(Received for publication, May 26, 1995; and in revised form, August 21, 1995)
From the
Bovine articular chondrocytes cultured in alginate beads were
used to study the effect of catabolic cellular mediators on CD44
expression. Treatment with either the 29-kDa fragment of fibronectin or
interleukin-1 results in a time- and dose-dependent inhibition of
proteoglycan synthesis as well as a stimulation in the expression of
CD44 mRNA level as determined by semi-quantitative polymerase chain
reaction following reverse transcription. No noticeable effect at 6 h
was observed. By 24 h, the major CD44 product (CD44H) from fibronectin
fragment-treated cultures showed an 8-fold increase; CD44H from
interleukin-1
-treated cultures showed a 6-fold increase as
compared to control cultures. In addition, a minor band, determined to
be an isoform of CD44, was also shown to be up-regulated by both
mediators. Stimulation of CD44 mRNA via interleukin-1 was also evident
by in situ hybridization studies of bovine as well as human
articular cartilage in organ culture. The increased in CD44 mRNA is
matched by an increase at the protein level as determined by Western
blot analysis. The Western blot reveals a doublet protein band at
80-90 kDa that corresponds to the molecular mass of CD44H.
Cultures incubated with fibronectin fragments for 24 h had an 8.0-fold
increase in CD44, while a 6.6-fold was observed for interleukin-1
.
Fluorescein-conjugated hyaluronan binding and internalization studies
indicate that the increase in CD44 protein, induced by
interleukin-1
, closely correlates with an increase in functional
hyaluronan receptors present at the chondrocyte cell surface. Taken
together these results indicate that conditions that up-regulate
chondrocyte catabolism also up-regulate the expression of CD44, a cell
surface hyaluronan receptor involved in hyaluronan endocytosis.
Articular cartilage is a specialized tissue that covers the ends
of bones to provide smooth articulation of the joints during
load-bearing and physical activities. It is composed of a small number
of living resident cells, the chondrocytes, embedded in an extensive
extracellular matrix. The chondrocytes maintain the composition of this
extracellular matrix by regulation of the synthesis and degradation of
the matrix components. The two major components of cartilage are type
II collagen (1) and aggrecan, the cartilage-specific
proteoglycan (PG)()(2, 3, 4) .
Aggrecan molecules, often as many as 50, interact with single filaments
of hyaluronan (HA) to form PG aggregates (2, 5, 6, 7, 8) with
molecular mass between 10
and 10
daltons(5, 6, 7, 9) , and this
interaction is further stabilized by link
protein(2, 10) . It is the presence of the PG
aggregates within the collagenous network that gives cartilage its
unique ability to resist compression. The aggrecan-rich matrix is
assembled and retained at the cell surface of chondrocytes via the
interaction of HA with CD44/HA receptors(11) . Thus, HA plays a
pivotal role in the organization and retention of aggrecan molecules
within cartilage extracellular matrix. Understanding the mechanism(s)
involved in HA turnover are therefore critical to a complete
understanding of cartilage turnover as a whole.
Aggrecan catabolism is thought to occur extracellularly and involve the proteolytic cleavage between the G1 and G2 domain of the core protein of PG; the resulting degradation products are rapidly lost from the matrix of cartilage(12, 13, 14) . Although it has been shown that the turnover of PG and HA is co-ordinately regulated, having similar half-lives in the range of 13-25 days, no HA degradation products could be detected either in the medium or within the extracellular matrix of the tissue(15, 16) . Furthermore, no enzymes for extracellular breakdown of cartilage HA have been documented(17) . However, a previous study (18) from our laboratory has demonstrated that chondrocytes do have the capacity to internalize and degrade HA to small oligosaccharides within a low pH lysosomal compartment. This endocytosis mechanism, resulting in the catabolism of HA, has also been shown to be mediated via cell surface CD44/HA receptors(18) .
