Collagenolytic Enzymes Research Articles

Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics.

Over the past decade, advances in molecular tools, stem cell differentiation, osteoclast and osteoblast signaling mechanisms, and genetically manipulated mice models have resulted in major breakthroughs in understanding osteoclast biology. This review focuses on key advances in our understanding of molecular mechanisms underlying the formation, function, and survival of osteoclasts. These include key signals mediating osteoclast differentiation, including PU.1, RANK, CSF-1/c-fms, and src, and key specializations of the osteoclast including HCl secretion driven by H+-ATPase and the secretion of collagenolytic enzymes including cathepsin K and matrix metalloproteinases (MMPs). These pathways and highly expressed proteins provide targets for specific therapies to modify bone degradation. The main outstanding issues, basic and translational, will be considered in relation to the osteoclast as a target for antiresorptive therapies.

Arfaptin 1 inhibits ADP-ribosylation factor-dependent matrix metalloproteinase-9 secretion induced by phorbol ester in HT 1080 fibrosarcoma cells

Matrix metalloproteinase-9 (MMP-9) is a collagenolytic enzyme secreted by cancer cells and involved in invasiveness and metastasis. Its secretion from human fibrosarcoma HT 1080 cells is markedly enhanced by phorbol 12-myristate 13-acetate (PMA) and abolished by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation. These results support a role for ARF in PMA-stimulated MMP-9 secretion. Overexpression of arfaptin 1, a 39 kDa ARF-binding protein that inhibits in vitro activation of cholera toxin ADP-ribosyltransferase and phospholipase D (PLD) by ARF, inhibited PMA-stimulated MMP-9 and PLD activation. These data are in agreement with previous results demonstrating a significant role for PLD in regulating MMP-9 secretion.

Characterisation of Collagenolytic Enzymes Produced by Thermophylic Actinomycetes

ABSTRACTThe aim of the present investigation was to characterize enzymes, produced by extremophilic microorganisms in soil samples, collected from different regions in Bulgaria. The cultivation conditions for obtaining high yield of collagenolytic activity were optimized for two active strains—A 35 and E 15. The molecular mass of the active fractions in the cultural liquid of both strains was determined by means of gel chromatography on Sephadex G-100 and were in the range from 40 to 140 kDa. The proteolytic enzymes were stable against high ethanol concentrations, which allows an effective elimination of different byproducts (pigments, nutrient medium components and metabolites). The electrophoretic patterns show a certain isomorphism of the fractions. The comparison with clostridiopeptidase A, as well as the inhibitory analysis show that these enzymes might be considered as “collagenase-like” enzymes. Preliminary experiments show that they could be applied for pre-treatment of collagen-rich raw materials, e.g. in the pelt industry.

Perspective of cytokine regulation for periodontal treatment: fibroblast biology.

Efforts to understand the pathogenesis of periodontal diseases have been underway for decades. Studies of immunological aspects in addition to the structural components of gingival fibroblasts showed that the fibroblasts actively participate in immune and inflammatory events in periodontal diseases. Future strategies for the prevention and treatment of periodontal diseases should biologically regulate fibroblast activities. These cells are surrounded by monocyte-derived proinflammatory cytokines such as interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and lymphocyte-derived interleukin-6 (IL-6) in inflamed gingival tissue. Recent anti-cytokine therapy for inflammatory diseases including rheumatoid arthritis aimed to inhibit the binding of cytokines to targeted cells such as fibroblasts and condrocytes. IL-1beta and TNF-alpha are thought to be therapeutic targets because these cytokines are essential for the initiation of inflammatory immune reactions and are produced for prolonged periods in inflammatory diseases. IL-6 is also a target, because it is abundantly present in inflammatory lesions and activates fibroblasts in the presence of soluble IL-6 receptor. In addition, these cytokines accelerate gingival fibroblasts to produce collagenolytic enzymes, resulting in tissue destruction. Soluble receptors for IL-1beta and TNF-alpha are suggested to be candidates for therapeutic molecules, but soluble receptor for IL-6 is suggested to be a factor-stimulating fibroblast. This paper will review the utilization of soluble receptors specific to inflammatory cytokines which potentially stimulate fibroblasts to regulate biological events involved in the pathogenesis of periodontal diseases.

Characteristics of collagenolytic enzymes secreted by deuteromycete fungi Aspergillus flavus.

