Latent Collagenase Research Articles

Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator.

To elucidate the mechanism of synovial damage in rheumatoid arthritis, we studied the activation of latent collagenases released from adherent rheumatoid synovial cells in culture. Latent enzyme was not complexed with alpha2 macroglobulin, the prinicpal proteinase inhibitor in serum, and could be activated by trypsin in the presence of alpha2 macroglobulin if sufficient proteinase was added to saturate inhibitor. Latent collagenase bound half as effectively to collagen fibrils as active enzyme. Plasmin was a threefold better activator of latent enzyme than trypsin and could be generated by addition of plasminogen to synovial-cell cultures. Production of both collagenase and plasminogen activator was inhibited by dexamethasone (10(-9) M). These studies emphasize in importance of control of activation in regulation collagenase activity, It is likely that rheumatoid synovium produces both latent collagenase and plasminogen activator; plasmin is activated from its zymogen, plasminogen, present in inflamed tissues, and in turn activates collagenase.

A Latent Collagenase from Embryonic Human Skin Explants

Collagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme. 1) Embryonic human skin explants showed a much higher capacity for collagenase production than did adult skin, on the basis of unit weight of tissue. 2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system. 3) The molecular weights of active and latent collagenases were about 40,000 and 50,000, respectively. 4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3 M. The degree of activation produced by this treatment was as high as that by limited proteolysis with trypsin. It was concluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex. 5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.

Evidence that latent collagenases are enzyme-inhibitor complexes

Specific collagenase from the culture media of various rabbit tissues and cells exists in active and latent forms. Latent collagenase is most effectively activated with 4-aminophenylmercuric acetate, a thiol-blocking reagent, strongly suggesting that latent forms are enzyme-inhibitor complexes. A collagenase inhibitor from bone cultures, which may be closely related to the inhibitor of such latent enzyme complexes, was partially characterized.

Changes in the collagenolytic activity released by primary VX-2 carcinoma cultures as a function of tumor growth

Serum-free media of minced tissue cultures of VX-2 rabbit carcinoma contained a specific collagenolytic activity capable of releasing soluble radioactive peptides from [14C]-labeled collagen fibrils. It was also capable of reducing the viscosity of acid-soluble collagen solutions by cleaving the tropocollagen (TC) molecules primarily at one site to TCA (75%) and TCB (25%) fragments. Three chromatographic fractions were separated by gel filtration: F1, (MW 85-110,000) present in larger amounts in early cultures of younger tumor tissue; F2, (MW-35-40,000) the major component with maximum production in the day 3 media of younger and advanced tumor tissues; F3, (MW 18-22,000) the minor component. Early cultures of younger tumor tissue contained a latent collagenase and were subject to trypsin activation suggesting the presence of inactive enzyme precursors or an enzyme-inhibitor complex.

A Complex of Collagenase with Low Molecular Weight Inhibitors in the Culture Medium of Embryonic Chick Skin Explants

Collagenase inhibitors with molecular weights of about 6,000 and 12,000 were isolated from latent chick skin collagenase treated with 3 M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.

Activation of alveolar macrophage collagenase by a neutral protease secreted by the same cell

MAMMALIAN collagenase is a proteolytic enzyme which specifically cleaves native triple helical collagen at physiological pH and temperature. As with other extracellular proteases, there is increasing evidence that this enzyme can be found in an inactive, latent form which may represent a proenzyme or an enzyme–inhibitor complex1–8. It has been known for some time that, like the zymogen forms of the pancreatic proteases, latent collagenase could be activated with low concentrations of trypsin2–5. The existence of inactive mammalian collagenase has been perplexing because this is often the major state in which the enzyme is found: it has not been known whether trypsin activation of latent collagenase has any physiological significance. Drawing parallels to the physiological activation of pancreatic chymotrypsinogen, procarboxypeptidase and proelastase by pancreatic trypsin, several investigators have suggested the existence of protease(s) that can activate latent collagenase in the physiological conditions in which collagenase functions1–3. The study reported here validates this concept by demonstrating that a single cell type, the alveolar macrophage, secretes an inactive form of collagenase that can be activated in physiological conditions by a protease secreted at the same time by the same cell type.

