Upregulation Of Cyr61 Research Articles

Upregulation of CYR61 by TGF-β and YAP signaling exerts a counter-suppression of hepatocellular carcinoma

TGF-β and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-β target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-β in liver cancer cells. In addition, TGF-β-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-β- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-β- or YAP-induced malignant transformation of HCC cells in vitro, and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-β and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Discovery of canine drug toceranib phosphate as a repurposed agent against human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is an aggressive malignancy with poor clinical outcomes. There are limited therapeutic options for those diagnosed with terminal HCC and therefore incorporating novel agents into standard-of-care regimens is urgently needed. In contrast to de novo drug discovery, the strategy of repurposing compounds initially designed to treat animals might yield substantial advantages in terms of efficacy and safety. Given the evidence for the clinical efficacy of toceranib phosphate (TOC) against canine carcinomas, we aimed to investigate its therapeutic effect on human HCC. The antitumor effects of TOC were evaluated using human HCC celllines and cell-line-derived xenograft models. Changes in autophagic response upon TOC exposure were quantified through immunoblotting and immunofluorescence analysis. The role of TOC-triggered autophagy was addressed via pharmacological and genetic inhibition. We demonstrated TOC exhibited potent antitumor activity against human HCC cells by stimulating apoptosis in vitro and in vivo by a concomitant increase in autophagic flux. Blocking the TOC-triggered autophagy inhibited cellular proliferation and decreased tumour burden, indicating a protective role of autophagy against TOC-mediated HCC cell death. This role played by TOC-induced autophagy was further linked to the inactivation of the Akt/mTOR pathway that could be attributed to the upregulation of Cyr61. Moreover, treatment with sorafenib plus TOC resulted in pronounced synergistic effects on HCC cells. Our results elucidate a newly identified therapeutic potential of TOC in treating HCC, sparking a growing interest in repurposing such canine drugs for human use.

CYR61, regulated by miR-22-3p and MALAT1, promotes autophagy in HK-2 cell inflammatory model.

BackgroundRenal tubular epithelial cells play an important role in renal function and are a major site of injury from inflammation. Emerging evidence suggests that CYR61 is involved in the regulation of autophagy. However, there are few studies on CYR61 in nephropathy and associated inflammation. This study aimed to clarify how CYR61 regulates autophagy in human renal epithelial cells while in an inflammatory state and regulates the upstream pathway of CYR61 levels.MethodsThe human renal tubular epithelial cells (HK-2) cell line treated by lipopolysaccharide (LPS) was used as an inflammatory model of human epithelial cells. Short hairpin RNA (shRNA) was used to down-regulate CYR61, and the changes in the transcription and expression levels of related molecules, as well as the morphological changes of HK-2 cells, were detected by quantitative real time-PCR (qRT-PCR), western blot (WB), and transmission electron microscopy. Either CYR61 or MALAT1 were up-regulated by overexpression vectors, or MALAT1 was down-regulated by miR-22-3p mimics. Subsequently, the levels of CYR61, MALAT1, related inflammatory factors, and autophagy factors were measured by qPCR, WB, and enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry and acridine-orange assay.ResultsWe observed that down-regulation of CYR61 could down-regulate 1B-light chain 3 (LC3) level and inhibit autophagy in the LPS-induced inflammation model of HK-2 cells. The expression levels of CYR61, Beclin1, Atg5, LC3, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were significantly increased by upregulating CYR61 or MALAT1 by overexpression vector, while the expression level of p62 was significantly decreased, intracellular reactive oxygen species (ROS) content was increased, and the proportion of autophagy and apoptosis was increased. The use of miR-22-3p mimics significantly reversed the changes induced by up-regulation of CYR61 or MALAT1 at the molecular and cellular levels.ConclusionsOur data indicated that CYR61 positively regulates autophagy of HK-2 cells under an inflammatory state, and was negatively regulated by miR-22-3p, while miR-22-3p and MALAT1 were negatively regulated by each other.

