Major Component Of Cartilage Matrix Research Articles

FSTL1 Promotes Inflammatory Reaction and Cartilage Catabolism through Interplay with NFκB Signaling Pathways in an In Vitro ONFH Model.

Osteonecrosis of the femoral head (ONFH) usually occurs in young people and is closely associated with autoimmune reactions. Follistatin-like 1 (FSTL1) was recently proven to participate in several inflammation-related diseases. The role of FSTL1 in ONFH is still unclear. Serum levels of FSTL1 were not significantly different in ONFH patients and healthy individuals. In contrast, elevated expression levels of FSTL1 were observed in degraded cartilage and synovial fluid in ONFH patients and in a cultured human primary chondrocyte model treated with interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Suppression of FSTL1 by FSTL1-siRNA downregulated the inflammatory response mediated by IL-1β or TNF-α in cultured human chondrocytes. In a human cartilage culture model, FSTL1 promoted the production of inflammatory cytokines and cartilage degradation enzymes. The activation of NFκB signaling pathway was detected in degenerated cartilage from ONFH patients and in FSTL1-treated chondrocytes. Additionally, administration of an NFκB inhibitor (JSH-23) significantly reduced the overexpression of inflammatory cytokines and protein degradation enzymes induced by FSTL1 and maintained the level of major cartilage matrix components (aggrecan and collagen II). In summary, FSTL1 was involved in the degeneration progression of the ONFH and might provide a novel direction for treating and curing ONFH.

Effects of FGF-2 and FGF receptor antagonists on MMP enzymes, aggrecan, and type II collagen in primary human OA chondrocytes

Objectives: Fibroblast growth factor (FGF)-2 is a member of the FGF family and is found in the synovial fluid of patients with osteoarthritis (OA). The aim of this study was to investigate the effects of FGF-2 on human OA cartilage/chondrocytes by examining the association between FGF-2 and the cartilage degrading enzymes matrix metalloproteinase (MMP)-1 and MMP-13 and the major cartilage matrix components aggrecan and collagen II.Method: Cartilage samples were obtained from 97 OA patients undergoing total knee replacement surgery. Cartilage tissue cultures were conducted and levels of FGF-2, MMP-1, and MMP-13 released into the culture medium were measured by immunoassay. The effects of FGF-2 on the expression of MMP-1, MMP-13, aggrecan, and collagen II were further investigated in cultures of primary human OA chondrocytes.Results: FGF-2, MMP-1, and MMP-13 were released into the culture medium from cartilage samples obtained from patients with OA. FGF-2 concentrations correlated positively with the concentrations of MMP-1 (r = 0.414, p < 0.001) and MMP-13 (r = 0.362, p < 0.001). FGF-2 also up-regulated the production of MMP-1 and MMP-13, and down-regulated the expression of aggrecan and collagen II, in human OA chondrocyte cultures. Furthermore, FGF receptor antagonists AZD4547 and NVP-BGJ398 down-regulated the expression of MMP-1 and MMP-13 and up-regulated aggrecan and collagen II both in the absence and in the presence of exogenous FGF-2.Conclusions: Our results suggest that, in contrast to its growth factor-like effects in some other tissues, FGF-2 induces catabolic effects in human OA cartilage. Moreover, FGF receptor antagonists showed promising beneficial effects on the balance of catabolic and anabolic factors within OA cartilage.

Perinatal Exposure to Vitamin A Differentially Regulates Chondrocyte Growth and the Expression of Aggrecan and Matrix Metalloprotein Genes in the Femur of Neonatal Rats1–3

Vitamin A (VA) and its active form, retinoic acid (RA), are regulators of skeletal development. In the present study, we investigated if maternal VA intake during pregnancy and lactation, as well as direct oral supplementation of neonates with VA + RA (VARA) in early life, alters neonatal bone formation and chondrocyte gene expression. Offspring of dams fed 3 levels of VA (marginal, adequate, and supplemented) for 10 wk were studied at birth (P0) and postnatal day 7 (P7). One-half of the newborns received an oral supplement of VARA on P1, P4, and P7. Tissues were collected on P0 and 6 h after the last dose on P7. Pup plasma and liver retinol concentrations were increased by both maternal VA intake and VARA (P < 0.01). Although maternal VA did not affect bone mineralization as assessed by von Kossa staining, newborn femur length was increased with maternal VA (P < 0.05). VARA supplementation of neonates increased the length of the hypertrophic zone only in VA-marginal pups, close to that in neonates from VA-adequate dams, suggesting VARA caused a catching up of growth that was limited by low maternal VA intake. Maternal diet did not alter type X nor type II collagen mRNA. However, VARA-treated pups from VA-supplemented dams had reduced mRNA for aggrecan, a major component of cartilage matrix, and increased mRNA for matrix metalloproteinase (MMP)13, which catalyzes the degradation of aggrecan and collagens. These results suggest that moderately high maternal VA intake combined with neonatal VARA supplementation can reduce the ratio of aggrecan:MMP, which may unfavorably alter early bone development.

