Chronic exposure of interleukin-13 suppress the induction of matrix metalloproteinase-1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt.

Peripheral blood mononuclear cells taken from patients with scleroderma express increased levels of interleukin (IL)-13. Moreover, the expression of matrix metalloproteinase-1 (MMP-1) from involved scleroderma skin fibroblasts is refractory to stimulation by tumour necrosis factor (TNF)-α. To elucidate the mechanism(s) involved, we examined the effect of IL-13 on TNF-α-induced MMP-1 expression in normal and scleroderma human dermal fibroblast lines and studied the involvement of serine/threonine kinase B/protein kinase B (Akt) in this response. Dermal fibroblast lines were stimulated with TNF-α in the presence of varying concentrations of IL-13. Total Akt and pAkt were quantitated using Western blot analyses. Fibroblasts were treated with or without Akt inhibitor VIII in the presence of IL-13 followed by TNF-α stimulation. MMP-1 expression was analysed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (anova) or Student's t-test. Upon TNF-α stimulation, normal dermal fibroblasts secrete more MMP-1 than systemic sclerosis (SSc) fibroblasts. This increase in MMP-1 is lost when fibroblasts are co-incubated with IL-13 and TNF-α. IL-13 induced a significant increase in levels of pAkt in dermal fibroblasts, while Akt inhibitor VIII reversed the suppressive effects of IL-13 on the response of cultured fibroblasts to TNF-α, increasing their expression of MMP-1. We show that IL-13 suppresses MMP-1 in TNF-α-stimulated normal and scleroderma dermal fibroblast. Akt inhibitor VIII is able to reverse the suppressive effect of IL-13 on MMP-1 expression and protein synthesis. Our data suggest that IL-13 regulates MMP-1 expression in response to TNF-α through an Akt-mediated pathway and may play a role in fibrotic diseases such as scleroderma.

Cyclic equibiaxial tensile strain induces both anabolic and catabolic responses in articular chondrocytes.

Mechanical disturbance is directly implicated in the development of osteoarthritis (OA) but the precise mode for degenerative changes is still largely unknown because of the complexity of the biomechanical and biochemical milieu in the articular joint. To investigate the effects of tensile strain on articular cartilage, cyclic equibiaxial tensile strain (CTS, 0.5 Hz, 10% strain) was applied to monolayer cultures of porcine articular chondrocytes by using a Flexercell strain unit. Overproduction of proinflammatory mediators and imbalanced expression of anabolic and catabolic genes were induced. The cellular secretion of nitric oxide (NO) and prostaglandin E(2) (PGE(2)), as well as the mRNA level of cyclooxygenase-2 (COX-2) were up-regulated in response to mechanical stimuli. Additionally, CTS resulted in an initial peak of anabolic response at 3 h of stretch with respect to the expression of type II collagen and aggrecan. After 12 h of CTS, the expression for these two cartilage-specific matrix proteins fell to control levels. A distinct catabolic response developed after 24 h of stretch with an increase in matrix metalloproteinase-1 (MMP-1). Interestingly, a parallel increase in transforming growth factor (TGF) beta3 was associated with the anabolic changes while an increase in expression of TGF beta1, the predominant isoform of the TGF family, appeared at 24 h. The expression at 24 h of MMP-1, an enzyme that degrades interstitial collagens as well as other cartilage matrix proteins and TGF beta1, may signify a shift towards matrix remodeling and potentially a change in matrix composition as a consequence of continuous CTS.

Regulation of cartilage collagenase by doxycycline.

