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It has been assumed that cleavage of the N-terminal propeptide domain of membrane type-1 matrix metalloproteinase (MT1-MMP) is required for enzyme function. We recently demonstrated that the propeptide domain of MT1-MMP is not cleaved and actually is required for function of the membrane-bound enzyme in transfected COS-1 cells (Cao, J., Drews, M., Lee, H. M., Conner, C., Bahou, W. F., and Zucker, S. (1998) J. Biol. Chem. 273, 34745-34752). In this report, we have inserted the cDNA encoding the signal and propeptide sequences of MT1-MMP (MT(1-109)) and the cDNA encoding propeptide-deleted mature MT1-MMP (MT delta pro) in expression vectors that were then transfected into matrix metalloproteinase-deficient COS-1 cells. Co-expression of both the mature sequence and the prosequence of MT1-MMP as independent polypeptides (in trans) in COS-1 cells resulted in reconstitution of MT1-MMP function in terms of facilitating (125)I-labeled tissue inhibitor of metalloproteinase 2 binding to transfected cells and subsequent activation of progelatinase A. Transfection of cells with either cDNA alone resulted in non-functional cells. These results are consistent with the propeptide sequence of MT1-MMP functioning as an intramolecular chaperone involved in protein folding and trafficking to the cell surface.

To elucidate the role of E-cadherin in matrix metalloproteinases (MMPs) expression, we transfected to squamous carcinoma cells with E-cadherin cDNA. HN5 cells and mock-transfected HN5-neo cells expressed proMMP-2 and active MMP-2. E-cadherin-transfected HN5-EC cells produced comparable proMMP-2 but low active MMP-2; and membrane type 1-MMP (MT1-MMP) mRNA declined. Phosphorylated ERK, a marker of mitogen-activated protein (MAP) kinase cascade, also declined in HN5-EC cells. The addition of anti-E-cadherin antibody resulted in the disappearance of these alterations in HN5-EC cells. These results suggest that E-cadherin suppresses MAP kinase cascade and down-regulates MT1-MMP.

Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM- 1, and integrin chains alpha2, alpha3, alpha4, alpha5, alpha6, alpha v, beta1, beta3 and beta7 failed to inhibit breast cancer cell migration through bone marrow fibroblasts. Inhibitors of matrix metalloproteases, 1, 10-phenanthroline, Ro-9790, TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers suggesting a role for these enzymes in the migration of breast cancer cells through bone marrow adherent layers. Co-culture of MDA-MB-231 cells and bone marrow fibroblasts resulted in augmentation of the levels of the matrix metalloproteases MMP-1 and MMP-2 in culture supernatants. Soluble factors produced by bone marrow fibroblasts were responsible for the increase in MMP-1 levels. However, maximal MMP-2 production was dependent on direct contract between the breast cancer cells and the bone marrow fibroblasts. Modulation of MMP production by cell-cell contact or soluble factors suggests a mechanism by which breast cancer cells can enhance their ability to invade the bone marrow microenvironment.

Matrix metalloproteinase-1 (MMP-1) plays an important role in atherosclerotic plaque rupture. The purpose of this study was to investigate the expression of MMP-1 by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cells) were cocultured. MMP-1 levels were measured by enzyme-linked immunosorbent assay. Collagenolytic activity was determined by fluorescent labeled-collagen digestion. Immunohistochemistry was performed to determine which types of cells produce MMP-1. Adding THP-1 cells to VSMCs markedly increased the MMP-1 levels and activity of the culture media. MMP-1 levels were maximal when the cellular ratio of THP-1 cells/VSMCs was 1.0. Immunohistochemistry revealed that both types of cells in the coculture produced MMP-1. Separated coculture experiments showed that both direct contact and a soluble factor(s) contributed to MMP-1 production. Neutralizing anti-interleukin (IL)-6 and tumor necrosis factor-alpha antibodies inhibited coculture conditioned medium-induced MMP-1 production by VSMCs and THP-1 cells. Protein kinase C inhibitors, tyrosine kinase inhibitors, and a mitogen-activated protein kinase inhibitor significantly inhibited MMP-1 production by cocultures. Direct cell-to-cell interaction between THP-1 cells and VSMCs enhanced MMP-1 synthesis in both types of cells. Increased local MMP-1 production and activity induced by monocyte-VSMC interaction play an important pathogenic role in atherosclerotic plaque rupture.