Pericyte FAK negatively regulates Gas6/Axl signalling to suppress tumour angiogenesis and tumour growth

The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK.

Cysteine-rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl-2 pathway.

Although the targeted tyrosine kinase inhibitor imatinib mesylate (IM) has achieved significant responses against CML in the clinical setting, a small proportion of patients fail to respond to IM treatment and their disease continues to progress, indicating resistance to IM therapy. As a secreted extracellular matrix protein, cysteine‐rich protein 61 (Cyr61) plays an important role in the resistance of solid tumors to chemotherapy, but its role in CML is unclear. In the present study, we observed that Cyr61 levels were upregulated in the plasma and bone marrow (BM) of patients with CML as well as in K562 cells. This upregulation of Cyr61 significantly decreased IM‐induced cellular apoptosis of K562 cells through nuclear factor kappa B/B‐cell lymphoma 2 pathways. Inhibition of Cyr61 restored the chemosensitivity of K562 cells to IM both in vitro and in vivo. Thus, our results showed for the first time that Cyr61 plays an important role in regulating the chemosensitivity of CML cells to IM, suggesting that selectively targeting Cyr61 directly or its relevant effector pathways may provide potential value in improving the clinical response of patients with CML to IM treatment.

Activation of WNT/β-Catenin Signaling Enhances Pancreatic Cancer Development and the Malignant Potential Via Up-regulation of Cyr61

Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of WNT signaling is also commonly observed in PDAC. To ascertain the impact of postnatal activation of WNT-stimulated signaling pathways in PDAC development, we combined the Elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and WNT1 stimulated the progression of premalignant pancreatic intraepithelial neoplasias (PanIN) and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A or RCAS-Wnt1 had reduced survival relative to RCAS-GFP-injected controls (P<.05). Ectopic expression of active β-catenin, or its DNA-binding partner TCF4, enhanced transformation associated phenotypes in PDAC cells. In contrast, these phenotypes were significantly impaired by the introduction of ICAT, an inhibitor of the β-catenin/TCF4 interaction. By gene expression profiling, we identified Cyr61 as a target molecule of the WNT/β-catenin signaling pathway in pancreatic cancer cells. Nuclear β-catenin and CYR61 expression were predominantly detected in moderately to poorly differentiated murine and human PDAC. Indeed, nuclear β-catenin- and CYR61-positive PDAC patients demonstrated poor prognosis (P<.01). Knockdown of CYR61 in a β-catenin-activated pancreatic cancer cell line reduced soft agar, migration and invasion activity. Together, these data suggest that the WNT/β-catenin signaling pathway enhances pancreatic cancer development and malignancy in part via up-regulation of CYR61.

Glucose tolerance in obese men is associated with dysregulation of some angiogenesis-related gene expressions in subcutaneous adipose tissue.

Obesity and its metabolic complications are one of the most profound public health problems and result from interactions between genes and environmental. The development of obesity is tightly connected with dysregulation of intrinsic gene expression mechanisms controlling majority of metabolic processes, which are essential for regulation many physiological functions, including insulin sensitivity, cellular proliferation and angiogenesis. Our objective was to evaluate if expression of angiogenesis related genes VEGF-A, CYR61, PDGFC, FGF1, FGF2, FGFR2, FGFRL1, E2F8, BAI2, HIF1A, and EPAS1 at mRNA level in adipose tissue could participate in the development of obesity and metabolic complications. We have shown that expression level of VEGF-A, PDGFC, FGF2, and FGFRL1 genes is decreased in adipose tissue of obese men with normal glucose tolerance (NGT) versus a group of control subjects. At the same time, in this group of obese individuals a significant up-regulation of CYR61, FGF1, FGFR2, E2F8, BAI2, and HIF1A gene expressions was observed. Impaired glucose tolerance (IGT) in obese patients associates with down-regulation of CYR61 and FGFR2 mRNA and up-regulations of E2F8, FGF1, FGF2, VEGF-A and its splice variant 189 mRNA expressions in adipose tissue versus obese (NGT) individuals. Thus, our data demonstrate that the expression of almost all studied genes is affected in subcutaneous adipose tissue of obese individuals with NGT and that glucose intolerance is associated with gene-specific changes in the expression of E2F8, FGF1, FGF2, VEGF-A, CYR61 and FGFR2 mRNAs. The data presented here provides evidence that VEGF-A, CYR61, PDGFC, FGF1, FGF2, FGFR2, FGFRL1, E2F8, BAI2, and HIF1A genes are possibly involved in the development of obesity and its complications.

