Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Collagenase activity in the normal rat myocardium

Summary Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart secctions were incubated under optimal conditions for collagenase activity (37°C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is. the result of collagen degradation by intrinsic collagenase of the myocardium.     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge

Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articulage were examined in detail in 7 joints within the age rangee 21 to 72 years. The results of preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented.Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in unixial compression perpendicular to the articular surface.The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres.At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was not significantly reduced by the action of cathepsin D.In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue.Biochemical analysis of the incubation media, and the specimens, reveled that a large proportion of the proteoglycans was released from the cartilage by each of the freeze enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase relesed between 5.3 and 27.8 per cent of the collagen after 24 h.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague–Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.     

Some newly characterized collagenases from procaryotes and lower eucaryotes

Summary Chemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed.The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed.The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed.The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence.The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the a chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.     

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.     

Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3)

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.     

Effects of products from macrophages, blood mononuclear cells and or retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied on cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of 3H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of alpha 1 chains; the amount of alpha 2 chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. alpha 2 chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen, and therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Heterogeneity of protein–polysaccharides of porcine articular cartilage. The chondroitin–sulphate proteins associated with collagen

Pig articular cartilage, from which protein-polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris-HCl buffer after bacterial collagenase digestion, followed by the same urea-sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein-polysaccharide preparation obtained previously. The protein-polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein-polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea-sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein-polysaccharides in all three extracts although somewhat shorter than in protein-polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein-polysaccharides in cartilage.     

The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.     

Analysis of the reducible components of the muscle protein, connectin: absence of lysine-derived cross-links

After exhaustive salt extractions of rabbit and human skeletal muscle, the amino acid compositions of the residual proteins were similar to those reported for connectin. Complete removal of collagen contamination was achieved only after treatment of the connectin preparations with bacterial collagenase. On reduction with KB³H₄, the small amounts of lysine-derived reducible cross-links that were present in the initial connectin preparations were completely absent after treatment with collagenase. In adult human connectin some hexitol-lysine derivatives were present after reduction. These results indicate that, in contrast to previous reports, connectin does not participate in the same lysyl oxidase-mediated cross-linking system that occurs in collagen and elastin.     

Macrophage-fibroblast interactions in collagenase production and cartilage degradation.

Rabbit bone-marrow macrophages and fibroblasts were cultured, independently or together, with pieces of 35S-labelled cartilage or at the surface of dried [14C]collagen gels. Each type of cell, cultivated alone, rapidly degraded the proteoglycan of cartilage, but only the fibroblasts degraded collagen. The co-culture of both types of cell had no consistent effect on the rate of proteoglycan degradation, but it stimulated the rate of collagen degradation. In parallel, the accumulation of collagenase in the culture fluid was enhanced but not that of neutral proteinase. Coinditioned media from macrophage cultures added to cultures of fibroblasts had the same effect as the living macrophages in stimulating the production of collagenase. Their action was itself enhanced when the macrophages had been activated by concanavalin A-stimulated spleen-cell factors. These data suggest that fibroblasts may act as effector cells in producing collagenase and degrading collagen in response to soluble factors released by macrophages under the control of lymphocyte factors.     

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.     

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.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.