Objective. Osteoarthritic (OA) cartilage destruction depends on collagen-and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1␤ (IL-1␤)-stimulated chondrocytes in vitro.Methods. Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1␤.Results. In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1␤. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly downregulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1␤. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1␤.Conclusion. Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1␤ stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.Osteoarthritic (OA) cartilage degeneration as well as cartilage destruction in rheumatoid arthritis depend, at least to a significant degree, on enzymatic degradation of matrix components. Two types of molecules, collagen fibrils and the proteoglycan aggrecan, represent the major targets in terms of enzymatic activity and functional loss of the cartilage matrix as a result of damage to these molecules. Whereas degradation of collagen fibrils leads to matrix instability with tissue swelling, degradation of proteoglycans leads to cartilage softening and loss of fixed charges (1), both of which are classic features of cartilage destruction.Catabolism of both types of molecules is supposed to involve different types of enzymes, largely from 2 protease families: matrix metalloproteinases (MMPs) and the "A disintegrin and metalloproteinases with thrombospondin type 1 motif" (ADAM-TS). The initial degradation of collagen fibrils (within the triple-helical region) depends on cleavage at the collagenase site, for which there exist 2 major candidate enzymes...

Objective. Despite many research efforts in recent decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. The present study was therefore initiated in order to identify differentially expressed disease-related genes and potential therapeutic targets.Methods. This investigation consisted of a large gene expression profiling study performed based on 78 normal and disease samples, using a custom-made complementary DNA array covering >4,000 genes.Results. Many differentially expressed genes were identified, including the expected up-regulation of anabolic and catabolic matrix genes. In particular, the down-regulation of important oxidative defense genes, i.e., the genes for superoxide dismutases 2 and 3 and glutathione peroxidase 3, was prominent. This indicates that continuous oxidative stress to the cells and the matrix is one major underlying pathogenetic mechanism in OA. Also, genes that are involved in the phenotypic stability of cells, a feature that is greatly reduced in OA cartilage, appeared to be suppressed.Conclusion. Our findings provide a reference data set on gene alterations in OA cartilage and, importantly, indicate major mechanisms underlying central cell biologic alterations that occur during the OA disease process. These results identify molecular targets that can be further investigated in the search for therapeutic interventions.

Abstract. The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the eDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by '~9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes.T H F. outer and inner membranes of the nuclear envelope are continuous with each other at distinct circular sites of "~90 nm in diameter, termed nuclear pores (15). The nuclear pore is occupied by the nuclear pore complex, a supramolecular ensemble of an estimated mass of--50-100 x 106 D (for reviews see 14, 28). Transport of proteins and nucleic acids in and out of the nucleus proceeds via the nuclear pore complex (7, 11).An integral membrane protein of an estimated 190 kD containing asparagine-linked, high mannose-type oligosaccharides (gpl90) has recently been shown by immunoelectron microscopy to be associated with the nuclear pore complex (19). The finding that gpl90 remains associated with nuclear pore complexes after solubilization of the nuclear envelope lipids with nonionic detergent suggests that gpl90 spans the pore membrane and possesses a domain exposed to the nuclear pore complex and a domain containing the asparagine-linked oligosaccharides exposed to the perinuclear cisternae. Association with the nuclear pore complex suggests that gpl90 may function in anchoring pore complex components to the pore membrane (19).Recently, Unwin and Milligan have examined the nuclear pore complex using Fourier averaging methods on negatively stained images (39). They observed two "ring" structures composed of eight globular subunits lying parallel to the nuclear envelope, one on the cytoplasmic face and the other on the nucleoplasmic face of the nuclear envelope. Between these two rings, a set of eight "spokes" radiate in from the vicinity of the outer and inner membrane junction toward the center of the pore. These spokes surround a central "plug" located in the center of the pore complex. The pore complex is thus positioned around two axes of symmetry. A central axis showing octahedral symmetry passes through the central plug perpendicular to the surface of the nuclear envelope. The other axis is perpendicular to the central axis and essentially divides the pore complex into oppositely facing halves. If gpl90 was involved in anchoring one each of the eight subunits of the inner and outer ring (or spokes) of the pore complex, one would expect 16 copies of gpl90 per pore complex in good agreement with the estimated number of 16-24 gpl90 (18).As a step toward an understanding of the function of gpl90, we deduced the complete primary structure of gpl90 from the eDNA sequence. The primary structure reveals a polyp...

Our data characterize SW1353 cells as a cell line with only a very limited potential to mimic PHCs, though SW1353 cells can be of value to study the induction of protease expression within cells, a phenomenon also seen in chondrocytes.