Inhibition of gelatinase B (matrix metalloproteinase-9) by dihydrolipoic acid

Alpha-lipoic acid (LA) is a disulphide-containing fatty acid that is absorbed from the diet and transported to tissues. Once it has been taken up by mammalian cells, LA is reduced to dihydrolipoic acid (DHLA), a vicinal dithiol, and rapidly effluxed into the extracellular milieu. We hypothesized that DHLA may be an effective inhibitor of human gelatinase B (GelB). Purified human GelB was incubated with 0 to 200 micromol/L DHLA, and residual enzyme activity was measured by HPLC using a fluorogenic substrate (matrix metalloproteinase substrate III). DHLA inhibited GelB in a dose-dependent fashion with an IC50 of 20 micromol/L. Oxidation of DHLA resulted in a loss of DHLA's capacity to inhibit GelB. The DHLA-mediated inhibition of GelB was independent of the zinc concentration in the reaction buffer. DHLA had no inhibitory effect on gelatinase A. Zymographs of activated neutrophil lysates demonstrated that higher concentrations of DHLA also prevent the activation of GelB proenzyme. Bronchoalveolar lavage fluid from mice fed a diet enriched with LA showed significantly increased GelB inhibitory capacity (p = 0.0002 vs. regular diet). We conclude that DHLA can modulate neutrophil-derived GelB activity through direct inhibition of enzyme activity and by preventing the activation of GelB proenzyme.     

Direct production of an activated matrix metalloproteinase-9 (gelatinase B) from mammalian cells

Matrix metalloproteinase-9 (MMP-9) is produced by the inactive proform and activated by a proteolytic process. However, it has not been reported to produce the active form directly from cells, which has hindered the research to elicit the physiological roles of this enzyme. In this study, we prepared mutant MMP-9 containing the furin-recognizing sequence in the prodomain and showed that the mutant MMP-9 was secreted as the active form directly from CHO-K1 cells and primary hepatocytes after the gene was transfected. The secreted MMP-9 showed proteolytic activity without further activation and degraded collagen IV in vitro. In addition, the transfection of the gene into the liver resulted in the efficient expression of active MMP-9 in the liver and the serum in vivo.     

Expression of matrix metalloproteinase-9 (gelatinase B) in benign, premalignant and malignant laryngeal lesions

The matrix metalloproteinases (MMPs) are a family of proteolytic zinc-containing enzymes, which are responsible for the breakdown of the extracellular matrix components in pathological and physiological conditions. They are involved in basement membrane disruption, stroma and blood vessel penetration, metastasis and more recently there is evidence that they participate in tumor growth and angiogenic events. Matrix metalloproteinase 2 and 9 (MMP 2 and 9) belong to the gelatinases, a subgroup of MMPs, and have the capacity to degrade the triple helix type IV collagen of basal lamina of the basement membrane. With the present study, we tried to demonstrate the expression of MMP-9 immunohistochemically, comparatively in benign, premalignant and malignant lesions of the larynx. We studied 154 laryngeal lesions including 55 squamous cell carcinomas, 8 in situ carcinomas, 54 cases of dysplasia (of low and intermediate grade), 13 papillomas and 24 cases of keratosis. Overexpression of MMP 9 was observed in 74.4% and 50% in invasive and in situ squamous cell carcinomas respectively. In dysplastic cases, in papillomas and in keratoses the percentage of overexpression was 62.9%, 61.53% and 54.16% respectively and the expression of MMP-9 was significantly higher in invasive squamous cell carcinomas compared to dysplasias (p=0.000004). Also significantly higher was the expression of MMP-9 in dysplastic cases compared to papillomas (p=0.023). The MMP-9 expression was related neither to survival nor to the other available clinicopathological parameters (tumor size, grade, clinical stage, lymph node status and patient age). In conclusion, our study indicates that the expression of MMP-9 is up-regulated in a stepwise fashion, with two main steps, the first one, when a dysplastic lesion evolves and the next one, when the dysplasia progresses to invasive carcinoma.     