Cysteine-Rich Protein 61 Plays a Proinflammatory Role in Obstructive Kidney Fibrosis

Cysteine-rich protein 61 (Cyr61) is a secreted matrix-associated protein that regulates a broad spectrum of biological and cellular activities. This study aimed to investigate the role of Cyr61 in progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) surgery in mice. The expression of Cyr61 transcripts and proteins in the obstructed kidneys were increased from day 1 and remained high until day 10 after surgery. Immunohistochemistry indicated that Cyr61 was expressed mainly in renal tubular epithelial cells. The upregulated Cyr61 in UUO kidneys was reduced in mice treated with pan-transforming growth factor-β (TGF-β) antibody. The role of TGF-β in tubular Cyr61 upregulation after obstructive kidney injury was further supported by experiments showing that TGF-β1 stimulated Cyr61 expression in cultured tubular epithelial cells. Notably, the upregulation of Cyr61 in UUO kidneys was followed by a marked increase in monocyte chemoattractant protein 1 (MCP-1) transcripts and macrophage infiltration, which were attenuated in mice treated with anti-Cyr61 antibodies. This proinflammatory property of Cyr61 in inducing MCP-1 expression was further confirmed in tubular epithelial cells cultured with Cyr61 protein. The anti-Cyr61 antibody in UUO mice also reduced the levels of collagen type 1-α1 transcripts, collagen fibril accumulation evaluated by picrosirius red staining, and the levels of α-smooth muscle actin (α-SMA) transcripts and proteins on day 4 after surgery; however, the antifibrotic effect was not sustained. In conclusion, the TGF-β-mediated increase in tubular Cyr61 expression involved renal inflammatory cell infiltration through MCP-1 induction during obstructive kidney injury. The Cyr61 blockade attenuated kidney fibrosis in the early phase, but the antifibrotic effect could not be sustained.

Epidermal growth factor upregulates endometrial CYR61 expression via activation of the JAK2/STAT3 pathway

Endometrial cysteine-rich protein 61 (CYR61, CCN1) is a growth factor-inducible gene whose expression is elevated during the proliferative phase of the menstrual cycle and which has been implicated in the pathogenesis of endometriosis. This study aimed to define the mediators of epidermal growth factor (EGF) signalling on CYR61 expression in spontaneously immortalised human endometrial epithelial cells (HES) as a model system. After 30 min of EGF treatment, the receptor was phosphorylated and internalised as well as mRNA CYR61 increased in HES cells. However, neither inhibition of C-terminal EGF receptor (EGFR)-phosphorylation nor blockage of the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was able to reduce CYR61 levels. Surprisingly, the HES cells showed upregulation of CYR61 mRNA expression after inhibition of the MAPK/ERK pathway when treated with EGF. Specific inhibitor studies identified the contribution of Janus kinase 2 (JAK2) and the signal transducer and activator of transcription protein STAT3 to the regulation of CYR61 expression. The JAK2/STAT3 interaction contributed to the basal expression of CYR61 and mediated EGF-driven regulation of CYR61 after 30 and 120 min of treatment. In summary, EGF-mediated CYR61 upregulation in HES cells involves STAT3 and is counter-regulated by the EGFR/MAPK/ERK pathway.

