MT1-MMP Transcripts Research Articles

Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

Invasive melanoma is an aggressive form of skin cancer with high incidence of mortality. The process of tumor invasion is a crucial primary step in the metastatic cascade, yet the mechanisms involved are still under investigation. Here we document a critical role for MLK3 (MAP3K11) in the regulation of melanoma cell invasion. We report the unexpected finding that cellular loss of MLK3 in melanoma cells promotes cell invasion. Cellular depletion of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. ERK hyperactivation leads to enhanced phosphorylation and inactivation of GSK3β and the stabilization of c-Jun and JNK activity. Blocking of ERK and JNK signaling as well as Hsp90 activity downstream of MLK3-silencing significantly reduces melanoma invasion. Furthermore, ERK activation in the aforementioned context is coupled to MT1-MMP transcription as well as the TOM1L1-dependent localization of the membrane protease to invadopodia at the invasive front. These studies provide critical insight into the mechanisms that couple MLK3 loss with BRAF hyperactivation and its consequence on melanoma invasion.

ELK3 Expression Correlates With Cell Migration, Invasion, and Membrane Type 1-Matrix Metalloproteinase Expression in MDA-MB-231 Breast Cancer Cells.

ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression.

Effect of Matrine on HPAC cell migration by down-regulating the expression of MT1-MMP via Wnt signaling.

AimThis study sought to explore the exact mechanism of Matrine inhibited migration and invasion of human pancreatic cancer cells.MethodsHPAC or Capan-1 cells were cultured in completed RPMI-1640 medium, contained with 50 μg/ml Matrine or 0.05 μg/ml docetaxel, respectively. Cell viability was evaluated by spectrophotometric analysis using MTT assay. Wound healing assay and transwell approach were used to detect the effects of Matrine on HPAC cell migration and invasion. Western Blot and RT-PCR were performed to detect the expressions of MT1-MMP, Wnt and β-Catenin. CHIP assay was used to detect whether the MT1-MMP transcription activity correlated with Wnt signaling pathway.ResultsMTT results indicated that cell proliferration was inhibited by Matrine at a range of concentrations, especially at high dose. We further found that Matrine treatment significantly induced cell migration and invasion decreased. Interestingly, the expression of MT1-MMP decreased evidently upon Matrine treatment, paralleled with the expressions of Wnt and β-Catenin detected by Western Blot and RT-PCR assay. Further analysis of MT1-MMP transcription activity revealed that Matrine reduced the expression of MT1-MMP mediated by Wnt signaling pathway.ConclusionMatrine play a vital role in inhibiting HPAC cellular migration and invasion through down-regulating the expression of MT1-MMP via Wnt signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0210-4) contains supplementary material, which is available to authorized users.

Vinculin negatively regulates transcription of MT1-MMP through MEK/ERK pathway

Vinculin regulates a variety of cellular functions partly through stabilization of tumor suppressor PTEN. In order to study the role of vinculin in tumor progression other than PTEN stabilization, vinculin was knocked down in PTEN-deficient squamous cell carcinoma HSC-4 cells. Knockdown of vinculin induced phenotypical change by reducing cell–cell and cell–extracellular matrix adhesions, and enhanced MT1-MMP expression at transcription level and subsequent cell migration. Up-regulation of MT1-MMP transcription by vinculin knockdown was abrogated by ERK inhibition. These results suggest that vinculin negatively regulates malignant phenotype of tumor cells including MT1-MMP transcription through MEK/ERK pathway.

Angiotensin II evokes angiogenic signals within skeletal muscle through co-ordinated effects on skeletal myocytes and endothelial cells.

Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle.

HPV16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases.