Extraction of collagenase from the involuting rat uterus

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90–95% recovery of the total active and latent collagenase activity in a 6000 × g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60°C for 4 main in 0.04 M Tris · HCl buffer, pH 7.5, containing 0.1 M CaCl 2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65–70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.

Synthesis and release of procollagenase by cultured fibroblasts.

An inactive collagenase was harvested from both serum-free and serum-supplemented fibroblast monolayer cultures in periods of active collagen synthesis. The latent collagenase did not hydrolyze collagen and did not bind the potent collagenase inhibitor alpha2-macroglobulin. Activation with trypsin imparted to the enzyme the ability to hydrolyze collagen at neutral pH in a typical manner and to form an inhibited complex with alpha2-macroglobulin. The molecular weights, determined by calibrated gel filtration, were 78,000 and 60,000 for the latent and active enzymes, respectively. The data indicate that collagenase is released from the cells in inactive form, as a zymogen.

Activation of latent bovine gingival collagenase

Latent collagenase from primary cultures of bovine gingiva was activated by brief exposure to trypsin and to a lesser extent by thiocyanate. The data suggest that activation by trypsin involves two distinct mechanisms: release of collagenase activity from high molecular weight inhibitor complexes and activation of latent collagenase (possibly a procollagenase) of approximately the same molecular size as active collagenase. Inhibition experiments confirmed that collagenase activity can be recovered from inactive inhibitor complexes, formed by the addition of whole serum or α 2-macroglobulin alone, after dialysis against thiocyanate or by brief exposure to trypsin.

Trypsin activation of latent collagenase from several mammalian sources

Latent collagenase, subject to activation by trypsin, was found in culture fluids of cells and tissues from several mammalian sources. The activation requires exposure to enzymatically active trypsin and cannot be achieved by inhibited or by heat-inactivated trypsin.

Connective tissue-degrading enzymes of human leukocytes.

Human leukocytes, when exposed to aggregated human gamma-globulin (AHGG) or immune complexes (isolated from RA synovial fluid) fixed to a cartilagenous surface, release neutral proteases that degrade the extracellular matrix of cartilage. The chondromucoprotein destruction by these proteases is suppressed by a variety of synovial fluids but is not susceptible to inhibition by trypsin, chymotrypsin, elastase inhibitors, or a combination of these agents. The inhibitory effect of synovial fluid can be reversed in the presence of increasing enzyme concentrations. Intact viable human polymorphonuclear leukocytes in the presence of AHGG also release a collagenase precursor that can be activated by limited proteolysis with trypsin or RA synovial fluids. Enzyme release (neutral proteases) by phagocytosing cells is inhibited by the antiinflammatory agents phenylbutazone and colchicine; these agents do not affect release of the collagenase precursor. However, the latent collagenase release is susceptible to inhibition when leukocytes are preincubated (prior to exposure to AHGG) with inhibitors of protein synthesis. Under these conditions, neutral protease release is unaffected.

Activation of latent collagenase by microbial plaque.

Sterile extracts of whole human microbial plaque released substantial latent collagenolytic activity in gingival collagenase preparations, homogenates of polymorphonuclear leukocytes, and tissue macrophages. The plaque activation factor was thermolabile, nondialyzable, and had essentially no inherent collagenolytic activity.

Synovial collagenase and joint diseases: the significancy of latent collagenase with special reference to rheumatoid arthritis.

Synovial collagenase and joint diseases: the significancy of latent collagenase with special reference to rheumatoid arthritis.

The release of collagenase as an inactive proenzyme by bone explants in culture.

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme-inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a ;procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.

A latent collagenase released by bone and skin explants in culture.

A latent collagenase released by bone and skin explants in culture.