This review of the literature supports the concept that ROS are not only deleterious agents involved in cartilage degradation, but that they also act as integral factors of intracellular signaling mechanisms. Further investigation is required to support the concept of antioxidant therapy in the management of joint diseases.

The role of endogenous inducers of inflammation is poorly understood. To produce the proinflammatory master cytokine interleukin (IL)-1␤, macrophages need double stimulation with ligands to both Toll-like receptors (TLRs) for IL-1␤ gene transcription and nucleotide-binding oligomerization domain-like receptors for activation of the inflammasome. It is particularly intriguing to define how this complex regulation is mediated in the absence of an infectious trigger. Biglycan, a ubiquitous leucine-rich repeat proteoglycan of the extracellular matrix, interacts with TLR2/4 on macrophages. The objective of this study was to define the role of biglycan in the synthesis and activation of IL-1␤. Here we show that in macrophages, soluble biglycan induces the NLRP3/ASC inflammasome, activating caspase-1 and releasing mature IL-1␤ without the need for additional costimulatory factors. This is brought about by the interaction of biglycan with TLR2/4 and purinergic P2X 4 /P2X 7 receptors, which induces receptor cooperativity. Furthermore, reactive oxygen species formation is involved in biglycan-mediated activation of the inflammasome. By signaling through TLR2/4, biglycan stimulates the expression of NLRP3 and pro-IL-1␤ mRNA. Both in a model of non-infectious inflammatory renal injury (unilateral ureteral obstruction) and in lipopolysaccharide-induced sepsis, biglycan-deficient mice displayed lower levels of active caspase-1 and mature IL-1␤ in the kidney, lung, and circulation. Our results provide evidence for direct activation of the NLRP3 inflammasome by biglycan and describe a fundamental paradigm of how tissue stress or injury is monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response. IL-1␤2 is a proinflammatory master cytokine produced by macrophages in response to inflammatory stimuli, such as LPS. The activity of IL-1␤ is regulated sequentially by synthesis of the 31-kDa precursor pro-IL-1␤, intracellular proteolytic conversion into active IL-1␤ (17 kDa) by the cysteine protease caspase-1, also known as IL-1-converting enzyme (1, 2), and by secretion of IL-1␤ (3). The synthesis of pro-IL-1␤ is initiated by Toll-like receptor (TLR) agonists, whereas ATP stimulates cleavage and maturation of IL-1␤ (4, 5). Activation of caspase-1 requires the assembly and activity of a cytosolic multiprotein complex known as the inflammasome, consisting of nucleotide-binding oligomerization-like receptor family members (NLRs; NLRPs (NLR family, pyrin domain-containing 3), NAIP (NLR family, apoptosis inhibitory protein), and NLRC4 (NLR family caspase recruitment domain-containing 4)) (6), generating functional caspase-1 p20 and p10 subunits (1,7,8). TLRs and NLRs contain leucine-rich repeats (LRRs), which are used as ligand-sensing motifs (9, 10). NLRP3, the best characterized member of NLRs, recruits caspase-1 to the inflammasome via the adapter molecule ASC (apoptosis-associated specklike protein containing caspase activation and r...

Abstract. The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.T o suit their functions, connective tissues must have distinct biomechanical properties that critically depend on the molecular structure of the components of the extracellular matrix as well as their supramolecular assembly. As the principal tensile elements, fibrils play a key role in structural stabilization. The distribution of the diameters and the organization of the fibrils into meshworks, fiber bundles, or highly ordered layers are characteristics of different tissues. For the stabilization of the tissue structure, fibrils require unique surface properties enabling them to specifically interact with themselves as well as other matrix components, such as proteoglycans and matrix glycoproteins. The morphological diversity of connective tissues is paralleled by a variety of the major molecular fibril components, the collagens (25). Recent reports have established the existence of heterotypic fibrils assembled from more than one collagen type (2,12,17,18,20,23,37,38). The interaction of different collagens during fibrillogenesis may well be crucial in the regulation of the fibril architecture and the modulation of the fibril surface properties.Cartilage is unique in that it contains a tissue-specific set of collagens (25); i.e., collagens II, IX, and XI. Collagen X is predominantly found in cartilage but also occurs at low levels in intramembranous bone (31). In cartilage, however, collagen X is restricted to hypertrophic zones and, therefore, may play a role in the transition of cartilage to bone (32). Collagen II is the major fibril component and is similar to collagens I and HI of other tissues in that the molecule essentially consists of a single uninterrupted helical domain 300 nm in length. Collagen II comprises three identical cd(II)-chains. Collagen XI probably is the structural analogue in cartilage to collagen V because, in the tissue form, both proteins contain a large amino-terminal noncollagenous domain in addition to a 300-nm triple helix (4, 26). Collagen XI is a heterotrimer (26). The od(XI)-and tx2(XI)-chains are structurally similar to the or(V)-and ot2(V)-chains (6, 11). Curiously, the ot3(XI)-chain is similar if not identical with an overglycosylated form of the txl(...

