Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion

When added to cultures of parathyroid hormone (PTH)- bones, dichloromethylenebisphosphonate (C12MBP) and 3-amino-1-hydroxypropydilene-1,1-bisphosphonate (AHPrBP) inhibit completely and in a parallel manner the development of resorption lacunae, the loss of calcium by the explants and their PTH-induced excretion of lysosomal hydrolases (β-glucuronidase and N-acetyl-β-glucosaminidase). The loss of collagen (hydroxyproline) by the bones is usually less inhibited than their loss of calcium and their heparin-induced excretion of collagenase is unaffected. To interpret these data, it is proposed that these bisphosphonates act more on the activity of osteoclasts, suppressing simultaneously their excretion of lysosomal enzymes and their erosion of mineralized bone matrix, than on that of other cell types (osteoblasts ?) responsible for collagenase production and the removal of uncalcified collagen.     

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an Mr in the range 15000-25000 and a pI corresponding to approx. pH 4.7. These biological and physiochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.     

A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.     

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an M_r in the range 15 000–25 000 and a pI corresponding to approx. pH 4.7. These biological and physicochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.     

Mammalian collagenase predisposes bone surfaces to osteoclastic resorption

Summary The cell-free endocranial surface of young adult rat parietal bones was used as a substrate for bone cell-derived mammalian collagenase. Incubation of parietal bones in a concentration of enzyme comparable to that secreted by osteoblastic cells in vitro caused destruction of surface osteoid, and resulted in exposure of mineral onto the bone surface. Bones so pre-treated were considerably more susceptible to osteoclastic resorption than bones preincubated in the absence of collagenase. These results are consistent with the view that the osteoid layer which covers bone surfaces acts as a barrier to osteoclastic contact with underlying, resorption — stimulating bone mineral; and that cells of the osteoblastic lineage induce osteoclastic resorption through collagenase secretion which, by digestion of the surface osteoid, exposes bone mineral to osteoclastic contact.     

Induction of collagenase secretion in human fibroblast cultures by growth promoting factors

In human foreskin fibroblast cultures, two proteins with Mr 60,000 and 55,000 were found to be induced about 3.5-fold by epidermal growth factor (EGF), platelet-derived growth factor, and beta-transforming growth factor. The induced proteins were identified as procollagenases by immunoprecipitation of induced medium with antibodies to purified human fibroblast collagenase. Collagenase enzyme activity in the medium from EGF-treated cultures was also induced at least 3-fold compared to control cultures. Induction of collagenase was dependent upon de novo protein and RNA synthesis and was observed in the medium 10 h after addition of EGF. Although these growth-promoting factors interact with separate membrane receptors, each induced the secretion of a common protein, suggesting that collagenase may be important in some aspect of mitogenesis, cell mobilization, and migration.     

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.     

Stimulation of collagenase production by collagen in mammalian cell cultures.

In this study, we investigated the possible regulatory role of collagen in collagenase production by cultured human skin fibroblasts and human and rabbit synovial cells. Addition of types I, II or III collagen in solution to the culture media markedly stimulated trypsin-activable collagenase activity in these cultures. In the human cell cultures the stimulatory effect of collagen was further enhanced by a soluble factor isolated from human monocyte culture media (Dayer, Russell and Krane, 1977). Both native and denatured forms of collagen stimulated enzyme production; their relative efficacy varied among the different types. The native form of both types I and II collagen showed a greater effect on collagenase production than the corresponding denatured form, whereas with type III collagen the denatured form was more effective.     

Direct extraction and assay of bone tissue collagenase and its relation to parathyroid-hormone-induced bone resorption.

A method has been developed for the quantitative extraction of collagenase from as little as one 19-day-fetal-mouse calvarium. About 20-40 munits of collagenase are extracted per mg of tissue, all in a latent form that, after proper activation, shows the typical properties of mammalian collagenase. Culturing the calvaria for 2 days with parathyroid hormone (PTH) increases their procollagenase content up to 3-fold and induces bone resorption. Both PTH effects are prevented by cycloheximide, but not by indomethacin. Calcitonin inhibits resorption without affecting the PTH-induced procollagenase synthesis. The role of this synthesis is discussed in relation to the mechanisms of bone resorption.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin.

1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.     

Isolation and immunochemical localization of collagenase in mouse peritoneal macrophages

Collagenase was isolated from the culture medium of thioglycollate-stimulated mouse peritoneal exudate macrophages. The macrophage collagenase activity was inhibited by goat anti-mouse bone collagenase antibody, indicating that macrophage collagenase immunologically cross-reacts with mouse bone collagenase. The enzyme was localized in mouse peritoneal macrophages by indirect immunofluorescent antibody technique. Distinct granular fluorescence was observed intracellularly in most thioglycollate-stimulated macrophages, whereas slight or no fluorescence was observed in non-stimulated control macrophages.     

Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts

The levels of collagenase and collagenase mRNA produced by early-passage (less than 40% of lifespan completed) and late-passage (greater than 80% of lifespan completed) cultures of human fibroblasts were analyzed. The constitutive levels of collagenase and collagenase mRNA produced by the late-passage cultures were 10-30 x greater than the levels observed in similarly treated early-passage cultures. Immunofluorescence analysis established that the percentage of collagenase-positive cells was also greater (77% vs. 4%) in the late-passage cultures. To determine whether the difference in collagenase production resulted from cell-derived regulatory factors, collagenase production was examined in cultures plated onto substrates coated with fibroblast extracellular matrix (ECM). Collagenase and collagenase mRNA production was enhanced in both types of cultures, although amounts produced by ECM-induced early-passage cultures was significantly less than that produced by similarly treated late-passage cultures. Collagen-coated substrates also induced collagenase synthesis.     

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.     

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.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.     

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.