A variety of agents, including the inflammatory cytokine interleukin-1 (IL-1) elicit an enhanced catabolic state of cartilage tissue (14, 19, 20, 21, 22) and have been used to study matrix turnover(19) . The principal effects of IL-1 on chondrocytes are enhanced matrix degradation due to the secretion of metalloproteinases and decreased PG synthesis. This up-regulation of cartilage matrix catabolism by IL-1 closely mimics the degradation of articular cartilage seen in osteoarthritis and rheumatoid arthritis(19) . Other potential cellular mediators with the capacity to modulate chondrocyte metabolism are fragments of the adhesive glycoprotein, fibronectin (FN-f). Addition of the 29-kDa FN-f to explant cultures of articular cartilage causes an increase in expression of extracellular metalloproteinases resulting in PG degradation and release from the cartilage as well as the release of chondrocyte-derived IL-1(23, 24) . Furthermore, PG synthesis is also inhibited. Similar fragments of FN have been identified in the synovial fluid of osteoarthritis and rheumatoid arthritis patients, bathing the underlying articular cartilage with these potent mediators(25) . Since HA and PG turnover are co-ordinately controlled(15, 16) , it was postulated that conditions that up-regulate PG turnover would also stimulate the mechanisms involved in the turnover of HA. Furthermore, if CD44-mediated endocytosis is the primary pathway leading to the catabolism of HA, CD44 expression would be similarly regulated by these cell mediator signaling pathways.
In the present study, it was
demonstrated that treatment of chondrocytes with either IL-1 or
the 29-kDa FN-f results in an inhibition of PG synthesis as well as an
increase in CD44 mRNA expression. Furthermore, this increase in CD44
mRNA is matched by an increase in CD44 at the protein level.
Fluorescein-HA binding studies indicate that the IL-1
-induced
increase in CD44 protein is closely correlated with an increase in
functional HA receptors present at the chondrocyte cell surface. Thus,
conditions that up-regulate chondrocyte metabolism, including the
elevation of extracellular matrix-degrading enzymes, also up-regulate
the expression of a cell surface HA receptor involved in HA
endocytosis.
To generate this internal standard, 1 µg of pCD44E was used as a template in polymerase chain reaction (PCR) mixture consisting of 2 mM magnesium chloride, 200 µM of each dNTP, 0.15 µM of each primers, and 2.5 units of AmpliTaq DNA. The DNA was denatured by heating at 95 °C for 2 min, followed by 30 cycles of 1 min at 95 °C, annealing at 55 °C, and extension at 72 °C for 1 min using a Perkin-Elmer thermal cycler. The amplified products were analyzed by agarose gel electrophoresis followed by staining with ethidium bromide. The 872-bp product was then purified using a Promega's Wizard DNA purification kit (Madison, WI) and its concentration determined by absorbance at 260 nm. Serial dilutions of the internal standard were made with sterile distilled water containing molecular biology grade glycogen (50 µg/ml) and stored at -20 °C.
Bovine articular
cartilage slices from 18-month-old steer were either fixed immediately
following dissection by immersing in 4% paraformaldehyde or cultured in
DMEM/Ham's F-12 supplemented with 10% FBS in the absence or
presence 1 ng/ml IL-1 for 48 h. Full thickness non-calcified human
articular cartilage was removed from the femoral condyle ankle joint of
a 54-year-old female donor obtained from the Regional Organ Bank of
Illinois, and slices were treated in the same manner except that
IL-1
was used in place of IL-1
(32) . Following
fixation in 4% paraformaldehyde, tissue slices were embedded in
paraffin, sectioned, and processed according to the in situ hybridization procedures described by Sandell et
al.(31) . Briefly, sections were treated with acetic
anhydride (0.25% in 0.1 M triethanolamine), dehydrated,
delipidated, and air-dried. The
S-labeled oligosaccharide
probe was added to the hybridization buffer containing 25% deionized
formamide, 10% dextran sulfate, 300 mM sodium chloride, 10
mM Tris, 1 mM EDTA, 1
Denhardt's, 0.5
mg of yeast tRNA/ml, and 10 mM dithiothreitol. A 60-µl
aliquot containing 2 pmol of probe/ml was applied to each slide. In
some instances, a mixture containing equal molar concentration of
labeled and unlabeled antisense probes was used in competitive assays.
The slides were incubated with probe overnight in a moist chamber at
30.9 °C, washed with four changes of 1
standard saline
citrate for 30 min each time at 47.8 °C, followed by two 90-min
washes at room temperature. The sections were dehydrated through a
graded series of alcohols containing 300 mM ammonium acetate,
dipped in NTB2 emulsion (Kodak) diluted with 600 mM ammonium
acetate, exposed for 4 days, and developed in D-19 developer (Kodak).