Collagen induced the synthesis of collagenolytic enzymes in the culture of Aspergillus flavus. Enzyme activity of the culture increased during storage and passage on a medium with this inductor. We developed a method for isolation and purification of collagenolytic enzymes and obtained two electrophoretically homogenous enzyme preparations belonging to neutral thermolabile collagenolytic metalloproteinases.

Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin.

Punch biopsies of human skin were obtained 1 day after irradiation with two minimal-erythema doses (MED) from either a UVB light source or a Solar Simulator and incubated in organ culture for 72 h. Organ culture fluids obtained at 24, 48 and 72 h were analyzed for collagenolytic activity and for reactivity with antibodies to matrix metalloproteinase-1 (MMP-1; interstitial collagenase) and MMP-13 (collagenase-3). High levels of collagenolytic activity were seen in organ culture fluid from skin exposed to either light source. MMP-1 was strongly induced in parallel, increasing from less than 100 ng/ml in organ culture fluid from control skin to approximately 1.1 microg/ml in culture fluid from UV-treated skin. Whereas most of the detectable MMP-1 in control culture fluid was represented by the latent form of the enzyme, approximately 50% of the enzyme was present as the active form in organ culture fluid of UV-exposed skin. In contrast, there was no detectable MMP-13 in control organ culture fluid and very little change after UV exposure (less than 100 ng/ml in both cases). Finally, neutralization studies with a blocking antibody to MMP-1 removed 95 +/- 4% of the collagenolytic activity in the organ culture fluid from UV-treated skin. These findings strongly implicate MMP-1 rather than MMP-13 as the major collagenolytic enzyme responsible for collagen damage in photoaging.

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on a DEAE-Sephadex A-50 and gel filtration chromatography on a Sephadex G-75 column. The purification and yield were 30.5-fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg 2+ and Zn 2+ ) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K m and V max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.

Textural Changes During Iced Storage of Salmon (Salmo salar) and Cod (Gadus morhua)

Textural properties, protein solubility, water holding capacity and activity of collagenolytic enzymes of salmon (Salmo salar) and cod (Gadus morhua) fillets were measured during iced storage. Breaking strength and hardness of fillets were reduced during iced storage. Elasticity of cod fillets was reduced, while cohesiveness of salmon increased during storage. Salmon was softer and less elastic than cod. During storage, total amount of extracted proteins was reduced for cod, and increased followed by a reduction for salmon. The fraction of salt soluble proteins increased during storage, and values for cod were higher than those for salmon. The activity of collagenolytic enzymes was higher in cod than salmon during the whole storage period.

Quantifying histological changes in archaeological bones using BSE–SEM image analysis

Recent years have seen rapid developments in the understanding of diagenetic changes to archaeological bones. In particular, the degradation or preservation of proteins and other biomolecules has been explored using an increasingly sophisticated battery of analytical techniques. Problems remain, however, in correlating these parameters with physical changes to bone that may be observed microscopically. This is due, in part, to the problems in reproducibly quantifying histological changes to archaeological bone. This paper introduces a novel method for the accurate quantification of these changes employing image analysis of SEM images. Self–consistency of results was tested using measurements of total calcium content at different magnifications. The term ‘bioerosion index’ is suggested for the measured parameter.

Purification and characterization of a collagenolytic protease from the filefish, Novoden modestrus.

A serine collagenolytic protease was purified from the internal organs of filefish, Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G- 150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and 55 degrees C. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

Expression of Matrix-Degrading Cysteine Proteinase Cathepsin K in Cholesteatoma

Cholesteatoma is a nonneoplastic lesion of the middle ear space or mastoid that is histologically characterized by a progressive bone erosion of the ossicles and surrounding bone. Several matrix-degrading enzymes have been implicated as mediators of this bone erosion. Because the novel cysteine proteinase cathepsin K has been shown to play a central role in bone resorption, we examined the expression of this enzyme in tissue specimens of cholesteatoma. Tissue specimens of 9 patients with cholesteatoma were obtained during middle-ear surgery. Expression of cathepsin K mRNA was determined by RT-PCR using specific primers. Immunohistochemical analysis of cathepsin K protein expression in tissue sections was performed by using the streptavidin-alkaline phosphatase technique. Expression of both cathepsin K mRNA and protein was detected in areas affected by cholesteatoma, whereas specimens of nonaffected ear cartilage and surrounding tissue were not positive. In addition, cathepsin K was detected in numerous multinucleated giant cells, particularly osteoclasts at the site of bone degradation. In contrast, keratinized squamous epithelium was negative for cathepsin K. These data demonstrate that the matrix-degrading cysteine proteinase cathepsin K may be involved in bone erosion in cholesteatoma. Strong expression of this collagenolytic enzyme in osteoclasts suggests that these cells are mainly involved in cathepsin K-mediated bone destruction.