Production of matrix metalloproteinases (MMPs) for degradation of extracellular matrix is a vital step in cancer metastasis. We investigated the effects of HPV16 oncoproteins (16E6, 16E6*I and 16E7), either individually or combined, on the transcription of 7 MMPs implicated in cervical cancer invasiveness. The levels of 7 MMPs reported to be increased in cervical cancer were determined in C33A stably expressing different HPV16 oncoproteins using quantitative RT-PCR and compared with invasion ability of cell lines using in vitro invasion and wound healing assays. Overexpression of MMP-2 and MT1-MMP was detected in HPV16E6E7 expressing cells which correlated with increased cell invasion. Combination of HPV oncoproteins always showed greater effects than its individual form. Inhibition of cell invasion using a specific MMP-2 inhibitor, OA-Hy, and anti-MT1-MMP antibody confirmed that invasion in these cells was dependent on both MMP-2 and MT1-MMP expression. Depletion of HPV16E6E7 by shRNA-mediated knock-down experiments resulted in decreased MMP-2 and MT1-MMP expression levels as well as reduced invasion ability which strongly suggested specific effects of HPV oncoproteins on both MMPs and on cell invasion. Immunohistochemistry study in invasive cervical cancers confirmed the enhanced in vivo expression of these two MMPs in HPV16-infected cells. In addition, possible sites required by HPV16E6E7 on the MMP-2 and MT1-MMP promoters were investigated and PEA3 (at −552/−540 for MMP-2, −303 for MT1-MMP) and Sp1 (at −91 for MMP-2, −102 for MT1-MMP) binding sites were shown to be essential for mediating their transactivation activity. In conclusion, our study demonstrated that HPV16E6 and E7 oncoproteins cooperate in promoting cervical cancer invasiveness by specifically upregulating MMP-2 and MT1-MMP transcription in a similar manner.

Membrane-Type-3 Matrix Metalloproteinase (MT3-MMP) Functions as a Matrix Composition-Dependent Effector of Melanoma Cell Invasion

In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

Abstract 3492: NET as a repressor on transcriptional regulation of MT1-MMP in endothelial cells

Abstract In mice deficient for the Ets family genes, disrupted angiogenesis and malformed vascular system inform critical role of Ets factors in vasculogenesis and angiogenesis. NET is a member of ETS transcription factors and acts as a repressor of transcription. To find role of NET in angiogenesis, angiogenesis related assay were carried out using NET specific siRNA. In addition, it was confirmed regulation of NET on transcription of angiogenesis-regulated genes. Our results showed that suppression of NET expression result in buildup of VEGF-induced tube formation. In addition, suppressed expression of NET induced expression of MT1-MMP. Induction of MT1-MMP expression by knock down of NET expression was not result direct effect of NET on transcription of MT1-MMP in endothelial cells. In endothelial cell, NET suppressed ETS-1 induced-transcriptional activity of MT1-MMP. Our results suggest that NET is a negative regulator of angiogenesis on indirect regulation of MT1-MMP and induction of tube formation by its expressional suppression. 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 3492. doi:10.1158/1538-7445.AM2011-3492

MT1-MMP association with membrane lipid rafts facilitates G-CSF−induced hematopoietic stem/progenitor cell mobilization

Soluble matrix metalloproteinases (MMPs) facilitate the egress of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF)-induced mobilization. Because membrane-type (MT)1-MMP, which is localized on the leading edge of migrating cells, activates the latent forms of soluble MMPs, we investigated its role in HSPC mobilization. We examined the effect of G-CSF on the expression of MT1-MMP and its activities (proMMP-2 activation and migration) in hematopoietic cells. We also investigated the subcellular localization of MT1-MMP and the signaling pathways that regulate its expression and function in hematopoietic cells after exposure to G-CSF. We found that G-CSF increases MT1-MMP transcription and protein synthesis in hematopoietic cells; proMMP-2 activation in cocultures of HSPC with BM fibroblasts; chemoinvasion across reconstituted basement membrane Matrigel toward a stromal cell-derived factor-1 gradient, which is reduced by small interfering RNA silencing of MT1-MMP; and localization of MT1-MMP to membrane lipid rafts through a mechanism that is regulated by the phosphatidylinositol 3-kinase signaling pathway. Disruption of raft formation (by the cholesterol-sequestering agent methyl-beta-cyclodextrin) abrogated phosphatidylinositol 3-kinase phosphorylation and MT1-MMP incorporation into lipid rafts resulting in reduced proMMP-2 activation and HSPC migration. G-CSF-induced upregulation of MT1-MMP in hematopoietic cells and its enhanced incorporation into membrane lipid rafts contributes to proMMP-2 activation, which facilitates mobilization of HSPC.