Sections were counterstained with cresyl violet acetate and were
photographed using Nikon Microphot-FXA microscope. Sections were also
viewed under phase contrast microscope whereby the hybridized probe to
mRNA within the cells was observed as grains localized over cells
within lacuna.
Bovine
chondrocytes cultured in alginate beads were treated with a range of
concentrations of the 29-kDa FN-f (0.01-0.5 µM) or
IL-1 (0.1-5.0 ng/ml) for 3 days. Both of these mediators
resulted in a dose-dependent inhibition on proteoglycan synthesis (Fig. 1A). At a concentration of 0.01 µM FN-f, an inhibition of 68% that of control was observed with
maximal inhibition of 80% attained at 0.1 µM. A similar
inhibition curve was observed with IL-1
cultures whereby
proteoglycan synthesis was reduced by 68% at 0.1 ng/ml and peaked at
0.2 ng/ml (75%) as shown in Fig. 1B. For all subsequent
experiments, maximal concentrations of either 0.1 µM FN-f
or 0.2 ng/ml IL-1
were used to induce the catabolic effects.
Figure 1:
Effects of different concentrations of
FN-f and IL- on proteoglycan synthesis. Bovine chondrocytes were
treated for 3 days with different concentrations of either FN-f ranging
from 0.01 to 0.5 µM (panel A) or IL-1
in the
range of 0.1-5.0 ng/ml (panel B). On day 3, the cells
were labeled with [
S]sulfate for 4 h and
incorporation into total proteoglycan was analyzed by Sephadex G-25.
Data represent the mean ± S.E. of duplicate
determinations.
In
addition, the inhibitory effects on proteoglycan synthesis were
time-dependent whereby as early as 24 h, 0.1 µM FN-f and
0.2 ng/ml of IL-1 resulted in approximately 50% inhibition of
proteoglycan synthesis (Table 1). This inhibition increased to
75% by 48 h, and remained relatively constant thereafter (Table 1).
No
noticeable effect of these two mediators on CD44 message level was
observed at 6 h (Fig. 2A). However, by 24 h, both FN-f-
and IL-1-treated cultures showed an increase in the amount of CD44
message level as compared to the control, and this stimulation was
maintained at the 48-h and 72-h time points. To ascertain that an
equivalent amount of RNA was used in the amplification process, RNA
from each experiment was subjected to RT-PCR using primers specific for
the ``housekeeping gene,'' GAPDH. The level of GAPDH product
remained nearly constant in the different culture conditions and during
the time course of the experiment (Fig. 2B).
Figure 2:
Time course of effects of FN-f and
IL-1 on CD44 and GAPDH mRNA level. Bovine chondrocytes in alginate
beads were incubated with either FN-f (0.1 µM) or
IL-1
(0.2 ng/ml) for 6, 24, 48, or 72 h. Total RNA from cultures
at each time point was isolated and subjected to RT-PCR with specific
primers for CD44 (A) and GAPDH (B). Sd,
X174/HaeIII DNA markers; C, control cultures; Fn, FN-f-treated cultures; IL, IL-1
-treated
cultures. The major CD44 product (arrow b, 600 bp), minor CD44
product (arrow a, 754 bp), and GAPDH product (arrowhead, 450 bp) are indicated.
In addition to the major CD44 product predicted at 600 bp (CD44H, arrow b), another band at 754 bp (arrow a) was detected. The 754-bp band was also up-regulated by the presence of both mediators. The major band at 600 bp was purified, sequenced via cycle sequencing, and found to match the published sequence data for bovine CD44 (data not shown). In addition, DNA sequencing data indicate that the sequence of the 754-bp minor product is identical to the bovine CD44H with the addition of an internal stretch of DNA highly homologous with the alternatively spliced human v10 exon(33, 34) . In order to confirm the identity of the 754-bp band as the alternatively spliced v10 isoform of CD44, restriction enzyme digestion with ApaLI, predicted to generate one cleavage within either the CD44H or CD44v10 isoforms, was performed. The two CD44 bands were separated by low melting agarose gel electrophoresis and each of the products purified. Following ApaLI restriction enzyme digestion, the 600-bp CD44 product yielded the predicted products: 350-bp and 250-bp products (Fig. 3A). The 754-bp CD44 product yielded an identical 350-bp digestion product as well as a 404-bp fragment. To further substantiate the identity of the 754-bp CD44 as an alternative spliced v10 isoform of CD44, the product was amplified using the same sense primer and a nested antisense primer specific for the sequenced bovine variant 10 exon. A predicted product of approximately 550 bp was generated, and restriction enzyme digestion with ApaLI yielded the predicted digestion products: 350 bp and 200 bp (Fig. 3B). These results are consistent with the larger CD44 product (754 bp) being an alternatively spliced v10 isoform of CD44.