Increased vascularity and spontaneous metastasis of breast cancer by hedgehog signaling mediated upregulation of cyr61

The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic hedgehog (SHH), plays an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively.Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.

Cyr61 is regulated by cAMP‐dependent protein kinase with serum levels correlating with prostate cancer aggressiveness

Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression. Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined. There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition. These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa.

Abstract 1414: Lysophosphatidic acid-induced Cyr61 is associated with prostate cancer progression

Abstract Lysophosphatidic acid (LPA) is a serum phospholipid that has been associated with progression in several types of cancer. Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. We have reported that Cyr61 expression in benign prostate cells is regulated by LPA (Endocrinology 2004). Furthermore that Cyr61 expression is significantly up-regulated in prostate cancer (PCa) and that the expression levels correlate with Gleason score (J Urol 2010). The aim of this study was to determine if LPA-induced Cyr61 up-regulation is associated with PCa progression. BPH1 was utilized as a benign prostate cell line and PC3 as a PCa cell line. In real-time PCR of untreated BPH1 and PC3 cells, Cyr61 expression ratios to TBP were 0.5 and 5.1, and these ratios after 2 hours of 1uM LPA under serum-free medium treatment were 1.8 and 44.3, respectively. These results indicated that the Cyr61 expression levels and their increase by LPA stimulation were higher in PCa cells than in benign prostate cells. To examine whether LPA promotes cell invasion, Matrigel invasion assays were performed for 72 hours with or without 1uM of LPA in the lower chamber. LPA stimulation increased the invaded cell number from 0.3±0.5 to 12.6±2.5 (p&amp;lt;0.001) in BPH1 cells and from 31.9±20.9 to 55.2±14.8 (p=0.005) in PC3 cells. These results indicate that LPA promotes cell invasion in both benign prostate and PCa cells. To examine whether LPA-induced Cyr61 is associated with cell invasion, the Cyr61 was knocked down by siRNA in PC3 cells. Invaded cell numbers were changed by 48 hours of LPA stimulation after scramble siRNA from 2.2±1.3 to 12.3±10.2 (p=0.002) and after Cyr61 targeting siRNA from 1.8±2.6 to 2.7±2.7 (p=0.235), respectively, revealing that Cyr61 down-regulation suppresses LPA induced cell invasion. To investigate the association between Cyr61 up-regulation and prostate carcinogenesis, Cyr61 over-expressing BPH1 cells (BPH1-Cyr61) were established and their cell proliferation was compared with vector only transfected BPH1 cells (BPH1-mock) by MTS assays. The cell proliferation ratio of BPH1-mock and BPH1-Cyr61 cells after 48 hours of incubation in serum-free medium was 1.24±0.05 and 1.40±0.02 (p=0.043), respectively, indicating that Cyr61 over-expression promoted cell proliferation of BPH1 in vitro. In conclusion, LPA induced Cyr61 up-regulation was suggested to be associated with PCa progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2011-1414

Relationship of pro-angiogenic factor Cyr61to colorectal cancer development and prognosis.

