Pro-inflammatory effects of matrix metalloproteinase 7 in acute inflammation.

Matrix metalloproteinase 7 (MMP7) is a member of the MMP family. In the small intestine, MMP7 is responsible for activating α-defensins, which are broad-spectrum anti-microbial peptides produced by the Paneth cells. We report that MMP7(-/-) mice are resistant to LPS-induced lethality and that this resistance is correlated with reduced levels of systemic cytokines. LPS induced the upregulation and activation of MMP7 in the small intestine, degranulation of the Paneth cells, and induction of intestinal permeability in MMP7(+/+) mice. In MMP7(-/-) mice, both LPS-induced intestinal permeability and consequent bacterial translocation to the mesenteric lymph nodes were reduced. Based on gene expression analysis and evaluation of intestinal damage, we attribute the protected state of MMP7(-/-) mice to reduced intestinal inflammation. Interestingly, we found that different α-defensins, namely Crp1 (DEFA1) and Crp4 (DEFA4), can stimulate IL-6 release in macrophages and ileum explants in a TLR4 independent way. We conclude that absence of MMP7 protects mice from LPS-induced intestinal permeability and lethality, and suggest that MMP7-activated α-defensins, in addition to their previously recognized bactericidal and anti-inflammatory roles, may exhibit pro-inflammatory activities in the intestine by activating macrophages and amplifying the local inflammatory response in the gut, leading to intestinal leakage and subsequent increase in systemic inflammation.

Metalloproteinases and their associated genes contribute to the functional integrity and noise-induced damage in the cochlear sensory epithelium.

Matrix metalloproteinases (MMPs) and their related gene products regulate essential cellular functions. An imbalance in MMPs has been implicated in various neurological disorders, including traumatic injuries. Here, we report a role for MMPs and their related gene products in the modulation of cochlear responses to acoustic trauma in rats. The normal cochlea was shown to be enriched in MMP enzymatic activity, and this activity was reduced in a time-dependent manner after traumatic noise injury. The analysis of gene expression by RNA sequencing and qRT-PCR revealed the differential expression of MMPs and their related genes between functionally specialized regions of the sensory epithelium. The expression of these genes was dynamically regulated between the acute and chronic phases of noise-induced hearing loss. Moreover, noise-induced expression changes in two endogenous MMP inhibitors, Timp1 and Timp2, in sensory cells were dependent on the stage of nuclear condensation, suggesting a specific role for MMP activity in sensory cell apoptosis. A short-term application of doxycycline, a broad-spectrum inhibitor of MMPs, before noise exposure reduced noise-induced hearing loss and sensory cell death. In contrast, a 7 d treatment compromised hearing sensitivity and potentiated noise-induced hearing loss. This detrimental effect of the long-term inhibition of MMPs on noise-induced hearing loss was further confirmed using targeted Mmp7 knock-out mice. Together, these observations suggest that MMPs and their related genes participate in the regulation of cochlear responses to acoustic overstimulation and that the modulation of MMP activity can serve as a novel therapeutic target for the reduction of noise-induced cochlear damage.

Alternative luminal activation mechanisms for paneth cell α-defensins.

Paneth cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(-/-) mice lack mature α-defensins in Paneth cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(-/-) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(-/-) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(-/-) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(-/-) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.

MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis.

AIMS: Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell-cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis. METHODS AND RESULTS: Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (approximately 35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice. CONCLUSION: This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.

Matrix metalloproteinase-7 facilitates immune access to the CNS in experimental autoimmune encephalomyelitis.

BACKGROUND: Metalloproteinase inhibitors can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Matrix metalloproteinase-9 (MMP-9) has been implicated, but it is not clear if other MMPs are also involved, including matrilysin/MMP-7 - an enzyme capable of cleaving proteins that are essential for blood brain barrier integrity and immune suppression. RESULTS: Here we report that MMP-7-deficient (mmp7-/-) mice on the C57Bl/6 background are resistant to EAE induced by myelin oligodendrocyte glycoprotein (MOG). Brain sections from MOG-primed mmp7-/-mice did not show signs of immune cell infiltration of the CNS, but MOG-primed wild-type mice showed extensive vascular cuffing and mononuclear cell infiltration 15 days after vaccination. At the peak of EAE wild-type mice had MMP-7 immuno-reactive cells in vascular cuffs that also expressed the macrophage markers Iba-1 and Gr-1, as well as tomato lectin. MOG-specific proliferation of splenocytes, lymphocytes, CD4+ and CD8+ cells were reduced in cells isolated from MOG-primed mmp7-/- mice, compared with MOG-primed wild-type mice. However, the adoptive transfer of splenocytes and lymphocytes from MOG-primed mmp7-/- mice induced EAE in naïve wild-type recipients, but not naïve mmp7-/- recipients. Finally, we found that recombinant MMP-7 increased permeability between endothelial cells in an in vitro blood-brain barrier model. CONCLUSION: Our findings suggest that MMP-7 may facilitate immune cell access or re-stimulation in perivascular areas, which are critical events in EAE and multiple sclerosis, and provide a new therapeutic target to treat this disorder.