There is accumulating evidence of complicated interactions among vascular cells, i.e. endothelial cells, smooth muscle cells and monocytes/macrophages, in the regulation of vascular function and remodeling. We have investigated the mechanisms responsible for matrix metalloproteinase (MMP)-1 expression by interactions between monocytes and vascular endothelial cells. THP-1 cells (human monocytic cell line) and human umbilical vein endothelial cells (HUVECs) were cocultured. MMP-1 levels in the culture medium were measured by enzyme-linked immunosorbent assays. Collagenolytic activity in the culture medium was measured by fluorescence labeled-collagen digestion. Immunohistochemistry using an anti-MMP antibody was carried out to determine which types of cell produce MMP-1. The addition of THP-1 cells to HUVECs for 48 h induced increases in MMP-1 levels and collagenolytic activity, which were 5- and 2-fold relative to those of HUVECs alone, respectively. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced MMP-1 production in the cocolture. Immunohistochemical analysis revealed that both types of cell produce MMP-1 in the coculture. Neutralizing anti-interleukin-1 beta and tumor necrosis factor- alpha antibodies inhibited MMP-1 production by the coculture. The Src kinase and MEK inhibitors significantly inhibited MMP-1 production by the coculture. Coculture of THP-1 cells and HUVECs induced significant increases in Src and mitogen activated protein (MAP) kinase activities. Enhanced MMP-1 expression induced by monocyte-endothelial cell interactions may play an important role in the pathogenesis of atherosclerosis and plaque rupture., (Copyright 2000 Academic Press.)

Objective: To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay., Sample Population: Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses., Procedure: RNA was extracted from samples by use of a modified Trizol procedure. Northern blot hybridization and the RT-PCR assay were performed; results were quantitated by use of glyceraldehyde 3-phosphate dehydrogenase as an internal standard., Results: Articular cartilage samples from joints with mild or moderate OA yielded less total RNA than samples from joints with severe OA. Northern blot hybridization indicated that type-II collagen mRNA expression in articular cartilage samples from joints with OA was significantly greater than expression in samples from healthy joints. The RT-PCR assay identified low levels of MMP3 mRNA expression in 4 of 8 sets of articular cartilage samples and 4 of 6 sets of synovial membrane samples, whereas Northern blot hybridization identified MMP3 mRNA expression in only 1 of 6 sets of articular cartilage samples and 1 of 6 sets of synovial membrane samples., Conclusions: A RT-PCR assay is more sensitive than Northern blot hybridization for detection of MMP3 mRNA expression in articular cartilage and synovial membrane and requires smaller samples.

Inflammatory cytokines play a major role in cartilage destruction in diseases such as osteoarthritis and rheumatoid arthritis. Because physical therapies such as continuous passive motion yield beneficial effects on inflamed joints, we examined the intracellular mechanisms of mechanical strain-mediated actions in chondrocytes. By simulating the effects of continuous passive motion with cyclic tensile strain (CTS) on chondrocytes in vitro, we show that CTS is a potent antagonist of IL-1 beta actions and acts as both an anti-inflammatory and a reparative signal. Low magnitude CTS suppresses IL-1 beta-induced mRNA expression of multiple proteins involved in catabolic responses, such as inducible NO synthase, cyclo-oxygenase II, and collagenase. CTS also counteracts cartilage degradation by augmenting mRNA expression for tissue inhibitor of metalloproteases and collagen type II that are inhibited by IL-1 beta. Additionally, CTS augments the reparative process via hyperinduction of aggrecan mRNA expression and abrogation of IL-1 beta-induced suppression of proteoglycan synthesis. Nonetheless, the presence of an inflammatory signal is a prerequisite for the observed CTS actions, as exposure of chondrocytes to CTS alone has little effect on these parameters. Functional analysis suggests that CTS-mediated anti-inflammatory actions are not mediated by IL-1R down-regulation. Moreover, as an effective antagonist of IL-1 beta, the actions of CTS may involve disruption/regulation of signal transduction cascade of IL-1 beta upstream of mRNA transcription. These observations are the first to show that CTS directly acts as an anti-inflammatory signal on chondrocytes and provide a molecular basis for its actions.

Interleukin (IL-)17 is a T cell-derived pro-inflammatory cytokine produced by RA synovium. We studied the role of IL-17 in the synovium cytokine network to determine whether it can influence the inflammatory and destructive pattern characteristic of RA. Herein, we investigated whether the production and action of MMP-1 and its inhibitor TIMP-1 could be modulated by IL-17 in the presence of pro-inflammatory cytokine (IL-1) and anti-inflammatory cytokines (IL-4, IL-13, IL-10). The effect of the blockade of endogenous IL-17 on the secretion of MMP-1 and TIMP-1 by RA synovium and matrix destruction was also studied. IL-17 increased the spontaneous production of MMP-1 by synoviocytes five-fold. IL-1 was more potent since it increased MMP-1 production nine-fold. Addition of IL-4, IL-13 and IL-10 to synoviocyte cultures reduced the spontaneous production of MMP-1 and induced TIMP-1 production by synoviocytes stimulated with IL-17 or/and IL-1beta. In the presence of anti-IL-17 blocking mAb, MMP-1 production and collagenase activity by RA synovium was reduced by 50% and associated with a 50% reduction in type I collagen C-telopeptide fragments (CTX) released in the supernatants, demonstrating the direct contribution of IL-17 in destruction. IL-17 and its producing T cells appear to contribute to the inflammatory process involved in the rheumatoid lesion., (Copyright 2000 Academic Press.)

