EXPRESSION OF CLUSTERIN IN THE SUPERFICIAL ZONE OF ARTICULAR CARTILAGE
*Khan, I; **Thomson, B; +*Archer, C W
+*Cardiff School of Biosciences, Cardiff, Wales. Cardiff School of Biosciences, Museum Avenue, PO Box 911, Cardiff , Wales, UK, CF10 3US, +44(0)29 20875206,
Fax: +44(0)29 20874594, [email protected]
Introduction: Articular cartilage is comprised of chondrocytes that organise
collagens, proteoglycans and non-collagenous proteins into a unique, highly
ordered structure (1). The composition, mechanical properties, organisation of
extracellular matrix, cell morphology and cell function vary with depth from
the articular surface. It is possible to identify four histologically distinct zones
parallel to the articular surface; the superficial zone, the transitional zone, the
radial or deep zone and the zone of calcified cartilage, and although the
boundaries between each zone are not well defined, extensive biological and
mechanical studies have demonstrated that zonal organisation has functional
significance (2). The superficial zone is important in many respects; firstly it
forms the fluid-tissue interface of articular cartilage in the synovial cavity and
as such, is generally thought to perform critical barrier functions. Secondly in
degenerative joint diseases surface fibrillation and cell death in the superficial
zone are one of the earliest pathological indicators of disease progression.
During growth of articular cartilage, cells of the superficial zone incorporate
radiolabelled thymidine and have extended cell cycle times indicative of
appositional as well as interstitial growth (3). The latter finding suggests that
cells of the superficial zone may harbour precursor or stem cells (4). Despite
our abundant knowledge of the biochemical, biomechanical, histological and
ultrastructural features of the superficial zone of articular cartilage, there has
been relatively little characterisation done at the level of gene expression.
Therefore in order to clarify and understand to a greater degree the role of the
superficial zone in articular cartilage development and disease, we conducted
a differential screen of an immature bovine articular chondrocyte cDNA
library to clone genes which are either predominately or specifically expressed
in the superficial chondrocytes.
Materials and Methods: Differential Screening: Approximately 2 x 103
plaque formimg units (pfu) of a bovine articular cartilage cDNA library were
plated separately onto three 150mm dishes containing NZYCM media. Two
replicates were made of each plate using nylon membrane. One replica filter
set was hybridised with 50ng 32PαdCTP radiolabeled cDNA from superficial
zone cells, the other set using 50ng radiolabeled cDNA from the deep zone
cells and incubated overnight at 65°C. Filters were washed to high stringency
(0.1xSSC) and then exposed to film. Comparison of autoradiograph films for
each set of replicate filters allowed the identification of differentially
transcribed gene clones. Northern Hybridisation: Five micrograms of total
RNA from superficial and deep zone cells was electrophoresised on a 1.5%
agarose gel in 10mM sodium phosphate buffer pH 7. The gel was blotted onto
nylon membrane and hybridised to full-length radiolabelled clusterin cDNA.
The blot was incubated at 65°C overnight and washed to high stringency
(0.1xSSC). In Situ Hybridisation: Articular cartilage from the
metacarpalphalangeal joint of immature (7 day old) and mature (1 year old)
bovine steers were fixed overnight in 4% paraformaldehyde/PBS, dehydrated
using a graded set of ethanol solutions, incubated in xylene for 30 mins and
embedded in paraffin wax. Ten micron sections were then digested with
proteinase K (20µg/ml) at 37°C for 30 mins. Acetylation of sections was
performed by a 10 min incubation in 0.1M tri-ethanolamine pH 8.0 buffer
containing 0.25% acetic anhydride. Digoxigenin labelled sense and antisense
probes were made by in vitro transcription of EcoR1 and Xho1 digested
plasmid pBKCMV 36.3 (clusterin), respectively. Sections were hybridised
with probe (approximately 100ng/ml in hybridisation buffer; 5 x SSC, 50%
formamide, 10% dextran sulfate and 10µg/ml salmon sperm DNA) overnight
at 42°C, then washed sequentially at 55°C for 30 mins each in 1 x SSC and
0.1 X SSC. For colour detection, tissue sections were incubated with
1.25units/ml alkaline phosphatase-conjugated anti-DIG polyclonal antibody in
PBS/1% BSA for 30 mins washed in PBS three times for 5 mins and
incubated in a solution of 0.2mM nitroblue tetrazolium salt, 0.2mM 5-bromo
4-chloro-3-indolyl phosphate, 5mM MgCl2 and 1mM levamisole for 24
hours.
Results: One cDNA sequence isolated in our screen for genes specifically
expressed in the superficial zone of articular cartilage was bovine glycoprotein
III or clusterin (5, 6). Our screening data was confirmed by conducting
Northern blot analysis using clusterin to probe total RNA from superficial and
deep zone cells. It was found that clusterin was predominately expressed by
cells of the superficial zone. In Situ hybridisation analysis was used to
visualise expression of clusterin transcripts in immature and mature articular
cartilage (mature cartilage shown in Figure 1). It was found that expression in
mature and immature cartilage was limited to the superficial zone and that
expression was seen in cells extending only 4-5 cell depths from the articular
surface.
Figure 1. In Situ hybridisation analysis of clusterin expression in full depth
mature bovine articular cartilage. Bar 10 microns.
Discussion: We have demonstrated that clusterin mRNA is differentially
expressed in articular cartilage and that it specifically transcribed by cells in
the superficial layer. Clusterin is a secreted, heterodimeric glycoprotein whose
function has yet to be conclusively demonstrated. Previous findings indicate
possible functions of clusterin in regulating complement, in lipid transport and
as an extracellular chaperone (6). Indeed, clusterin expression has been shown
to be highest in those tissues which serve to protect cells and underlying cells
from surface-active components in biological fluids (7). In tissues subject to
oxidative stress or injury resulting in cell death there is an association with
upregulation of clusterin in surviving cells and overexpression of clusterin has
also been reported in a number of pathophysiological conditions such as
neurodegenerative diseases, renal diseases and athersclerosis (6). In the light
of these findings, clusterin may potentially be useful as a marker of tissue
responses to injury in articular cartilage.
References: 1) Buckwalter JA and Hunziker EB., (1990) In Biology of the
Synovial Joint. Amsterdam: Harwood Academic Publishers; pp. 75-100. 2)
Meachim G, Stockwell RA., (1973) In Adult Articular Cartilage. Kent
(UK):Piman Medical Publishing; pp. 1-67. 3) Archer CW et al, (1994) Ann
Rheum Dis 53, 624-630. 4) Hayes A et al, (2000) in press. 5) Palmer DJ and
Christie DL., (1990) J Biol Chem 265, 6617-6623. 6) Wilson MR and
Easterbrook-Smith SB., (2000) Trends Bichem 25, 95-98. 7) Aronow BJ et
al, (1993) PNAS 90, 725-729.
**Smith & Nephew plc, Group Research Center, York Science Park,
Heslington, York.
0386
Poster Session - Cartilage Matrix Biology - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California