Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.     

Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel

An improved method is described for culturing primary rat brain capillary endothelial cells (RBCEC) on glass, covered by Matrigel. The procedure using Matrigel yields spindle-shaped endothelial cells exhibiting close cell-cell appositions seen on electron microscopic sections. These cells permanently express tight junction proteins ZO-1, claudin-5 and the adherent junction protein beta-catenin, as revealed by immunofluorescence. Furthermore, glass coverslips covered with Matrigel provide a stable and low-background fluorescent base for microfluorimetric calcium measurements. By this method, hereby we show that the PAR-4 agonist peptide induces transient [Ca2+]i changes with different kinetics compared to that due to activation of the PAR-1 receptor. This indicates that RBCE cells grown on Matrigel express PAR-4 receptors.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Increased expression of matrix metalloproteinase-2 (MMP-2) predicts tumour recurrence and unfavourable outcome in non-small cell lung cancer

The purpose of this study was to analyse the expression of matrix metalloproteinase-2 (MMP-2) and its extracellular matrix metalloproteinase inducer (EMMPRIN) in non-small cell lung cancer (NSCLC), and to evaluate their significance to predict tumour behaviour. The study consists of 212 patients treated by the resection of the tumour. Tumour samples were stained immunohistochemically, and the expression of MMP-2 and EMMPRIN was evaluated both in tumour cells and in peritumoural stromal tissue. The results were compared with clinicopathological factors and survival of the patients. High expression of MMP-2 in tumour cells was found in 83 out of 191 cases (44%). Adenocarcinomas showed more often high expression of MMP-2 as compared with squamous cell or large cell carcinomas (p=0.001). High cancer cell associated MMP-2 expression was associated with increased tumour recurrence (p=0.001). Tumour stroma showed positive staining in 162 (98%) cases and was considered highly stained in 120 (72%) cases. The high stromal MMP-2 expression was noticed more often among large cell carcinomas as compared with other histological types (p=0.007). High cancer cell associated EMMPRIN expression was found in 115 (61%) cases and was associated only with high MMP-2 expression in tumour cells (p=0.006). In overall survival (OS) and disease free survival (DFS) analyses, type of tumour (p=0.001 and p=0.0004), advanced stage (p=0.001 and p=0.013) and high MMP-2 expression in tumour cells (p=0.018 and p=0.001) were associated with poor survival. Also, high stromal MMP-2 expression was related to poor outcome in both OS and DFS analyses (p=0.010 and 0.045, respectively). In multivariate analysis, stromal MMP-2 expression retained its prognostic value to predict OS and DFS (p=0.028 and p=0.039, respectively), together with tumour type and stage (p=0.017, p=0.001 and p=0.021, p=0.008, respectively). The present study shows the significant prognostic value of MMP-2 in NSCLC suggesting that the use of MMP-2 is valuable in determining the patients with more aggressive disease.     

METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer

N6-methyladenosine (m6A) is a well-known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT-qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down-regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock-down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16-mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.     

The localization of thrombospondin-1 (TSP-1), cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG) TSP receptor, and matrix metalloproteinase-9 (MMP-9) in colorectal cancer

Thrombospondin-1 (TSP-1) is a 450 kDa matrix bound glycoprotein involved in tumor invasion, metastasis, and angiogenesis. One of the receptors involved in TSP-1 mediated tumor cell adhesion and metastasis is the cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG) receptor. One mechanism of TSP-1 in promoting tumor cell metastasis involves the up-regulation of matrix metalloproteinase-9 (MMP-9) expression, specifically through the CSVTCG TSP-1 receptor. TSP-1 and its CSVTCG receptor has been implicated in tumor progression in a variety of cancers including breast adenocarcinomas, head and neck squamous cell carcinomas, and pancreatic carcinomas. In this study, we examined 99 cases of colorectal cancer by immunohistochemical analysis to investigate 1) the localization of TSP-1 and CSVTCG TSP-1 receptor, 2) the relationship with MMP-9, and 3) the correlation of expression with clinical staging. Strong expression of TSP-1 was observed in the submucosa or the serosa adjacent to the tumor. Positive staining for CSVTCG TSP-1 receptor was observed in tumor cells and microvessels. MMP-9 was also expressed in tumor cells. In addition, staining intensity of CSVTCG TSP-1 receptor was higher in poorly differentiated adenocarcinoma than well or moderately differentiated adenocarcinoma. Tumors in which inflammatory cells stained strongly for CSVTCG TSP-1 receptor correlated with decreased incidence of distant metastasis and angiogenesis. These data were consistent with our previous studies for breast, pancreatic, and head and neck carcinoma. They suggest an important role for TSP-1 and CSVTCG TSP-1 receptor in tumor progression in colorectal cancer.     