Objective: To investigate the regulation of perimenstrual MMP-1 expression in human endometrium., Design: In vitro study utilizing epithelial-stromal co-cultures., Setting: Cell Biology Unit, International Institute of Cellular and Molecular Pathology, and Departments of Pathology and Gynecology, Saint-Luc University Clinics, Louvain University Medical School, Brussels, Belgium., Methods: Contact-dependent and contact-independent co-cultures were established and resulting MMP-1 gene and protein expression was analyzed by RNase protection assays and soluble-collagen assays., Results: MMP-1 expression in endometrial fibroblasts is markedly stimulated by epithelial cell-conditioned medium. This stimulation can be prevented by antibodies directed against interleukin 1 alpha (IL-1 alpha). Ovarian steroids inhibit MMP-1 production by IL-1 alpha-stimulated fibroblasts in vitro., Conclusion: Taken together, our results suggest that epithelium-derived IL-1 alpha is the most important paracrine induced of MMP-1 in endometrial fibroblasts. However, IL-1 alpha-stimulated MMP-1 production in the human endometrium is effectively blocked by ovarian steroids. We believe that this mechanism responsible for the MMP-1 repression that occurs when systemic sex steroid concentrations are high and the MMP-1 production and activity during the perimenstrual phase when estrogen and progesterone concentrations are low.

Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C chemokine superfamily, has recently been shown to be involved in the pathogenesis of tissue fibrosis. In vitro studies demonstrated that MCP-1 up-regulates type I collagen gene expression via endogenous production of TGF-beta in rat lung fibroblasts. We here show that recombinant human MCP-1 affects gene expression of interstitial collagenase (matrix metalloproteinase-1 (MMP-1)) in primary human skin fibroblasts and a stable fibroblast cell line. MMP-1 mRNA was induced by MCP-1 (10 ng/ml) as early as 6 h and reached a maximal expression at 24 h. MCP-1 also caused an increase of MMP-2 mRNA expression in both types of fibroblasts at 48 h. Interestingly, tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was also up-regulated by MCP-1, and TIMP-1 mRNA expression peaked at 48 h in both types of fibroblasts. Immunoblot analysis demonstrated increased levels of MMP-1 and TIMP-1 protein in the culture supernatants of primary fibroblasts stimulated with MCP-1. In addition, MCP-1 strongly induced IL-1 alpha mRNA expression in dermal fibroblasts in parallel with the induction of MMP-1. Preincubation with IL-1 receptor antagonist almost completely abrogated the expression of MMP-1 mRNA, and partially inhibited MMP-1 synthesis induced by MCP-1. Transient transfection of primary skin fibroblasts with a MMP-1 promoter-reporter construct indicated a dose-dependent increase in promoter activity by MCP-1 stimulation. These data demonstrate that MCP-1 up-regulates MMP-1 mRNA expression and synthesis in human skin fibroblasts at a transcriptional level and provide evidence that this is mediated by an IL-1 alpha autocrine loop.

Matrix metalloproteinase-1 (MMP-1) may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture. We investigated the effect of the calcium channel blockers amlodipine and nifedipine on the expression of MMP-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in endothelial cells (ECs). MMP-1 and TIMP-1 levels in conditioned media of human vascular ECs were measured by enzyme-linked immunosorbent assay. Collagenolytic activity was determined by fluorescence-labeled collagen digestion. The addition of interleukin-1beta (IL-1beta) increased MMP-1 levels in the culture media of ECs. Amlodipine, but not nifedipine, significantly decreased MMP-1 levels in IL-1beta-stimulated ECs. TIMP-1 levels also were significantly increased by IL-1beta, and its expression was slightly decreased by amlodipine, not by nifedipine. Amlodipine significantly inhibited collagenolytic activity in the culture media of IL-1beta-stimulated ECs, whereas nifedipine showed no significant effect on the activity. Our findings revealed that amlodipine, but not nifedipine, inhibits IL-1beta-induced MMP-1 expression in human ECs.