Abstract 2369: The adhesion molecule MCAM/MUC18 regulates the expression of Id-1 and contributes to melanoma invasion and metastasis

Abstract The acquisition of the metastatic phenotype of melanoma is associated with increase of the melanoma cell adhesion molecule MCAM/MUC18. We have previously shown that MUC18 plays a major role in promoting cellular invasion by regulating MMP-2 expression and activity. Here, we aim to establish the molecular mechanism by which MUC18 regulates melanoma cell invasion to promote tumor growth and metastasis. Utilizing lentivirus-incorporated small hairpin RNA, we stably silenced MUC18 expression in the metastatic melanoma cell lines A375SM and C8161 and conducted cDNA gene expression microarray analyses. We identified the Inhibitor of DNA Binding-1 (Id-1) to be down regulated by 5.7 fold as a result of MUC18 silencing. Id-1 expression has been shown to correlate with the progression of melanoma and to promote cell invasion by regulating MT1-MMP. We have found that Id-1 expression decreased significantly after MUC18 silencing in both A375SM and C8161, thereby corroborating our microarray results. Additionally, we found that MUC18 regulates Id-1 expression at the transcriptional level through regulation of ATF-3. ATF-3 was upregulated by 6 fold in our cDNA microarray analysis. ChIP analysis showed increase binding of ATF-3 to the Id-1 promoter after MUC18 silencing. Moreover, mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18 silenced cells. To further confirm the specificity of regulation of Id-1 and ATF-3 by MUC18, we rescued MUC18 and found that their expression was indeed inverted. Moreover, overexpression of Id-1 in MUC18 silenced cells demonstrated rescue of the invasive phenotype as well as increased activity of MMP-2 and the transcription of MT1-MMP. Collectively, our results suggest that MUC18 contributes to the malignant phenotype of melanoma via regulation of Id-1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2369.

The Essential Roles of TIMP-2, MT1-MMP, and MMP-2 in the Invasive Capacity of SHI-1 Leukemic Cells.

