Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation

Abstract

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-α and/or interleukin-1 at 48 and 72?h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%–93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.     

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Abstract

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH₂-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1?µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10?µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10?µM SB203580 strongly inhibited the collagen cleavage compared with 1?µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.     

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH₂-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10µM SB203580 strongly inhibited the collagen cleavage compared with 1µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.     

The effects of dexamethasone in vitro on the production of collagenase and inhibitor by synovial and cartilage explants from the joints of rabbits with a proliferative arthritis

Summary Using a rabbit model arthritis we have investigated the ability of dexamethasone to alter the production of collagenase and the specific metallo-proteinase inhibitor TIMP by explants of synovium and cartilage in vitro. The patterns of collagenase and TIMP production by untreated explants from arthritic joint tissues in culture were similar to those described previously [1, 2]. Dexamethasone dramatically altered the patterns of production of collagenase and TIMP. At a dose of 10 nM, or above, the patterns of production by treated synovium resembled those of normal rabbit synovium: collagenase production was suppressed and TIMP increased compared with untreated arthritic synovium. The levels of latent collagenase in cartilage also fell with increasing doses of dexamethasone and TIMP levels were higher, although normal levels were not reached.These experiments have been conducted as a prelude to testing the effects of various anti-rheumatic drugs in vivo, and attempting to correlate changes in clinical parameters with the subsequent production of collagenase and TIMP in vitro. The data are discussed in relation to the therapeutic use of corticosteroids and to their mode of action on joint tissues.     

Autoimmune reaction to type II collagen and cartilage degeneration in MRL/Mp-<Emphasis Type="Italic">lpr/lpr</Emphasis> mouse

The early phase of cartilage degeneration was immunohistochemically examined in order to clarify the importance of autoimmune reaction against type II collagen in MRL/Mp-lpr/lpr (MRL/l) mouse in an experimental model of rheumatoid arthritis (RA). Anti-type II collagen antibodies were detectable in 3-week-old mice and preceded the appearance of rheumatoid factors. Furthermore, mesenchymal cells and tartrate-resistant acid phosphatase-positive cells began to accumulate remarkably in the periphysis, a fibrochondro-osseous area in the bone marrow vicinity. The numbers of these cells increased with mice age, together with serum levels of anti-type II collagen antibodies. Immunostaining of the periphysis revealed expression of type II collagen, IgG, C3, Mac-3, MHC class II antigen Ia, and cathepsin-L. Osteoclast-like cells and macrophage infiltration into the lesion area were confirmed by transmission electron microscopy. The results indicate that cartilage degeneration in MRL/l mouse may originate in the periphysis and progress via a pathway independent of synovial invasion.     

Study of the effect of a glycosaminoglycan-peptide complex on the degradative enzyme activities in human osteoarthritic cartilage

Summary The in vitro collagenolytic and proteoglycanasic activity from human fibrillated osteoarthritic cartilage was determined using labelled proteoglycans and type II collagen as substrates. In vitro, a glycosaminoglycan-peptide complex (GP-C Rumalon^®) induced a dose-dependent inhibition of both collagenolytic and proteoglycanasic activities while sodium salicylate and indomethacin had only a weak suppressive effect on proteoglycanase. Phospholipase A₂ activity was unmodified by GP-C suggesting that the effect of the drug on degradative enzymes was unrelated to prostaglandin formation.     

Antibodies to type II collagen and their association with HLA DR1 alleles in Japanese patients with rheumatoid arthritis

 To investigate whether immunological responses to type II collagen (CII) play an important role in the pathogenesis of rheumatoid arthritis (RA), the presence of anti-CII antibodies was examined by enzyme immunoassay in 130 Japanese patients with RA, 10 systemic lupus erythematosus (SLE) patients, and 30 healthy subjects. In addition, the HLA-DRB1 genes of 40 RA patients were determined, and their association with positive findings of anti-CII antibodies was examined. A significantly high frequency of positive findings of anti-CII antibodies was detected in sera from RA patients (19%, P < 0.05) in comparison with that in sera from healthy subjects (3%). High frequencies of DRB1^*0405 and 0101 alleles were observed in the 40 RA patients examined (40.0% and 30.0%, respectively). Patients with DRB1^*0101 had a significantly higher rate of positive findings of anti-CII antibodies than those without DRB1^*0101 (66.7% and 28.6%, respectively, P < 0.05). No such association was observed for DRB1^*0405. From these findings, we suggest that immunological responses to CII may play an important role in the development of arthritis in some RA patients. Received: April 9, 2002 / Accepted: June 13, 2002 Acknowledgment We thank Dr. T. Sakamaki, Division of Clinical Research, Sakura National Hospital, Japan, for technical assistance and useful advices on HLA typing. Correspondence to:T. Sumida     

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase

OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.     