Monocytes/macrophages are directly involved in tissue remodeling and tissue destruction through the release of matrix metalloproteinases (MMP). In the present study, we examined the effect mediated by contact of polarized Th cells with mononuclear phagocytes on the production of MMP-1, MMP-9, and their inhibitor. Plasma cell membranes from Ag-activated Th1 and Th2 cells were potent inducers of MMP-1 production by THP-1 cells. Cell membrane-associated TNF was found to be only partially involved in MMP-1 induction by both Th1 and Th2 cells. In Th2 cells exclusively, membrane-associated IL-4 induced MMP-1 production by THP-1 cells. This membrane-associated IL-4 effect was additive to that of TNF and was specifically observed on MMP-1 as MMP-9 production was concomitantly inhibited. Similarly, soluble IL-4 induced THP-1 cells to produce MMP-1, its effect proving additive to that of soluble TNF and to that of cell membranes of mitogen-activated HUT-78 cells. Its activity was blocked by IL-4 neutralization, and was unaffected by the presence of indomethacin. These effects on THP-1 cells were observed at protein and mRNA levels. Although inhibitory on freshly isolated peripheral blood monocytes, soluble IL-4 enhanced T cell-induced MMP-1 and inhibited MMP-9 production both at protein and mRNA levels in monocytes cultured for 7 days in the presence of GM-CSF. Thus, in contrast with previously reported effects, Th2 and IL-4 specifically induce MMP-1 production by mononuclear phagocytes at various stages of differentiation. This IL-4 activity may be relevant to pathological conditions dominated by Th2 inflammatory responses, resulting in tissue remodeling and destruction.

This study tests the hypothesis that alpha(v)beta(3) integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that beta(3) integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the beta(3) integrin-blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8+/-30.8 cells/mm(2) in control rats to 10.29+/-7.03 cells/mm(2) in F11-treated rats (P=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed alpha(v)beta(3) integrin receptors, whereas adult SMCs did not. Stimulation of newborn (alpha(v)beta(3)+) SMCs with osteopontin, a matrix ligand for alpha(v)beta(3), increased MMP-1 production from 114.4+/-35.8 ng/mL at 0 nmol/L osteopontin to 232.5+/-57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC alpha(v)beta(3) integrin receptors play an important role in regulating migration by stimulating SMC MMP production.

Background: beta2-Microglobulin (beta2m) amyloidosis is a destructive articular disease that causes significant morbidity in patients undergoing hemodialysis. The amyloid deposits contain beta2m, some of which is altered with advanced glycation end products (AGE-beta2m). The deposits are located principally in joint structures, with adjacent degradation of cartilage and bone. We hypothesized that one of the mechanisms by which beta2m induces joint destruction is to induce the release of matrix metalloproteinase-1 (MMP-1), but not tissue inhibitor of metalloproteinase-1 (TIMP-1), from synovial fibroblasts., Methods: To test this hypothesis and determine the role of AGE-beta2m, we incubated human osteoarthritic synovial fibroblasts in the presence and absence of beta2m and AGE-beta2m and measured the release of interstitial collagenase (MMP-1) and/or TIMP-1 by enzyme-linked immunosorbent assay and Northern blot analysis., Results: beta2m and AGE-beta2m at 10 and 25 microg/mL induced the release of MMP-1 from human osteoarthritic synovial fibroblasts at 24 hours. In contrast, there was no increased release of TIMP-1, leading to an increase in the MMP-1/TIMP-1 ratio indicative of uncontrolled collagenolysis. A similar dose response was observed at 48 hours, except that AGE-beta2m had no effect over control cultures. MMP-1 mRNA expression by Northern blot analysis paralleled these findings. The source of the fibroblasts did not alter the results. Finally, we demonstrated that doxycycline, a treatment for arthritis, can inhibit the release of MMP-1 from synovial fibroblasts incubated with beta2m., Conclusion: beta2m, at physiologically relevant concentrations, induces the release of MMP-1 without concomitant release of TIMP-1 from human synovial fibroblasts, leading to uncontrolled collagenolysis. The alteration of beta2m with AGE did not alter this effect at 24 hours, but blocked the effect at 48 hours. These findings may account for the tissue destruction seen in beta2m amyloidosis.

Objective: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis., Methods: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13., Results: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results., Conclusion: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Background: The process of proteolysis is important at several stages of the metastatic cascade. A balance between the expression of the genes encoding endogenous proteinases and inhibitors exists and when the production of proteinases exceeds that of inhibitors proteolysis occurs., Aims: To determine whether differences in the profile and activity of proteinases and inhibitors exist within breast tumour tissue (n = 51), surrounding background breast tissue (n = 43), normal breast tissue from breast reduction mammoplasty operations (n = 10), and cells of the breast cancer cell line, MCF-7., Methods: Proteinase (matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, urokinase-type plasminogen activator (uPA), and tissue-type PA (tPA)) and inhibitor (tissue inhibitor of metalloproteinases; TIMP-1 and TIMP-2) expression and proteinase activity were compared using substrate zymography, western blotting, immunohistochemistry, and quenched fluorescent substrate hydrolysis., Results: The presence of all proteinases and inhibitors was greater in breast tumour tissue when compared with all other types of breast tissue (p < 0.05). The activity of total MMPs as determined by quenched fluorescent substrate hydrolysis was also greater in breast tumours (p < 0.05)., Conclusions: There is increased proteolysis in human breast tumours when compared with other breast tissues.