Abstract 3119Poster Board III-56The frequency of extramedullary infiltration (EMI) in acute myeloblastic leukemia (AML) is reported up to 40% and most prevalent in myelomonoblastic and monoblastic subtypes of AML (M4 and M5 according to FAB classification). The majority of patients with EMI suffered poor prognosis. To explore mechanism underlying EMI, we analyzed SHI-1 cells, a highly invasive human acute monocytic leukemia cell line, and other leukemia cells such as NB4, K562, U937 and THP-1 cells. qRT-PCR revealed that the transcription of TIMP-2, MMP-2 and MT1-MMP in SHI-1 cells were all higher than other leukemic cells(P<.05). Western blotting analysis showed that expression of these genes in SHI-1 cells were also significantly higher than that in other leukemic cells at the protein level (P<.01). By using cell migration assay to determine the invasive capability of leukemic cells, and BMSCs co-culture to mimic the microenvironment of bone marrow, we found that the invasion rates of NB4, K562, U937 and THP-1 cells were only 5% to 60% of SHI-1 cells(P<.05). To elucidate the individual contribution of TIMP-2, MMP-2 and MT1-MMP to the invasive capability of SHI-1 cells, RNA interference (RNAi) was applied to individually knock down these genes. As determined by qRT-PCR 24 hours after transfection, the transcription of theses genes was inhibited to 85% to 98%, respectively, when compared with control cells transfected with non–target-directed siRNA(P<.001). Immunoblot analysis on cell lysates revealed a minimal expression of these genes at protein level. Cells being treated with RNAi were applied to the transwell assay to detect their migratory potential. We found that the down-regulation of TIMP-2, MMP-2 and MT1-MMP significantly decreased the invasion rates of SHI-1 cells by 60%-70%, 50%-60% and 40%-50%, respectively (P<.05). These findings suggested that all these three genes enhance the process of SHI-1 cells invasion. To determine the precise role of TIMP-2 in this specific leukemic cell, SHI-1 cells were transfected with human TIMP-2 cDNA. Following G418 selection and limited dilution, three stable subclones (S1, S2 and S3) were isolated and expanded. qRT-PCR analysis showed that the mRNA level of TIMP-2 increased about 3 fold, 2 fold and 1.5 fold in S1, S2 and S3 cells, respectively(P<.05), whereas the MMP-2 and MT1-MMP almost unchanged. Western blotting further exhibited that protein levels of TIMP-2 in S1, S2 and S3 cells increased 2.6 fold, 1.5 fold and 1.3 fold, respectively(P<.01) when compared with the control SHI-1 cells, while no difference of MMP-2 and MT1-MMP protein level could be found between three subclone cells and SHI-1 cells. These subclone cells co-cultured with BMSC were then subjected to transwell assay to detect the influence of TIMP-2 up-regulation on cell invasion. We found that the invasion rates of these subclone cells elevated about 1.5 fold to 2.5 fold (P<.05). To explore the underlying mechanism of high expression of TIMP-2, MMP-2 and MT1-MMP promoting SHI-1 cells invasion, by using zymography to detect proMMP-2 secretion and MMP-2 activation, we found that released proMMP-2 (72KDa) as well as activated MMP-2 (62KDa) from SHI-1 cells were both higher than that in other leukemic cells (P<.05). Furthermore, three TIMP-2 transfected SHI-1 subclones demonstrated about 1.5 fold to 3.0 fold (P<.01) increase in activated MMP-2 while the proMMP-2 secretion remained unchanged. Down-regulating the expression of MMP-2, but not MT1-MMP and TIMP-2, led to a markedly decline of secreted proMMP-2. Activated MMP-2 had not been found in the supernatants any of these transfected cell culture. These results suggested that up-regulation of TIMP-2 as well as elevation of MMP-2 and MT1-MMP-2 expression will promote SHI-1 cell invasion, at least partially via the increase of MMP-2 activation. In conclusion, our study demonstrated that constitutively high expression of MMP-2, MT1-MMP and TIMP-2 in SHI-1 leukemic cells facilitated cell invasion by promoting MMP-2 activation. Up-regulation of TIMP-2 exhibited not a repressive but an active effect on SHI-1 leukemic cells invasion. An elevated expression of TIMP-2 in AML patients may have higher risk of EMI, particularly in patients in company with high levels of MMP-2 and MT1-MMP. We suggest that a careful analysis of TIMP/MMP ratio in vivo may be useful to monitor and predict extramedullary infiltration of leukemia cells in AML patients. DisclosuresNo relevant conflicts of interest to declare.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Abstract 3536Poster Board III-473Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures:No relevant conflicts of interest to declare.

Differential role of β‐catenin in VEGF and histamine‐induced MMP‐2 production in microvascular endothelial cells

