Migration rate of rabbit bone-marrow stromal cells and rabbit dermal fibroblasts in different gels and activity of their MMPS

This work deals with the study of the migration rate of rabbit cells in collagen and fibrin gels. It has been shown that dermal fibroblasts more actively migrate in collagen gel, whereas in fibrin gel bone-marrow stromal cells do. In the study of activities of matrix metalloproteinases (MMPs) that are synthesized by cells in the process of cultivation, MMP-2 activity in cells of both types was established as being higher during migration in collagen gel, while MMP-9 activity was so during migration in fibrin gel. The different cell migration rates may be due to the cell properties, to the activity of their synthesized MMP, and to the effect up this process of the cell microenvironment (collagen or fibrin).     

Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus

Postnatal development of the mouse uterus involves differentiation and development of the endometrial glands as well as the myometrium. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in extracellular matrix breakdown and morphogenesis of many epitheliomesenchymal organs. As a first step to understanding their roles in postnatal mouse uterine development, MMPs and TIMPs found to be expressed in the neonatal mouse uterus by microarray analysis were localized by in situ hybridization. The MMP-2 mRNA was detected only in the uterine stroma, whereas the MMP-10 mRNA was present only in the uterine epithelium from Postnatal Day (PND) 3 to PND 9. All other MMPs (MMP-11, MMP-14, and MMP-23) as well as TIMP-1, TIMP-2, and TIMP-3 were detected in both epithelial and stromal cells of the endometrium, but not in the myometrium. Uterine extracts were then analyzed by gelatin and casein gel zymography to detect active gelatinases and stromelysins, respectively. Five major gelatinase bands of activity were detected and inhibited by the MMP inhibitors, EDTA or 1,10-phenanthroline, but not by PMSF, a serine protease inhibitor. Western blot analysis confirmed the presence of MMP-2 and MMP-9 proteins in the uterus. Immunoreactive MMP-9 protein was detected only in the endometrial stroma, whereas immunoreactive MMP-2 protein was detected in both the stroma and epithelium of the uterus. Casein zymography detected three major bands of activity ( approximately 54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.     

明胶酶谱实验方法的优化简

目的：优化明胶酶谱实验方法,并检测不同浓度凝血酶刺激人皮肤成纤维细胞分泌的明胶酶B（MMP-9）活性的变化。方法：用含明胶的聚丙烯酰胺凝胶电泳分开上清中各蛋白条带,经2．5％TritonX-100洗脱、明胶酶缓冲液孵育、考马斯亮蓝染色液染色及脱色液脱色后,经光密度扫描记录,应用ImageJ软件进行密度计量分析。结果：凝血酶刺激人皮肤成纤维细胞分泌MMP一9具有浓度依赖性。结论：利用优化的明胶酶谱实验方法可做出高质量的明胶酶谱图片,对检测明胶酶活性有重要意义。     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

Production of matrix metalloproteinases in specific subpopulations of human-patient breast cancer invading in three dimensional culture system

This article aims at investigating the effect of production of matrix metalloproteinases (MMP) in human breast cancer tissues by means of three dimensional culture system. Thirty-nine tumour samples were taken from breast cancer patients. The tumour blocks were cultured on sponge gel using the three dimensional culture system. Breast cancer cells began shedding into the culture medium after 24 hours of culture. The cells were stained with trypan blue dye to assess viability on days 2, 4, 6 and 8. The culture medium was collected at these time points and tested for matrix metalloproteinases (MMP) 1,2,3 and 9 activities. There was a progressive increase in migration of cancer cells into the gel and culture medium from day 2 to day 8 and the interval difference was statistically significant (F ratio=4.06; p=0.008). The levels of all the MMPs tested were also significantly raised (P<0.05 for all the MMPs tested). When the levels of MMPs were correlated with the metabolic activities in the gel, medium and tumour block, cells in block show no correlation whereas cells in gel correlated significantly with MMP-1 and MMP-3. Cancer cells in the culture medium correlated with MMP-9. In conclusion, there is a progressive migration of cancer cells outside the tumour block. The migration into the gel and culture medium is associated with progressive and differential production of MMPs. It is likely that the three dimensional culture model assists in the selection of different subpopulations of cancer cells with different invasion properties as exemplified by the differential production of MMP.     

Matrix metalloproteinase inhibitors attenuate endotoxemia induced cardiac dysfunction: A potential role for MMP-9

Enhanced cardiac generation of peroxynitrite contributes to septic cardiomyopathy. Since matrix metalloproteinases (MMPs) are activated in vitro by peroxynitrite, we hypothezised that MMPs may contribute to cardiac mechanical dysfunction in sepsis. Rats were injected (i.p.) with either lipopolysaccharide (LPS, 4mg/kg) or vehicle. MMP inhibitors, either Ro 31-9790 (20 mg/kg), doxycycline (4mg/kg), or vehicle were administered i.p. 30 min after LPS. At 6 h, when the symptoms of endotoxemia peak, hearts were excised and perfused as working hearts with Krebs-Henseleit buffer at 37°C. Cardiac work (cardiac output x peak systolic pressure product) was measured. Perfusate and ventricle samples were analyzed by gelatin zymography to quantify MMP activity.Cardiac function was significantly depressed in LPS-treated rats compared to control rats (control: 55 ± 4, LPS: 26 ± 6 mmHg*mL*min^–1). LPS also caused a loss of 72 kDa MMP-2 activity in the ventricles and the perfusate. Although MMP-9 activity was not detected in the ventricles, LPS resulted in an increase in perfusate 92 kDa MMP-9 activity. The MMP inhibitors significantly improved cardiac function of LPS-treated rats (Ro31-9790: 38 ± 3, doxycycline: 51 ± 3 mmHg*mL*min^–1), had no effect on the loss of MMP-2 activity, and significantly reduced the MMP-9 activity in the perfusate. These results demonstrate, for the first time, that LPS induced cardiac dysfunction is associated with a loss in ventricular MMP-2 activity and the release of MMP-9 from the heart. MMP inhibitors can significantly preserve cardiac mechanical function during septic shock.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