Epidemiologic studies have indicated the association between tobacco smoking and skin aging, but the exact mechanism of tobacco smoke-induced premature skin aging is currently unknown. In this study, we investigated the alterations of collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human fibroblasts treated with tobacco smoke extract. Human fibroblasts were exposed to different concentrations of water-soluble extract from tobacco smoke. Human fibroblasts irradiated with ultraviolet A1 (UVA1) were used as positive controls because the mechanism of UVA1-mediated MMP expression has been well characterized. The expression of MMP and TIMP was analyzed semiquantitatively following reverse transcriptase-polymerase chain reaction. Production of type I and type III collagens was detected by Western blotting and biosynthesis of new collagen was assessed by 3H-proline incorporation. Upon treatment with tobacco smoke extract or UVA1 irradiation, the expression of MMP-1 and MMP-3 mRNA was significantly increased in a dose-dependent manner. Maximum induction was observed with 25 microl/ml tobacco smoke extract. In contrast, the expression of TIMP-1 and TIMP-3 mRNA remained unchanged. Western blotting of the supernatant revealed that type I and type III collagens were decreased as compared with untreated controls. Collagen biosynthesis was significantly reduced by 40.1% following treatment with 25 microl/ml tobacco smoke extract. Sodium azide, L-ascorbic acid and Trolox (a water-soluble vitamin E) prevented both the UVA1- and the tobacco-induced alteration of MMP-1. These observations suggest that the imbalance of connective tissue matrix components might contribute to the molecular basis for premature skin aging in smokers. They also suggest that reactive oxygen species including singlet oxygen mediate this process.

Background: Matrix metalloproteinases (MMPs), enzymes capable of degrading collagens and other extracellular matrix components, have been implicated in gastric ulcer formation. However, the effect on MMP expression of Helicobacter pylori, also implicated in these lesions, has not been studied to our knowledge., Aim: To seek links between H. pylori and MMP expression likely to affect gastric ulcer formation. After fibroblasts from human gastric wall were cocultured with H. pylori. concentrations of MMP-1 and -2 in the medium were determined by enzyme-linked immunosorbent assays., Results: Whereas MMP-1 was not detected in media from fibroblasts or H. pylori culture alone, MMP-1 was detected in cocultures (P<0.01). Similar amounts of MMP-2 were detected in medium from fibroblasts cultured alone and with H. pylori. No MMP-2 production by H. pylori cultured alone was detected., Conclusions: MMP-1 appears to be important in gastric ulcer pathogenesis, and MMP-1 induction by H. pylori may impede gastric ulcer healing.

The newly emerging astacin metalloproteinase family comprises multiple members with diverse functions. Most recently, the development-related functions have been attributed to both (1) proteolytic cleavage and subsequent release of active TGF-beta-like growth factors from latent inhibitory complexes and (2) modification of extracellular matrix (ECM) assembly and composition. We previously identified and purified hydra metalloproteinase 1 (HMP-1), a developmentally important astacin proteinase that functions in head regeneration and transdifferentiation of tentacle battery cells (L. Yan et al., 1995, Development 121, 1591-1602). In the present study, further cloning revealed that HMP-1 is produced as a secreted zymogen with a conserved hydrophobic signal sequence and a putative propeptide. The processed HMP-1 is composed of a characteristic astacin proteinase domain and a unique Cys-rich C-terminus. With this simple domain structure, HMP-1 represents an ancestral astacin proteinase. Consistent with its role in head regeneration, HMP-1 mRNA is expressed at highest levels by endodermal cells at the apical pole of the body column just inferior to the base of tentacles, the region of active cell differentiation or transdifferentiation. A modified immunocytochemical procedure demonstrated that HMP-1 protein can be localized not only to ECM of tentacles as we previously reported, but also to endodermal cells of the body column in a pattern similar to its mRNA distribution. The localization of HMP-1 protein in tentacles was confirmed using an enzymatic approach. A translocation of HMP-1 protein from cells in the body column to the extracellular milieu in tentacles further suggests that HMP-1 is a secreted protein. HMP-1 expression undergoes extensive regulation at the transcriptional level both temporally and spatially during head regeneration. The involvement of HMP-1 in this morphogenetic process is further supported by the blockage of head regeneration with localized antisense treatment. Taken together, these results suggest that HMP-1 is a secreted astacin metalloproteinase that has an important role in regulating hydra head morphogenesis potentially through its differential expression along the body axis., (Copyright 2000 Academic Press.)

