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.     

Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling

Endothelial cell (EC)-derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin α(V) . MPs isolated from EC-conditioned medium (Sup) were significantly enriched for matrix-altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP-1 and TIMP-2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP-2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP-2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies.     

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.     

Proteolytic action of kallikrein-related peptidase 7 produces unique active matrix metalloproteinase-9 lacking the C-terminal hemopexin domains

The gelatinases, matrix metalloproteinase (MMP)-9 and -2, are produced as latent, inactive enzymes that can be proteolytically activated by a number of proteases. In many normal and pathological conditions, where the expression of MMPs is deregulated, changes in the expression of other proteases have also been reported. Human kallikrein-related peptidase 7 (KLK7), a chymotryptic-like serine protease, is overexpressed in many different types of neoplastic conditions, which have also been shown to express high levels of both MMP-9 and -2. Since the activation of MMPs by KLK7 has never been examined, we sought to determine whether KLK7 can activate these MMPs. To test this hypothesis KLK7 was incubated with the recombinant MMPs and the products of the reaction were analyzed for their activity. Incubation of proMMP-9 with KLK7 resulted in the production of a novel truncated, active MMP-9 lacking the C-terminal hemopexin domains. In contrast, KLK7 degraded, but did not activate, proMMP-2. The novel activation of proMMP-9 by KLK7 was further confirmed using conditioned medium prepared from an MMP-9-expressing cell line, MDA-MMP-9. Our results clearly establish that KLK7 activates proMMP-9 to produce a novel truncated, active MMP-9 product not generated by other proteases. These findings suggest that KLK7 may play an important role in the activation of MMP-9 in tumors that express high levels of both these proteases and the resulting truncated MMP may possess altered substrate specificities compared with full-length MMP-9 activated by other proteases.     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

DIDS Prevents Ischemic Membrane Degradation in Cultured Hippocampal Neurons by Inhibiting Matrix Metalloproteinase Release

During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ～20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus-evoked release of von Willebrand Factor from human umbilical vein endothelial cells. We conclude that DIDS inhibits MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.     

Activation of GABA‐A receptor ameliorates homocysteine‐induced MMP‐9 activation by ERK pathway

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase‐9 (MMP‐9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ‐aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA‐A receptor in Hcy‐induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP‐9 activity via the extracellular signal‐regulated kinase (ERK) signaling pathway and by inhibition of GABA‐A receptors, thus behaving as an inhibitory neurotransmitter. Hcy‐induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′‐dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate‐oxidase‐4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP‐9, measured by gelatin zymography. The GABA‐A receptor agonist, muscimol ameliorated the Hcy‐mediated MMP‐9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase‐4 (TIMP‐4). Treatment of the endothelial cell with muscimol restored the levels of TIMP‐4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy‐induced MMP‐9 via ERK signaling pathway. These results suggest that Hcy competes with GABA‐A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. J. Cell. Physiol. 220: 257–266, 2009. © 2009 Wiley‐Liss, Inc.     

Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.     

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.     

Activated protein C directly activates human endothelial gelatinase A

Angiogenesis (formation of new blood vessels) occurs in a number of diseases such as cancer and arthritis. The matrix metalloproteinase (MMP), gelatinase A, is secreted by endothelial cells and plays a vital role during angiogenesis. It is secreted as a latent enzyme and requires extracellular activation. We investigated whether activated protein C (APC), a pivotal molecule involved in the body's natural anti-coagulant system, could activate latent gelatinase A secreted by human umbilical vein endothelial cells (HUVEC). APC induced the fully active form of gelatinase A in a dose (100-300 nM)- and time (4-24 h)-responsive manner. The inactive zymogen, protein C, did not activate gelatinase A when used at similar concentrations. APC did not up-regulate membrane type 1 MMP (MT1-MMP) mRNA in HUVEC. In addition, the MMP inhibitor, 1, 10-phenanthroline (10 nM), was unable to inhibit APC-induced activation. These results suggested that MT1-MMP was not involved in the activation process. APC activation of gelatinase A occurred in the absence of cells, indicating that it acts directly. APC may contribute to the physiological/pathological mechanism of gelatinase A activation, especially during angiogenesis.     

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.     

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage

Background

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.

Methods

The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.

Results

TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.

Conclusion

TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.

General significance

Proteolytic cleavage of TAA90K may have functional implications in colon cancer.     

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.     

Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.     

Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA

Homology screening for human membrane-type MMP (MT–MMP) was carried out, and cDNA encoding a soluble type of MT3–MMP (SM3), which is considered to be an alternatively spliced variant of MT3–MMP, was obtained. SM3 had a novel sequence consisting of 50 amino acids after Lys407 instead of amino acids containing the transmembrane domain of MT3–MMP. When SM3 tagged with a FLAG epitope (SM3–flag) was expressed in COS-7 cells, SM3–flag was present in the conditioned medium in its activated form. The enzymatic activity of SM3 was studied using a recombinant enzyme expressed in E. coli (SM3-e). The fluorogenic peptide substrate hydrolyzing activity of SM3-e was inhibited by EDTA and by the tissue inhibitor of metalloproteinase-2 (TIMP-2), whereas TIMP-1 had only relatively weak inhibitory ability. SM3-e was able to activate proMMP-2, and this activity was also inhibited by TIMP-2 but not by TIMP-1. SM3-e was able to cleave type III collagen, and also digested fibronectin. In view of the homology of the primary structures, MT3–MMP was considered to have the same catalytic activity as SM3. The results of studies of SM3's activity on extracellular matrix (ECM) protein suggests that MT3–MMP plays a role in ECM turnover not only by activating proMMP-2 but also by acting directly on ECM macromolecules.     

Reduced nonprotein thiols inhibit activation and function of MMP-9: implications for chemoprevention

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur "blockage" of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular-extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.     

Xanthine oxidase activates pro-matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells through non-free radical mechanisms

Reactive oxygen species (ROS) have been implicated in the regulation of matrix metalloproteinases (MMPs). The xanthine/xanthine oxidase (X/XO) reaction has been widely used as a source of exogenous ROS in studying MMPs, but commercial XO has also been known to be contaminated by proteolytic activity, and MMPs are protease sensitive substrate. We have investigated the activation of proMMP-2 by X/XO in cultured vascular smooth muscle cells (SMCs). SMCs were incubated with X/XO (unpurified or purified) or XO alone for 24h. X/XO activated proMMP-2 in a dose-dependent manner. A similar profile was observed using XO. Purified XO produced lower amounts of active MMP-2 compared to unpurified XO. EPR study showed that X/XO, not XO itself, produced superoxide anion, which was completely scavenged by SOD. However, X/XO-induced proMMP-2 activation could not be inhibited by combination of SOD and catalase. Incubation with XO either in cell-free conditioned media or in cells resulted in similar amounts of active MMP-2, suggesting that membrane-type-MMPs were not involved in proMMP-2 activation. This was further confirmed by the lack of inhibitory effect of hydroxamate MMP inhibitor, BB1101. Aprotinin blocked unpurified XO-induced proMMP-2 activation in a dose-dependent manner, demonstrating the proteolytic activity contained in XO is essential. We conclude that proteolytic activity contained in XO, rather the ROS derived from X/XO, is responsible for proMMP-2 activation in cultured SMCs. The results also suggest that caution needs to be taken when interpreting the reported results on activation of MMPs where X/XO had been used as an "authentic" source of superoxide anion.     

Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.