Increases in endothelial cell permeability and production of matrix-degrading enzymes are two early steps in the angiogenic process. Factors such as vascular endothelial growth factor (VEGF) and histamine induce the angiogenic process through alterations in both permeability and proteolysis. We hypothesized that beta-catenin acts as a positive regulator of MMP-2 and MT1-MMP transcription following VEGF or histamine stimulation. Rat microvascular endothelial cells were exposed to VEGF or histamine overnight and MMP-2 protein production was assessed by gelatin zymography. Latent MMP-2 protein levels were increased following VEGF and histamine treatment as were MMP-2 mRNA transcript levels. Endothelial cells exposed to VEGF and histamine had an increased level of nuclear beta-catenin, which was sensitive to inhibition of the PI3-kinase signaling pathway. Promoter assays indicated increased transcriptional activity of both MMP-2 and MT1-MMP in endothelial cells co-transfected with luciferase reporter constructs and beta-catenin. Inhibition of beta-catenin signaling with inhibitor of catenin and T cell factor (ICAT) revealed that the VEGF-induced increase in MMP-2 mRNA is beta-catenin dependent. Interestingly, while MMP-2 mRNA levels increased in response to histamine H1 or H2 receptor activation, significantly larger increases were observed in cells co-treated with ICAT and histamine or the histamine receptor agonists, HTMT and dimaprit. While both VEGF and histamine increase nuclear beta-catenin and MMP-2 production, the role of beta-catenin in MMP-2 regulation differs between the two stimuli.

C‐Jun regulates MMP‐2 and MT1‐MMP mRNA expression in endothelium.

Angiogenesis is the formation of new capillaries from pre-existing ones. Proteolysis of the basement membrane and the interstitial matrix is mediated by the matrix metalloproteinases (MMPs) and is critical to the angiogenic process. We showed previously that reorganization of the actin cytoskeleton induces the expression of MMP-2 and membrane type (MT)1-MMP through the activation of JNK. Here we examined the involvement of the transcription factor c-Jun, a downstream effector of JNK. We hypothesized that c-Jun regulates the transcription of both MMP-2 and MT1-MMP. Treatment of skeletal muscle endothelial cells (SMEC) with small interference (si)RNA targeting c-Jun decreased basal levels of c-Jun protein by 40% and resulted in decreased basal MMP-2 and MT1-MMP mRNA expression. Activation of JNK by anisomycin increased MMP-2 and MT1-MMP mRNA expression and this induction was attenuated by c-Jun siRNA treatment. Depolymerization of the actin cytoskeleton, which increases MMP-2 protein production and activation, and both were attenuated with siRNA treatment. Vascular endothelial growth factor (VEGF) induction of MMP-2 mRNA expression was repressed by c-Jun siRNA. Our results point to c-Jun as the transcription factor regulating MMP-2 and MT1-MMP mRNA expression downstream of JNK. Funded by NSERC and CIHR.

Endothelial cell regulation of matrix metalloproteinases

The process of sprouting angiogenesis requires that the endothelial cells degrade the basement membrane matrix and migrate into the interstitial matrix. Matrix metalloproteinases are enzymes capable of cleaving numerous extracellular matrix proteins. Increased production and activity of matrix metalloproteinases in any cell type is associated with a more migratory and invasive phenotype. This paper describes results of recent in-vitro studies of the regulation of transcription and activation of MMP-2 and MT1-MMP in endothelial cells, as well as studies that examined roles of matrix metalloproteinases in activity-induced angiogenesis.

Gestational profile of matrix metalloproteinases in rat uterine artery.

Mechanisms underlying structural reorganization of the uterine artery in pregnancy remain largely unknown. Matrix metalloproteinases (MMPs) which are involved in degradation of vascular wall matrix are likely to play a key role. In this investigation of rat uterine artery, key MMPs and the specific tissue inhibitors of MMPs (TIMPs) together with three housekeeping genes were studied before, during and after pregnancy, using real time PCR. Data were analysed by partial least squares analysis as well as by conventional univariate methods. Each gene studied [MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, membrane-type 1 (MT1)-MMP, TIMP-1, TIMP-2, GAPDH, cyclophilin and beta-actin] increased in late pregnancy (day 21). MMP-2, MT1MMP, MMP-3 and TIMP-1 transcripts were also elevated at day 7. TIMP-1 and MMP-3 mRNA expression returned to virgin control values in the post-partum, whereas others remained elevated or increased further (MMP-9, MMP-13). Gelatin zymography showed maximum elevation of MMP-2 at day 21. A novel 43-45 kDa gelatinolytic doublet was observed which increased in density with gestation and may represent an active MMP-2 fragment. Together, these data strongly suggest that MMPs and TIMPs are likely to play an important role in remodelling uterine arteries in rat pregnancy and may represent means by which vasodilatation is maintained in later pregnancy. Continued elevated levels of some MMPs post-partum may contribute to vessel regression and return to a non-pregnant physiological state.