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as basigin or CD147, is a glycoprotein that is enriched on the surface of tumor cells and stimulates production of several matrix metalloproteinases by adjacent stromal cells. In this study, we have found that EMMPRIN not only stimulates the production of interstitial collagenase (MMP-1) but also forms a complex with MMP-1 at the tumor cell surface. Complex formation was demonstrated by phage display, affinity chromatography, and immunocytochemistry. Presentation of MMP-1 complexed to EMMPRIN at the tumor cell surface may be important in modifying the tumor cell pericellular matrix to promote invasion.

Objective: Interferon-beta (IFNbeta) treatment is emerging as a potentially effective form of therapy in various immune-mediated conditions. This study evaluated the effects of IFNbeta therapy on the cell infiltrate, cytokine profile, and expression of metalloproteinase 1 (MMP-1) in synovial tissue from patients with rheumatoid arthritis (RA). To further assess the mechanism of action, in vitro experiments were conducted to determine the effects of IFNbeta on the production of MMP-1, MMP-3, tissue inhibitor of metalloproteinases 1 (TIMP-1), and prostaglandin E2 (PGE2) by human fibroblast-like synoviocytes (FLS)., Methods: Eleven patients were treated for 12 weeks with purified natural fibroblast IFNbeta (Frone; Ares-Serono, Geneva, Switzerland) subcutaneously 3 times weekly with the following dosages: 6 million IU (n = 4), 12 million IU (n = 3), and 18 million IU (n = 4). Synovial biopsy specimens were obtained by needle arthroscopy at 3 time points: directly before and at 1 month and 3 months after initiation of treatment. Immunohistologic analysis was performed using monoclonal antibodies specific for the following phenotypic markers and mediators of joint inflammation and destruction: CD3, CD38, CD68, CD55, tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-6, MMP-1, and TIMP-1. In addition, we measured the production of MMP-1, MMP-3, TIMP-1, and PGE2 by RA FLS and dermal fibroblasts in the presence and absence of IFNbeta., Results: A statistically significant reduction in the mean immunohistologic scores for CD3+ T cells and the expression of MMP-1 and TIMP-1 at 1 month, CD38+ plasma cells and the expression of IL-6 at 3 months, and the expression of IL-1beta at both 1 and 3 months was observed in synovial tissue after IFNbeta treatment. The scores for CD68+ macrophages and TNFalpha expression also tended to decrease, but the differences did not reach statistical significance. The in vitro experiments revealed inhibition of MMP-1, MMP-3, and PGE2 production by RA FLS, whereas TIMP-1 production was only slightly decreased. These effects were more consistent in RA FLS than in dermal fibroblasts., Conclusion: The changes in synovial tissue after IFNbeta treatment and the in vitro data support the view that IFNbeta therapy has immunomodulating effects on rheumatoid synovium and might help to diminish both joint inflammation and destruction. Larger well-controlled studies are warranted to show the efficacy of IFNbeta treatment for RA.

Purpose: To determine whether conjunctivochalasis, denoting redundant, loose, nonedematous inferior bulbar conjunctiva, is associated with increased expression and activity of matrix metalloproteinases (MMPS) over their tissue inhibitors (TIMPs)., Methods: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. Gelatin and casein zymography and quantitative collagenase activity assay were performed in the serum-free conditioned media., Results: Compared with normal conjunctival fibroblasts from six subjects, conjunctivochalasis fibroblasts from eight patients showed markedly increased transcript expression of MMP-1 (5- to 32-fold) and MMP-3 (4 to 30-fold), whereas that of MMP-2, TIMP-1, TIMP-2, and uPA was similar between the two groups. Protein levels were increased in the serum-free conditioned media of conjunctivochalasis fibroblasts for MMP-1 (3.5- to 7.6-fold) and MMP-3 (2.3- to 13-fold), determined by ELISA and Western blot analysis. There was increased caseinolytic activity of MMP-3 and collagenolytic activity of MMP-1 (2.2-fold) by conjunctivochalasis fibroblasts, whereas no difference was noted between these two types of fibroblasts in the protein and gelatinolytic activity of MMP-2 or expression of TIMP-1 and TIMP-2 proteins, although that of TIMP-1 transcript was slightly higher in some conjunctivochalasis fibroblasts. No expression of MMP-9 was detected., Conclusions: Overexpression of MMP-1 and MMP-3 mRNA by conjunctivochalasis fibroblasts is correlated with their increased protein levels and proteolytic activities. Collectively, these data help explain how conjunctivochalasis manifests excessive degradation of the conjunctival matrix and Tenon's capsule.

Neutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1beta mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1beta signal transduction.

Background: Degradation of basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be an essential step in the complicated process of hematogenous metastasis. MMP-1 is a member of collagenases, a family of MMPs that degrades collagens type I, II, and III, main components of the interstitial stroma. The purpose of this study was to investigate the expression of MMP-1 in colorectal cancer and its correlation with hematogenous metastasis. Patients and Methods. We examined 133 cases of colorectal cancer (Dukes A: 72; Dukes B: 26; Dukes C: 23; Dukes D: 12). Sections were cut from formalin-fixed, paraffin-embedded samples containing the deepest site of cancer invasion and stained immunohistochemically with a monoclonal antibody to MMP-1. According to the area of the tumor that was stained, patients were divided into high- and low-MMP-1 expression groups., Results: MMP-1 expression was observed in the cytoplasm of cancer cells, some stromal cells, and a few normal epithelial cells of colonic mucosa. High MMP-1 expression was found in 47 (35.3%) cases and low in 86 (64.7%). Hematogenous metastasis was identified in 14 (29.8%) of high-MMP-1 groups and 12 (13.9%) of low-MMP-1 groups. MMP-1 expression significantly correlated with hematogenous metastasis of colorectal cancer, but no correlation was found between MMP-1 expression and the other clinicopathological features investigated., Conclusions: MMP-1 expression may be a novel marker for hematogenous metastasis of colorectal cancer, and its inhibition may be a strategy for prevention of metastasis.

Cartilage is a specialized connective tissue. It contains few cells into an extracell matrix. The matrix mainly constituents are collagen and proteoglycans. Its degradation depends on synoviocytes activity, that secrete metalloproteases, agents to proteoglycans catabolism. There are two types of synoviocytes: macrophagics (type "A:') and fibroblastics (type "B"). The proteoglycan destruction can be LT-dependent or LT-independent. The aim of this work is synoviocytes function ex vívo study, free immune system influence. In order to do it, heparinized synovial fluid samples were obtained from 6 osteoarthritic (OA) and 6 arthritic (RA) both sex untreated patients, diagnosed according ACR criteria, which disease duration was longer than 6 months. Patients average age was 70 +/- 2 years. Control samples were synovial fluid from traumatic arthritis or non inflammatory bone-muscle pathology. Synovial fluid was centifugated at 1500 g for 30 minutes to isolate synoviocytes. Sediment containing cells was 6 hs incubed with Dulbecco-Eagles media, that has HEPES Gibco (26 mM); NaHCO3 (0.5 g/I); glutamine (2 mM), streptomicine (100 mg/l), G-penicillin (1 U/ml); anphotericine B (2.5 mg/l). Cells calification and viability were cytopathologically determined. Before and after incubation, collagenase activity was measured by ELISA-double-sandwich, using 10 micrograms/ml monoclonal anti-MMPs in phosphate-buffer-saline. The antigen-antibody complex production with inespecific proteins was blocked by bovine seric albumine. Streptavidin peroxidase was added and washed with 2,2,azin,di(3-ethyl-benztazoilinsuiphonic) acid to develop color. The link of labeled antibody by absorbance at 410 nm was determined in ELISA-spectrophothometer. RA patients earlier MMPs synoviocytes production was 1373 +/- 115 ng/ml. Then 6 hs incubation 2143 +/- 132 ng/ml was reached. The increase (56%) had high significance (p < 0.0001). OA earlier MMPs cells production was 276 +/- 23 ng/ml, but after incubation it reached 542 +/- 47 ng/ml. (96% increased with highly significativa difference too: p < 0.0001). Microscopic study was carried out before and after incubation, and shows a lot of synoviocytes with plenty of cytopiasme when the collagenase leveis were highest. On the contrary, when low MMPs production by synovial fluid, as no incubated osteoarthritic material, a few cells containing picnotics nucleous were observed. Significant quantitative differences in AR and OA enzymatic secretion were observed. Although in rheumatoid arthritic MMPs leveis synoviocytes production were 4.6 times than OA levels, after 6 hs incubation percentage of increase in OA cells secretion was highest. Described results confirm MMP-1 synthesis by synoviocytes, and these levels correlate with inflammation, more pronounced in acute (RA) than chronic pathology (OA). Synoviocyte incubation let us to test disease changes in synovial fluid according to cells number and phagocytic activity. Authors agree to assert that synovial fluid may reflect what is happening in an articular cartilago, because SF provides markers of joint disease. MMPs are giving information about pathways involved in OA and RA cartilage degradation.