Figure 3:
Restriction enzyme and nested PCR
analysis of CD44 isoforms. The two CD44 PCR products depicted in Fig. 2A were isolated and purified. In the experiment
depicted in panel A, each product was digested with ApaLI, the digests separated on 4% polyacrylamide gels, and
the products visualized by ethidium bromide staining. Lanes 1 and 4 represent X174 DNA/HaeIII markers; lane 2, ApaLI digest of 600-bp isoform of CD44; lane 3, ApaLI digest of 754-bp isoform of CD44. In
the experiment depicted in panel B, the purified 754-bp
product was subjected to nested PCR using a bovine CD44v10-specific
antisense primer. The nested PCR product is depicted in lane
5. This PCR product was again subjected to ApaLI
digestion (lane 6). The major CD44 undigested product (arrow b, 600 bp) and the minor CD44 product (arrow
a, 754 bp) are indicated.
The major 600-bp CD44 product was further quantified using purified CD44E as an internal standard in semi-quantitative PCR. It was first important to determine that the amplification efficiencies of the target and the internal standard were similar. To this end, equimolar quantities of target and internal standard, CD44E were co-amplified in the presence of radiolabeled sense primer. The results shown in Fig. 4indicate that the amplification efficiencies of the target (600 bp) and the internal standard, CD44E (872 bp) were similar at every PCR cycle. Hence, CD44E can be utilized as an internal standard in assessing the amount of CD44 message by semi-quantitative PCR.
Figure 4:
Kinetic amplification of CD44 target cDNA
and competitor, CD44E. Equimolar amount (0.1 attomole) of target cDNA
() and competitor, CD44E (
) were co-amplified in the
presence of
-
S-labeled sense primer. An aliquot was
removed from each amplification cycle beginning from cycle 25 to 30.
The products were resolved on a 4% polyacrylamide gel. Following gel
electrophoresis, the products were excised and the amount of
radiaoctivity determined by scintillation
counting.
To
determine the relative increase in the amount of CD44 mRNA, aliquots of
cDNA from control, FN-f, and IL-1 samples were co-amplified with
serial dilutions of known amount of internal standard, CD44E. Fig. 5shows a representation of semi-quantitative PCR carried
out on 24-h samples that were coamplified using 2-fold serial dilutions
of the internal standard. The initial amounts of target and the
internal standard products are equal in those reactions where the molar
ratio of the scanned products are equal. Since the amount of internal
standard added to the PCR reaction is known, the initial amount of
target can be determined. The results from duplicate samples are that
FN-f treatment resulted in an 8-fold increase in CD44 message and
IL-1
treatment, a 6-fold increase.
Figure 5:
Semi-quantitative PCR analysis of the
effects of FN-f and IL-1 in CD44 mRNA. Total RNA (0.5 µg) from
24 h cultures was reverse transcribed and the resultant cDNA diluted.
An equivalent aliquot of each diluted cDNA sample was co-amplified with
2-fold serial dilutions of CD44E (0.00625-0.08 attomole) for 30
cycles. The products were separated on 1% agarose and visualized by
ethidium bromide staining. Lanes 1-5 represent control
cultures; lanes 6-10, FN-f-treated cultures; lanes
11-15, IL-1
-treated cultures. The CD44 products (arrow a, 872 bp; arrow b, 754 bp; arrow c,
600 bp) are indicated.
Figure 6:
Effects on FN-f and IL-1 on CD44
expression at the protein level. Total protein was extracted from
bovine chondrocytes cultured in the absence or presence of FN-f or
IL-1
for 3 days. Equivalent amount of total protein was separated
on 10% SDS-PAGE and transferred to nitrocellulose and probed with
either monoclonal antibody IM 7.8.1 or irrelevant rat isotype control
IgG
. Lanes 1 and 4 represent control
cultures; lanes 2 and 5, FN-f-treated cultures; lanes 3 and 6, IL-1
-treated cultures. Doublet
CD44 protein bands are indicated by arrows.