In vitro migration of melanoblasts requires matrix metalloproteinase-2: implications to vitiligo therapy by photochemotherapy.

It is known that the migration of melanocyte precursors (melanoblasts) from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial to the repigmentation of vitiliginous skin treated with photochemotherapy (PUVA), but such migratory cells must penetrate extracellular matrix tissue barriers in vivo. To test the hypothesis that matrix metalloproteinases (MMPs) are required for this process, we determined whether cultured melb-a cells, an immortal line of melanoblasts isolated from neonatal mouse epidermis, express and secrete MMPs and whether a synthetic metalloproteinase inhibitor, GM6001 (Galardin), inhibits their migratory behavior in vitro. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine the patterns of MMP expression by melanoblasts at the mRNA and protein levels, respectively. The proteolytic activities of MMPs secreted into the culture medium were assessed by gelatin zymography. The capacity of melanoblasts to migrate on fibronectin, laminin or laminin-5 substrates was estimated using Transwell migration assays. The results show that MMP2, MMP9 and MT1-MMP transcripts are expressed by these melanoblasts, but only MMP2 is secreted and activated in the extracellular environment. Although the therapeutic efficacy of PUVA in stimulating repigmentation of vitiliginous skin might derive from direct effects of UVA and/or 8-methoxypsoralen (8MOP), recent studies have shown that keratinocyte-derived factors induced by ultraviolet radiation, especially alpha-melanocyte stimulating hormone (alpha MSH), play a major role in regulating melanocyte function. Therefore, we also examined whether 8MOP and/or alphaMSH are involved in the up-regulation of MMP2 expression in melanoblasts. Western blotting and zymographic analyses revealed that MMP2 synthesis and secretion were induced by 8MOP and/or by alpha MSH. This induction of MMP2 resulted in significant increases of migration by melanoblasts on laminin or on laminin-5 substrates, while concomitant treatment with GM6001 blocked that induced migration. Taken together, these results suggest the importance of MMP2 in melanoblast migration and in the response to PUVA therapy.

Balance between activation and inhibition of matrix metalloproteinase-2 (MMP-2) is altered in colorectal tumors compared to normal colonic epithelium.

Matrix metalloproteinase-2 (MMP-2) is overexpressed in human cancers and facilitates tumor growth and metastasis. It is synthesized as an inactive proenzyme that is activated by membrane-type matrix metalloproteinase-1 (MT1-MMP) and inhibited by tissue inhibitor of metalloproteinase-2 (TIMP-2). We hypothesized that there is an imbalance between the expression of TIMP-2 and the expression of MMP-2 and MT1-MMP that favors activation of MMP-2 in malignant colon tumors compared to normal colonic tissue. Specimens of colon tumors and of adjacent normal mucosa were obtained from 22 patients at the time of surgical resection. MMP-2, MT1-MMP, and TIMP-2 RNA transcripts were measured in each sample using a quantitative reverse transcriptase polymerase chain reaction assay. We observed that MMP-2 RNA levels were significantly elevated in tumors compared to normal tissue (P = 0.039). In addition, the TIMP-2:MMP-2 ratio was twofold lower (P = 0.001) and the TIMP-2:MT1-MMP ratio was 1.5-fold lower (P = 0.003) in tumors compared to normal mucosa. These results suggest that the balance between genes that activate and inhibit MMP-2 is shifted toward activation in colon tumors. The abnormal expression of gene products that regulate MMP-2 activity may be an important early step in the malignant transformation of colon cancer and may provide a useful target for new chemoprevention and adjuvant treatment strategies.

