TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type 1 matrix metalloproteinase-dependent mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated as a physiological activator of progelatinase A (MMP-2). We previously reported that plasmin treatment of cells results in proMMP-2 activation and increased type IV collagen degradation. Here, we analyzed the role of MT1-MMP in plasmin activation of MMP-2 using HT-1080 cells transfected with MT1-MMP sense or antisense cDNA. Control, vector-transfected cells that expressed endogenous MT1-MMP, and antisense cDNA transfectants with very low levels of MT1-MMP did not activate proMMP-2. Conversely, cells transfected with sense MT1-MMP cDNA expressed high MT1-MMP levels and processed proMMP-2 to 68/66-kDa intermediate activation products. Control cells and MT1-MMP transfectants had much higher levels of cell-associated MMP-2 than antisense cDNA transfectants. Addition of plasmin(ogen) to control or MT1-MMP-transfected cells generated active, 62-kDa MMP-2, but was ineffective with antisense cDNA transfectants. The effect of plasmin(ogen) was prevented by inhibitors of plasmin, but not by metalloproteinase inhibitors, implicating plasmin as a mechanism for proMMP-2 activation independent of the activity of MT1-MMP or other MMPs. Plasmin-mediated activation of proMMP-2 did not result from processing of proMT1-MMP and did not correlate with alpha(v)beta(3) integrin or TIMP-2 levels. Thus, plasmin can activate proMMP-2 only in the presence of MT1-MMP; however, this process does not require the catalytic activity of MT1-MMP.     

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis

Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.     

Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion

Activation of proMMP-2 by MT1-MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP-2 bound to MT1-MMP on the cell surface acts as a receptor for proMMP-2. Subsequently, adjacent TIMP-2-free MT1-MMP activates the proMMP-2 in the ternary complex. In this study, we demonstrate that MT1-MMP forms a homophilic complex through the hemopexin-like (PEX) domain that acts as a mechanism to keep MT1-MMP molecules close together to facilitate proMMP-2 activation. Deletion of the PEX domain in MT1-MMP, or swapping the domain with the one derived from MT4-MMP, abolished the ability to activate proMMP-2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1-MMP lacking the catalytic domain (MT1PEX-F) efficiently inhibited complex formation of the full-length enzymes and activation of pro MMP-2. Furthermore, expression of MT1PEX-F inhibited proMMP-2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1-MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.     

Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.     

Design,synthesis and biological evaluation of bifunctional inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP)

Collagen degradation and proMMP-2 activation are major functions of MT1-MMP to promote cancer cell invasion. Since both processes require MT1-MMP homodimerization on the cell surface, herein we propose that the use of bifunctional inhibitors of this enzyme could represent an innovative approach to efficiently reduce tumor growth. A small series of symmetrical dimers derived from previously described monomeric arylsulfonamide hydroxamates was synthesized and tested in vitro on isolated MMPs. A nanomolar MT1-MMP inhibitor, compound 6, was identified and then submitted to cell-based assays on HT1080 fibrosarcoma cells. Dimer 6 reduced MT1-MMP-dependent proMMP-2 activation, collagen degradation and collagen invasion in a dose-dependent manner with better results even compared to its monomeric analogue 4. This preliminary study suggests that dimeric MT1-MMP inhibitors might be further developed and exploited as an alternative tool to reduce cancer cell invasion.     

Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of membrane-type 1 matrix metalloproteinase.

The formation of multimeric complexes by membrane-type 1 matrix metalloproteinase (MT1-MMP) may facilitate its autocatalytic inactivation or proMMP-2 activation on the cell surface. To characterize these processes, we expressed various glutathione S-transferase/MT1-MMP fusion proteins in human HT-1080 fibrosarcoma cells and SV40-transformed lung fibroblasts and analyzed their effects on MT1-MMP activity and potential homophilic interactions. We report here that MT1-MMP is expressed on the cell surface as oligomeric 200--240-kDa complexes containing both the active 60-kDa and autocatalytically processed 43-kDa species. Overexpression of a glutathione S-transferase/MT1-MMP fusion protein containing the transmembrane and cytoplasmic domains of MT1-MMP inhibited the phorbol 12-myristate 13-acetate-induced autocatalytic cleavage of endogenous MT1-MMP to the 43-kDa species, but not proMMP-2 activation. On the other hand, a similar fusion protein with the hemopexin, transmembrane, and cytoplasmic domains inhibited proMMP-2 activation in a dominant-negative fashion. These results suggest that both the autocatalytic cleavage of MT1-MMP and proMMP-2 activation may be regulated by oligomerization through the cytoplasmic and hemopexin domains. Indeed, either domain, when attached to the cell membrane by a transmembrane domain, formed stable homophilic complexes. Copurification of MT1-MMP with these fusion proteins correlated with their cell-surface co-localization. Thus, MT1-MMP oligomerization through the hemopexin, transmembrane, and cytoplasmic domains controls its catalytic activity.     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

miRNA-105 and -128 function as rheostats modulating MMP-2 activities by downregulation of TIMP-2 and upregulation of MT1-MMP

