Paradigmatic identification of MMP-2 and MT1-MMP activation systems in cardiac fibroblasts cultured as a monolayer

Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Characterization of a truncated recombinant form of human membrane type 3 matrix metalloproteinase.

Membrane type 3 matrix metalloproteinase (MT3-MMP), an activator for the zymogen of MMP-2 (proMMP-2, or progelatinase A), is known to be expressed in human placenta, brain, lung and rat vascular smooth muscle cells, but information about its biochemical properties is limited. In the present study, we expressed and purified a truncated form of MT3-MMP lacking the transmembrane and intracytoplasmic domain (DeltaMT3) and characterized the enzyme biochemically. DeltaMT3 digested type III collagen into characteristic 3/4- and 1/4-fragments by cleaving the Gly781-Ile782 and Gly784-Ile785 bonds of alpha1(III) chains. Although DeltaMT3 did not have such an activity against type I collagen, it attacked the Gly4-Ile5 bond of the triple helical portion of alpha2(I) chains, leading to removal of the crosslink containing N-terminal telopeptides. By quantitative analyses of the activities of DeltaMT3 and a similar deletion mutant of MT1-MMP (DeltaMT1), DeltaMT3 was approximately fivefold more efficient at cleaving type III collagen. DeltaMT3 also digested cartilage proteoglycan, gelatin, fibronectin, vitronectin, laminin-1, alpha1-proteinase inhibitor and alpha2-macroglobulin into almost identical fragments to those given by DeltaMT1, although carboxymethylated transferrin digestion by DeltaMT3 generated some extra fragments. The activity of DeltaMT3 was inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2) and TIMP-3 in a 1 : 1 stoichiometry, but not by TIMP-1. ProMMP-2 was partially activated by DeltaMT3 to give the intermediate form. These results indicate that, like MT1-MMP, MT3-MMP exhibits proteolytic activities against a wide range of extracellular matrix molecules. However, differences in the proMMP-2 activation and tissue distribution suggest that MT3-MMP and MT1-MMP play different roles in the pathophysiological digestion of extracellular matrix.     

Type I collagen-induced pro-MMP-2 activation is differentially regulated by H-Ras and N-Ras in human breast epithelial cells

Tumor cell invasion and metastasis are often associated with matrix metalloproteinases (MMPs), among which MMP-2 and MMP-9 are of central importance. We previously showed that H-Ras, but not N-Ras, induced invasion of MCF10A human breast epithelial cells in which the enhanced expression of MMP-2 was involved. MMP-2 is produced as a latent pro-MMP-2 (72 kDa) to be activated resulting the 62 kDa active MMP-2. The present study investigated if H-Ras and/or N-Ras induces pro-MMP-2 activation of MCF10A cells when cultured in two-dimensional gel of type I collagen. Type I collagen induced activation of pro-MMP-2 only in H-Ras MCF10A cells but not in N-Ras MCF10A cells. Induction of active MMP-2 by type I collagen was suppressed by blocking integrin alpha2, indicating the involvement of integrin signaling in pro-MMP-2 activation. Membrane-type (MT)1-MMP and tissue inhibitor of metalloproteinase (TIMP)-2 were up-regulated by H-Ras but not by N-Ras in the type I collagen-coated gel, suggesting that H-Ras-specific up-regulation of MT1-MMP and TIMP-2 may lead to the activation of pro-MMP-2. Since acquisition of pro-MMP-2 activation can be associated with increased malignant progression, these results may help understanding the mechanisms for the cell surface matrix-degrading potential which will be crucial to the prognosis and therapy of breast cancer metastasis.     

Functional interplay between type I collagen and cell surface matrix metalloproteinase activity

Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves beta(1) integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both alpha(2)beta(1) and alpha(3)beta(1) integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of alpha(3)beta(1) strongly promotes activation of pro-MMP-2, whereas alpha(2)beta(1) integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.     

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)₂/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)₂/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.     

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.     

TIMP independence of matrix metalloproteinase (MMP)-2 activation by membrane type 2 (MT2)-MMP is determined by contributions of both the MT2-MMP catalytic and hemopexin C domains

The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP.MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2.     

TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras—extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.     

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.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.     

Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation

Previous studies have shown that membrane type 1-matrix metalloproteinase (MT1-MMP) (MMP-14) initiates pro-MMP-2 activation in a process that is tightly regulated by the level of tissue inhibitor of metalloproteinase (TIMP)-2. However, given the difficulty in modulating TIMP-2 levels, the direct effect of TIMP-2 on MT1-MMP processing and on pro-MMP-2 activation in a cellular system could not be established. Here, recombinant vaccinia viruses encoding full-length MT1-MMP or TIMP-2 were used to express MT1-MMP alone or in combination with various levels of TIMP-2 in mammalian cells. We show that TIMP-2 regulates the amount of active MT1-MMP (57 kDa) on the cell surface whereas in the absence of TIMP-2 MT1-MMP undergoes autocatalysis to a 44-kDa form, which displays a N terminus starting at Gly(285) and hence lacks the entire catalytic domain. Neither pro-MT1-MMP (N terminus Ser(24)) nor the 44-kDa form bound TIMP-2. In contrast, active MT1-MMP (N terminus Tyr(112)) formed a complex with TIMP-2 suggesting that regulation of MT1-MMP processing is mediated by a complex of TIMP-2 with the active enzyme. Consistently, TIMP-2 enhanced the activation of pro-MMP-2 by MT1-MMP. Thus, under controlled conditions, TIMP-2 may act as a positive regulator of MT1-MMP activity by promoting the availability of active MT1-MMP on the cell surface and consequently, may support pericellular proteolysis.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.     