Preeclampsia-associated decrease of potential collagenolytic and gelatinolytic activities in the wall of the umbilical cord vein

Preeclampsia is the most common pathological syndrome associated with pregnancy. It is accompanied by remodelling of the extracellular matrix of the umbilical cord. A decrease of collagen content in the umbilical cord vein was described. This decrease may result from reduced collagen biosynthesis or enhanced collagen degradation. It was decided to evaluate whether or not this phenomenon is associated with alterations in the activities of collagenolytic, gelatinolytic and non-specific proteolytic enzymes that may be involved in collagen degradation, as well as the activity of prolidase which provides proline as a substrate for collagen biosynthesis. Studies were performed on the umbilical cord veins of newborns delivered by healthy mothers and those with preeclampsia. The control vein extract, activated with trypsin, degraded reconstituted collagen fibres (64.4±2.9 nmol Hyp×mg −1 protein), whereas the preeclamptic material demonstrated only a trace activity. The venous wall extract contained a latent form of gelatinase that might have been activated by trypsin and 4-aminophenylmercuric acetate. A decrease in the gelatinolytic and proteolytic activities of preeclamptic vein extract at neutral pH was found. Prolidase activity was almost 3-fold lower in the preeclamptic extract (240.6±29.3 nmol Pro×min −1×mg −1 protein) in comparison to the control (608.2±63.7 nmol Pro×min −1×mg −1protein). It was concluded that the umbilical cord vein contains a latent form of gelatinase A. The decrease in prolidase activity may reduce collagen biosynthesis, resulting in a decrease of this protein in the preeclamptic umbilical cord vein.

Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

Vitamins C and E: Missing links in preventing preterm premature rupture of membranes?

We propose that generation of reactive oxygen species may be a potentially reversible pathophysiologic pathway leading to preterm premature rupture of the membranes. Reactive oxygen species generated by the body’s response to diverse insults such as infection, cigarette smoking, bleeding, or cocaine use can activate collagenolytic enzymes and impair fetal membrane integrity. Vitamin E, a lipid-soluble antioxidant, inhibits membrane-damaging effects of reactive oxygen species–induced lipid peroxidation. Vitamin C, a water-soluble antioxidant in plasma, stimulates and protects collagen synthesis while recycling vitamin E. Prior evidence shows that (1) damage by reactive oxygen species can impair fetal membrane integrity, (2) reduced midgestation levels of vitamin C are associated with preterm premature rupture of membranes, and (3) these vitamins can be safely and effectively absorbed and delivered to gestational tissues. Current prenatal vitamin preparations contain vitamins C and E in concentrations that are less than 1/3 and 1/10, respectively; these levels have been suggested for effective antioxidant protection. We hypothesize that increased dietary consumption or supplementation of vitamins C and E during pregnancy may reduce physiologically the risks of that portion of preterm premature rupture of membranes that is mediated by excessive or undamped peroxidation of fetal membranes. This hypothesis, if confirmed, should stimulate initiation of therapeutic trials to test the efficacy of enhanced supplementation with vitamins C and E during pregnancy to prevent preterm premature rupture of membranes. (Am J Obstet Gynecol 2001;185:5-10.)

Inhibition of Type I Procollagen Synthesis by Damaged Collagen in Photoaged Skin and by Collagenase-Degraded Collagen in Vitro

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Control of 92 kDa Collagenase Secretion in Mammalian Cells by Modulation of AP-1 Activity: an Experimentally based Theoretical Study

Collagenolytic enzymes control cell migration through connective tissues. They appear to be of crucial importance for angiogenesis, tumor metastasis or wound repair. A well-documented stimulation pathway of collagenase secretion, either by natural (cytokines) or synthetic (phorbol esters) molecules, acts through activation of the proto-oncogen activating protein 1 (AP-1). Interestingly, this nuclear factor enhances its own synthesis. It also modulates the activity of different genes, including the one coding for 92 kDa gelatinase. We developed a mathematical model to describe this pathway. It led us to conjecture the existence of an hysteresis cycle for PMA-stimulated collagenase secretion, which was experimentally demonstrated later in MDBK cells in culture. We also modified our model to simulate the behavior of tumoral cells expressing AP-1. In this case, the system becomes highly unstable and, once stimulated, cannot be brought back to rest. This approach paved the way for the understanding and the control of mammalian cell processes, connective tissue maintenance or metastasis dissemination.