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that remodel and degrade the extracellular matrix. Of various MMPs, MMP-2 plays an important role in tumor metastasis. Recently, microRNAs with pro- or anti-metastatic effects were collectively referred to as metastamiRs. We screened 215 human miRNA mimics for modulators of MMP-2 activities in HT-1080 cells, and found that miR-105 and miR-128 promote MMP-2 activities. Bioinformatics analysis predicted that miR-105 and miR-128 both bind to the 3′ untranslated region (UTR) of TIMP-2, an inhibitor of MMP-2 activities. This prediction was verified by reduced luciferase activity in HT-1080 cells co-transfected with miR-105 or miR-128 mimics and plasmids encoding luciferase fused to 3′ UTR of TIMP2. In addition, Western blotting showed that transfection of HT-1080 cells with miR-105 or miR-128 suppressed TIMP-2 levels and enhanced levels of MT1-MMP, an activator of MMP-2 activities. The mechanism by which miR-128 upregulates MT1-MMP was determined to be downregulation of PRKD1, an inhibitor of MT1-MMP, at least in part. Cell invasion assays using Matrigel demonstrated that HT-1080 cells transfected with miR-105 or miR-128 are more invasive as compared to control cells. Taken together, these findings show that miR-105 and miR-128 are metastamir promoting MMP-2 activities via simultaneously downregulating TIMP-2 and upregulating MT1-MMP, and may provide a platform for the development of therapeutics against metastasis.     

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.     

Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.     

Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier

Cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier.     

Cellular activation of proMMP-13 by MT1-MMP depends on the C-terminal domain of MMP-13

Procollagenase-3 (proMMP-13) can be activated by soluble or cell associated membrane type matrix metalloproteinase 1 (MT1-MMP). In this study we show that the cell based activation of proMMP-13 by MT1-MMP was dependent on the C-terminal domain, as delta(249-451) proMMP-13, which lacks the haemopexin domain, and a chimaera from N-terminal MMP-13 and C-terminal MMP-19 (proMMP-13/19) were not processed by MT1-MMP expressing cells. Only the initial cleavage at Gly(35)-Ile(36) was dependent on MT1-MMP activity, as conversion to the active enzyme (Tyr(85) N-terminus) required a functional MMP-13 active site. Unlike proMMP-2 activation, this process was independent of tissue inhibitor of metalloproteinase-2 (TIMP-2) as MT1-MMP expressing cells from the TIMP-2-/- mouse efficiently activated proMMP-13.     

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly to promote cell motility

Fibronectin (FN) matrix assembly is an essential process in normal vertebrate development, which is frequently lost in tumor cells. Here we show that membrane-type 1 matrix metalloproteinase (MT1-MMP) regulates FN matrix assembly. MT1-MMP knockdown induced FN assembly in breast carcinoma cells. Ectopic expression of MT1-MMP reduced specifically the assembled FN matrix level without affecting whole FN production in fibroblasts. Treatment of fibrosarcoma HT1080 cells with dexamethasone (DEX) enhanced FN synthesis, resulting in short fibrils but not dense matrix formation. Combined treatment of DEX and MT1-MMP inhibitor accelerated FN matrix assembly, which mediated cellular adhesion and reduced cell migration and invasion. These results indicate that MT1-MMP stimulates cell migration and invasion by negatively regulating FN assembly.     

Phosphorylation of membrane type 1-matrix metalloproteinase (MT1-MMP) and its vesicle-associated membrane protein 7 (VAMP7)-dependent trafficking facilitate cell invasion and migration

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.     

Progesterone differentially regulates the membrane-type matrix metalloproteinase-1 (MT1 -MMP) compartment of proMMP-2 activation in MG-63 cells.

Osteoblast-derived matrix metalloproteinases (MMPs) are considered to play a crucial role in bone formation and initiation of bone resorption by degrading the bone matrix. MMP-2 is constitutively secreted in a latent zymogen by osteoblasts, and requires the process of activation mediated by membrane-type matrix metalloproteinase-1 (MT1-MMP)/tissue inhibitor of metalloproteinase (TIMP-2) complex in the cell surface. Bone is one target tissue for progestins. In the present study, we observed the effects of progesterone on proMMP-2 activation and MT1-MMP expression, and also TIMP-2 levels in osteoblastic MG-63 cells. Gelatin zymograms and ELISA showed that progesterone have no effects on proMMP-2 activation. Using Western immunoblot analysis, we unexpectedly found that treatment with increasing doses of progesterone in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein, and after 48h treatment, progesterone at 10(-8)M increased MT1-MMP protein level. Confocal immunohistochemistry analysis also confirmed that progesterone induced MT1-MMP expression in MG-63 cells. The results of Northern blot analysis showed that progesterone at 10(-8)M increased MT1-MMP protein levels after 48 h treatment. We also found that TIMP-2 levels were undetectable in MG-63 cells. In conclusion, progesterone increases MT1-MMP protein and mRNA levels in MG-63 cells, but has no effects on proMMP-2 activation, which is partly attributable to the undetectable levels of tissue inhibitor of metalloproteinase-2 (TIMP-2). Our studies suggest that TIMP-2 is involved in proMMP-2 activation, and regulation of MT1-MMP by progesterone may contribute to its actions on bone formation.     

Cell surface activation of progelatinase A （proMMP—2） and cell migration

Gelatinase A (MMP-2) is considered to play a critical role in cell migration and invasion.The proteinase is cerceted from the cell as an inactive zymogen.In vivo it is postulated that activation of progelationase A (proMMP-2) takes place on the cell surface mediated by membrane-type matrix metalloproteinases (MT-MMPs).Recent studies have demonstrated that proMMP-2 is recruited to the cell surface by interacting with tissue inhibitor of metalloproteinases-2 (TIMP-2) bound to MT1-MMP by forming a ternary complex.Free MT1-MMP closely located to the ternary complex then activates proMMP-2 on the cell surface.MT1-MMP is found in cultured invasive cancer cells at the invadopodia.The MT-MMP/TIMP-2/MMP-2 system thus provides localized expression of proteolysis of the extracellular matrix required for cell migration.