Endoglin regulates cyclooxygenase-2 expression and activity

The endoglin heterozygous (Eng(+/-)) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng(+/-) mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E(2) were observed in the Eng(+/-) mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng(+/-) but not in Eng(+/+) mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng(+/+) mice. N(omega)-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-beta1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng(+/-) mice.     

MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer

BACKGROUND AND AIMS: microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression negatively. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNA to tumor biology has not been established. METHODS: We evaluated the expression of miRNA in human hepatocellular cancer (HCC) by expression profiling, and defined a target gene and biologically functional effect of an up-regulated miRNA. RESULTS: miR-21 was noted to be highly overexpressed in HCC tumors and cell lines in expression profiling studies using a miRNA microarray. Inhibition of miR-21 in cultured HCC cells increased expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, and decreased tumor cell proliferation, migration, and invasion. In contrast-enhanced miR-21 expression by transfection with precursor miR-21 increased tumor cell proliferation, migration, and invasion. Moreover, an increase in cell migration was observed in normal human hepatocytes transfected with precursor miR-21. PTEN was shown to be a direct target of miR-21, and to contribute to miR-21 effects on cell invasion. Modulation of miR-21 altered focal adhesion kinase phosphorylation and expression of matrix metalloproteases 2 and 9, both downstream mediators of PTEN involved in cell migration and invasion. CONCLUSIONS: Aberrant expression of miR-21 can contribute to HCC growth and spread by modulating PTEN expression and PTEN-dependent pathways involved in mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion.     

Phospholipase Cgamma1 signalling regulates lipopolysaccharide-induced cyclooxygenase-2 expression in cardiomyocytes

Lipopolysaccharide (LPS) induces cyclooxygenase-2 (COX-2) expression in cardiomyocytes, which plays a role in myocardial depression during endotoxemia. The purpose of this study was to investigate the role of phosphatidylinositol (PI)-phospholipase Cgamma1 (PLCgamma1) in cardiac COX-2 expression in vitro and in vivo. In cultured mouse neonatal cardiomyocytes, LPS increased PLCgamma1 phosphorylation and COX-2 expression. Knockdown of PLCgamma1 with specific siRNA or inhibition of PI-PLC with U73122 attenuated COX-2 mRNA and protein expression induced by LPS (1 microg/ml). PLCgamma1 activation by LPS also increased ERK1/2 MAPK phosphorylation, and inhibition of ERK1/2 MAPK blocked the effect of PLCgamma1 on COX-2 expression. Furthermore, activation of PLCgamma1 is a consequence of the Src family activation since inhibition of Src abrogated whereas over-expression of Src enhanced PLCgamma1 phosphorylation and COX-2 expression in LPS-stimulated cardiomyocytes. To investigate the role of PLCgamma1 in endotoxemia, wild-type and PLCgamma1(+/-) adult mice were pre-treated with U73122, or its inactive analog, U73343 (9 mg/kg, i.p.), or vehicle for 15 min followed by LPS (4 mg/kg, i.p.) for 4 h. U73122 or heterozygous deletion of PLCgamma1 decreased cardiac COX-2 expression. The phosphorylation of ERK1/2 MAPK induced by LPS was also attenuated in U73122- or PLCgamma1(+/-) compared to U73343-treated or wild-type littermate hearts, respectively. In conclusion, our study suggests that PLCgamma1 signalling represents a novel pathway regulating cardiac COX-2 expression during LPS stimulation. The Src family is responsible for PLCgamma1 activation, which signals the ERK1/2 MAPK pathway, resulting in COX-2 production in LPS-stimulated cardiomyocytes.     

