Download Matrix deposition by articular cartilage chondrocytes treated with

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.