Mechanical forces-induced human osteoblasts differentiation involves MMP-2/MMP-13/MT1-MMP proteolytic cascade

Matrix metalloproteinase (MMP) family proteins play diverse roles in many aspects of cellular processes such as osteoblastic differentiation. Besides, mechanical forces that occur in 3D collagen gel promote the osteoblastic phenotype and accelerate matrix mineralization. Although MMPs have been involved in bone differentiation, the proteolytic cascades triggered by mechanical forces are still not well characterized. In this study, we have investigated the contribution of both proteolytic cascades, MMP-3/MMP-1 and MMP-2/MMP-13/MT1-MMP in the differentiation of human osteoblasts cultured in a floating type I collagen lattice (FL) versus an attached collagen lattice (AL). Compared to AL, contraction of human osteoblasts-populated FL led to a fast (1 day) induction of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteoprotegerin (OPG), and Runx-2 expression. At day 4, osteocalcin (OC) overexpression preceded the formation of calcium-containing nodule formation as assessed by X-ray analyses. MMP-1 and MMP-3 were produced to similar extent by cells cultured in FL and AL, whereas contraction of collagen lattices triggered both mRNA overexpression of MMP-2, MMP-13, and MT1-MMP (i.e., MMP-14), and their activation as evidenced by Western blotting or zymographic analyses. Down-regulating MT1-MMP expression or activity either by siRNA transfection or supplementation of culture medium with TIMP-1 or TIMP-2 highlighted the contribution of that enzyme in OC, ALP, and OPG expression. MMP-2 and MMP-13 were more directly involved in BSP expression. So, these results suggest that the main proteolytic cascade, MMP-2/MMP-13/MT1-MMP, and more particularly, its initial regulator MT1-MMP is involved in osteoblast differentiation through mechanical forces.     

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.     

Type I collagen-induced MMP-2 activation coincides with up-regulation of membrane type 1-matrix metalloproteinase and TIMP-2 in cardiac fibroblasts

Migration of cardiac fibroblasts is implicated in infarct healing and ventricular remodeling. Activation of matrix metalloproteinases induced by three-dimensional type I collagen, the principal component of the myocardial interstitium, is hypothesized to be essential for this migration. By utilizing primary cultures of cardiac fibroblasts and collagen lattice models, we demonstrated that type I collagen induced MMP-2 activation, and cells undergoing a change from isometric tension to mechanical unloading were associated with increased levels of total and active MMP-2 species. The collagen-induced MMP-2 activation coincided with up-regulated cellular levels of both membrane type 1-matrix metalloproteinase (MT1-MMP) and TIMP-2. A fraction of cellular membrane prepared from cells embedded in the collagen lattice containing active MT1-MMP and TIMP-2 was capable of activating pro-MMP-2, and exogenous TIMP-2 had a biphasic effect on this membrane-mediated MMP-2 activation. Interestingly, the presence of 43-kDa MT1-MMP species in a fraction of intracellular soluble proteins prepared from monolayer cells but not cells embedded in the lattices indicates that MT1-MMP metabolizes differently under the two different culture conditions. Treatment of cells embedded in the lattice with furin inhibitor attenuated pro-MT1-MMP processing and MMP-2 activation and impeded cell migration and invasion. These results suggest that the migration and invasion of cardiac fibroblasts is furin-dependent and that the active species of MT1-MMP and MMP-2 may be involved in both events.     

Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins

Culture of human dermal fibroblasts within a three-dimensional matrix composed of native type I collagen fibrils is widely used to study the cellular responses to the extracellular matrix. Upon contact with native type I collagen fibrils human skin fibroblasts activate latent 72-kDa type IV collagenase/ gelatinase (MMP-2) to its active 59- and 62-kDa forms. This activation did not occur when cells were cultured on plastic dishes coated with monomeric type I collagen or its denatured form, gelatin. Activation could be inhibited by antibodies against MT1-MMP, by the addition of TIMP-2 and by prevention of MT1-MMP processing. MT1-MMP protein was detected at low levels as active protein in fibroblasts cultured as monolayers. In collagen gel cultures, an increase of the active, 60-kDa MT1-MMP and an additional 63-kDa protein corresponding to inactive MT1-MMP was detected. Incubation of medium containing latent MMP-2 with cell membranes isolated from fibroblasts grown in collagen gels caused activation of the enzyme. Furthermore, regulation of MT1-MMP expression in collagen cultures seems to be mediated by alpha2beta1 integrins. These studies suggest that activation of the proMMP-2 is regulated at the cell surface by a mechanism which is sensitive to cell culture in contact with physiologically relevant matrices and which depends on the ratio of proenzyme and the specific inhibitor TIMP-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.