446 Background: Angiogenic factorCysteine-rich 61 (Cyr61) is a member of the CCN protein family that has been implicated in diverse biological processes such as cell adhesion, proliferation, angiogenesis, and tumorigenesis. An altered expression of Cyr61 is found to be associated with several human cancers. However, the correlation of expression of Cyr61 protein and clinical features of colorectal cancer remains unknown. Methods: Cyr61 expression in colorectal cancer and normal tissues was evaluated by Western blot analysis. Immunohistochemical staining was carried out using tissue microassay (TMA) to examine the expression status of Cyr61. Correlations of Cyr61 over-expression with various clinicopathologic factors were also determined. Statistical analysis was performed to explore the links between expression of the Cyr61 and clinicopathological parameters. Results: On Western blot analysis Cyr61 up-regulation was observed in colorectal cancer tissues (17/21, 80.9%). In 234 colorectal cancers, tumor tissue microarray revealed significantly up-regulated Cyr61 protein expression in colorectal cancer tissues versus normal tissues adjacent to tumor. Cyr61 expression was high in 136 of 234 cases of colorectal carcinomas (58.1%). Cyr61 over-expression was significantly associated with TNM stage (p=0.012) and regional lymph node involvement (p=0.018). Kaplan-Meier survival analysis showed that over-expression of Cyr61 was related to poor survival of colorectal cancer patients (p=0.031). But significant associations were not found between CYR61 expression versus tumor grade, age and gender. Conclusions: Our results suggest that Cyr61 is highly expressed in colorectal carcinomas and Cyr61 may play a role in the progression of colorectal cancers. Also, Cyr61 might be a new molecular marker to predict the prognosis and serve as valuable targets for therapeutic intervention of patients with colorectal carcinoma. No significant financial relationships to disclose.

Regulation of the Matricellular Proteins CYR61 (CCN1) and NOV (CCN3) by Hypoxia-Inducible Factor-1α and Transforming-Growth Factor-β3 in the Human Trophoblast

It is known that a hypoxic environment is critical for trophoblast migration and invasion and is fundamental for appropriate placental perfusion. Because cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3) are expressed in the extravillous trophoblast and expression levels are deregulated in preeclampsia, we investigated their regulation properties in first-trimester placental explants and in JEG3 choriocarcinoma cells upon a physiological low oxygen tension of 1-3%. In placental explants, both proteins were expressed in the extravillous trophoblast cells and were increased upon hypoxia. JEG3 cells revealed a significant up-regulation of CYR61 and NOV intracellular as well as secreted protein upon hypoxic treatment accompanied by the stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha). Treatment with dimethyloxalylglycine to mimic hypoxia and silencing of HIF-1alpha using small interfering RNA revealed that only the increase in intracellular protein expression seems to be dependent on HIF-1alpha but obviously not the secretion process. Moreover, recombinant TGF-beta3 was able to further enhance the amount of intracellular CCN proteins as well as secreted CYR61 levels under hypoxia. These results indicate that low oxygen levels trigger elevation of intracellular as well as secreted CYR61 and NOV protein probably in two independent pathways. Addition of recombinant CYR61 and NOV proteins increases migration as well as invasion properties of JEG3 trophoblast cells, which strengthen their role in supporting trophoblast migration invasion properties. In summary, CYR61 and NOV are regulated by HIF-1alpha and TGF-beta3 in the trophoblast cell line JEG3, and their enhanced secretion could be implicated in appropriate placental invasion.

Extracellular Matrix Associated Protein CYR61 is Linked to Prostate Cancer Development

The cancer cell microenvironment includes complex interactions between the cell and the extracellular matrix. Expression of the CCN family of extracellular matrix associated proteins is often modified in disease states. Depending on cancer type these changes are linked with enhanced or inhibited tumor growth. We characterized Cyr61 in prostate cancer. Cyr61 is an integrin binding matricellular protein with altered expression in many cancer types. Cyr61 expression in prostate cancer, benign prostatic hyperplasia and normal tissues was evaluated by microarray analysis, quantitative real-time polymerase chain reaction and tissue microarray. Immunoblots were analyzed to assess endogenous protein expression in prostate cancer cell lines. On genomic analysis Cyr61 up-regulation was observed in prostate cancer tissue and in normal prostate tissue adjacent to tumor vs that in prostate donor tissue. In 174 matched tumors and normal prostate tissues adjacent to tumor tissue microarray revealed significantly up-regulated Cyr61 protein expression in cancer tissue vs normal prostate tissue adjacent to tumor. Also, increased Cyr61 expression correlated with Gleason sum 8 or greater cancer. Staining in high grade prostatic intraepithelial neoplasia was moderately up-regulated vs that in normal prostate tissue adjacent to tumor but generally less intense than in carcinoma tissue. In addition to the correlation with more advanced disease, the strong association between Cyr61 expression and prostate cancer supports the potential usefulness of Cyr61 as a novel biomarker for prostate cancer. This warrants further analysis to determine the mechanisms by which Cyr61 may contribute to prostate cancer development and progression.

