Preferential binding of collagenase to alpha 2-macroglobulin in the presence of the tissue inhibitor of metalloproteinases

The binding of collagenase to both alpha 2-macroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to alpha 2-macroglobulin although no transfer of collagenase to alpha 2-macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule.     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

Collagen binding site in collagenase can be determined using the concept of sense-antisense peptide interactions.

Tissue degradation and invasion are hallmarks of the metastatic phenotype. While several extracellular matrix components can be digested by proteases, degradation of interstitial collagen is selectively initiated by collagenase. It is obvious that inhibitors of collagenase activity would be extremely useful in preventing tissue destruction and tumor cell invasion and thus prove invaluable therapeutic agents. We describe here the possible development of such inhibitors through the use of the principle of complementary hydropathy. A peptide was deduced from the nucleotide sequence complementary to that coding for the region in interstitial collagen surrounding the bond between Gly775 and Ile776 which is cleaved by the enzyme. Labeled collagen binds specifically and quantitatively to this peptide. A polyclonal mouse serum raised against this peptide recognized purified human collagenase, was able to immunoprecipitate collagenase from cultured human keratinocyte supernatants and was effective in inhibiting collagenolytic activity with a K(iapp) = 0.3 microM.     

The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

Tissue collagenase: a simplified, semiquantitative enzyme assay

Tissue collagenase activity from the ulcerating rabbit cornea has been quantitated in a sensitive capillary tube assay system with an unlabeled, native collagen substrate. In this assay system, initial rates of gel lysis are proportional to enzyme concentration over a defined range of enzyme concentrations. Increased sensitivity to enzyme with an unlabeled substrate has been achieved by restricting diffusion of enzyme to one dimension, in a capillary gel. Corneal collagenase activity has been measured at concentrations down to 0.1 μg/μl. In addition to its high sensitivity to enzyme, the precision and simplicity of the assay and minimal equipment requirements all recommend its use for routine screening of biological fluids for collagenase activity and in the investigation of the effects of inhibitors and stimulators of collagenase activity.     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase.

1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.     

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.     

Collagenase production by smooth muscle: correlation of immunoreactive with functional enzyme in the myometrium

A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti-collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti-smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra- or extra-cellularly.     

Isolation and characterisation of a chicken gelatinase (type IV collagenase).

The proform of chick gelatinase (type IV collagenase) was isolated and purified to a high specific activity of 12,071 U/mg from cultured embryonic skin fibroblasts stimulated with cytochalasin-B. The enzyme was activated in the presence of 4-aminophenylmercuric acetate with a fall in molecular weight from 66,000-58,000 on non-reducing polyacrylamide gel electrophoresis and was active over the pH range of 6.0-8.9 against a number of substrates. Further biochemical characterisation showed that the organomercurial activated form of the enzyme behaved like a typical mammalian gelatinase, actively degrading gelatin, soluble type I collagen, collagenase generated type I fragments, type IV collagen (producing 3/4 and 1/4 fragments) and type V collagen, whilst having little effect on laminin. The enzyme was inhibited by metal chelators such as EDTA and 1,10-phenanthroline, but not by inhibitors is suggested that this may be TIMP-2. An antiserum was raised to the proenzyme and was found to localise intra- and extra-cellularly in both tissue sections and cell cultures.     

Nature of the inhibitory effect of collagenase on phosphodiesterase activity

The level of phosphodiesterase (PDE) activity is lower in collagenase-isolated human fat cells than in adipose tissue fragments. The inhibition is not species-specific since collagenase also inhibits PDE in rat adipose tissue and bovine heart. In subcellular fractions from isolated fat cells, the PDE activities were lowest in the plasma membrane-enriched fractions and highest in the cytosolic fractions. This is opposite to PDE in subcellular fractions obtained from adipose tissue fragments. In dose-response experiments, collagenase inhibited particulate PDE to a much larger extent than it inhibited soluble PDE. The extracellular activities of PDE were completely eliminated by collagenase. Repeated washings or reincubation of the isolated fat cells did not restore the PDE activity. A purified collagenase with low specific protease activity reduced the PDE activity in isolated fat cells to a lesser extent than did a collagenase with high specific protease activities. Collagen and several protease inhibitors were ineffective in preventing the reduction of PDE after exposure to collagenase. It is concluded that nonspecific proteases in the collagenase preparations used for fat cell isolation interact with particulate and soluble PDE causing an irreversible inhibition of PDE activity in isolated fat cells. Of the various forms of PDE, plasma membrane-associated PDE seems most sensitive to collagenase.     

