MMP‐2 and MMP‐9 as prognostic markers for the early detection of urinary bladder cancer

The present study assessed protein and gene expression levels of tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 in urine and blood samples of 50 patients with bladder carcinoma. The expression of TIMP‐2, MMP‐2, and MMP‐9 levels with tumor stage and grade was also assessed. Results showed that the expression levels of MMP‐2 and MMP‐9 in both blood and urine were significantly elevated in group 1 when compared with groups 2 and 3 healthy subjects. The discriminatory ability in the diagnosis of bladder carcinoma of MMP‐2 and MMP‐9 expression was confirmed by receiver operating characteristic curve analysis that revealed a sensitivity and specificity of 100%. MMP‐2 and MMP‐9 levels were not correlated with grade or stage of the tumor. With respect to TIMP‐2 blood and urine levels, results showed a significant decrease in gene expression levels in bladder carcinoma group, whereas, TIMP‐2 protein showed a significant increase in bladder carcinoma.     

Regulation of matrilysin expression in endothelium by fibroblast growth factor-2

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.     

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.     

Virus survival of RNA silencing without deploying protein-mediated suppression in Nicotiana benthamiana

Unstimulated human fibrosarcoma cells (HT1080) constitutively secrete matrix metalloproteinase 2 (MMP 2) as a proenzyme requiring proteolytic cleavage by membrane type-1 MMP (MT1 MMP) for activation. Physiological and pharmacological stimuli induce clustering of MT1 MMP/tissue inhibitor of MMP 2 "receptors", promoting binding and activation of MMP 2. We now report that cholesterol depleted HT1080 cells accumulated MT1 MMP on the cell surface and activated MMP 2. A specific inhibitor of mitogen activated protein kinase kinase 1/2 inhibited both MMP 2 activation and extracellular signal-related kinase phosphorylation induced by cholesterol depletion. Our data indicate that the cholesterol content of unstimulated cells is critical for secretion of MMP 2 as an inactive zymogen and control of pericellular proteolysis.     

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex.     

MT1‐MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1‐MMP is a membrane‐associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion‐promoting, pro‐tumorigenic MMP in cancer. We show that deregulation of MT1‐MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1‐MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1‐MMP in melanoma pathogenesis. We demonstrate that MT1‐MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1‐MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1‐MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1‐MMP is required to sustain RAC1 activity and promote MT1‐MMP‐dependent cell motility. These data highlight a novel MT1‐MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.     

Inhibitory effect of the carnosine–gallic acid synthetic peptide on MMP‐2 and MMP‐9 in human fibrosarcoma HT1080 cells

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine–gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP‐2 and MMP‐9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP‐2 and MMP‐9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)–uPA receptor signaling pathways to inhibit MMP‐2 and MMP‐9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP‐2 and MMP‐9‐mediated health problems such as metastasis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH(2)-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.     

Development and in vitro efficacy of novel MMP2 and MMP9 specific doxorubicin albumin conjugates.

Two doxorubicin albumin conjugates (A-DP1 and A-DP2), which differ in their substrate specificity for the matrix metalloproteinases MMP2 and MMP9, were prepared by binding maleimide doxorubicin peptide derivatives to the cysteine-34 position of human serum albumin. The incorporated octapeptide, Gly-Pro-Gln-Arg-Ile-Ala-Gly-Gln, in A-DP2 is not cleaved by activated MMP2 and MMP9 in contrast to Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln incorporated in A-DP1 that is cleaved efficiently by activated MMP2 and MMP9 liberating a doxorubicin tetrapeptide. A-DP1 showed antiproliferative activity in a murine renal cell carcinoma line in the low micromolar range (IC(50) value approximately 0.2 microM).     

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.     

Heterogeneity of serum gelatinases MMP‐2 and MMP‐9 isoforms and charge variants

The matrix metalloproteinases (MMPs) gelatinase A (MMP‐2) and gelatinase B (MMP‐9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2‐DZ) as an extension of classic monodimensional zymography (1‐DZ) to analyse the complete pattern of isoforms and post‐translational modifications of both MMP‐9 and MMP‐2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP‐2 isoforms, and at least three different isoforms and two different states of organization of MMP‐9 (the multimeric MMP‐9 and the N‐GAL‐MMP‐9 complex) were observed. In addition, 2‐DZ revealed for the first time that all MMP‐9 and MMP‐2 isoforms actually exist in the form of charge variants: four or five variants in the N‐GAL complex, more charge variants in the case of MMP‐9; and five to seven charge variants for MMP‐2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP‐9) and phosphorylation (MMP‐2) contributed to molecular heterogeneity. The detection of charge variants of MMP‐9 and MMP‐2 in MS serum samples illustrates the power of 2‐DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.     