PGF2α stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation

Prostaglandin-F2α (PGF2α) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of β-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.

Premenstrual Regulation of the Pro-Angiogenic Factor CYR61 in Human Endometrium

The pro-angiogenic factor cysteine-rich protein 61 (CYR61/CCN1) mediates different signals in tumorigenesis, angiogenesis and is involved in the pathogenesis of endometriosis. In this study we investigated the temporal and spatial expression pattern in human endometrium during the menstrual cycle and its possible regulation mechanisms in the premenstrual phase. CYR61 transcript expression showed two distinct periods of elevated levels in the proliferative phase and in menstrual effluents. Because the menstrual breakdown of the functionalis is triggered by cytokines, prostaglandins (PGs), as well as hypoxia, we used a benign endometrial cell line to investigate if CYR61 is regulated by these factors. Hypoxic conditions transiently induced CYR61 mRNA levels and enhanced the secretion of the CYR61 protein into the medium. The hypoxia-inducible factor (HIF) 1alpha mediated this effect on CYR61 as evidenced by dimethyloxalylglycine treatment and by HIF1alpha short interfering RNA. CYR61 mRNA expression was further regulated by IL-1, TNFalpha, PGE2, and PGF2alpha. In addition, TNFalpha and PGE2 elevated significantly CYR61 cellular protein levels in well-oxygenated cells but had only a slight effect on the quantity of secreted protein. Moreover, PGE2 combined with hypoxic conditions increased CYR61 mRNA and protein levels synergistically, whereas the combination with TNFalpha abolished the CYR61 levels induced by hypoxia. Together, the up-regulation of CYR61 by hypoxia via HIF1alpha, TNFalpha, and PGE2 could represent possible mechanisms for the CYR61 increase at the onset of menstruation. The opposite effect of TNFalpha combined with hypoxia on CYR61 up-regulation could contribute to a balanced expression level of this angiogenic factor in the endometrium.

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.

Up-Regulation of cyr61 in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats

In the present study, we applied a fluorescent differential display method to mRNAs from aortae of spontaneously hypertensive rats (SHRs), stroke-prone spontaneously hypertensive rats (SPSHRs), and their parental strain, Wistar Kyoto rats (WKYRs), to identify the genes involved in the development of hypertension. Through this screen we came across a gene that is consistently up-regulated in hypertensive rats. Nucleotide sequence determination of the corresponding cDNA revealed that the gene is the rat orthologue of cyr61. Northern blot analysis showed that cyr61 expression increases in SHR and SPSHR before the onset of hypertension and is sustained thereafter at higher levels than in age-matched WKYRs. In situ hybridization analysis demonstrated that cyr61 is expressed strongly in smooth muscle cells (SMCs) in media of SHR and SPSHR but not WKYR aorta. Fluorescent in situ hybridization mapped the cyr61 gene to rat chromosome 1p12–13, which is located in close proximity to a recently defined quantitative trait locus including NHE3 Na+/H+ exchanger. Overexpression of the cyr61 gene in stably transfected rat SMC line A7r5 caused rather inhibitory effects on the proliferation and DNA and protein synthesis. Our results thus demonstrate for the first time that cyr61 can also act as a growth inhibitor in SMC of genetically hypertensive rats. This may reveal a new route for investigation of the pathogenesis of hypertension.

Comparison of Prostaglandin F2α, Bimatoprost (Prostamide), and Butaprost (EP2 Agonist) on Cyr61 and Connective Tissue Growth Factor Gene Expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.