Th2 cell membrane factors in association with IL-4 enhance matrix metalloproteinase-1 (MMP-1) while decreasing MMP-9 production by granulocyte-macrophage colony-stimulating factor-differentiated human monocytes

Monocytes/macrophages are directly involved in tissue remodeling and tissue destruction through the release of matrix metalloproteinases (MMP). In the present study, we examined the effect mediated by contact of polarized Th cells with mononuclear phagocytes on the production of MMP-1, MMP-9, and their inhibitor. Plasma cell membranes from Ag-activated Th1 and Th2 cells were potent inducers of MMP-1 production by THP-1 cells. Cell membrane-associated TNF was found to be only partially involved in MMP-1 induction by both Th1 and Th2 cells. In Th2 cells exclusively, membrane-associated IL-4 induced MMP-1 production by THP-1 cells. This membrane-associated IL-4 effect was additive to that of TNF and was specifically observed on MMP-1 as MMP-9 production was concomitantly inhibited. Similarly, soluble IL-4 induced THP-1 cells to produce MMP-1, its effect proving additive to that of soluble TNF and to that of cell membranes of mitogen-activated HUT-78 cells. Its activity was blocked by IL-4 neutralization, and was unaffected by the presence of indomethacin. These effects on THP-1 cells were observed at protein and mRNA levels. Although inhibitory on freshly isolated peripheral blood monocytes, soluble IL-4 enhanced T cell-induced MMP-1 and inhibited MMP-9 production both at protein and mRNA levels in monocytes cultured for 7 days in the presence of GM-CSF. Thus, in contrast with previously reported effects, Th2 and IL-4 specifically induce MMP-1 production by mononuclear phagocytes at various stages of differentiation. This IL-4 activity may be relevant to pathological conditions dominated by Th2 inflammatory responses, resulting in tissue remodeling and destruction.     

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.     

hsa-miR-125a-5p通过上调Rock-1表达促进胃癌细胞侵袭

摘要：目的 探讨hsa-miR-125a-5p表达对胃癌细胞侵袭能力的影响及其作用机制。方法 在胃癌MKN45细胞株中瞬时转染hsa-miR-125a-5p-mimics使其表达明显上调,采用Transwell侵袭实验检测细胞的侵袭能力。然后,通过micRNA靶基因预测软件获得hsa-miR-125a-5p的潜在靶基因Rock-1,以抗体阻断及Western blot进行验证。结果 与对照组相比,经瞬时转染后hsa-miR-125a-5p在胃癌MKN45细胞株中表达明显增加;Western blot结果显示hsa-miR-125a-5p表达上调后Rock-1表达明显上调,Transwell实验结果表明MKN45细胞的侵袭能力明显增强（P<0.05）。而hsa-miR-125a-5p表达上调后阻断Rock-1表达,细胞的侵袭能力明显减弱（P<0.05）。结论 hsa-miR-125a-5p促进胃癌细胞的侵袭,其机制与Rock-1通路有关。     

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.     

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.     

Predictive value of protease-activated receptor-2 (PAR2) in cervical cancer metastasis

Metastasis is the primary cause of an unfavourable prognosis in patients with malignant cancer. Over the last decade, the role of proteinases in the tumour microenvironment has attracted increasing attention. As a sensor of proteinases, proteinase-activated receptor 2 (PAR₂) plays crucial roles in the metastatic progression of cervical cancer. In the present study, the expression of PAR₂ in multiple types of cancer was analysed by Gene Expression Profiling Interactive Analysis (GEPIA). Kaplan-Meier plotter was used to calculate the correlation between survival and the levels of PAR₂, Grb-associated binding protein 2(Gab2) and miR-125b. Immunohistochemistry (IHC) was performed to examine PAR₂ expression in a tissue microarray (TMA) of CESCs. Empower Stats was used to assess the predictive value of PAR₂ in the metastatic potential of CESC. We found that PAR₂ up-regulation was observed in multiple types of cancer. Moreover, PAR₂ expression was positively correlated with the clinicopathologic characteristics of CESC. miR-125b and its target Gab2, which are strongly associated with PAR₂-induced cell migration, are well-characterized as predictors of the prognostic value of CESC. Most importantly, the Cancer Genome Atlas (TCGA) data set analysis showed that the area under the curve (AUC) of the PAR₂ model was significantly greater than that of the traditional model (0.833 vs 0.790, P < .05), demonstrating the predictive value of PAR₂ in CESC metastasis. Our results suggest that PAR₂ may serve as a prognostic factor for metastasis in CESC patients.     

Thrombin and its protease-activated receptor-1 (PAR1) participate in the endothelial-mesenchymal transdifferentiation process

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.     

Proteinase-activated receptor 1 (PAR-1) and cell apoptosis

This review summarizes the main aspects and newest findings of how proteinase-activated receptor 1 (PAR-1) may modulate programmed cell death. Activation of PAR-1 has been found to induce or inhibit apoptosis in a variety of cells, depending on the dosage of its physiological agonist thrombin, or that of synthetic receptor activators. To date, cellular targets for PAR-1-mediated effects on apoptosis include neuronal, endothelial, and epithelial cells, fibroblasts, and tumor cells. The signaling pathways involved in the induction or prevention of apoptosis by PAR-1 activation are diverse, and include JAK/STAT, RhoA, myosin light chain kinase, ERK1/2, and various Bcl-2 family members. In view of the well-established involvement of microbial proteinases in host tissue malfunction, the article also elaborates on the possible significance of PAR-1 activation for the pathogenesis of infectious disease.     

A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at -1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.