The role of plasminogen activators in the regulation of connective tissue metalloproteinases.
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Animals↗
Biomedical subjects
J J Reynolds
Publications and source records attributed to J J Reynolds.
Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases.
The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely.
Animals↗
Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.
The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.
Amino Acid Sequence↗
Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines.
Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Immunolocalization of metalloproteinases and TIMP in normal and pathological tissues.
Molecular and biochemical studies have made substantial contributions to the understanding of metalloproteinases (MPs) and their natural inhibitor TIMP (tissue inhibitor of metalloproteinases) but knowledge of their specific roles in tissue breakdown in vivo is still meagre. A major problem is that there are few techniques available that can detect small amounts of these entities at the sites of resorption. One approach to this problem is to prepare specific polyclonal antisera for use in immunolocalization studies on cells and tissues ex vivo. Another is to develop model systems of rapid matrix destruction. Examples of these techniques are presented and discussed in relation to other studies. In many situations unique patterns of synthesis have been observed, consistent with specific roles for the individual MPs and biochemical data. Active collagenase can be localized to extracellular components in tissues where rapid destruction is taking place. Thus both approaches are proving invaluable in defining the roles of MPs and TIMP in normal and pathological situations.
Animals↗
The origin of matrix metalloproteinases and their familial relationships.
New computer comparisons of the sequences of mammalian matrix metalloproteinases have established for the first time strong links with bacterial metalloproteinases. We also propose that there are five groups in the family of matrix metalloproteinases, although only three are as yet well-characterized as proteins, and discuss their origin and relationships with other zinc containing proteases.
Amino Acid Sequence↗
Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes.
We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.
Binding Sites↗
The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1.
Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.
Amino Acid Sequence↗
Bone-derived growth factors modulate collagenase and TIMP (tissue inhibitor of metalloproteinases) activity and type I collagen degradation by mouse calvarial osteoblasts.
The finding that osteoblasts synthesize collagenase has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby preparing the underlying mineralized bone for osteoclastic action. To further understand the mechanisms regulating osteoid removal, mouse calvarial osteoblasts were cultured on 14C-labelled type I collagen films and the abilities of (i) bovine bone matrix extracts and (ii) purified or recombinant human growth factors, to modify their collagenolytic behaviour were investigated. EDTA/Tris-HCl extracts of bone matrix containing growth factor activity, exerted a dose-dependent inhibition of type I collagenolysis by osteoblasts stimulated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, 10 ng/ml). Inhibition was accompanied by a reduction in collagenase activity and an increase in free TIMP (tissue inhibitor of metalloproteinases) in the culture medium. Transforming growth factor-beta, epidermal growth factor, platelet-derived growth factor and the acidic and basic fibroblast growth factors all mimicked these effects. In contrast, insulin-like growth factors-I and -II did not inhibit type I collagenolysis, only partially inhibited collagenase activity, and did not stimulate TIMP production by either 1,25(OH)2D3-treated or untreated cells. These findings provide additional evidence for the tight control exerted on the proteolytic activity of osteoblasts and the importance of TIMP in its regulation. They suggest strongly that the conversion (coupling) of the initial resorptive phase of the bone remodelling cycle to one of deposition, may be mediated by polypeptide growth factors either produced locally by osteoblasts, or released by proteolysis from the bone matrix.
Animals↗
Porcine collagenase from synovial fibroblasts: cDNA sequence and modulation of expression of RNA in vitro by various cytokines.
Collagenase is a metalloproteinase that is important in extracellular matrix turnover and is produced by synovial fibroblasts in response to various cytokines and growth factors. Porcine collagenase cDNA was cloned and the sequence shows a 469-amino acid (AA) peptide with high homology to the human and rabbit enzyme (84% and 83.4% respectively). Predicted amino acid sequence from position #99-114 agree well with previously obtained NH2-terminal AA sequence data of purified mature, active pig collagenase. Using the cloned porcine cDNA as a probe in Northern analysis, it was found that IL-1, TNF and EGF enhanced 24-hour steady state mRNA levels while TGF-beta inhibited basal expression of collagenase. When added 10 hours previously, TGF-beta partially inhibited the induction of collagenase by TNF and EGF, but did not affect induction by IL-1.