Regulation of human skin collagenase activity by hydrocortisone and dexamethasone in organ culture

Hydrocortisone and dexamethasone (9α-fluoro, 16α-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid synovium and rat uterus. Hydrocortisone is maximally inhibiting at 10^?7M and dexamethasone at 10^?8M in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids.     

New thiol inhibitors of Clostridium histolyticum collagenase. Importance of the P3' position

An extensive series of synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) was prepared, and the inhibitory constants were determined on the Clostridium histolyticum collagenase. Among the factors which control the optimal binding of these inhibitors, we found that the presence of a free C-terminal carboxylate group in the position P3' of the compounds is of primary importance. In general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of magnitude. We observed also that the enzyme favored the inhibitors having a long linear apolar or basic side-chain at the position P3'. The present data suggest a large S3' subsite of the C. histolyticum collagenase. The compound which contains a homoarginine residue at the P3' position with a Ki of 0.2 microM proved to be the most potent synthetic inhibitor known to date for the C. histolyticum collagenase.     

Serine proteinase activation of latent human skin collagenase

Latent and active collagenase were demonstrated following direct extraction from normal skin homogenates with 0.1M calcium chloride at 60 degrees C. 83% of the collagenase activity was in latent form and could be maximally activated with trypsin. Partial activation of the latent enzyme could also be demonstrated by incubation of the skin extract without added trypsin. This endogenous activation was inhibited by the addition of soya bean trypsin inhibitor, trasylol, di-isopropylphosphofluoridate and phenylmethanesulphonylfluoride, none of which inhibited collagenase directly. This suggests that the skin extracts contain a collagenase activating enzyme with the inhibition profile of a serine proteinase. A chymotryptic proteinase with a similar inhibition profile was extracted from normal human skin and partially purified. This enzyme activated fibroblast procollagenase derived from tissue culture of normal skin. The procollagenase was also partially activated by plasmin and chymotrypsin. This is the first demonstration of a collagenase activating enzyme in human skin and raises the possibility that collagenase activation by this mechanism may be responsible for collagen degradation in some disease processes.     

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

A modified collagenase assay method based on the use of p-dioxane.

Radioactive collagen synthesized by human skin fibroblasts in monolayer culture was used as a substrate for collagenase. The high specific activity of this substrate (75,000 cpm/μg) and the use of p-dioxane as a precipitant of the undigested collagen permit this enzyme to be assayed with collagen in solution at 35°C and pH 7.5. The dilutions used are sufficient to prevent the collagen molecules from aggregating, thus precluding the use of inhibitors of gel formation which tend to decrease the activity of the enzyme. Using a 1-h incubation, the procedure is reproducible (SD ± 2.3%) and linear over the range from 10 to 100 ng of bacterial collagenase. Vertebrate collagenase activity is also easily measured with this method.     

Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE₂)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE₂. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacinblocked cultures, indicating a PGE₂-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE₂ synthesis differed. Dexamethasone inhibited PGE₂ synthesis, which resulted in the suppression of collagenase. However, PGE₂ production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE₂ levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE₂ or phospholipase A₂. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.     

Metalloproteinase inhibitors from bovine cartilage and body fluids

Inhibitors of the mammalian metalloproteinases, collagenase, proteoglycanase and gelatinase were isolated from bovine cartilage (extracts and culture medium) and bovine amniotic fluid and serum. These inhibitors either bind or do not bind to concanavalin-A--Sepharose, with Mr (gel filtration) of about 30 000 and 20 000, respectively. Cartilage and chondrocyte culture media contained only concanavalin-A-binding inhibitors whereas cartilage extracts contained only a non-binding inhibitor: serum and amniotic fluid contained both forms of inhibitory activities. In moist biochemical respects, particularly in their abilities to inhibit metalloproteinases, all of the inhibitors were found to be similar. It is concluded that the forms of the inhibitors that differ in Mr may be closely related to the tissue inhibitor of metalloproteinases (TIMP) previously purified from rabbit and human sources. These findings help to clarify other studies on collagenase inhibitors and support the concept that TIMP-like inhibitors may be important in the control of connective tissue degradation.     

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.