Breast cancer. Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease.
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To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development. (+info)
Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.
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Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation. (+info)
The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts.
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Cyr61 is a heparin-binding, extracellular matrix-associated protein of the CCN family, which also includes connective tissue growth factor, Nov, WISP-1, WISP-2, and WISP-3. Cyr61 is capable of multiple functions, including induction of angiogenesis in vivo. Purified Cyr61 mediates cell adhesion and induces adhesive signaling, stimulates cell migration, enhances cell proliferation, and promotes cell survival in both fibroblasts and endothelial cells. In this study, we have used cDNA array hybridization to identify genes regulated by Cyr61 in primary human skin fibroblasts. The Cyr61-regulated genes fall into several groups known to participate in processes important for cutaneous wound healing, including: 1) angiogenesis and lymphogenesis (VEGF-A and VEGF-C); 2) inflammation (interleukin-1beta); 3) extracellular matrix remodeling (MMP1, MMP3, TIMP1, uPA, and PAI-1); and 4) cell-matrix interactions (Col1alpha1, Col1alpha2, and integrins alpha(3) and alpha(5)). Cyr61-mediated gene expression requires heparin binding activity of Cyr61, cellular de novo transcription, and protein synthesis and is largely dependent on the activation of p42/p44 MAPKs. Cyr61 regulates gene expression not only in serum-free medium but also in fibroblasts cultured on various matrix proteins or in the presence of 10% serum. These effects of Cyr61 can be sustained for at least 5 days, consistent with the time course of wound healing in vivo. Interestingly, Cyr61 can interact with transforming growth factor-beta1 to regulate expression of specific genes in an antagonistic, additive, or synergistic manner. Furthermore, we show that the Cyr61 gene is highly induced in dermal fibroblasts of granulation tissue during cutaneous wound repair. Together, these results show that Cyr61 is inducibly expressed in granulation tissues after wounding and that Cyr61 activates a genetic program for wound repair in skin fibroblasts. We propose a model in which Cyr61 integrates its activities on endothelial cells, fibroblasts, and macrophages to regulate the processes of angiogenesis, inflammation, and matrix remodeling in the context of cutaneous wound healing. (+info)
Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer.
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Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60-90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G(1) arrest, prominently up-regulated expression of p53 and p21(WAF1), and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC. (+info)
Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in primary breast cancers associated with more advanced features.
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To gain insight into the role of the CCN genes in human breast carcinomas, we quantified connective tissue growth factor (CTGF), WISP-1, CYR61, and human NOV (NOVH) mRNA expression levels in samples from 44 primary breast tumors and seven normal breasts using quantitative real-time PCR assay. Overexpression of CTGF, WISP-1, CYR61, and NOVH was found in 55 (24 of 44), 46 (20 of 44), 39 (17 of 44), and 11% (5 of 44) primary breast tumors, respectively. Statistical univariate analysis was performed to explore the links between expression of the CCN genes and clinical and pathological parameters. Interestingly, significant associations were found between CTGF expression versus stage, tumor size, lymph node status, and age at diagnosis; WISP-1 mRNA levels versus stage, tumor size, lymph node, and HER-2/neu overexpression; and CYR61 expression with stage, tumor size, lymph node, age, and estrogen receptor expression. In contrast to CTGF, WISP-1, and CYR61, no significant correlation was found between NOVH expression and any of the clinical and pathological factors. Furthermore, multivariate classification tree model analysis showed that stage and lymph node involvement were important for predicting CTGF expression in breast cancers; the stage, age, and HER-2/neu status were key factors for WISP-1 expression; and the stage, age, and estrogen receptor were valuable predictors for CYR61 expression. In summary, these results suggest that CTGF, WISP-1, and CYR61 may play a role in the progression of breast cancer and might serve as a valuable tool for monitoring tumor status of breast cancer patients. (+info)
Expression and regulation of Cyr61 in human breast cancer cell lines.