Effects of fukinolic acid and cimicifugic acids from Cimicifuga species on collagenolytic activity.

The inhibitory collagenolytic activity (47-64% inhibition in 0.22-0.24 microM) of fukinolic acid and cimicifugic acids A, B, and C, which are esters of fukiic acid (3',4'-dihydroxybenzyl tartaric acid) was more potent than that (20-37% inhibition in 0.23-0.24 microM) of cimicifugic acids D, E, F, which are esters of pscidic acid (4'-hydroxybenzyl tartaric acid). Since fukiic acid showed weaker inhibition, and caffeic acid, ferulic acid, isoferulic acid, and p-coumaric acid showed far weaker activities, the entire structures of fukinolic acid and cimicifugic acids A, B, and C proved to be responsible for the inhibitory activities. Trypsin and pronase E hydrolyzed collagen nonselectively alone or in addition to collagenase. These collagenolytic activities were also inhibited by fukinolic acid. These results show that fukinolic acid may inhibit either the collagenolytic activities specific to collagenase or nonspecific to other emzymes. The present studies suggest the potential effect of fukinolic acid and cimicifugic acids of Cimicifuga rhizomes in preventing collagen degradation by collagenases or collagenolytic enzymes under pathological conditions, wound healing, or inflammation.

Identification of the 183RWTNNFREY191Region as a Critical Segment of Matrix Metalloproteinase 1 for the Expression of Collagenolytic Activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.

Interleukin 8 expression in human myometrium: changes in relation to labor onset and with gestational age.

Preterm labor remains the major cause of perinatal mortality and morbidity in normally formed babies. The mechanisms involved in the onset of preterm labor are poorly understood, mainly because the mechanisms initiating term labor remain ill-defined. Human myometrial samples were collected at cesarean delivery from preterm (26-36 weeks gestation) and term (37-41 weeks) women. Women at term were either non-laboring or laboring. The expressions of interleukin-8 (IL-8) mRNA and protein were measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of both IL-8 mRNA and protein significantly increased in the term laboring group, compared with either the term non-laboring or preterm groups. Levels of IL-8 expression did not alter with advancing gestational age. The increased expression of IL-8 in laboring myometria at term supports the hypothesis that up-regulation of IL-8 has a role in the initiation of labor in association with an influx of neutrophils and the release of their collagenolytic enzymes into uterine tissues.

Protease expression in human aneurysmal and occlusive atherosclerotic aortas

Abstract Background Atherosclerosis is implicated in the pathogenesis of abdominal aortic aneurysm (AAA), yet often causes aortic occlusive disease (AOD). Proteolysis of elastin and collagen, the major structural proteins of the aortic wall, must occur for an aneurysm to dilate. The matrix metalloproteinases (MMPs) are a class of enzymes that degrade elastin and collagen in the extracellular matrix and are believed to play a central role in the pathogenesis of AAA. However, some MMPs have also been found in atherosclerotic occlusive disease, in the absence of dilating disease. Differences in the expression of MMPs may determine whether an atherosclerotic aorta dilates or occludes. The expression of MMPs and their natural inhibitors (TIMPs) in human abdominal aortic aneurysm and stenosing atherosclerotic aortas was measured. Methods Samples of aortic wall were obtained from eight patients with AAA and eight with AOD. Expression of the MMPs was estimated with the reverse transcription polymerase chain reaction using a synthetic multicompetitor standard as a robust internal control. The standard allowed assay of 14 MMPs and three TIMPs. Results AAA wall expressed significantly more of the collagenolytic enzyme stromelysin 1 (MMP-3) and TIMP-3 than AOD wall (stromelysin 1: mean(s.d.) log (MMP-3/GAPDH) −1·97(0·52) in AAA versus −3·36(0·77) in AOD, P < 0·01; TIMP-3: −1·79(0·69) versus −2·84(0·78), P < 0·01). The elastolytic enzymes matrilysin, macrophage metalloelastase (MMP-7 and MMP-12), gelatinases 2 and 9, and membrane-type metalloproteinase 1 were expressed in both AAA and AOD. Conclusion There is expression of most elastolytic and collagenolytic MMPs in both aneurysms and occlusive disease, but expression of stromelysin 1 is greatly increased in aneurysms. Stromelysin 1 may be responsible for collagen breakdown in aneurysm disease and provide a target for pharmacological intervention.