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

IntroductionDeletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).MethodsThe effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.ResultsrhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.ConclusionOur findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.

Agrin is highly expressed by chondrocytes and is required for normal growth

Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction. Consistent with this role, mice deficient of agrin die at birth due to respiratory failure. Here we examined the early postnatal development of agrin-deficient mice in which perinatal death was prevented by transgenic expression of neural agrin in motor neurons. Such transgenic, agrin-deficient mice were born at Mendelian ratio but exhibited severe postnatal growth retardation. Growth plate morpholgy was markedly altered in these mice, with changes being most prominent in the hypertrophic zone. Compression of this zone was not caused by reduced viability of hypertrophic chondrocytes, as no differences in the apoptosis rates could be observed. Furthermore, deposition of the major cartilage matrix components collagen type II and aggrecan was slightly reduced in these mice. Consistent with a role for agrin in skeletal development, we show for the first time that agrin is highly expressed by chondrocytes and localizes to the growth plate in wild-type mice. Our data show that agrin is expressed in cartilage and that it plays a critical role in normal skeletal growth.

TIMP-3 Is a Potent Inhibitor of Aggrecanase 1 (ADAM-TS4) and Aggrecanase 2 (ADAM-TS5)

The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K(i) values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.

Expression of collagen types IX and XI and other major cartilage matrix components by human fetal chondrocytes in vivo.

Coordinate differentiation of the chondrocytes plays a crucial role during skeletal development. In the cascade of endochondral bone formation, mature chondrocytes of the fetal growth plate represent metabolically highly active cells. They show high expression levels of the major cartilage matrix genes, collagen types II, IX, and XI, the major cartilage proteoglycan aggrecan, and proteoglycan link protein. The strongest signals are found in areas of maximal growth, the proliferative and upper hypertrophic zones. The major cartilage matrix components are co-expressed by the chondrocytes of the resting and proliferative zones. Type X collagen is restricted to lower hypertrophic chondrocytes. Interestingly, in the lower hypertrophic zone type IX collagen, but not type II and XI collagen, mRNA expression is downregulated, indicating a discoordinate expression of these collagen types in hypertrophic chondrocytes. The results of this study confirm the strict zonal differentiation pattern of chondrocytes in the developing fetal growth plate, which can be monitored by the expression patterns of its major expression products, the collagen subtypes and aggrecan and proteoglycan link protein.

Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion.

During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.

Acid mucopolysaccharides in the development of the Pacific great skate, Raja binoculata

ABSTRACT Histochemical treatments specific for hyaluronic acid, C-S-A/C, C-S-B and heparin were applied to a series of Pacific great skate (Raja binoculata) embryos from cleavage to immediate prehatching stages, in order to characterize histochemically the AMPS present in the embryos and to study the events leading to AMPS localization in the adult. A progression was observed: (a) intracellular neutral polysaccharides in cleavage stages, (b) a combination of extracellular polysaccharides and weakly acidic AMPS (hyaluronic acid) associated with cell processes in neurulating stages, (c) extracellular strongly acidic AMPS (chondroitin sulphates) in later stages, particularly in areas of cartilage development. In neurulating embryos hyaluronic acid appeared in quantity between adjacent tissues, suggestive of some developmental significance for this compound. Hyaluronic acid also appeared after neurulation in close association with developing gut and mesonephros. Histochemical localization of these compounds in immediate prehatching stages agreed with previously reported biochemical analyses of shark skin and cartilage, i.e. C-S-B occurred in skin and C-S-A/C was a major component of cartilage matrix.

The significance of the histochemical reaction for carboxyl groups of protein in cartilage matrix.

A major component of cartilage matrix reacting in the histochemical reaction for protein-bound carboxyl groups is the protein moiety of chondromucoprotein. C-terminal carboxyl groups in cartilage matrix are too sparse to contribute to the histochemical reaction; the side-chain carboxyls are responsible for all of the color developed. It is suggested that variation in staining reaction in the matrix reflects differences in the state of aggregation of chondromucoprotein. Thus the presence of an intense reaction for protein-bound carboxyl groups in cartilage matrix may be explained on the basis of aggregation of chondromucoprotein into high molecular weight complexes, yielding a high local concentration of requisite side-chain carboxyl groups for dye-coupling.