Matrix metalloproteinase 7 is required for tumor formation, but dispensable for invasion and fibrosis in SMAD4-deficient intestinal adenocarcinomas.

Expression of matrix metalloproteinase 7 (MMP7) is increased in the human colorectal carcinomas, and correlates with malignant progression. However, its contribution to colon cancer pathogenesis is not understood thoroughly. To investigate the roles of MMP7 in colon cancer progression, we introduced an Mmp7 knockout mutation into the cis-Apc/Smad4 mutant mouse, a model of invasive colon cancer in which SMAD4-dependent TGF-beta family signaling is inactivated. We demonstrate here that lack of MMP7 reduces the number and size of tumors in the cis-Apc/Smad4 mice. On the other hand, MMP7-deficiency does not affect the depth of tumor invasion, number of stromal fibroblasts or levels of extracellular matrix components in the tumors. These results indicate that MMP7 is required for tumor formation, but not for the invasion or fibrosis of the colon cancer whose SMAD4-dependent TGF-beta family signaling is blocked.

Sulindac treatment alters collagen and matrilysin expression in adenomas of ApcMin/+ mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) have shown potential as chemopreventive agents against cancer formation, especially colorectal cancers. However, the mechanisms by which these drugs act are not fully understood. In this study, Apc(Min/+) mice, a genetic model of human familial adenomatous polyposis, were treated with sulindac, and these mice demonstrated tumor reduction of >80%, consistent with previous reports. Gene microarray analyses of RNA from adenoma-derived dysplastic epithelial cells revealed that collagen genes, viz. Col1a2, Col5a2, Col6a2 and Col6a3, were upregulated, and matrilysin matrix metalloproteases-7 (Mmp7) was downregulated, in sulindac-treated mice. Reverse transcription-polymerase chain reaction validated gene expression of the Col6a2 subunit of collagen VI and of Mmp7. Confocal microscopy and immunofluorescence showed that within the tumors of non-treated mice, collagen VI was present in low amounts, but was enhanced within the tumors of sulindac-treated mice. Collagens I and V demonstrated similar patterns, but were not as prominent as collagen VI. Mmp7 was found in 'hot spot' areas within the tumors of Apc(Min/+) mice treated with the vehicle, but was greatly diminished in those mice treated with sulindac. Studies with Apc(Min/+)/Mmp7(-/-) double-deficient mice demonstrated the reciprocal relationships of Mmp7 expression and the levels of these three collagens in vivo. The results of this study demonstrated that sulindac was effective in increasing the expression of different collagens and decreasing the expression of Mmp7, effects that may contribute to altered tumor burden in cancer patients undergoing NSAIDs treatments.

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion.

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.

Insights from knock-out models concerning postischemic release of TNFalpha from isolated mouse hearts.

The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) is controversially discussed in ischemia/reperfusion damage of the heart. Purpose of this study was to elucidate cellular sources of TNFalpha and parameters which possibly influence its release in the heart following ischemia. Isolated hearts of mice were subjected to 15 min of global ischemia and 90 min of reperfusion. We employed hearts of various mice knock-out strains (interleukin-6(-/-), matrix metalloprotease-7(-/-), mast-cell deficient WBB6F1-Kit(W)/Kit(W-v), TNF-R1(-/-)) and wildtype mice, the latter perfused without and with infusion of cycloheximide or TNFalpha-cleaving-enzyme inhibitor (TAPI-2). Normoxic control hearts showed basal release of TNFalpha during the whole experiment. Immunohistology identified cardiac mast cells, macrophages and endothelial cells as main sources. TNFalpha release was stimulated during postischemic reperfusion, occurring in a two-peak pattern: directly after ischemia (0-10 min) and again after 60-90 min. The first peak mainly reflects tissue washout of TNFalpha accumulated during ischemia. The second, protracted peak arose continuously from the basal level and was abolished by protein synthesis inhibitor cycloheximide. Both properties are characteristic for de novo synthesis of TNFalpha, e.g., in cardiac muscle cells. However, immunohistological staining for TNFalpha failed in cardiomyocytes after 90 min of reperfusion. In contrast to hearts of TNF-R1(-/-) and Kit(W/W-v)-mice, those of IL-6(-/-) and MMP-7(-/-) mice lacked the late TNFalpha peak. TAPI did not suppress release of TNFalpha. While autostimulation via TNF-R1 also does not seem obligatory and mast cell can be ignored as source of the second peak, IL-6 may support de novo synthesis of TNFalpha. Additionally, TNFalpha release may essentially involve cleavage of membrane bound TNFalpha by MMP-7.