Figure 7:
Detection of CD44 mRNA by in situ hybridization. Bovine as well as human articular cartilage slices
were either fixed immediately following dissection with 4%
paraformaldehyde or cultured in DMEM/F-12 supplemented with 10% FBS in
the absence or presence of IL-1 for 48 h. The tissue slices were
processed for in situ hybridization whereby CD44 mRNA
expression was detected using S-labeled antisense CD44
probe. The photomicrographs depict: uncultured bovine articular
cartilage fixed immediately following dissection (panel A),
bovine articular cartilage cultured in DMEM/F-12 supplemented with
either 10% FBS (panel B) or 10% FBS and 1.0 ng/ml IL-1
(panel C), and human articular cartilage cultured in 10% FBS
and IL-1
(panel D). Magnification,
200. As
controls, tissue sections were hybridized with equimolar concentration
of
Slabeled and unlabeled antisense CD44 probes
(photomicrographs in panels A and C, inset).
Photomicrographs (Fig. 7, A and C, inset) depict slices incubated with
-
S-labeled antisense probe in the presence of an
equimolar concentration of unlabeled antisense probe. The unlabeled
probe, even though not in excess, successfully competed for the
majority of the staining associated with the chondrocytes. In situ hybridization results do not allow for accurate quantification of
IL-1
-induced enhancement in CD44 expression. Nonetheless, these
results are consistent with the alginate cell cultures studies
described above.
Since the antisense primer used for RT-PCR and in situ also anneals to human CD44 mRNA, slices of a sample of normal human cartilage taken from the ankle of a 54-year-old female donor, were processed for in situ hybridization as described for bovine tissue. Similar staining and localized of grain development was observed in sections of human cartilage tissue (Fig. 7D), as compared to the bovine tissue (Fig. 7, A-C). This suggests that human chondrocytes also express CD44 mRNA.
Previous studies demonstrated that the HA- and PG-rich pericellular matrix of chondrocytes is bound or tethered to the cell surface via interaction with specific HA binding sites, termed HA receptors(11, 36) . The HA receptors expressed on chondrocytes have properties similar to HA receptors present on many transformed cell types such as the human bladder carcinoma cell line, HCV-29T(37, 38) . In more recent studies, it has become evident that the HA receptors expressed on many tumor cells are identical to the lymphocyte homing receptor, CD44(38, 39, 40) . In an effort to better correlate CD44 with chondrocyte function, we demonstrated that COS-7 cells transfected with a plasmid containing the gene for CD44H gain the capacity to assemble chondrocyte-like pericellular matrices in the presence of HA and chondrocyte-derived aggregating PG(29) . In addition to the role of chondrocyte HA receptors in matrix assembly, these HA receptors also appear to participate in the catabolism of HA, mediated via a receptor-coupled endocytosis mechanism(18) . Under some conditions, HA receptor function in catabolism may predominate over its role in matrix assembly. Furthermore, the binding of HA to the surface of chondrocytes and its subsequent endocytosis can be blocked by anti-CD44 antibodies (18) . All of these results provide indirect evidence that on chondrocytes, as with other transformed cell types, CD44 functions as the primary receptor for HA. In the present study we demonstrate that bovine articular chondrocytes transcribe mRNA for CD44, which is then translated into functional CD44/HA receptor proteins at the cell surface. In addition, the expression of CD44/HA receptors is regulated by potent cellular mediators of chondrocyte metabolism.
The turnover of newly synthesized HA and PG in radiolabeled explant cultures has been shown to be co-ordinately regulated, with nearly identical half-lives in the range of 13-25 days(15, 16) . The turnover of aggrecan is believed to involve proteolytic cleavage within the interglobular domain of the core protein via an enzyme termed ``aggrecanase,'' and/or stromelysin, resulting in the release of the chondroitin sulfate rich domain from the cartilage(12, 14) . However, in the HA/PG turnover studies, HA degradation products were neither found in the tissue nor shed into the medium. These results, in addition to the lack of any detectable extracellular enzymatic activity toward HA(17) , have led to the hypothesis that HA turnover must occur via another mechanism, such as endocytosis and degradation within the chondrocyte itself. Previous work from our laboratory demonstrated that chondrocytes have the capacity to internalize HA via a receptor-mediated endocytosis mechanism, resulting in its complete degradation(18) . The binding and endocytosis of HA by bovine chondrocytes were inhibited, both by HA hexasaccharides and by IM 7.8.1 anti-CD44 monoclonal antibodies. A similar endocytosis mechanism, also inhibited by anti-CD44 antibodies, has been demonstrated in the catabolism of HA by macrophages(41) .