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We have shown that Cyr61, an angiogenic regulator, is overexpressed in invasive and metastatic human breast cancer cells and tumor biopsies. We have further demonstrated that Cyr61 promotes acquisition of estrogen-independence and anti-estrogen resistance in vivo in breast cancer cells. Moreover, we have demonstrated that Cyr61 induces tumor formation and tumor vascularization in vivo, events mediated through the activation of the MAPK and the Akt signaling pathways. Here we investigate how Cyr61 expression is regulated in both estrogen receptor (ER)-positive and ER-negative breast cancer cells. We demonstrate that Cyr61 mRNA and protein expression is inducible by estrogen and anti-estrogens in ER-positive breast cancer cells. We show that a labile protein as well as a negative regulator might be involved in Cyr61 expression in estrogen-dependent breast cancer cells. Other important regulators of Cyr61 expression in breast cancer cells that we found are the phorbol ester TPA, vitamin D, and retinoic acid. TPA causes positive regulation of Cyr61 expression in ER-positive MCF-7 cells. Vitamin D induces a transient stimulatory effect on Cyr61 gene expression. Lastly, retinoic acid has a negative effect on Cyr61 expression and downregulates its expression in MCF-7 cells. Interestingly, most of these effects are not seen in aggressive breast cancer cells that do not express ER and express high levels of Cyr61, such as the MDA-MB-231 cells. Our results are in agreement with our knowledge that Cyr61 promotes tumor growth, and that tumor-promoting agents have a positive impact on cells that express low levels of Cyr61, such as the ER-positive breast cancer cells; however, these agents have no significant effect on cells that express high levels of Cyr61. Our findings suggest an association between increased Cyr61 expression and an aggressive phenotype of breast cancer cells. (+info)
Identification of integrin alpha(M)beta(2) as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions.
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Cysteine-rich 61 (Cyr61, CCN1) and connective tissue growth factor (CTGF, CCN2) are growth factor-inducible immediate-early gene products found in blood vessel walls and healing cutaneous wounds. We previously reported that the adhesion of endothelial cells, platelets, and fibroblasts to these extracellular matrix-associated proteins is mediated through integrin receptors. In this study, we demonstrated that both Cyr61 and CTGF are expressed in advanced atherosclerotic lesions of apolipoprotein E-deficient mice. Because monocyte adhesion and transmigration are important for atherosclerosis, wound healing, and inflammation, we examined the interaction of THP-1 monocytic cells and isolated peripheral blood monocytes with Cyr61 and CTGF. THP-1 cells and monocytes adhered to Cyr61- or CTGF-coated wells in an activation-dependent manner and this process was mediated primarily through integrin alpha(M)beta(2). Additionally, expression of alpha(M)beta(2) on human embryonic kidney 293 cells resulted in enhanced cell adhesion to Cyr61. Consistent with these data, a GST-fusion protein containing the I domain of the integrin alpha(M) subunit bound specifically to immobilized Cyr61 or CTGF. We have also investigated the requirement of cell surface heparan sulfate proteoglycans (HSPGs) as coreceptors for monocyte adhesion to Cyr61. Pretreatment of monocytes with heparin or heparinase I resulted in partial inhibition of cell adhesion to Cyr61. However, monocytes, but not fibroblasts, were capable of adhering to a Cyr61 mutant deficient in heparin binding activity. Collectively, these results show that activated monocytes adhere to Cyr61 and CTGF through integrin alpha(M)beta(2) and cell surface HSPGs. However, unlike fibroblast adhesion to Cyr61, cell surface HSPGs are not absolutely required for this adhesion process. (+info)
Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II.
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BACKGROUND: The renin-angiotensin system is thought to be involved in development and progression of arteriosclerosis, thereby contributing to adverse cardiovascular events. To elucidate the role of angiotensin II (Ang II) at a cellular level, we analyzed the Ang II-induced gene expression profile. METHODS AND RESULTS: Genes induced on Ang II stimulation (10(-7) mol/L, 45 minutes) in rat smooth muscle cells were analyzed by polymerase chain reaction selected subtraction. In addition to known genes, such as interleukin 6, leukemia inhibitory factor, and c-fos, we identified CYR61, an angiogenic immediate early gene. Northern blot analysis revealed a rapid 2.5-fold increase of CYR61 transcript levels by Ang II, peaking at 30 minutes, which was blunted by Ang II type 1 receptor blockade. Exposure of rat aortic rings to Ang II (30 minutes) revealed a 2-fold, and intraperitoneal injection of Ang II (30 minutes) in mice a 3-fold, increase of aortic CYR61 transcripts. In arteriosclerotic aortas of apolipoprotein E-deficient mice, CYR61 transcripts confirmed by in situ hybridization and proteins shown by immunohistochemistry were elevated, whereas they were hardly detectable in wild types. In human carotid atherectomies and arteriosclerotic coronary arteries, immunohistochemical analysis revealed expression of CYR61 within connective tissue in neointima, adventitia, and surrounding small capillaries and blood vessels, colocalized with ACE and Ang II. Normal human arteries showed no significant staining for CYR61. CONCLUSIONS: CYR61, an angiogenic factor, is induced by Ang II in vascular cells and tissue. The expression of CYR61, colocalized with Ang II and ACE, in small vessels of human arteriosclerotic lesions is consistent with the notion that the activated renin-angiotensin system may contribute to plaque neovascularization by enhancing regulators of microvessel formation and cell proliferation. (+info)