MicroRNA-15a positively regulates insulin synthesis by inhibiting uncoupling protein-2 expression

MicroRNAs are small noncoding RNAs that have been highly conserved during evolution and have been implicated to play an important role in many diseases, including diabetes. Several reports indicated the function of miRNAs in insulin production. However, the mechanisms by which miRNAs regulate this process remain poorly understood. Here we found that the expression of miR-15a was up-regulated in the presence of high glucose for 1 h, whereas prolonged periods of high glucose exposure resulted in depressed expression of miR-15a, and the change in expression levels of miR-15a coincided with insulin biosynthesis. Moreover, ectopic expression of miR-15a promoted insulin biosynthesis in MIN6 cells, whereas its repression was sufficient to inhibit insulin biosynthesis. Further, we verified that miR-15a directly targeted and inhibited uncoupling protein-2 (UCP-2) gene expression. miR-15a mimics inhibited UCP-2 3′UTR luciferase reporter activity. Western blot analysis showed that miR-15a inhibited endogenous UCP-2 protein levels, and resulted in the increase in oxygen consumption and reduced ATP generation. This study suggests miR-15a is a mediator of β cell function and insulin biosynthesis, thus offering a new target for the development of preventive or therapeutic agents against diabetes.     

微小RNA miR-199a／a^＊模拟物转染肝癌细胞HepG2中Met原癌基因的表达

目的探讨在肝癌细胞系HepG2中,Met是否为miR-199a／a^＊的靶基因。方法将miR-199a／a^＊的模拟物及阴性对照物分别转染至HepG2细胞中,转染后48h及72h分别提取RNA及蛋白行RT—PCR及Western Blot检测Met的表达。结果与阴性对照相比,转染Up-miR-199a^＊组的MetmRNA（P〈0．05）和蛋白（P〈0．001）表达水平均显著下调;而Up—miR-199a组与阴性对照组相比无统计学差异。结论肝癌细胞HepG2中miR-199a^＊负调控Met的mRNA及蛋白表达。     

Significance of cyclooxygenase—2 expression in human primary hepatocellular carcinoma

AIM：To clarife the significance of cyclooxygenase-2(COX-2)expression in human primary hepatcellular carcinoma(HCC)and adjacent nontumorous tissues.METHODS;TheCOX-2protein and mRNA were investigated in 27HCC tissues with adjacent nontumorous tissues,and 5histologically normal liver tissues,using immunohistochemistry and in situ hybridization.RESULTS:The well-differentiated HCC expressed COX-2protein(5.68±1.19)more strongly than moderated HCC(3.43±1.98)and poor differentiated HCC(3.33±1.50)(P<0.05 respectively),adjacent nontumorous tissues(4.93±1.05)and normal live tissues(3.20±1.92)(P<0.01 respectively);More intensive staining of COX-2in adjacent nontumorous tissues was observed than that in normal liver tissues(P<0.05).There was no significant difference among adjacent nontumorous tissues,moderately differentiated HCC and poorly differentiated HCC(P>0.05).The expression of COX-2mRNA was observed in the cytoplasm of the cells of HCC and of gtthe hepatocytes in adjacent nontumorous tissues in which COX-2 protein was positive.CONCLUSION:The overexpression of COX-2 in well-differentiated HCsuggets that COX-2 may play a role in the early stages of hepatocarcinogensis.     

Protease-activated receptor-2 regulates cyclooxygenase-2 expression in human bile duct cancer via the pathways of mitogen-activated protein kinases and nuclear factor kappa B

Background/purpose

Recent studies have suggested that protease-activated receptor-2 (PAR-2) activity correlates with cell proliferation and tumor growth, and its activation induces expression of cyclooxygenase-2 (COX-2). However, no previous reports have investigated PAR-2 signaling pathways in bile duct cancer. The aim of this study was to determine whether PAR-2 activation can regulate COX-2 expression via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-??B) in human bile duct cancer cells.

Methods

We immunohistochemically examined PAR-2 and COX-2 expression in 104 resected human specimens of extrahepatic bile duct cancer. We then determined how inhibitors of MAPKs and NF-??B signaling pathways influence COX-2 expression under PAR-2 activation in HuCCT1 and TKKK, human bile duct cancer cell lines.

