB-regulated anti-apoptotic genes were examined, and we found that only XIAP showed a significant 3-4-fold increase in mRNA and protein in Cyr61-overexpressing cells but not in neo control cells. Treatment with inhibitor of apoptosis protein (XIAP)-specific antisense, but not sense, oligonucleotides abolished the apoptosis resistance of the Cyr61-overexpressing cells. At the same time, transfection of these stable cells with DN-IkappaB to block NF-kappaB activity also effectively reduced the elevated XIAP level. Function-neutralizing antibodies to alpha(v)beta(3) and alpha(v)beta(5) could inhibit Cyr61-mediated NF-kappaB activation as well as XIAP expression. Taken together, our data suggested that Cyr61 plays an important role in resistance to chemotherapeutic agent-induced apoptosis in human breast cancer MCF-7 cells by a mechanism involving the activation of the integrins/NF-kappaB/XIAP signaling pathway.     

Determination of a Potential Role of the CCN Family of Growth Regulators in Connexin43 Transfected C6 Glioma Cells

Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.     

Determination of a potential role of the CCN family of growth regulators in connexin43 transfected C6 glioma cells

Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.     

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.     

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.