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

The kinetics of triple-helix formation in type I11 pN-collagen, type I11 collagen and a quarter fragment of type I11 collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25 'C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4-15 "C) were mismatched structures of lower stability formed. At 25 'C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy-terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper-like fashion. This gives rise to zero-order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis --$ trans isomerization of peptide bonds. A model mechanism was developed which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis --$ trans isomerization k = 0.01 5 s-' and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuartion.The three major types of collagen found in interstitial tissues have a uniform and linear triple-helical conformation. The molecules are comprised of three a chains and more than 95% of each a chain consists of a repeating triplet sequence in which glycine is every third amino acid and the other two positions are frequently proline and hydroxyproline. Each of the three chains is folded into a left-handed poly-(proline) I1 helix and these three helices are wrapped around each other into a right-handed super-helix [ I , 21. Formation of the triple helix in vivo occurs at the level of procollagen, the precursor of collagen which contains additional peptide sequences fat both the amino and carboxy ends of the a chains [3 -81. Intermediate forms of procollagen, commonly referred to as pN-collagens [4], can be isolated from diseased or Abbreviations. Type I11 pN-collagen, type I11 collagen which still, contains the precursor-specific amino-terminal portion of type I11 procollagen; CD, circular dichroism.Enzymes. Trypsin (EC 3.4.21.4); pepsin (EC 3.4.23.1).fetal tissues. These pN-collagens have been convenient sources to study the structure of amino-terminal propeptides which consist of a non-collagenous and a short triple-helical domain [6 -81. This precursorspecific triple helix is composed of 45 triplets and separated from the main triple helix (about 1000 triplets) by a small non-helical segment. The globular carboxy-terminal propeptides lack a collagenous structure and possess disulfide bridges connecting the ...

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of ϳ20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.In vertebrates, hyaline cartilage occurs in specialized regions of the skeleton and comprises the structural tissue of other organs (e.g. the cartilaginous rings of the trachea or the avian sclera). In the skeleton, cartilaginous regions either are permanent (e.g. in articular joints) or form a transient tissue template during bone development. The main functions of hyaline cartilage are biomechanical in nature and, essentially, are performed by its extracellular matrix, which occupies the major fraction of the tissue volume. Two matrix suprastructures can easily be distinguished in cartilage by electron microscopy. First are the collagen-containing fibrils with a periodic banding pattern of D ϭ 67 nm. The other component is the electronlucent extrafibrillar matrix, which is rich in immobilized anionic charges. Despite a high degree of molecular organization of its major components, aggrecan and hyaluronan, the extrafibrillar cartilage matrix has no conspicuous morphological features. However, the anionic charges cause extensive binding of water, which results in the generation of substantial osmotic pressure. The fibrils serve as the essential tensile elements of the tissue and contain the swelling pressure exerted by the extrafibrillar matrix.Cartilage fibrils vary in their molecular organization, their width, and their orientation in the tissue in order to resist forces generated by external load. In adult articular cartilage, for example, thin fibrils near the joint cavity preferentially run parallel to the surface, since lateral forces predominate in this region. In the interterritorial regions of the deep zones, in contrast, wider fibrils are arranged perpendicularly to the surface to strengthen the tissue in the direction along the axis of the bones. A prominent feature of the fibrils in developing and in immature cartilage is their strictly uniform diameter of about 20 nm and their more random orientation.Cartilage fibrils are often referred to as collagen II fibr...

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Abstract. It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril. major question in cell biology is how individual macromolecules combine to form the often large and complex supramolecular structures of the extracellular matrix. Approaches to this problem include the direct microscopic visualization of tissue sections aided by chemical stains and immunological tools, reconstitution and binding studies with purified components in solution followed by analysis of the resulting products, and, where possible, direct x-ray analysis of highly ordered arrays in situ.This strategy is particularly well exemplified by the many detailed studies of collagen fibrils and fibrillogenesis. This work has resulted in an understanding of the interactions that govern lateral association of fibrillar collagens (for recent reviews see references 3, 7, and 19). The quarter stagger model that arose from these studies has been considerably refined since its introduction, but still remains as the cornerstone of current models as it explains how fibrils formed in vitro from purified collagen molecules give rise to the characteristic staining patterns observed in the electron microscope. Although the fibril staining patterns produced in vitro match those seen in vivo, the diameter regulation of collagen fibrils in vivo and their complexity, including association with other components, especially proteoglycans, are features not reproduced by mixing solutions of collagens in vitro. In summary, two critical questions remain: how is the construction of fibrils regulated in vivo and what role might other molecules play in these processes?This problem is particularly well illustrated in cartilage. The morphology of fibrils reconstituted from purified type II collagen in solution is vastly different from that of cartilage fibrils in situ. Large tactoidal aggregates with d-periodic staining patterns are formed under appropriate reconstitution conditions (21). In contrast, fibrils from chicken embry...