Cellular mediators
such as IL-1 trigger a cascade of intracellular signaling events that
result in an enhanced catabolic state of chondrocytes(42) .
This enhanced catabolic state is characterized by inhibition of
synthesis of matrix macromolecules, coupled with an increase in matrix
turnover. In this study it was demonstrated that IL-1 causes a
time-, as well as dose-dependent inhibition of PG synthesis. These
results are in agreement with the work of other
investigators(43, 44) . IL-1 also stimulates the
expression of proteases such as the matrix metalloproteinases
stromelysin (MMP-3) and vertebrate collagenase (MMP-1), as well as
tissue plasminogen activator(45, 46) , resulting in
enhanced degradation of matrix macromolecules(47) . Inhibition
of PG synthesis was also observed previously in bovine explant cultures
treated with specific concentration range of 29-kDa FN-f(48) .
In the present study, alginate cultures of isolated bovine articular
chondrocytes exhibit inhibition of PG synthesis upon treatment with
29-kDa FN-f within a similar concentration range. Other effects of FN-f
on chondrocytes mirror those observed for IL-1-induced catabolism,
including the expression of metalloproteinases and the resultant
degradation and release of PG from the cartilages(26) . It has
been postulated that some of FN-f catabolic effects are mediated via
endogenous IL-1, as FN-f have been shown to induce the release of IL-1
from chondrocytes (24) . Thus, both IL-1
and FN-f are
potent cellular mediators and were used in this study to determine the
effect of elevating the catabolic state of chondrocytes on the
expression of CD44/HA receptors.
We have developed a
semi-quantitative PCR procedure to better quantify relative changes in
CD44 mRNA expression. With this procedure, it was demonstrated that
chondrocytes cultured with FN-f had an 8-fold increase in CD44 mRNA,
while the message was augmented by 6-fold in IL-1-treated
chondrocytes. Nevertheless, it should be noted that this method
quantifies only the amount of cDNA present in a given sample. If the
efficiency of reverse transcription is less than 100%, this would
result in an underestimate of the total amount of mRNA present. Reverse
transcription-PCR of GAPDH mRNA was used to verify that the relative
amount of mRNA in each sample was equivalent. However, even though
little change in GAPDH product was observed from sample to sample (Fig. 2B), we cannot rule out the possibility that IL-1
or FN-f also affect the expression of GAPDH mRNA.
In addition to the
standard isoform of CD44, termed CD44H, additional isoforms of CD44
exist that are generated by the alternative splicing of 10 variant
exons(33) . In our chondrocyte culture system, we have detected
a minor CD44 product that is 154 bp larger than the major 600-bp
product, CD44H, and identified this product as CD44v10. Interestingly,
the mRNA for this 754-bp minor product was also stimulated by both
cellular mediators, IL-1 and FN-f. Restriction enzyme analysis
helped to confirm the minor band as an isoform of CD44 with the 154-bp
additional sequence at the predicted site for insertion of variant
exons (33) (i.e. within the 250-bp CD44H restriction
fragment; Fig. 3). Although variant isoform expression is
observed in some normal tissues(49, 50) , variant
isoform expression is most often associated with tumor progression,
particularly increase in metastatic
potential(51, 52) . Nonetheless, little is known about
the function or regulation of any of the variant CD44
isoforms(33) . The role of this CD44v10 isoform expressed by
chondrocytes will be the focus of further investigations.
By in
situ hybridization, unamplified CD44 mRNA was detected within
bovine as well as human articular cartilage. Detection in bovine
cartilage immediately fixed following dissection indicates that CD44
mRNA is being continuously expressed in normal adult tissue.