Wound healing response is a major contributor to the severity of cutaneous leishmaniasis in the ear model of infection

In the conventional mouse model for cutaneous leishmaniasis involving infection with stationary phase Leishmania major promastigotes at the base of the tail, mice congenic for leishmaniasis resistance loci designated lmr1,2,3 cured their lesions more rapidly and laid down more ordered collagen fibres than the susceptible parental BALB/c mice, while the opposite was the case for the congenic mice carrying the susceptibility loci on the resistant C57BL/6 background. In that model, we showed that wound healing and not T cell responses played a major role in determining the resolution of skin infection. Here, we show a similar disease phenotype in the mouse model that mimics more closely the situation in humans, that is, strictly intradermal infection in the ear pinna with small numbers of metacyclic promastigotes. The data show that at the site of infection the innate and adaptive immune responses act in concert to clear parasites, and induce tissue repair and wound healing. Importantly, the data show that the host responses controlled by the lmr loci, which act locally to control infection in the skin, are distinct from the host responses operating systemically in the draining lymph node.     

Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets

One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.     

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.     

Overweight, insulin resistance and type II diabetes in type I Gaucher disease patients in relation to enzyme replacement therapy

Type I Gaucher disease, a lysosomal storage disorder is associated with metabolic abnormalities such as high resting energy expenditure, low circulating adiponectin and peripheral insulin resistance. Treatment with enzyme replacement therapy (enzyme therapy) leads to a decrease in resting energy expenditure, but its influence on weight and risk of development of type II diabetes is unknown.We studied the BMI, prevalence of overweight, insulin resistance and type II diabetes in untreated and enzyme therapy treated Gaucher patients before and after several years of follow-up and compared this to data on healthy subjects from literature.We established that in untreated Gaucher patients the prevalence of overweight is lower than in the general population. Long-term treatment with enzyme therapy induces a larger than average weight gain leading to a similar prevalence of overweight in enzyme therapy treated patients and the general population. The prevalence of type II diabetes increases significantly during treatment with enzyme therapy, resulting in a comparable prevalence of type II diabetes in enzyme therapy treated patients and the general population.     

Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13

OBJECTIVE: To determine the sites of cleavage and denaturation of type II collagen (CII) by collagenase(s) in healthy and osteoarthritic (OA) human articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 (MMP-1) and MMP-13. METHODS: Single (per subject) full-depth specimens from femoral condylar cartilage were isolated from articulating surfaces at autopsy from 8 subjects without arthritis and during arthroplasty from 10 patients with OA. Fixed frozen sections of cartilage were examined by immunoperoxidase localization, using antibodies to the collagenase-generated cleavage site in CII, to an intrachain epitope recognized only in denatured CII, and to MMP-1 and MMP-13 (proenzyme, activated enzyme, or enzyme/inhibitor complex). RESULTS: Staining for collagen cleavage, denaturation, and both MMPs was weak to moderate and was frequently observed in pericellular sites in cartilage from younger, nonarthritic subjects. In specimens from older subjects, this staining was often more widespread and of greater intensity. Similar staining was usually, but not always, seen for all antibodies. In OA cartilage, staining was often stronger and more intense than that in normal cartilage from older subjects, and the distribution of staining was often similar for the different antibodies. Pericellular staining in the deep zone was frequently more pronounced in arthritic cartilage and extended to territorial and sometimes interterritorial sites. In very degenerate specimens, staining was distributed throughout most of the cartilage matrix. CONCLUSION: These observations provide evidence for the presence of limited cleavage and denaturation of CII restricted to mainly pericellular and superficial sites in cartilage from younger, healthy subjects, where MMP-1 and MMP-13 are also selectively localized. Collagen degradation is more extensive and often more pronounced in cartilage from older, nonarthritic subjects. Characteristic changes in early OA are similar to those seen with aging in cartilage from older, healthy subjects, with collagen damage and collagenases concentrated closer to the articular surface. There was usually a close correspondence between the cleavage and denaturation of CII and the sites at which these collagenases were detected, suggesting that both MMPs are involved in the physiology and pathology. There was no evidence that the damage to CII is ordinarily initiated in sites other than at and near the articular surface and around chondrocytes.     