Results

PAR-2 and COX-2 proteins were immunohistochemically recognized in 63 and 57% of specimens and were significantly correlated. PAR-2 agonist peptide activated mRNA and protein expression of COX-2 in HuCCT1 and TKKK. Pharmacologic blockade of p44/42 or p38 MAPK significantly inhibited PAR-2-activated mRNA and protein expression of COX-2 in both cells. COX-2 protein expression was also inhibited by the blocker of NF-??B pathway in both cells.

Conclusions

PAR-2 may regulate COX-2 expression in human bile duct cancer via the MAPKs and NF-??B pathways.     

Mitochondrial dysfunction activates cyclooxygenase 2 expression in cultured normal human chondrocytes

OBJECTIVE: Mitochondrial alterations play a key role in the pathogenesis of osteoarthritis (OA). This study evaluated a potential role of mitochondrial respiratory chain (MRC) dysfunction in the inflammatory response of normal human chondrocytes. METHODS: Commonly used inhibitors of the MRC were utilized to induce mitochondrial dysfunction in normal human chondrocytes. Levels of prostaglandin E(2) (PGE(2)) protein and expression of cyclooxygenase 2 (COX-2) and COX-1 messenger RNA (mRNA) and protein were analyzed. To identify the underlying mechanisms responsible for PGE(2) liberation, reactive oxygen species (ROS) were measured. Inhibitors of ROS, including vitamin E, and inhibitors of mitochondrial Ca(2+) and NF-kappaB were used to test their effects on the MRC. RESULTS: Antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) significantly increased the levels of PGE(2) (mean +/- SEM 505 +/- 132 pg/50,000 cells and 288 +/- 104 pg/50,000 cells, respectively, at 24 hours versus a basal level of 29 +/- 9 pg/50,000 cells; P < 0.05) and increased the expression of COX-2 at both the mRNA and protein levels. Expression of COX-1 did not show any modulation with either inhibitor. Further experiments revealed that antimycin A and oligomycin induced a marked increase in the levels of ROS. Production of PGE(2) and expression of COX-2 protein were inhibited by antioxidants, vitamin E, and mitochondrial Ca(2+) and NF-kappaB inhibitors. The response to blockers of mitochondrial Ca(2+) movement showed that ROS production was dependent on mitochondrial Ca(2+) accumulation. CONCLUSION: These results strongly suggest that, in human chondrocytes, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to PGE(2) production via mitochondrial Ca(2+) exchange, ROS production, and NF-kappaB activation. These data may prove valuable for a better understanding of the participation of mitochondria in the pathogenesis of OA.     

Correlation between expression of cyclooxygenase-2 and angiogenesis in human gastric adenocarcinoma

AIM: To evaluate the expression of cyclooxygenase (COX2) and the relationship with tumor angiogenesis and advancement in gastric adenocarcinoma.METHODS: Immunohistochemical stain was used for detecting the expression of COX-2 in 45 resected specimens of gastric adenocarcinoma; the monoclonal antibody against CD34 was used for displaying vascular endothelial cells, and microvascular density (MVD) was detected by counting of CD34-positive vascular endothelial cells. Paracancerous tissues were examined as control.RESULTS: Immunohistological staining with COX-2-specific polyclonal antibody showed cytoplasmic staining in the cancer cells, some atypical hyperplasia and intestinal metaplasia,as well as angiogenic vasculature present within the tumors and prexisting vasculature adjacent to cancer lesions. The rate of expression of COX-2 and MVD index in gastric cancers were significantly increased, compared with those in the paracancerous tissues (77.78 vs 33.33 %, 58.13±19.99 vs 24.02±10.28, P＜0.01, P＜0.05, respectively). In 36 gastric carcinoma specimens with lymph node metastasis, the rate of COX-2 expression and MVD were higher than those in the specimens without metostasis (86.11 vs 44.44 %,58.60±18.24 vs 43.54±15.05, P＜0.05, P＜0.05, respectively).The rate of COX-2 expression and MVD in the specimens with invasive serosa were significantly higher than those in the specimens without invasion to serosa (87.88 vs 50.0 %,57.01±18.79 vs42.35±14.65, P＜0.05, P＜0.05). Moreover,MVD in COX-2-positive specimens was higher than that in COX-2-negative specimens (61.29±14.31 vs 45.38±12.42,P＜0.05). COX-2 expression was positively correlated with MVD (r=0.63, P＜0.05).CONCLUSION: COX-2 expression might correlate with the occurance and advancement of gastric carcinoma and is involved in tumor angiogenesis in gastric carcinoma. It is likely that COX-2 by inducing angiogenesis can be one of mechanisms which promotes invasion and metastasis of gastric carcinoma. It may become a new therapeutic target for anti-angiogenesis.     

MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1

Adhesion molecules expressed by activated endothelial cells play a key role in regulating leukocyte trafficking to sites of inflammation. Resting endothelial cells normally do not express adhesion molecules, but cytokines activate endothelial cells to express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which mediate leukocyte adherence to endothelial cells. We now show that endothelial cells express microRNA 126 (miR-126), which inhibits VCAM-1 expression. Transfection of endothelial cells with an oligonucleotide that decreases miR-126 permits an increase in TNF-alpha-stimulated VCAM-1 expression. Conversely, overexpression of the precursor to miR-126 increases miR-126 levels and decreases VCAM-1 expression. Additionally, decreasing endogenous miR-126 levels increases leukocyte adherence to endothelial cells. These data suggest that microRNA can regulate adhesion molecule expression and may provide additional control of vascular inflammation.     

Enhanced cyclooxygenase-2 expression in sporadic and familial adenomatous polyposis of the human colon

BACKGROUND: The cyclooxygenase (COX) enzymes exist in two related but unique isoforms (COX-1 and COX-2) and catalyze the formation of prostaglandins (PGs). COX-1 is constitutively expressed, and is responsible for the synthesis of PGs necessary for gastroprotection and normal renal function. The COX-2 isoform is important in a variety of pathophysiological conditions such as inflammation and tumorigenesis. Numerous studies report that regular use of non-steroidal anti-inflammatory drugs (NSAIDs) can decrease the incidence of some tumor types, including gastrointestinal polyposis. METHODS: In this study, we evaluated COX-1 and COX-2 expression in 30 polyps collected from 10 patients with familial adenomatous polyposis (FAP) and in 18 polyps collected from 18 patients with sporadic adenomatous polyposis (SAP) using COX-1 or COX-2 isoform-specific antibodies. All tissues were formalin-fixed and paraffin-embedded. Immunoreactivity was detected using tyramide signal amplification and evaluated utilizing an immunohistochemical scoring system. RESULTS: COX-2 was minimally detected in the distant non-neoplastic epithelium, which also served as an internal negative control. In comparison, all polyps collected from SAP or FAP patients overexpressed COX-2 in the neoplastic epithelial cells (P < or = 0.002). Additionally, pronounced COX-2 expression was observed in the stromal cells underlying and adjacent to adenomatous lesions. COX-1 immunoreactivity was weak to mild throughout each tissue evaluated and did not change in the neoplastic or stromal cells of the polyps. CONCLUSIONS: COX-2 expression is upregulated in the adenomatous epithelium of SAP and FAP, while the COX-1 isoform appears to be constitutively expressed at low levels in both neoplastic and non-neoplastic regions. The differential expression of COX-1 and COX-2 in these neoplasms suggests that COX-2 rather than COX-1 may play a role in adenoma formation and/or growth in cases of SAP and FAP in humans.     