Amino Acid Sequence↗
Binding of latent and high Mr active forms of stromelysin to collagen is mediated by the C-terminal domain.
A specific high-titre polyclonal antiserum to recombinant human prostromelysin was raised in a sheep and shown by immunoblotting to detect latent prostromelysin, high and low Mr active forms and the C-terminal domain. This antiserum was used to demonstrate by indirect immunofluorescence that latent and active high Mr prostromelysin bind to reconstituted collagen fibrils, and to other extracellular matrix components in tissues ex vivo but that active low Mr stromelysin does not. Isolation of the C-terminal domain was carried out to demonstrate that stromelysin binding was through this domain. By use of an antiserum to the tissue inhibitor of metalloproteinases (TIMP) it was shown that TIMP is unable to bind to reconstituted collagen fibrils. TIMP, however, will bind when active high Mr stromelysin is present but not if latent prostromelysin is bound. We conclude that stromelysin has different binding specificities from those previously documented for collagenase; only active collagenase binds to reconstituted collagen fibrils. However, TIMP binds to the active forms of both stromelysin and collagenase when these are bound to the collagen fibrils. These results have important implications for the interpretation of immunolocalization data in establishing the roles of metalloproteinases and their inhibitors in vivo.
Amino Acid Sequence↗
Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.
Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo rôle of gelatinases is discussed.
Amino Acid Sequence↗
Metalloproteinases and tissue damage.
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Amino Acid Sequence↗
Clinical dimensions of discharge planning with the person with AIDS.
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Acquired Immunodeficiency Syndrome↗
Osteopetrotic (grey-lethal) bone produces collagenase and TIMP in organ culture: regulation by vitamin A.
Evidence has recently accumulated suggesting that osteoblasts play a direct role in bone resorption by producing collagenase. In this paper we describe studies carried out with explants of bone from osteopetrotic grey lethal (gl/gl) mice and show that despite the lack of osteoclastic activity the production of both active and latent collagenase and its specific inhibitor TIMP (tissue inhibitor of metalloproteinases) is similar to that of normal bones. Synthesis of collagenase was stimulated by the bone resorptive agent vitamin A (retinol); concomitantly, TIMP levels fell to zero and active enzyme was detected in the culture medium. This work supports the view that bone collagenase is produced by cells other than osteoclasts, since the response of the osteoblastic population to resorptive signals appears normal.
Animals↗
Immunolocalization of collagenase and tissue inhibitor of metalloproteinases (TIMP) in mechanically deformed fibrous joints.
To investigate the effects of mechanical deformation on matrix degradation in fibrous joints, coronal suture explants from neonatal rabbits were stressed in vitro for 24 hours in an established tooth-movement model system. The metalloproteinase collagenase (CL) and its inhibitor, TIMP (tissue inhibitor of metalloproteinases), were immunolocalized in two ways by a two-step indirect technique: (1) extracellularly by immunoprecipitation at the site of secretion, and (2) intracellularly by incubation of the explants with the ionophore monensin. Immunoprecipitates of CL and TIMP were distributed throughout the sutural and periosteal tissues of nonstressed explants. In stressed explants, however, CL immunoprecipitates were predominantly associated with an area of rounded cells between the bone ends. In explants treated with monensin a significant increase in the number of CL-positive cells was observed in this cellular area; active enzyme was suggested by the demonstration of CL bound to collagen. Extracellular TIMP was not seen within the area of rounded cells of stressed explants, but intracellular TIMP was detectable; this suggests that insufficient TIMP was available to immunoprecipitate with anti-TIMP, probably because it had become irreversibly complexed with active CL. Since the area of rounded cells corresponds to the site of increased cell proliferation in this and other animal models of tooth movement, these data suggest that collagenase production and cell proliferation might be correlated. We speculate that matrix degradation is an essential prerequisite for cell proliferation as it creates room to accommodate an increase in cell population.