Matrix metalloproteinase (MMP)-7 activates MMP-8 but not MMP-13.

Matrix Metalloproteinases (MMPs) are a class of zinc-dependent enzymes that degrade extracellular matrix components, particularly collagen. MMPs have been implicated in a diverse list of pathological processes, including cancer and cardiovascular disease. Recent efforts to bring MMP inhibitors to clinical trials, however, have proved disappointing. These failures are attributed, in part, to the non-selective nature of current inhibitors. The possibility also exists, however, that inhibition of a particular MMP type will lead to feedback accumulation of parallel MMP members. MMP-7, also known as matrilysin, has a broad list of substrates, including denatured collagen and other MMPs involved in the collagenolytic pathway, namely MMP-1, MMP-2, and MMP-9. Whether the additional collagenases, MMP-8 and MMP-13, are also activated by MMP-7 has not been explored. We show here that recombinant active MMP-7 was able to process MMP-8 to its active form in vitro, but did not activate MMP-13. In the left ventricles of mice lacking the MMP-7 gene, MMP-8 levels increased while MMP-13 levels decreased in vivo. The switch in MMP profile was not accompanied by a change in left ventricular dimensions or wall thickness. Together, these data suggest that MMP-8 is an in vivo substrate of MMP-7, and that the accumulation of pro-MMP-8 in the absence of MMP-7 downregulates pro-MMP-13 levels in order to maintain baseline collagenolytic function. The interplay between MMP-8 and MMP-13 suggest that these MMPs may play reciprocal roles. The design of selective MMP inhibitors, therefore, must take into consideration changes in parallel MMP types as a potential compensatory mechanism.

Corneal neovascularization after excimer keratectomy wounds in matrilysin-deficient mice.

PURPOSE: Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing. METHODS: Matrilysin-deficient mice (n = 17) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic factors, the levels of basic fibroblast growth factor (bFGF) were compared with those of vascular endothelial growth factor (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV). RESULTS: The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% +/- 5.2% and 18.7% +/- 5.8% at days 3 and 7, respectively, compared with 5.3% +/- 2.4% and 5.5% +/- 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 +/- 0.55 mm(2) and 0.77 +/- 0.34 mm(2), respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV). CONCLUSIONS: Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.

Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin.

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.

The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis.

BACKGROUND: The Fas ligand/Fas receptor (FasL/Fas) system is an important mediator of apoptosis in the immune system where the juxtaposition of cells expressing the cell-surface ligand induces the apoptotic pathway in Fas-expressing lymphocytes. The FasL/Fas system has also been shown to be involved in apoptosis in epithelial tissues, including the involuting rodent prostate. FasL can be shed through the action of an hitherto unidentified metalloproteinase to yield soluble FasL (sFasL), although the biological activity of sFasL has been disputed. RESULTS: Here we report that the matrix metalloproteinase matrilysin can process recombinant and cell-associated FasL to sFasL, and that matrilysin-generated sFasL was effective at inducing apoptosis in a target epithelial cell population. In the involuting mouse prostate, FasL and matrilysin colocalized to the cell surface in a restricted population of epithelial cells. Mice deficient in matrilysin demonstrated a 67% reduction in the apoptotic index in the involuting prostate compared with wild-type animals, implicating matrilysin in this FasL-mediated process. CONCLUSIONS: The results show that a functional form of sFasL was generated by the action of the metalloproteinase matrilysin, and suggest that matrilysin cleavage of FasL is an important mediator of epithelial cell apoptosis.