New signaling pathways from cancer progression modulators to mRNA expression of matrix metalloproteinases in breast cancer cells

We observed previously that each of seven cancer progression inhibitors suppresses the mRNA expression of some matrix metalloproteinases (MMPs), but stimulates that of others, in breast cancer cells. In the present study we tested the effect of overexpressing other cancer modulators on MMP expression. The MMPs tested are MMP1, MMP2, MMP7, MMP13, MMP14, MMP16, MMP19, and MMP25. The proteins that were overexpressed are cancer inhibitors (NME, DRG1, IL10), enhancers (SOD2, FAK, IL17, and CREB), and proteins that suppress cancer progression in cells of some cancers and promote it in others (FUT1, integrin beta3, serpin E1, TIAM1, and claudin 4). Unexpectedly, all of them only lowered MMP mRNA expression, mainly of MMP16, MMP2, and MMP13, in breast cancer cells. Signaling from SOD2 uncoupled the accumulation of two MMP16 mRNA splice variants, suggesting signaling to a late step in MMP16 mRNA accumulation, such as MMP16 mRNA stabilization or late mRNA processing. Signaling that modulates MMP expression differed widely among the total population of MDA-MB-231 cells and single-cell progenies cloned from that population. It also differed substantially between cells of two metastatic breast basal adenocarcinomas, MDA-MB-231 and MDA-MB-468. The present study detected 37 new signaling pathways from cancer progression modulators located upstream of MMP mRNA expression in human breast cancer cells. Our siRNA-induced MMP knockdown data support the interpretation that signaling from MMP19, MMP1, MMP7, MMP12, MMP14, and MMP11 each stimulates the mRNA expression of other MMPs in breast cancer cells.     

基质金属蛋白酶2、9和血管内皮生长因子在碱烧伤小鼠角膜组织中的表达

采取滤纸片放置法,用1mol／L的NaOH在小鼠角膜中央进行碱烧伤,建立动物模型。取烧伤前和烧伤后的小鼠角膜制备组织切片,用抗MMP2、抗MMP9和抗VEGF的抗体分别进行免疫组化染色,检测它们在碱烧伤前后的小鼠角膜组织中的蛋白表达情况;提取碱烧伤前和碱烧伤后不同时间点的小鼠角膜组织的总RNA并进行逆转录,以获得的逆转录产物作为模板,用特异的引物和探针进行实时定量PCR,分别检测MMP2、MMP9和VEGF在碱烧伤前后的小鼠角膜组织中转录水平的变化;提取碱烧伤前后小鼠角膜的蛋白组分,用明胶底物酶谱的方法检测其中MMP2和MMP9的活性变化。结果表明,浸透1mol／L的NaOH的滤纸小片,对小鼠角膜进行碱烧伤可以有效地诱导角膜新生血管形成,烧伤后3-4d就有明显的新生血管形成,至第8、9d左右开始退行。免疫组化结果显示,正常角膜中没有MMP9和VEGF表达,MMP2表达极低;碱烧伤后3种因子的表达均上升。实时定量PCR的结果显示MMP2在碱烧伤后表达升高,第3d达到最高随后下降;MMP9在正常角膜组织中没有表达,碱烧伤后第1d达到最高之后开始下降;VEGF在正常角膜中也没有表达,碱烧伤后第4d达到高峰之后开始下降。以上结果说明碱烧伤后的角膜组织中MMP2、MMP9和VEGF的表达均经历先上升后下降的变化,与角膜愈合及新生血管发育和退化过程相吻合。     

Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2 ubiquitination and degradation

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is a downstream effector of Rho that plays an important role in the tumorigenesis and progression of hepatocellular carcinoma (HCC). Matrix metalloproteinase 2 (MMP2) is a master regulator of tumor metastasis. In this study, we investigated the collections of Rock2 and MMP2 in HCCs and determined the potential role and molecular mechanism of Rock2 in MMP2-mediated invasiveness and metastasis. We found that Rock2 and MMP2 were markedly overexpressed in HCCs compared with the corresponding adjacent tissues, where a positive correlation in their expression was found. The knockdown of Rock2 significantly decreased MMP2 expression and inhibited the invasion and metastasis of HCC in vitro and in vivo. Additionally, the upregulation of MMP2 rescued the decreased migration and invasion induced by the knockdown of Rock2, whereas the knockdown of MMP2 decreased Rock2-enhanced HCC migration and invasion. Mechanistically, Rock2 stabilized MMP2 by preventing its ubiquitination and degradation. Together, our results link two drivers of invasion and metastasis in HCC and identify a novel pathway for MMP2 control.     

The amount and activity of active matrix metalloproteinase 13 is suppressed by estradiol and progesterone in human pelvic floor fibroblasts

As a key degrader of fibrillar collagens, matrix metalloproteinase 13 (MMP13), may contribute to the progression of pelvic organ prolapse. Here we aimed to define the regulation of MMP13 by estradiol and progesterone in the vaginal supportive tissues. Fibroblasts cultured from the arcus tendineous fasciae pelvis of three pre- and three postmenopausal women with prolapse were treated with 17-beta-estradiol (E2), progesterone (P4), E2 + P4, or E2 + ICI 182,780 (ICI). Collagenase inhibitor I (CI) and MG-132 were employed to investigate the mechanism of MMP13 degradation into inactive fragments (fragmentation) by hormones. The regulation of MMP13 in vivo was assessed by comparing tissues of ovariectomized (ovx) vs. sham-operated rats. Expression of MMP13 (proenzyme and active and fragment forms) was quantitated by Western immunoblotting, and MMP13 enzymatic activity was measured using a substrate degradation assay. The amount of cellular active MMP13 and MMP13 proteolytic activity decreased in the presence of hormones. The decrease was paralleled by increased proenzyme and fragment forms. MG-132, not CI, suppressed cellular MMP13 fragmentation. Active MMP13 increased in rats following ovx and was suppressed by E2 + P4 supplementation. Active MMP13 is suppressed in vivo and in vitro by estradiol and progesterone, suggesting a protective effect against vaginal supportive tissue deterioration.