John J. Jeffrey

Initiation of Osteoclast Bone Resorption by Interstitial Collagenase

Osteoclasts form an acidic compartment at their attachment site in which bone demineralization and matrix degradation occur. Although both the cysteine proteinases and neutral collagenases participate in bone resorption, their roles have remained unclear. Here we show that interstitial collagenase has an essential role in initiating bone resorption, distinct from that of the cysteine proteinases. Treatment of osteoclasts with cysteine proteinase inhibitors did not affect the number of resorption lacunae (“pits”) formed on the surface of dentine slices, but it generated abnormal pits that were demineralized but filled with undegraded matrix. Treatment with metalloproteinase inhibitors did not alter the qualitative features of lacunae, but it greatly reduced the number of pits and surface area resorbed. Treatment of bone cells with an inhibitory anti-rat interstitial collagenase antiserum reduced bone resorption markedly. In the presence of collagenase inhibitors, resorption was restored by pretreatment of dentine slices with rat interstitial collagenase or by precoating the dentine slices with collagenase-derived gelatin peptides or heat-gelatinized collagen. Immunostaining revealed that interstitial collagenase is produced at high levels by stromal cells and osteoblasts adjacent to osteoclasts. These results indicate that interstitial collagenase can function as a “coupling factor,” allowing osteoblasts to initiate bone resorption by generating collagen fragments that activate osteoclasts.

Collagenase production by human skin fibroblasts.

Abstract Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.

Human Skin Fibroblast Collagenase: Interaction with Substrate and Inhibitor

Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.

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

Abstract 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.

Studies on uterine collagenase in tissue culture: II. Effect of steroid hormones on enzyme production

Abstract Cultures of post-partum rat uterus normally synthesize and release into the culture medium a specific neutral collagenase. When progesterone is added to the medium of these cultures at a concentration of 5 · 10 −5 M, collagenase activity is completely abolished. This concentration of steroid corresponds to a tissue concentration of 1 · 10 −8 M. Progesterone itself has no direct inhibitory effect on collagenase activity. The potent progesterone analogue 6α-methyl, 17α-acetoxy progesterone completely inhibits the appearance of collagenase activity at 1 · 10 −6 M in culture. Neither estradiol nor testosterone diminishes collagenase activity in vitro . The appearance of hydroxyproline-containing peptides in the medium of the progesterone cultures is diminished by 85% relative to controls, indicating that collagen catabolism in the tissue is prevented at the same time that collagenase activity is undetectable. The ability of progesterone to suppress collagenase activity is stimulated by the presence of estradiol in the culture medium, suggesting the possibility that a specific progesterone receptor mediates this effect of progesterone in the uterus.

An extractable collagenase from crustacean hepatopancreas

Abstract An enzyme capable of degrading native collagen under physiologic conditions has been extracted from the hepatopancreas of a crustacean, Uca pugilator . The collagenase acts on native collagen fibrils and on collagen in solution. At 25° the enzyme is capable of producing a marked reduction in the specific viscosity of collagen with no loss of optical rotation, indicating the ability of this enzyme to cleave the native collagen helix without producing denaturation. Collagenolytic activity, as investigated by disc electrophoresis, results in the production of numerous new components below the original α band. Three fragments, 75% (TC A 75 ), 70% (TC A 70 ) and 67% (TC A 67 ) the molecular length from the “A” or N-terminal end of the molecule, have been identified in electron micrographs of segment-long-spacing crystallites prepared from enzyme-collagen reaction mixtures. Crude collagenase preparations from this digestive organ also contain significant chymotrypsin- and trypsin-like activities. The cleavage by hepatopancreas extracts of peptide bonds in the nonhelical, N-terminal region of collagen, at or near the intramolecular cross-link, may be due to the action of chymotrypsin on collagen. Chymotrypsin, however, is unable to degrade α chains further at 25°. Trypsin has little effect on the viscosity of collagen in solution at concentrations equivalent to those present in hepatopancreas extracts. At these concentrations trypsin is unable to reduce the content of cross-linked components in collagen or catalyze cleavages in the native helix. In addition, the ability of hepatopancreas extracts, but not chymotrypsin or trypsin, to degrade collagen in fibrillar form further indicates the presence of a true collagenase in crustacean hepatopancreas.

Immunologic relationship of purified human skin collagenase to other human and animal collagenases

Abstract Antibodies prepared to a number of animal and human collagenases have been used to investigate the relationships between these enzymes. The human skin collagenase used as an immunogen was one of two chromatographically separable peaks of collagenolytic activity obtained from culture media of normal human skin. On the basis of their electrophoretic mobility on polyacrylamide gel, they have been designated as fast-moving or slow-moving human skin collagenase. The fast moving enzyme was further purified and used to produce an anti-human skin collagenase antibody. Anti-fast-moving human skin collagenase antibody gives a reaction of complete identity on immunodiffusion with slow-moving skin collagenase as well as with preparations of human gingival and rheumatoid synovial collagenases. In addition, anti-fast-moving human skin collagenase antibody produces complete inhibition of these other human collagenases, suggesting that these enzymes are closely related proteins. Antibodies to tadpole and rat uterine collagenases are immunologically not identical when compared with one another or to the human skin enzyme. Crustacean hepatopancreas and bacterial collagenases also fail to react with anti-fast-moving human skin collagenase antibody, further indicating species specificity among collagenases.

Purification and properties of rat uterine procollagenase

A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5-10 nM) of dextran sulfate. Resultant eluates contain 8-10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of Mr approximately 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of Mr approximately 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ion-exchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 degrees C is approximately 3000 micrograms collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.

The function of Ca2+ in the action of mammalian collagenases

Abstract The removal of extrinsic Ca2+ from human skin, rat skin, and postpartum rat uterus collagenases results in a reversible loss of enzymatic activity, which becomes irreversible with increasing length of Ca2+-free incubation at physiological temperature and pH. Ca2+ is necessary for thermostabilization both in the presence and absence of the collagen substrate. Enzymes from all three sources display linear rates of reaction at Ca2+ concentrations from 0.5 to 20 m m and are half-maximally activated at 0.5 m m Ca2+. The increase in collagenase activity with increasing Ca2+ concentration is associated with an increase in thermostabilization. Ba2+ and Sr2+ are effective substitutes for Ca2+ in human skin collagenase but not in the collagenases from rat tissues. These studies also indicate that Ca2+ plays no role in the binding of collagenases to their substrate.

Evidence for mammalian collagenases as zinc ion metalloenzymes

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.