A residue in the S2 subsite controls substrate selectivity of matrix metalloproteinase-2 and matrix metalloproteinase-9

Matrix metalloproteinase (MMP)-2 and MMP-9 are closely related metalloproteinases that are implicated in angiogenesis. The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for understanding the structural basis of substrate recognition by the MMP family. Although the two MMPs exhibit some overlap in substrate recognition, our recent work showed that MMP-2 can cleave a set of peptide substrates that are only poorly recognized by MMP-9 (Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P(2) position of these peptide substrates dramatically reduced their selectivity for MMP-2. Inspection of the corresponding S(2) pocket of the substrate-binding cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2 contains Glu. Here, we test the hypothesis that this conservative substitution has a role in substrate selectivity. Mutation of Glu(412) in MMP-2 to Asp significantly reduced the hydrolysis of selective substrates, with only a minor effect on hydrolysis of non-selective substrates. The predominant effect of the mutation is at the level of k(cat), or turnover rate, with reductions reaching as high as 37-fold. The residues that occupy this position in other MMPs are highly variable, providing a potential structural basis for substrate recognition across the MMP family.     

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.     

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.     

Role of metalloproteinases at the onset of liver development

At the onset of liver development, the hepatic precursor cells, namely, the hepatoblasts, derive from the ventral foregut endoderm and form a bud surrounded by a basement membrane (BM). To initiate liver growth, the hepatoblasts migrate across the BM and invade the neighboring septum transversum mesenchyme. In the present study, carried out in the mouse embryo, we searched for effectors involved in this process and we examined the role of matrix metalloproteinases (MMPs). We found expression of a broad range of MMPs, among which MMP-2 was predominantly expressed in the septum transversum and MMP-14 in the hepatoblasts. Using a new liver explant culture system we showed that inhibition of MMP activity represses migration of the hepatoblasts. We conclude that MMPs are required to initiate expansion of the liver during development and that our culture system provides a new model to study hepatoblast migration.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Relationship between expression of matrix metalloproteinases and migration of epidermal and in vitro generated Langerhans cells

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowski's base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.     

Role of the hemopexin domain of matrix metalloproteinases in cell migration

The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non-proteolytic activities of MMPs are just beginning to be understood. Herein, we evaluated the role of proMMPs in cell migration. Employing a Transwell chamber migration assay, we demonstrated that transfection of COS-1 cells with various proMMP cDNAs resulted in enhancement of cell migration. Latent MMP-2 and MMP-9 enhanced cell migration to a greater extent than latent MMP-1, -3, -11 and -28. To examine if proteolytic activity is required for MMP-enhanced cell migration, three experimental approaches, including fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant MMP cDNAs, and addition of hydroxamic acid-derived MMP inhibitors, were employed. We demonstrated that the proteolytic activities of MMPs are not required for MMP-induced cell migration. To explore the mechanism underlying MMP-enhanced cell migration, structure-function relationship of MMP-9 on cell migration was evaluated. By using a domain swapping approach, we demonstrated that the hemopexin domain of proMMP-9 plays an important role in cell migration when examined by a transwell chamber assay and by a phagokinetic migration assay. TIMP-1, which interacts with the hemopexin domain of proMMP-9, inhibited cell migration, whereas TIMP-2 had no effect. Employing small molecular inhibitors, MAPK and PI3K pathways were found to be involved in MMP-9-mediated cell migration. In conclusion, we demonstrated that MMPs utilize a non-proteolytic mechanism to enhance epithelial cell migration. We propose that hemopexin homodimer formation is required for the full cell migratory function of proMMP-9.     

Determination of matrix metalloproteinase activity using biotinylated gelatin

Matrix metalloproteinases (MMPs) and, specifically, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are strongly associated with malignant progression and matrix remodeling. These enzymes are a subject of intensive studies involving screening of comprehensive chemical libraries of synthetic inhibitors. There is no simple method available for measurement of activity of gelatinases and related MMPs. Here, we report a simple, inexpensive, and highly sensitive assay for MMP activity. The assay performed in a 96-well microtiter plate format employs biotin-labeled gelatin (denatured collagen type I) as a substrate. Following the substrate cleavage, only the proteolytic fragments bearing biotin moieties are captured by streptavidin coated on the plastic surface and the captured fragments with at least two biotin molecules should be revealed by streptavidin conjugated with horseradish peroxidase. The frequency of lysine residues is low in collagen type I relative to the MMP cleavage sequences (PXGX). Accordingly, the majority of the cleavage products must be devoid of biotin or possess only one biotin group. Both of these types of fragments cannot be recognized by the horseradish peroxidase-streptavidin conjugate. Therefore, higher gelatinolytic activity is associated with lower signal in the assay. This 2-h assay allows identification of gelatinolytic activity of MMP-2 in concentrations as low as 0.16 ng/ml. The sensitivity of this ELISA-like assay is comparable to that of gelatin zymography, a method widely used to detect gelatinases. However, in contrast to zymography, the assay directly measures the enzymatic activity of MMP samples. The gelatinolytic activity assay permits efficient analyses and screening of the MMP inhibitor panels and allows quantitation of gelatinolytic activity of various MMPs in solution as well as on cell surfaces.     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.