OBJECTIVE: To investigate the ability of doxycycline to modulate collagenases, cytokines, and cytokine receptors in chondrocytes from osteoarthritic (OA) cartilage. METHODS: Chondrocytes were isolated from human OA cartilage and treated with doxycycline. Synthesis of collagenases, cytokines, and cytokine receptors was quantified by Northern and Western blot analysis and RNase protection assay. RESULTS: We observed significant inhibition of matrix metalloproteinases (MMP-1) and MMP-13 mRNA and protein production by chondrocytes, isolated from OA cartilage, after treatment with doxycycline. The decrease in collagenase protein level paralleled a decrease in mRNA for these enzymes, suggesting a transcriptional/posttranscriptional level of control. In addition, treatment with 10 microg/ml doxycycline resulted in 2.2-fold upregulation of transforming growth factor (TGF-beta3) and a significant decrease of interleukin 1alpha (IL-1alpha), IL-1beta, and IL-6 mRNA. Upregulation of TGF-beta RI and TGF-beta RII was also detected. These cytokines are known to affect collagenase expression and could contribute to inhibition of MMP-1 and MMP-13 production by OA chondrocytes. A decrease in IL-1alpha, IL-1beta, and IL-6 would reduce stimulation of MMP production, while an increase in TGF-83 would lead to downregulation of local proinflammatory cytokine production as well as of the collagenases themselves. CONCLUSION: Our findings show that a decrease in MMP-1 and MMP-13 collagenase production by articular chondrocytes in response to treatment with doxycycline can be explained by a regulatory effect of doxycycline on the production of cytokine and cytokine receptors.

Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen.

BACKGROUND/AIMS: Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis. Reversion of this disease has not been fully accomplished. Physiologically, collagen is degraded by interstitial collagenases, neutrophil collagenase (MMP-8) being the most active against type I collagen. Introduction of MMP-8 gene into liver cells could be an advantageous tool to potentiate fibrosis degradation. METHODS: We cloned latent and active MMP-8 genes in prokaryotic and eukaryotic expression vectors and an adenoviral vector. Transfection of MMP-8 in HepG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral transduction, and cells and culture supernatant were harvested 72 h after transfection for analysis of MMP-8 expression by reverse transcription-polymerase chain reaction and collagenolytic activity. RESULTS AND CONCLUSIONS: We show that a truncated neutrophil collagenase (tMMP-8) lacking a portion of the carboxy terminus and with an intact aminoterminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. Both MMP-8 mRNA (latent and active) were detected by polymerase chain reaction in cells transfected with CaPO4, P-L and adenoviral transduction; however, relative expression of MMP-8 was enhanced when the plasmid was delivered as a P-L complex and increased by adenoviral infection. Finally, a-tMMP-8 cDNA was cloned in a vector under transcriptional control of a regulated promoter (PEPCK-a-tMMP-8). HepG2 cells transfected with the PEPCK-a-tMMP-8 plasmid DNA up-regulated expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP and glucagon, while stimulation with insulin slightly down-regulated its expression. Recombinant MMP-8 expressed by HepG2-transduced cells can efficiently degrade soluble type I collagen, which is potentially useful for gene transfer therapies.

Tissue factor pathway inhibitor binds to platelet thrombospondin-1.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that down-regulates tissue factor-initiated blood coagulation. The most biologically active pool of TFPI is associated with the vascular endothelium, however, the biochemical mechanisms responsible for its cellular binding are not entirely defined. Proposed cellular binding sites for TFPI include nonspecific association with cell surface glycosaminoglycans and binding to glycosyl phosphatidylinositol-anchored proteins. Here, we report that TFPI binds specifically and saturably to thrombospondin-1 (TSP-1) purified from platelet alpha-granules with an apparent K(D) of approximately 7.5 nm. Binding is inhibited by polyclonal antibodies against TFPI and partially inhibited by the B-7 monoclonal anti-TSP-1 antibody. TFPI bound to immobilized TSP-1 remains an active proteinase inhibitor. Additionally, in solution phase assays measuring TFPI inhibition of factor VIIa/tissue factor catalytic activity, the rate of factor Xa generation was decreased 55% in the presence of TSP-1 compared with TFPI alone. Binding experiments done in the presence of heparin and with altered forms of TFPI suggest that the basic C-terminal region of TFPI is required for TSP-1 binding. The data provide a mechanism for the recruitment and localization of TFPI to extravascular surfaces within a bleeding wound, where it can efficiently down-regulate the procoagulant activity of tissue factor and allow subsequent aspects of platelet-mediated healing to proceed.

Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices.