Animals↗
The release of interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma by cultured peripheral blood mononuclear cells from patients with periodontitis.
The extracellular release of IL-1 beta by cultured peripheral blood monocytes from 26 periodontitis patients and 26 control subjects was measured by radioimmunoassay. Unstimulated monocytes from periodontitis patients released significantly more IL-1 beta than controls during 24 h of culture; there was a wide variation in the amount of IL-1 beta released (0.45-13.00 ng/ml per 10(6) cells) which did not correlate with either the degree of bone loss or pocket formation observed clinically. When stimulated with lipopolysaccharide (LPS; Actinobacillus actinomycetemcomitans; 5 micrograms/ml) monocytes from periodontitis patients produced significantly more IL-1 beta than those from control subjects. Monocyte culture supernatants from another 10 periodontitis patients and 10 control subjects were also assayed for both IL-1 beta and TNF-alpha by enzyme-linked immunosorbent assays. Spontaneous and LPS-stimulated (Bacteroides gingivalis; 5 micrograms/ml) IL-1 beta release were again significantly higher for periodontitis patients. TNF-alpha was detected in the periodontitis cultures (0-765 pg/ml per 10(6) cells), but the mean value was not significantly different from controls. LPS-stimulated TNF-alpha release, however, was significantly higher than for control subjects, and there was a strong correlation between spontaneous IL-1 beta and TNF-alpha release by monocytes from the periodontitis group. Measurement of interferon-gamma (IFN-gamma) in lymphocyte cultures from these patients by immunoradiometric assay showed that IFN-gamma levels in periodontitis cultures were consistently low, but not significantly so when compared to controls; both groups responded equally to concanavalin-A (5 micrograms/ml). Although the precise roles of IL-1 beta and TNF-alpha in periodontitis remain unclear, these data provide evidence that both cytokines may participate in the pathogenesis of the disease.
Adolescent↗
Type I collagen degradation by mouse calvarial osteoblasts stimulated with 1,25-dihydroxyvitamin D-3: evidence for a plasminogen-plasmin-metalloproteinase activation cascade.
To understand the mechanisms regulating osteoid removal by osteoblasts, mouse calvarial osteoblasts were grown on 14C-labelled type I collagen films and stimulated with 1,25-dihydroxyvitamin D-3 (2.5.10(-8) M) for 48-72 h. In the presence of 5% non-inhibitory rabbit serum this resulted in a 2-3-fold increase in collagen degradation and a dramatic change in osteoblast morphology, when compared with untreated osteoblasts. Collagenolysis was accompanied by increased synthesis and release of latent collagenase, gelatinase and stromelysin and a concomitant decrease in their specific inhibitor, TIMP (tissue inhibitor of metalloproteinases). In serum-free medium, osteoblasts failed to degrade collagen, but their ability to lyse collagen could be restored by adding plasminogen (5 micrograms/ml) to the cultures. Plasminogen-dependent collagenolysis was inhibited by human recombinant TIMP (5 units/ml), demonstrating that plasmin, derived from plasminogen, activated latent collagenase and did not itself degrade collagen. Plasminogen activator production was confirmed by culturing osteoblasts on 125I-labelled fibrin plates. Comparison with urokinase-type and tissue-type plasminogen activator standards suggested that osteoblast plasminogen activator was predominantly cell-associated and likely to be of the urokinase type. Immunocytochemistry indicated that osteoblasts also constitutively produce plasminogen activator inhibitor-1. These findings provide evidence for the involvement of a plasminogen-plasmin-latent metalloproteinase activation cascade in type I collagen degradation by osteoblasts, and for its regulation by TIMP and plasminogen activator inhibitor-1.
Animals↗