We have established a highly-metastatic cell line (designated as HNOS) and a non-metastatic cell line (designated as SAT) derived from human oral cavity squamous cell carcinoma (SCC). Both lines were transplantable in nude mice. The invasive activity through Matrigel-coated membrane of HNOS cells was also higher than that of SAT cells. mRNA of TIMP-1 was expressed in SAT cells but not in HNOS cells. Metastatic and invasive abilities were suppressed by the overexpression of TIMP-1 in HNOS cells. These results suggest that TIMP-1 may have an important role in inhibiting invasion and metastasis of human oral cavity SCC.

Objective: To investigate the expression of mRNA for TGF-beta, MMP-1 and TIMP-1 and their effects on the formation of hypertrophic scar (HTS)., Methods: In situ hybridization and image analysis were used in this study., Results: The expressions of mRNA for TGF-beta and TIMP-1 in HTS was greatly increased compared with those of normal skin. The average integral optical density of mRNA for TGF-beta and TIMP-1 increased by 8 and 7 folds (P < 0.001) related to normal skin and the expression of them showed highly positive correlation., Conclusions: The findings suggest that TGF-beta promotes the expression of TIMP-1 and may be a possible factor for hypertrophic scar formation.

Objective: The purpose of this study was to examine how gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) is involved in the molecular mechanism of cartilage degradation in rheumatoid arthritis (RA) with special reference to the GLS-induced gene expression and protein synthesis of matrix metalloproteinase (MMP)-1 (collagenase-1) and MMP-3 (stromelysin-1)., Methods: Fibroblast-like synoviocytes (FLSs) obtained from RA patients were cultured and stimulated by GLS. Changes in the expression levels of GLS, MMP-1 and MMP-3 were assessed by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) for GLS, and by RT-PCR and enzyme-linked immunosorbent assay for MMPs and tissue inhibitor of metalloproteinase 1., Results: GLS demonstrated a self-induction of mRNA in cultured RA FLSs. GLS evoked a dose-dependent induction of MMP-1 and MMP-3 mRNAs, and subsequently their extracellular secretion., Conclusion: These findings suggest that GLS is a plausible pathogenic factor causing the extensive joint destruction in RA mediated via MMPs.

Hereditary gingival fibromatosis (HGF) is characterized by an excess accumulation of extracellular matrix (ECM) resulting in a generalized and fibrotic enlargement of the gingiva. To investigate some of the regulatory features of this condition, gingival fibroblasts from normal gingiva (NG) and HGF were examined for the expression and production of matrix metalloproteinases (MMPs) and their inhibitors, tissue matrix metalloproteinases inhibitor (TIMPs). Our results, obtained from 2 different assays, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography, clearly demonstrated that the expression and production of MMP-1 and MMP-2 was significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher to those from HGF. Addition of antibodies against transforming growth factor-beta 1 (TGF-beta 1), which is produced in greater amounts by HGF fibroblasts, resulted in a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas TIMP-1 and TIMP-2 expressions were unaffected. These patterns of expression and production suggest that enhanced TGF-beta 1 production reduce the proteolytic activities of HGF fibroblasts, which favor the accumulation of ECM.

Nonenzymatic glycation of extracellular matrix (ECM) proteins is increased in diabetes mellitus and aging and triggers cellular events leading to an imbalance in ECM homeostasis. We studied the influence of collagen glycation on matrix metalloproteinase production by dermal fibroblasts using the model of lattice cultures. Contraction of glycated collagen lattices was strongly reduced when compared to controls. Meanwhile, fibroblasts synthesized lower amounts of interstitial collagenase (MMP-1). Gelatinase A (MMP-2) production was not modified, but its activation was strongly inhibited. These effects were independent from the intensity of lattice contraction and from any simultaneous modification of tissue inhibitors of metalloproteinase (TIMP-1 and 2) production. These results demonstrate that the impaired ability of fibroblasts to remodel and contract a glycated extracellular matrix coincides with a decrease in MMP production., (Copyright 1999 Academic Press.)

Expression of matrix metalloproteinases (MMPs)-1 and -3 in fibroblasts is upregulated by pro-inflammatory cytokines and growth factors during proliferative inflammatory processes, including wound healing and rheumatoid arthritis. The Activator Protein-1 (AP-1) transcription factor is essential but, we show here, not sufficient for upregulation because platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), which strongly activate AP-1, poorly induce MMP-1 and -3. Interleukin-1alpha, which activates nuclear factor-kappaB (NF-kappaB), synergistically upregulates MMP-1 and -3 expression in the presence of bFGF or PDGF. Adenovirus mediated overexpression of IkappaBalpha, the inhibitor of NF-kappaB, completely suppresses MMP-1 and -3 protein and mRNA expression. Hence, we show for the first time that (NF-kappaB) activity is also essential for MMP-1 and -3 upregulation., (Copyright 1999 Academic Press.)