Type II tropocollagen molecules were reacted with matrix metalloproteinase 8 (MMP-8) and the binding sites as well as the cleavage site of MMP-8 were detected on individual molecules using atomic force microscopy (AFM). Approximately 300-nm-long coiled-coil tropocollagen molecules were straightened and immobilized on an atomically flat surface for detection by AFM. The direct visualization of individual collagen molecules revealed heterogeneous characteristics of MMP-8:collagen complexes. We observed that there existed multiple MMP-8 nonspecific binding sites on the collagen molecules, but cleavage always took place at a unique site. When collagen molecules, straightened and immobilized on the surface, were reacted with MMP-8, a site of cleavage appeared as a gap in stretched molecules. This is the first report to visually show direct collagenase:collagen interactions using AFM. The described AFM-based analysis has potential as a protein analysis tool for understanding a complex mechanism of enzyme:substrate interactions.

Autocrine regulation of collagenase 3 (matrix metalloproteinase 13) during osteoarthritis.

OBJECTIVE: To correlate the increased collagenase production previously seen in chondrocytes isolated from osteoarthritic (OA) lesions and the expression of cytokines and cytokine receptors. METHODS: Chondrocytes were isolated from OA cartilage and characterized for synthesis of collagenases, cytokines, and cytokine receptors by Northern and Western blot analyses, RNA protection assay, and flow cytometry. RESULTS: Chondrocytes located in cartilage proximal to the macroscopic OA lesions bound more tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) compared with chondrocytes isolated from morphologically normal cartilage from the same joint. In response to TNFalpha stimulation, messenger RNA (mRNA) levels for the IL-1 receptor I (IL-1RI), IL-1RII, TNF receptor II (TNFR II), and IL-6 receptor as well as the level of proinflammatory cytokines, such as IL-1alpha, IL-1beta, lymphotoxin beta, TNFalpha, and IL-6, also increased. In contrast, treatment with transforming growth factor beta1 (TGFbeta1) resulted in down-regulation of matrix metalloproteinase 1 (MMP-1) and MMP-13 concomitant with a reduction in the levels of mRNA for IL-1RI, IL-1RII, TNFRI, and TNFRII and proinflammatory cytokine levels. In contrast, the levels of mRNA for TGFbeta receptor I, TGFbeta1, and TGFbeta3 were up-regulated. CONCLUSION: These data show that TGFbeta1 has antagonistic effects upon OA chondrocytes, in contrast to the effects seen with TNFalpha. The cyclical course of OA, where a period of active disease is followed by a period of remission, can be explained by a sequential pattern of cytokine stimulation followed by a feedback inhibition of autocrine cytokine production and cytokine receptor expression, thus affecting collagenase synthesis.

Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme.

OBJECTIVE: To investigate the inhibition of matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 by doxycycline, and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS: Recombinant human MMP-1 (collagenase 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain, and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen, a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS: The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline, while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast, in experiments with the peptolide substrate, neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline, while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8, inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION: Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8.

Differential patterns of response to doxycycline and transforming growth factor beta1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes.

OBJECTIVE: To investigate the ability of doxycycline, transforming growth factor beta1 (TGFbeta1), and phorbol myristate acetate (PMA) to modulate collagenase synthesis in osteoarthritic (OA) chondrocytes. METHODS: Levels of fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) proteins and messenger RNA (mRNA) were measured in chondrocytes isolated from involved and uninvolved areas of OA cartilage and from normal human chondrocytes, after treatment with doxycycline, TGFbeta1, and PMA. RESULTS: Chondrocytes isolated from cartilage immediately adjacent to the OA lesion had, on average, 1.8-3.9-fold higher basal levels of MMP mRNA. These cells down-regulated collagenase proteins and mRNA upon incubation with TGFbeta1. In contrast, chondrocytes from areas located more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGFbeta1. Discoordinate regulation was observed after stimulation with PMA, with an increase in MMP-1 and MMP-8 but a decrease in MMP-13. Incubation of OA chondrocytes with doxycycline (1-10 microg/ml), at pharmacologically achievable levels, decreased levels of mRNA of all 3 collagenases, but not G3PDH. In addition, doxycycline inhibited the increase in mRNA for these enzymes in normal chondrocytes stimulated with tumor necrosis factor alpha. CONCLUSION: These findings suggest that regulation of MMP-1, MMP-8, and MMP-13 in OA chondrocytes, although mediated by differing pathways, can be decreased by treatment with doxycycline at low concentrations. Our data provide a rationale for the use of doxycycline in the treatment of OA.

Collagenase expression by normal human eosinophils.