Vascular endothelial growth factor up-regulates cyclooxygenase-2 expression in human endothelial cells

We investigated the effects of vascular endothelial growth factor (VEGF) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in human microvascular endothelial cells (HMEC-1). Treatment of HMEC-1 with VEGF resulted in a dose- and time-dependent up-regulation of COX-2 mRNA and protein levels. This up-regulation was accompanied by a 1.6-fold increase in PGE(2) synthesis. Pretreatment of HMEC-1 with a selective COX-2 inhibitor, NS-398, abolished VEGF-induced PGE(2) synthesis, suggesting specific up-regulation of COX-2 activity by VEGF in HMEC-1. Transient transfection assays using deletion mutants of the human COX-2 promoter fused to the luciferase reporter gene indicated critical requirement of a regulatory region spanning -828/-123 bp for VEGF induction of COX-2 promoter activity in HMEC-1. Site-directed mutation analysis demonstrated that a GATA cis-acting element at -685/-680 bp was essential for VEGF- induced COX-2 promoter activity in HMEC-1. These observations are of particular importance given the recent demonstrations of critical requirement of COX-2 isoenzyme for tumor growth and angiogenesis.     

Integrin expression by human articular chondrocytes

Objective. To perform a comprehensive analysis of the integrin forms expressed by normal human articular chondrocytes. Methods. Cartilage sections and collagenase-released chondrocytes were probed with a comprehensive panel of integrin isoform–specific monoclonal antibodies (MAb), using in situ immunohistochemistry techniques, indirect immunofluorescence and flow cytometry, and immunoprecipitation/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Results. Chondrocytes in cartilage sections reacted with MAb specific for the α₅, α_v, and β₁ integrin subunits and the α_vβ₃ and α_vβ₅ heterodimers. They also reacted with a polyclonal antibody specific for the intracytoplasmic portion of the α₁ subunit. MAb specific for the α_v subunit reacted more strongly with chondrocytes near the articular surface than with those in deeper layers of cartilage, and the α_vβ₃-specific MAb reacted exclusively with chondrocytes within the most superficial 30 μm of cartilage. Flow cytometric analysis and SDS-PAGE analysis of immunoprecipitates prepared from extracts of cell-surface radioiodinated chondrocytes confirmed the above observations, and additionally revealed the presence of the α₃β₁ integrin. Conclusion. Normal human articular chondrocytes prominently display substantial quantities of the α₁β₁, α₅β₁, and α_vβ₅ integrin heterodimers, as well as lesser quantities of the α₃β₁ and α_vβ₃ heterodimers, The α_v subunit–containing integrins are detected more readily on the more superficial chondrocytes than on chondrocytes deep within cartilage. These observations provide the basis for analysis of the role of chondrocyte integrins in cartilage homeostasis and in the pathogenesis of joint diseases.     

Clinicopathologic significance of expression of cyclooxygenase-2 in human esophageal squamous cell carcinoma

BACKGROUND/AIMS: The focus of studies on cyclooxygenase-2 (COX2) have been on its ability to mediate the biological behavior of human tumors including tumorigenesis, tumor progression, apoptosis and differentiation. The aim of the current study was to elucidate a further finding on the clinicopathologic significance of immunohistochemical expression of COX2 in esophageal squamous cell carcinoma. METHODOLOGY: The immunohistochemical expression of COX2 was examined for 68 specimens of esophageal squamous cell carcinoma (ESCC) and the correlation of COX2 expression with clinicopathologic features was examined. COX2 immunoreactivity was weak in 27 (40%) and strong in 41 (60%) of the carcinomas. RESULTS: The proportion of poorly differentiated SCCs among tumors with a strong expression of COX2 (34%, 14 of 41) was significantly higher than among tumors with a weak expression of COX2 (19%, 3 of 14, p = 0.02). The depth of the tumors (p = 0.0004), lymph node metastasis (p = 0.009) and the stage of the tumors (p = 0.001) were advanced significantly more progressively in ESCCs with a strong COX2 expression. Moreover, survival was significantly reduced (p = 0.02) among patients with strong COX2 expression when compared with the COX2 weak group. CONCLUSIONS: This study showed that strong expression of COX2 was correlated with tumor progression and poor differentiation in ESCC.