Coordinated Induction of Extracellular Proteolysis Systems During Experimental Autoimmune Encephalomyelitis in Mice

Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) are considered to play an important role in the pathogenesis of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is widely used as an animal model of multiple sclerosis. Whereas several studies have addressed the expression of various MMPs and their inhibitors in the pathogenesis of EAE, the expression of the molecules of the PA system during EAE has not been reported previously. The present study was undertaken to investigate the expression of the molecules of the PA system (tPA, uPA, PAI-1, uPAR, LRP), as well as several members of the MMP family and their inhibitors in the course of actively induced EAE in BALB/c mice. During clinical EAE, the PA system was up-regulated in the central nervous system at several levels. Induction of expression of tPA and PAI-1 transcripts was detected in activated astrocytes in the white matter. Inflammatory cells expressed uPA receptor, uPAR. In situ zymography demonstrated the presence of increased tPA and uPA activities in the areas of the inflammatory damage. Accumulation of fibrin, fibronectin, and vitronectin immunoreactivity was seen in perivascular matrices of symptomatic animals. In addition, transcription of MT1-MMP and metalloelastase (in inflammatory cells), and TIMP-1 (in activated astrocytes) was induced during EAE. Increased gelatinolytic activity was detected at the sites of inflammatory cell accumulation by in situ zymography of fluorescently labeled gelatin; substrate gel zymography identified the up-regulated gelatinolytic activity as gelatinase B. Overall, our study demonstrates concurrent induction of PA and MMP systems during active EAE, supporting further the concept that the neuroinflammatory damage in EAE involves altered balance between multiple extracellular proteases and their inhibitors.

In Vivo Migration of Transplanted Myoblasts Requires Matrix Metalloproteinase Activity

Muscle cell migration and extracellular matrix remodeling are essential aspects of muscle development and regeneration. In this study, using a new technique to assess in vivo myoblast migration, we have confirmed previous results showing that the C2C12 myoblast cell line exhibits a higher migratory capacity than primary myoblasts. To test the hypothesis that matrix metalloproteinases (MMPs) are required for the migration of C2C12 myoblasts, we determined whether a synthetic metalloproteinase inhibitor, BB94 (Batimastat), inhibited this process in vivo. Pretreatment with BB94 for 3 days decreased the C2C12 migration at 2 days after cell injection. Since MMP expression is thus necessary for myoblast migration, we have undertaken the identification and characterization of the MMPs expressed by the C2C12 cell line. An RT–PCR assay was used to determine the pattern of MMP mRNA expression by the C2C12 cell line. The proteolytic activities of the MMPs secreted in the culture medium were also assessed by gelatin zymography. The results showed that MMP2 (gelatinase A, 72-kDa type IV collagenase) and MT1-MMP transcripts were expressed by this cell line; however, only MMP2 was secreted and was able to be activated in the extracellular environment. This cell line failed to express MMP9 (gelatinase B, 92-kDa type IV collagenase), stromelysine 2, or stromelysine 3. Our observation that the membrane type MMP (MT1-MMP) transcript is also expressed by the C2C12 suggests that the MMP2 proform (pro-MMP2), may be activated by the MT1-MMP. This possibility is supported by our observation that the pretreatment of C2C12 with concanavalin A (which is known to induce the expression of MT1-MMP) resulted in the processing of pro-MMP2 to its mature form, in a dose-dependent manner. Overexpression and activation of MMP2 in normal myoblasts showed significant increased migration of mouse myoblasts in vivo. Our finding that MMP2 and MT1-MMP gene are coexpressed by C2C12 myoblasts could account for the high migratory capacity of C2C12. Together these results supported the importance of MMP2 and its activation by MT1-MMP for myoblast migration.