Collagenolytic activity was detected in extracts from human blood eosinophilic granulocytes. To characterize this collagenase, we compared extracts from isolated populations of eosinophils and neutrophils. Significant collagenase activity against type I and II collagens was present in extracts from both cell populations. Although collagenase activity was present in eosinophils, the cells did not stain with antibodies specific for fibroblast, neutrophil collagenase, or collagenase-3. In contrast, neutrophils immunostained positively with antibody to neutrophil collagenase. Western blot analysis confirmed the presence of immunoreactive protein in neutrophil extracts but not in the eosinophil extracts. Reverse transcription-polymerase chain reaction using primers specific for all three known collagenases of an eosinophil cell suspension from peripheral blood that had 3% contamination with immature neutrophils showed a polymerase chain reaction product only with neutrophil collagenase oligonucleotide primers, but not with fibroblast collagenase or collagenase-3 primers. Eosinophil collagenase would appear to have a unique antigenic structure and may represent a new enzyme.

Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro.

The present study was designed to determine the influences of vascular endothelial growth factor (VEGF) on cell proliferation and the release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) from human dermal microvascular endothelial cells. Treatment of cultures with 10 ng/ml or more of VEGF significantly increased cell proliferation. The effect of VEGF treatment on the levels of specific MMPs and TIMPs in the media was subsequently examined in cultures that were treated with 10 ng/ml VEGF. Zymography and Western blot analyses demonstrated that gelatinase A levels in the media were increased by VEGF treatment. Collagenase was detected by Western blots in both VEGF-treated and untreated culture media, but the levels were not significantly increased by the VEGF treatment. An ELISA assay confirmed that VEGF treatment significantly increased gelatinase A levels but did not significantly increase collagenase levels. Western blot and ELISA data showed that VEGF treatment significantly decreased TIMP-1 and TIMP-2 levels compared to untreated cultures. The data suggest that VEGF may modulate endothelial cell-derived MMP activity by: (1) increasing the abundance of gelatinase A; (2) disinhibiting gelatinase A by decreasing the abundance of TIMP-2; and (3) disinhibiting preexisting collagenase by reducing levels of TIMP-1. These actions could contribute to the ability of VEGF to promote endothelial cell invasion of new territory.

Osteoarthritic lesions: involvement of three different collagenases.

OBJECTIVE: To assess the presence of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion. METHODS: Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis. RESULTS: MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor alpha stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13. CONCLUSION: These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.

Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent.

OBJECTIVE: To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. METHODS: After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 microM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. RESULTS: At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. CONCLUSION: The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular in some pathological situations, at lower pH the effect of doxycycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.

Chondrocyte matrix metalloproteinase-8. Human articular chondrocytes express neutrophil collagenase.

This study confirms that normal human articular chondrocytes express neutrophil collagenase or matrix metalloproteinase-8 (MMP-8), a gene product previously thought to be expressed exclusively by neutrophil leukocytes. Both MMP-8 protein and mRNA were present in articular cartilages collected from normal human donors. Cartilage extracts were assayed by immunoblotting and by analysis of enzymatic activity on gelatin-substrate gels. Latent MMP-8 extracted from cartilage has a molecular mass of 55 kDa; active MMP-8 was identified at 46 and 42 kDa. In the absence of a reducing agent, MMP-8 migrated in a high molecular mass complex above 200 kDa. Northern blotting results demonstrated the expression of MMP-8 in chondrocytes, which could be up-regulated by stimulation with interleukin-1 beta. In addition, reverse transcription-polymerase chain reaction using nested primers and in situ hybridization revealed the presence of MMP-8 mRNA in chondrocytes. The presence of both MMP-8 protein and message in cartilage supports the concept that neutrophil collagenase could be the enzyme described as "aggrecanase".

Activation of recombinant human neutrophil procollagenase in the presence of doxycycline results in fragmentation of the enzyme and loss of enzyme activity.

OBJECTIVE: To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. METHODS: Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C-acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10-phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. RESULTS: Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments while trypsin cleavage yield 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 microM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. CONCLUSION: These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.

Chondrocyte matrix metalloproteinase-8: up-regulation of neutrophil collagenase by interleukin-1 beta in human cartilage from knee and ankle joints.

