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R M Hembry

Publications and source records attributed to R M Hembry.

At least 19 recordsLinked to original sources
PubMed1992

Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity.

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.

Adipose Tissue↗
PubMed1992

Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver.

Hepatic lipocytes play a central role in the pathogenesis of liver fibrosis, both via production of extracellular matrix proteins and through secretion of matrix metalloproteinases. In this study, we have characterized lipocyte expression and release of tissue inhibitor of metalloproteinases-1 (TIMP-1), an important inhibitor of metalloproteinase activity, whose role in liver has not previously been examined. TIMP-1 was immunolocalized to human lipocytes, and secretion of TIMP-1 was confirmed by ELISA of culture media; (mean +/- SD) 159 +/- 79 ng of TIMP-1/10(6) cells per 24 h. Evidence for functional inhibitory activity of released TIMP-1 was obtained by (a) reverse zymography that demonstrated a single inhibitor band, M(r) 28 kD, that co-migrated with a TIMP-1-positive control sample; and (b) unmasking of inhibited gelatinase activity in lipocyte medium by separating it from TIMP-1 using gelatin sepharose chromatography; gelatinase activity in chromatographed medium increased more than 20-fold, compared with unfractionated medium, and could be reinhibited by adding back fractions that contained inhibitor. By Northern analysis, freshly isolated human lipocytes exhibited low levels of mRNA expression for TIMP-1, but this increased markedly relative to beta-actin expression with lipocyte activation during cell culture. We conclude that human hepatic lipocytes synthesize TIMP-1, a potent metalloproteinase inhibitor, and that TIMP-1 expression increases with lipocyte activation. These data indicate that hepatic lipocytes can regulate matrix degradation in the liver, and suggest that expression of TIMP-1 by activated lipocytes may contribute to the progression of liver fibrosis.

Adipose Tissue↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Proteinases in rheumatoid arthritis.

The role of proteinases in the extracellular breakdown of the connective tissue matrix during disease processes is outlined. Focussing on the matrix metalloproteinases, we present an overview of the mechanisms regulating their activity. Methods of assessing their role in disease processes is exemplified by new data on stromelysin activity in remodelling joint tissues in both model systems and human disease.

Animals↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.

Animals↗
PubMed1989

Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts.

The effect of various metalloproteinase-inhibiting compounds on collagen phagocytosis by fibroblasts was studied in cultured periosteal tissue. Evidence is presented indicating that neither anti-collagenase nor anti-stromelysin interfere with the uptake of collagen fibrils from the extracellular space and their intracellular digestion. Similar results were obtained with tissue inhibitor of metalloproteinases (TIMP). In the presence of the proteinase inhibitor leupeptin, a compound which strongly inhibits the intracellular degradation of phagocytosed collagen, a time-dependent increase in the amount of internalized collagen was found. This increase proved to be similar in explants treated as well as in those not treated with the metalloproteinase-inhibiting compounds. It is concluded that enzymes, such as collagenase and stromelysin, do not play a crucial role in the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. If these enzymes are involved it must be prior to these events. Based on the morphometric data the intralysosomal degradation time of collagen was calculated to be about 30 minutes. A comparison with findings in the literature on collagen metabolism in the periodontal ligament of the rat molar suggests that all collagen degraded may pass through the phagolysome pathway during physiological turnover and remodelling.

Animals↗
PubMed1989

The degradation of collagen in pig synovium in vitro and the effect of colchicine.

Colchicine induced a rapid destruction of the collagenous matrix of pig synovial explants in culture in the presence of serum. The most efficacious doses were 0.01-0.1 micrograms/ml (2.5 x 10(-8) M - 2.5 x 10(-7) M). The histological progression of the tissue breakdown induced by colchicine was very similar, although faster, to that described for other agents (Fell et al., 1986), with cells having basophilic nuclei accumulating in areas of fibril degradation. The loss of collagen correlated with an increase in collagenase production and at the peak of resorption (6 to 8 days) active collagenase was present in the culture media. Immunocytochemical methods demonstrated active collagenase bound to collagen fibrils after only 4 days in culture, before significant collagen degradation could be observed histologically. Collagen breakdown was completely inhibited by cortisol, and partially inhibited by indomethacin: only the inhibition by indomethacin could be reversed by exogenous prostaglandin E2. Vinblastine at a higher dose was as effective as colchicine but the lumicolchicines, which do not disrupt microtubules, were ineffective. Although the precise mechanism of action of colchicine is unknown, this culture system provides a useful in vitro model for increasing our understanding of the cellular mechanisms of tissue breakdown and for elucidating the roles of active collagenase and related metalloproteinases.

Animals↗
PubMed1989

Collagenase is expressed by rabbit VX2 tumour cells in syngeneic and xenogeneic hosts.

Specific antisera for the connective tissue metalloproteinases, collagenase, gelatinase (type IV collagenase) and stromelysin were used to study their respective localizations in both rabbit primary VX2 tumours and in lung metastatic deposits (frozen immediately after excision). Collagenase was found within some cells of the primary tumour and also bound to the extracellular matrix at discrete sites. Previous studies suggest that this matrix staining represents active enzyme. Stromelysin and gelatinase had a more limited distribution, particularly the latter, but both showed cell and matrix staining. In the lung metastases collagenase and stromelysin occurred less frequently, although both cell and matrix staining were observed; gelatinase was not seen. When rabbit VX2 cells were transplanted into nude mice they grew as a discrete nodule. Cells within this nodule stained with the antiserum to collagenase, which recognizes rabbit but not mouse enzyme, and thus demonstrated that cells of tumoural origin synthesize collagenase in vivo. Stromelysin was also co-localized with collagenase in some tumour cells.

Animals↗