Gastric gelatinase B/matrix metalloproteinase-9 is rapidly increased in Helicobacter felis-induced gastritis

It has previously been shown that matrix metalloproteinase-9 (MMP-9) levels, originating from macrophages, are considerably increased in human Helicobacter pylori-associated gastritis. Here, the early kinetics of the MMP-9 response resulting from Helicobacter infection in C57BL/6 and MMP-9 knock-out mice using the murine Helicobacter felis model were examined. H. felis infection induced severe gastritis in the murine stomach at just 2 weeks after infection. Before gastritis, an increase was observed in MMP-9-positive cells detected by immunohistochemistry in the basal lamina propria. This finding was corroborated by gelatin zymography of stomach samples. As the gastritis increased so did the concentration of MMP-9 and the incidence of gastric MMP-9-positive cells, their location corresponding to that of macrophages. In contrast, systemic levels of MMP-9 remained unchanged. When MMP-9-deficient mice were infected with H. felis, no significant difference in gastritis development was detected compared with disease development in wild-type animals. We conclude that MMP-9 production is an early event in the response to gastric Helicobacter infection, a feature that may favor the recruitment of immune cells early during infection. At later stages, however, the increased levels of MMP-9 may damage the integrity of the stomach mucosa.     

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL

Detection of matrix metalloproteinase (MMP) activities in the urine from patients with a variety of cancers has been closely correlated to disease status. Among these activities, the presence of a group of high molecular weight (HMW) MMPs independently serves as a multivariate predictor of the metastatic phenotype (). The identity of these HMW MMP activities has remained unknown despite their novelty and their potentially important applications in non-invasive cancer diagnosis and/or prognosis. Here, we report the identification of one of these HMW urinary MMPs of approximately 125-kDa as being a complex of gelatinase B (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL). Multiple biochemical approaches verified this identity. Analysis using substrate gel electrophoresis demonstrated that the 125-kDa urinary MMP activity co-migrates with purified human neutrophil MMP-9 x NGAL complex. The 125-kDa urinary MMP-9 x NGAL complex was recognized by a purified antibody against human NGAL as well as by a monospecific anti-human MMP-9 antibody. Furthermore, these same two antibodies were independently capable of specifically immunoprecipitating the 125-kDa urinary MMP activity in a dose-dependent manner. In addition, the complex of MMP-9 x NGAL could be reconstituted in vitro by mixing MMP-9 and NGAL in gelatinase buffers with pH values in the range of urine and in normal urine as well. Finally, the biochemical consequences of the NGAL and MMP-9 interaction were investigated both in vitro using recombinant human NGAL and MMP-9 and in cell culture by overexpressing NGAL in human breast carcinoma cells. Our data demonstrate that NGAL is capable of protecting MMP-9 from degradation in a dose-dependent manner and thereby preserving MMP-9 enzymatic activity. In summary, this study identifies the 125-kDa urinary gelatinase as being a complex of MMP-9 and NGAL and provides evidence that NGAL modulates MMP-9 activity by protecting it from degradation.     

Expression of matrix metalloproteinase 9 (MMP-9/gelatinase B) in adenocarcinomas strongly correlated with expression of immune response genes

Matrix metalloproteinases (MMPs) are endopeptidases considered to be important regulators of the microenvironment of cancer. While MMPs are traditionally associated with the extracellular matrix (ECM), here we provide new evidence from an analysis of gene expression profiles from human tumor tissue that MMP-9 (gelatinase B) is associated with elements of the immune system in a way analogous to the association of other MMPs, such as MMP-2 (gelatinase A), with components of the ECM. An analysis of three independent microarray datasets of lung adenocarcinomas from previous studies [Nat. Med. 8, 816-824 (2002); Proc. Natl. Acad. Sci. USA 98, 13790-13795 (2001); Proc. Natl. Acad. Sci. USA, 98, 13784-13789 (2001)] showed that, in each dataset, out of the set of genes with significant correlations in mRNA expression to the expression of MMP9 (P < 0.005), a highly disproportionate number were found to be annotated in the Locuslink database as having a role in the anti-pathogen response. By comparison, out of the set of genes significantly correlated with the expression of MMP2, a highly disproportionate number were known components of the ECM. The same patterns observed in the lung data for both MMP2 and MMP9 were found as well in an additional published dataset of colon and ovarian adenocarcinomas [Am. J. Pathol. 159, 1231-1238 (2001)]. The results of this study suggest a greater functional role for MMP-9 in the immune response to cancer than what may previously have been thought.     

Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inflammatory cytokines: role in cell migration

Mast cells are key effectors in the pathogenesis of inflammatory and tissue destructive diseases such as rheumatoid arthritis (RA). These cells contain specialized secretory granules loaded with bioactive molecules including cytokines, growth factors, and proteases that are released upon activation. This study investigated the regulation of matrix metalloproteinase MMP-9 (gelatinase B) in human mast cells by cytokines that are known to be involved in the pathogenesis of RA. Immunohistochemical staining of synovial tissue showed abundant expression of MMP-9 by synovial tissue mast cells in patients with RA but not in normal controls. The expression, activity, and production of MMP-9 in mast cells was confirmed by RT-PCR, zymography, and Western blotting using cord blood-derived human mast cells (CB-HMC). Treatment of CB-HMC with TNF-alpha significantly increased the expression of MMP-9 mRNA and up-regulated the activity of MMP-9 in a time- and dose-dependent manner. By contrast, IFN-gamma inhibited MMP-9 mRNA and protein expression. The cytokine-mediated regulation of MMP-9 was also apparent in the human mast cell line (HMC-1) and in mouse bone marrow-derived mast cells. Furthermore, TNF-alpha significantly increased the invasiveness of CB-HMC across Matrigel-coated membranes while the addition of IFN-gamma, rTIMP-1, or pharmacological MMP inhibitors significantly reduced this process. These observations suggest that MMP-9 is not a stored product in mast cells but these cells are capable of producing this enzyme under inflammatory conditions that may facilitate the migration of mast cell progenitors to sites of inflammation and may also contribute to local tissue damage.     

Deficiency in matrix metalloproteinase gelatinase B (MMP-9) protects against retinal ganglion cell death after optic nerve ligation

Loss of retinal ganglion cells is the final end point in blinding diseases of the optic nerve such as glaucoma. To enable the use of mouse genetics to investigate mechanisms underlying ganglion cell loss, we adapted an experimental model of optic nerve ligation to the mouse and further characterized post-surgical outcome. We made the novel finding that apoptosis of retinal ganglion cells correlates with specific degradation of laminin from the underlying inner limiting membrane and an increase in gelatinolytic metalloproteinase activity. These changes co-localize with a specific increase in levels of the matrix metalloproteinase, gelatinase B (GelB; MMP-9). Using a transgenic mouse line harboring a reporter gene driven by the GelB promoter, we further show that increased GelB is controlled by activation of the GelB promoter. These findings led us to hypothesize that GelB activity plays a role in ganglion cell death and degradation of laminin. Applying the genetic approach, we demonstrate that GelB-deficient mice are protected against these pathological changes. This is the first report demonstrating a causal connection between GelB activity and pathological changes to the inner retina after optic nerve ligation.     

Simultaneous presence of unsaturation and long alkyl chain at P'1 of Ilomastat confers selectivity for gelatinase A (MMP-2) over gelatinase B (MMP-9) inhibition as shown by molecular modelling studies

Structural analogues of Ilomastat (Galardin), containing unsaturation(s) and chain extension carrying bulky phenyl group or alkyl moieties at P'1 were synthesized and purified by centrifugal partition chromatography. They were analyzed for their inhibitory capacity towards MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14, main endopeptidases involved in tumour progression. Presence of unsaturation(s) decreased the inhibitory potency of compounds but, in turn increased their selectivity for gelatinases. 2b and 2d derivatives with a phenyl group inhibited preferentially MMP-9 with IC50 equal to 45 and 38 nM, respectively, but also display activity against MMP-2 (IC50 equal to 280 and 120 nM, respectively). Molecular docking computations confirmed affinity of these substances for both gelatinases. With aims to obtain a specific gelatinase A (MMP-2) inhibitor, P'1 of Ilomastat was modified to carry one unsaturation coupled to an alkyl chain with pentylidene group. Docking studies indicated that MMP-2, but not MMP-9, could accommodate such substitution; indeed 2a proved to inhibit MMP-2 (IC50=123 nM), while displaying no inhibitory capacity towards MMP-9.     

Matrix metalloproteinase-9 (gelatinase B) deficiency leads to increased severity of Staphylococcus aureus-triggered septic arthritis

Matrix metalloproteinases constitute a family of structurally related endopeptidases that are crucial for the normal turnover of the extracellular matrix. Elevated levels of MMP-9 have been demonstrated in synovial fluids of rheumatoid arthritis patients, and a correlation with the severity of the disease has been described. The aim of this study was to explore the impact of MMP-9 expression on joint inflammation and destruction in a model of bacterially induced septic arthritis. MMP-9 knock-out mice and C57Bl6 congenic controls were inoculated intravenously or intra-articularly with Staphylococcus aureus. Arthritis was evaluated clinically and by means of histology. Zymographic analyses were performed to study ex vivo induction of MMP-9 following exposure to S. aureus. The MMP-9 knock-out mice displayed a significantly higher frequency and severity, but not destructivity, of arthritis than did the wild-type mice. The knock-out mice also proved to harbour an increased number of bacteria locally in joints and systemically in kidneys, possibly by impaired extravasation and recruitment of leukocytes and a deficient early defence against infection. Our findings indicate that deficiency in MMP-9 increases the degree of joint inflammation due to decreased bacterial clearance.