Nonetheless, organ culture of cartilage tissue slices in medium
containing only FBS resulted in a substantial increase of CD44 message (Fig. 7B). This expression was further elevated by the
addition of IL-1 to the organ cultures (Fig. 7C).
These results document the presence of CD44 mRNA in chondrocytes within
their native matrix environment. The observations are consistent with,
and support, our results obtained by RT-PCR of RNA derived from
cultured bovine articular chondrocytes. In addition, the in situ hybridization results suggest that CD44 mRNA expression by
chondrocytes is not uniform within the tissue. This was most evident in
the example of human articular cartilage shown in Fig. 7D. The expression appears to be enhanced in the
cells of the superficial layer as compared to cells within deeper
layers. This may reflect the differential role of superficial
chondrocytes in matrix catabolism, differential responsiveness of
superficial cells to cellular mediators, or inherent differences in
chondrocyte metabolism within the different layers(20) .
Based on the nucleotide sequence of CD44, a transmembrane protein of
37 kDa is predicted. However, because the protein contains a high
degree of N- and O-linked carbohydrate substitution,
its apparent molecular mass is typically estimated as approximately 85
kDa, depending on the cell type studied(53, 54) . By
Western blot analysis, doublet bands, within the range of 80-90
kDa, were detected using either of the anti-CD44 monoclonal antibodies
IM 7.8.1 or KM 201. Both of these antibodies were raised against murine
lymphocyte CD44 but have been shown to block the putative CD44-mediated
functions on bovine chondrocytes(18) . In a recent study,
Mikecz et al. determined the epitope on murine CD44 recognized
by the IM 7.8.1 antibody(55) . This 13-amino acid epitope is
also present in the predicted bovine CD44 sequence with three
conservative amino acid substitutions. It is therefore likely that this
antibody does, in fact, specifically recognize bovine CD44. The nature
of the doublet CD44 bands observed in extracts of bovine chondrocytes
remains to be determined. The bands may represent identical gene
products that vary in post-translational glycosylation or processing.
Alternatively, they may represent expression of two different CD44
mRNAs (e.g. expression of CD44H and CD44v10). On the other
hand, no higher molecular mass CD44 bands indicative of
glycosaminoglycan chain addition were apparent in the detergent
extracts of bovine chondrocytes, as has been observed in other cell
types(51, 56) . Treatment with either FN-f or
IL-1 resulted in a time-dependent increase in the expression of
the doublet CD44 bands. Western blot quantification of CD44
immunoreactive protein (Fig. 6) paralleled closely the onset of
CD44 mRNA up-regulation by these cellular mediators. These results
would suggest that 1) there is little intracellularly stored CD44
protein available to respond rapidly to mediator-induced changes in
chondrocyte metabolism, and (2) chondrocytes have little stored
mRNA for CD44 protein enhancement. It is therefore likely that
transcriptional controls regulate CD44 expression.
Osteoarthritis is a degenerative disease characterized by an imbalance in chondrocyte metabolism(19, 57) . Catabolic processes are thought to exceed biosynthesis, resulting in depletion of critical components of the extracellular matrix. Many of these metabolic characteristics can be modelled experimentally by treatment of chondrocytes or intact cartilage with cellular mediators such as FN-f or IL-1. We have suggested in previous work that CD44/HA receptors participate in the turnover and degradation of HA(18) . If this hypothesis is correct, elevating the catabolic state of chondrocytes should result in enhanced HA turnover. We would also predict that CD44 expression will be elevated in human osteoarthritic cartilage. The application of in situ hybridization techniques to the study of human cartilage will help elucidate this hypothesis. In the present study, we observe that both FN-f and IL-1 treatment results in increased mRNA for CD44, increased CD44 protein and increased HA binding capacity at the surface of chondrocytes. In addition, the amount of fl-HA that accumulated within the treated chondrocytes was also increased (Table 2). Additional studies will be required to determine whether increased expression of CD44 actually affects an increased capacity and/or increased rate of HA endocytosis. Nonetheless, the increased accumulation of fl-HA suggests that more HA is being internalized, destined for degradation within the lysosomal compartment. CD44/HA receptors thus represent part of the delivery mechanism used by chondrocytes to bring extracellular HA into intracellular organelles. In future studies we will investigate whether treatment of chondrocytes with cellular mediators results in changes in the levels of lysosomal hyaluronidase activity involved in the actual degradation of internalized HA.