Expression of type I and type III collagens during the course of dimethylnitrosamine-induced hepatic fibrosis in rats

ABSTRACT— Aims/Background: We wished to clarify the mechanisms that account for the increase in hepatic collagen accumulation during hepatic fibrosis. Methods: The gene expression of type I and type III procollagens and matrix metalloproteinase-1 (MMP-1) was measured by Northern blot analysis; immunolocalization of both types of collagen was estimated by indirect immunohistochemical assay; and the hepatic content of collagen and malondialdehyde (MDA), a product of lipid peroxidation, were assayed in hepatic fibrosis induced in rats with a single dose of dimethylnitrosamine (DMN). Results: During the experimental period, more type I procollagen mRNA was found than type III procollagen mRNA. The immunoreactive intensity of type I collagen was greater in necrotic areas near central veins 3 days after DMN treatment than it was on day 9, whereas the type III collagen immunodeposition for the latter period of the hepatic fibrosis was stronger than it was on day 3. As compared with controls, hepatic collagen content increased significantly after 3 days and continued, increasing gradually, as did type I and III procollagen mRNA levels. On day 14, fibrosis was greatest and both types of procollagen gene expression were at their highest, and type I and III procollagen mRNA levels and hepatic collagen content increased as the dosage of DMN was raised. MMP-1 mRNA levels increased early in hepatic fibrogenesis, and increased on day 14 when DMN dosages were low. Hepatic MDA levels increased rapidly for 3 days after DMN treatment, remaining significantly higher than control values and showing a significant increase even in response to low DMN doses on day 14. Conclusions: Our results suggested that fibrotic liver collagen content may make its first notable increase due in part to the balance between type I collagen and MMP-1 expression rates. Also, lipid peroxidation may be important in the mechanism of hepatofibrogenesis.     

Toxicity of complement for chondrocytes. A possible source of cartilage degradation in inflammatory arthritis

Summary Cartilage from patients with rheumatoid arthritis and from animals with antigen-induced arthritis is frequently contaminated with complement-containing immune complexes. A possible role for complement activation in cartilage degradation was modeled in vitro by exposing cultured bovine chondrocytes to homologous serum, and determining cytotoxicity by monitoring the release of intracellular ⁵¹Cr. Complement activation was found to be cytotoxic, having maximal effect at 20–30% serum by 18 h. Serum toxicity was ablated by heat (50°C, 20 min) or methylamine treatment but not by EGTA, suggesting that in these experiments activation occurred by the alternate route. The implications of the results are discussed in relation to ultrastructural evidence for the involvement of complement in the pathogenesis of cartilage degradation in inflammatory arthritis.     

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of ...     

Frequency of type I and II diabetes in newly diagnosed diabetic patients: Measuring C-Peptide level

AimsDiabetes mellitus is a metabolic disease that manifested as hyperglycemia due to the defect in secretion or function of insulin. This study aimed was to survey about frequency type I and II diabetes in newly diagnosed diabetic patients base on c-peptide and anti-glutamate acid decarboxylase (GAD) tests.Materials & methodsThis study was conducted as a prospective study on 70 diabetic patients aged 15–45 years old who referred to diabetes clinics in Ahvaz city during 2012–2014 and their diabetes was diagnosed for the first time, but their type of diabetes was not clinically definitive. Patients with anti-GAD positive and fasting C-peptide level of less than 0.65 were diagnosed as type I diabetes. Patients with anti-GAD negative fasting C-peptide level of greater than or equal to 0.65 were considered as type II diabetes.ResultsEighty two patients (49 males and 33 females) with a mean age of 21.64 ± 4.36 years (range 15–34) and a mean BMI of 22.05 ± 4.41 kg/m² (range 14–18) were studied. Twenty three patients (28.5%) had type I diabetes and 59 patients (71.95%) had type II diabetes. In patients with type I diabetes, the mean BMI was 24.86 ± 2.36 kg/m² and the number of patients with family history (56.22%) was higher. In type II diabetic patients, the number of women (62.71%) was higher than that of men.ConclusionAnti-GAD test can be used as a predictive test for early diagnosis of disease and screening of people with a diagnosis of diabetes based on the type of diabetes.     

儿童与成人大骨节病关节软骨X型胶原表型和bFGF的表达

目的探讨儿童与成人大骨节病关节软骨中X型胶原表型和bFGF表达的分布特点。方法用单克隆和多克隆免疫组化法检测大骨节病与对照组关节软骨X型胶原表型和bFGF表达。结果(1)儿童大骨节病关节软骨深层X型胶原表型表达率(35.0%)比儿童对照组(63.0%)明显减少(P<0.01);(2)儿童大骨节病关节软骨中层X型胶原表型表达(0.0%)较成人大骨节病组(34.0%)减弱(P<0.01);(3)儿童大骨节病软骨表层bFGF表达率(6.0%)低于对照组(21.0%)(P<0.01),而中、深层表达(分别为12.0%、37.0%)与对照组(无表达)相比明显增多(P<0.01)。成人大骨节病关节软骨bFGF表达阳性率(分别为47.0%、37.0%、11.0%)与对照组(分别为19.0%、15.0%、1.0%)比较明显增多;(4)成人大骨节病关节软骨bFGF阳性表达在表、中层表达明显高于儿童组(P<0.01),深层儿童大骨节病组高于成人组(P<0.01)。结论X型胶原表型和bFGF表达在儿童与成人大骨节病关节软骨的分布不同。