The loss of aggrecan from articular cartilage may lead to the development of osteoarthritis (OA). Degradation products of human aggrecan, generated in vivo by enzymatic cleavages, have been identified in synovial fluid of patients with rheumatoid arthritis and OA. One matrix metalloproteinase (MMP), stromelysin (MMP-3), and an unidentified proteinase called "aggrecanase" are believed to generate these products in pathologic conditions. Thus far, only one proteinase, neutrophil collagenase (MMP-8), has been shown in vitro to be capable of cleavage of the aggrecan molecule at the "aggrecanase" site. In this study, we compare the presence and distribution of MMP-3 and MMP-8 in cartilages from two different joints of normal human donors. We determined whether mRNA for MMP-8 is expressed in normal human articular cartilage from different joints. In addition, we compared differences in MMP-8 and MMP-3 gene expression between human ankle and knee cartilage after in vitro stimulation by interleukin (IL)-1 beta. These two joints were chosen because the incidence of symptomatic and radiographic OA varies between the different joints. The knee is the most frequently involved joint, whereas the ankle (talocrural) joint is relatively rarely affected. Message for MMP-8 was detected in untreated cartilage from normal knee joints, but not in untreated cartilage of normal ankle joints. Message for MMP-3 was detectable in most of the knee and ankle cartilages. Messenger RNA expression for both MMPs could be up-regulated by IL-1 beta. The highest doses of IL-1 beta appeared to be most effective in stimulation of mRNA for MMP-3, whereas MMP-8 expression was more sensitive to lower doses of IL-1 beta. The fact that ankle cartilage with a low incidence of OA does not express MMP-8, whereas knee cartilage with a high incidence of OA does not express MMP-8, whereas knee cartilage with a high incidence of OA does constitutively express MMP-8, suggests that MMP-8 might be one of the key enzymes in the pathogenesis of osteoarthritis. This is further supported by our finding that the earliest signs of cartilage degradation were very similar to those found in IL-1 beta-treated explants.

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.

Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure, expression of cDNA in E. coli and enzymic properties.

Human neutrophils can be triggered to release the collagenolytic metalloenzymes, interstitial collagenase and 92 kDa type IV collagenase/gelatinase. We have isolated and sequenced a 2.3 kb cDNA from a chronic granulocytic leukemia cDNA library that encodes for human neutrophil type IV collagenase. With the exception of one amino-acid substitution at position 280 (Arg-->Gln), the deduced amino-acid sequences of neutrophil gelatinase are identical to the amino-acid sequences of the enzyme isolated from fibrosarcoma cells. Expression of the cDNA in E. coli yielded a 72 kDa protein having a gelatinolytic activity on zymogram gel. The recombinant enzyme was activated with APMA and trypsin. The activation was accompanied by a reduction in molecular weight of approximately 10 kDa; such a reduction is characteristic of matrix metalloproteinases. The recombinant gelatinase cleaved native type V and XI collagens. Native type I collagen was not a substrate for the enzyme. These data suggest that native and recombinant 92 kDa type IV collagenase produced in E. coli have similar biochemical properties. The successful expression of the collagenase in a prokaryotic system will greatly facilitate the structure-function characterization of the enzyme and allow a more precise analysis of its physiological and pathological roles.

Susceptibility of type I collagen containing mutated alpha 1(1) chains to cleavage by human neutrophil collagenase.

Two members of the matrix metalloproteinase family which can cleave native types I, II and III triple helical collagens are collagenases from fibroblasts and neutrophils. These enzymes are the products of different genes which share structural motifs but are only 57% identical. In this study, we determined the site of cleavage in the alpha 1(I) chains and showed that the neutrophil collagenase acted at the same site as the fibroblast collagenase. We also used collagens as substrates which were generated by site-directed mutagenesis of the murine Col1a1 gene and found that the pattern of susceptibility to cleavage by purified neutrophil collagenase was indistinguishable from that previously described for the fibroblast collagenase. Collagens containing substitutions of Pro for Ile-776 (P1) were not cleaved; whereas those containing substitutions of Met for Ile-776 were cleaved. Type I collagen which contained alpha 1(I) chains in which there were double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were also not cleaved. These type I collagens contained wild type alpha 2(I) chains as well as mutant alpha 1(I) chains in the mixed helical trimers; the alpha 2(I) chain in the trimers containing the resistant alpha 1(I) chains were also not cleaved by the neutrophil collagenase.