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A I26 Biochemical Society Transactions (2002)Volume 30, Part 6 35 Matrix deposition by articular cartilage chondrocytes treated with COL9AI and C O L l l A l antisense oligonucleotides. A. Vaughan-Thomas, R.D. Young, S.J. Gilbert, G.B.M. Davies, D.J. Mason and V.C. Duance Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, CFIO 3US. Collagen types IX and XI are present in cartilage collagen fibrils and are implicated in regulation of fibril morphology and matrix interactions. We used col9al and colf la1 antisense oligonucleotides to investigate matrix deposition by bovine chondrocytes. c D N A fragments encoding the N-terminal regions of bovine alpha 1 (IX) and alpha 1 (XI) collagen chains were cloned, sequenced and used to design modified antisense oligonucleotides. Each oligonucleotide reduced the expression of the appropriate collagen by chondrocytes maintained at high density. Combinations of four oligonucleotides were used in a further experiment. Following transfection of freshly isolated chondrocytes in high density culture overnight, chondrocytes were pelleted by centrifugation and incubated in serum-free medium supplemented with ITS and ascorbate for 12 days. Cell pellets were analysed by electron microscopy and immunofluorescence. While the collagens were still detectable in the matrix by immunofluorescence, fibrils of large diameter were observed in the matrix deposited by antisense oligonucleotide treated cultures. Manipulation of C O L 9 A l also clearly affected lateral association of microfibrils. We conclude that both collagen types IX and XI regulate fibril morphology. Their expression is important therefore in ensuring a functionally robust matrix. 36 Erythropoietin modulates Interleukin-I p induced nitrite production in rheumatoid synovial fibroblasts Y. Patel, P.J. Coussons and R. Grant u, Department of Sport, Exercise and Biomedical Science, University of Luton, Park Square, Luton, Bedfordshire, LU13JlJ Erythropoietin, a haemopoietic growth factor and a primary regulator of erythropoiesis, is widely used to treat anaemia in various chronic complications of rheumatoid arthritis (RA). Fibroblast-like cells, found in the pannus tissue of joints are thought to contribute to the inflammatory pathology of RA. Thus, for the current study a Swiss 3T3 fibroblast monolayer was chosen as a model system for the investigation of the mechanism of action of recombinant human E P O (rHuEPO), during treatment of anemia in RA. The results show that over a three day period r H u E P O (25pU/ml), both alone and in combination with the pro-inflammatory cytokine interleukin 1 p (IL-IP), induced significant production of nitrite. This is a substrate for nitrogen oxide ( N O ) synthesis by nitrogen oxide synthase (NOS), which is a well documented mediator of metalloproteinase-mediated tissue remodelling activity in RA. It therefore appears that, through modulation of nitrite and subsequent NOS-dependent NO production, r H u E P O may influence tissue remodelling in connective tissues, independently of its established erythropoetic role. 0 2002 Biochemical Society 37 Gap junctions and strain response in tendon cells A.D. W a , M. Benjamin, J.R. Ralphs School of Biosciences (BIOSI2), Cardiff University, PO Box 911, Cardiff CFIO 3US Tendons transfer tensile load from muscle to bone. They are made of longitudinally running collagen fibres, interspersed with rows of tendon cells. Tendon cells are in contact longitudinally within rows, and laterally between rows via cell processes. The cells modify the extracellular matrix according to the mechanical load they experience. Gap junctions consisting of connexins 32 and 43 occur between cells within a row, whereas only those containing connexin 43 occur where lateral cell processes meet. We hypothesise that gap junctions mediate signals between cells under mechanical strain and coordinate ECM changes. Here we examine effects of gap junction blockers o n collagen synthesis by tendon cells under cyclic tensile load. The blockers were a biomimetic peptide, G A P 27, that prevents formation of all gap junction channels, and specific downregulation of connexin 32 o r 43 using liposomally delivered antisense D N A . GAP27 reduced collagen synthesis under load by 50% compared to controls. A similar effect was observed when connexin 32 was downregulated. In contrast, downregulation of connexin 43 increased collagen secretion by around 100%. Thus communication via connexin 43 gap junctions is inhibitory and via connexin 32 stimulatory to collagen synthesis. The balance of inhibitory and stimulatory signals will lead to an appropriate coordinated tissue response under load. 38 Identification of a type IX collagen interaction with fibronectin: a molecular bridge in articular cartilage? P. Callender, A. Vaughan-Thomas, D.J. Mason & V.C. Duance CTBL, School of Biosciences, Cardiff University, Card# CFIO 3US In articular cartilage type IX is covalently cross-linked to the surface of heterotypic collagen fibrils and has a large N-terminal globular domain (NC4) which protrudes away from the surface of the fibril into the surrounding matrix. This makes the N C 4 domain an ideal candidate for matrix interactions important for cartilage integrity. To identify protein interactions of the N C 4 domain of type IX collagen we have used a yeast-two hybrid system (Matchmaker 3, Clontech). The N C 4 domain was used as a bait protein in a screen of 6.5 x lo5 human chondrocyte c D N A library transformants. 73% of the interacting clones were identified as human fibronectin (FN). F N is alternatively spliced to produce 24 different isoforms. A cartilage specific isoform exists that lacks the variable region and an immediately C-terminal constant region, present in all other isoforms. PCR analysis suggests that this constant region of F N may be important in the N C 4 - F N interaction. Immunohistochemical analysis of bovine cartilage sections confirmed the co-localisation of type IX collagen and F N to the pericellular matrix. The predominant receptor on the surface of chondrocytes is the a 5 p l integrin, which is specific for F N and has recently been described as a mechanoreceptor. It is therefore possible that the identified NC4-FN interaction is important in regulating cartilage matrix homeostasis and that a loss of this interaction may predispose articular cartilage to OA-like degradation.