Journal of Histochemistry and Cytochemistry, Vol. 46, 641-652, May 1998, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

An Essential Role for the Interaction Between Hyaluronan and Hyaluronan Binding Proteins During Joint Development

Gary P. Dowthwaitea, Jo C. W. Edwardsb, and Andrew A. Pitsillidesa
a Department of Veterinary Basic Sciences, The Royal Veterinary College, University of London, London, United Kingdom
b Department of Rheumatology Research, Faculty of Clinical Sciences, University College London,, London, United Kingdom

Correspondence to: Gary P. Dowthwaite, Dept. of Veterinary Basic Sciences, The Royal Veterinary College, University of London, London NW1 0TU, UK.


  Summary
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Summary
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Materials and Methods
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Discussion
Literature Cited

We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HA–HABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HA–HABP interactions play a major role in the initial separation events occurring during joint formation. (J Histochem Cytochem 46:641–651, 1998)

Key Words: hyaluronan, CD44, joint development, actin capping proteins, chick, immunocytochemistry


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The development of diarthrodial joints has been divided into two basic phases, the initial formation of the cartilaginous anlagen and the subsequent formation of the joint space and its associated synovial lining between these anlagen (for review see Archer et al. 1994 ). It is widely accepted that the specification of sites of both cartilaginous differentiation and joint formation are genetically determined (Archer et al. 1994 ). However, although many mechanisms have been proposed to account for the initial separation of opposed cartilage elements during the process of joint cavitation (Millaire 1947 ; Andersen and Bro-Rasmusen 1961; Mitrovic 1971 , Mitrovic 1972 , Mitrovic 1974 ; Nalin et al. 1995 ), the precise nature of joint morphogenesis remains enigmatic.

In adult joints the cavity is filled with viscous synovial fluid, which contains a high concentration of hyaluronan (HA) (Balazs et al. 1967 ). We have shown that the synovial lining cells of normal adult joints contain high activity of uridine diphosphoglucose dehydrogenase (UDPGD), an essential enzyme required for HA production (DeLuca et al. 1975 , DeLuca et al. 1976 ,1984) compared with the subintimal synovial cells (Pitsillides and Blake 1992 ; Wilkinson et al. 1992 ; Pitsillides et al. 1993 ), suggesting that in adult joints the phenotype of the synovial lining cells is related to their proposed function in HA synthesis (Pitsillides et al. 1993 ).

Subsequently, we found that during embryonic joint development, cells at the developing articular surfaces of both human and chick joints resemble those found in adult synovium. They contain elevated UDPGD activity per cell, as well as elevated immunocytochemical labeling for molecules associated with HA synthesis, compared with neighboring epiphyseal chondrocytes (Edwards et al. 1994 ; Pitsillides et al. 1995 ). Furthermore, use of the biotinylated HA binding region has shown that increased levels of histochemical labeling for free HA is predominant within the developing interzone before cavitation and within the cavity itself after joint cavity formation (Craig et al. 1990 ; Pitsillides et al. 1995 ). These results suggest that joint cavity development may depend, in part, on the specific differentiation of cells situated at the borders of presumptive joint spaces to a phenotype characteristically associated with the differential synthesis of increased levels of HA (Pitsillides et al. 1995 ).

Previous studies have shown that HA in conjunction with cell surface HA binding proteins (HABPs) is capable of facilitating cell separation in vitro (Toole 1981 ). Briefly, it was suggested that variations in local concentrations of extracellular HA are capable of promoting either cell aggregation (at low HA concentrations) or cell separation (at high HA concentrations) between adjacent cells expressing HABPs by a process involving HABP saturation (Toole 1981 ). We have recently shown that, in addition to increased UDPGD activity and other indicators of elevated HA synthesis, cells at the articular surface of developing human joints also express the cell surface HABP CD44 (Edwards et al. 1994 ). As a consequence, we postulated that the differential synthesis of HA and its association with cell surface HABPs plays a primary role in synovial joint cavitation and is a prerequisite for continued joint function.

The aim of this study was to establish the temporospatial expression of several cell surface HABPs during the process of joint cavitation in developing chick joints, and to determine the potential ligand binding status of these HABPs using biotinylated HA (Melrose et al. 1996 ). Moreover, on the basis of observations demonstrating that the interaction between the actin cytoskeleton and HABPs is a prerequisite for effective ligand binding (Isacke 1994 ), we also describe the distribution of a family of actin capping, CD44-linking proteins, i.e., ezrin, radixin, and moesin (collectively referred to as the ERM proteins) (Sato et al. 1992 ; Tsukita et al. 1994), and that of polymerized actin itself, during diarthrodial joint formation in chick embryos.

Finally, because it is known that small HA chains containing less than six disaccharide repeats are capable of displacing the HA-containing pericellular coat of cultured chondrocytes (Knudson and Knudson 1991 ), the effect of exogenously applied oligosaccharides of HA (HAO) on the formation of joint cavities was assessed using a slow-release bead delivery system in ovo.


  Materials and Methods
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Materials and Methods
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Tissue Preparation
Hind limbs (n = 6 from each stage) were removed from white leghorn chick embryos between Days 6 and 18 (stages 30–44) (Hamburger and Hamilton 1951 ), allowing knee, metatarsal–phalangeal (MTP), and proximal and distal interphalangeal (PIP and DIP) joints to be examined. Tissues were processed for immunocytochemistry as previously described (Pitsillides et al. 1995 ).

Immunocytochemical Staining for HABPs and ERM Family Members
Primary Antibodies. A panel of monoclonal and polyclonal antibodies to various HABPs and ERM family members was used in this study (Table 1). Antibody 30189 (a kind gift from Dr. S. Tsukita, Kyoto University, Japan) was raised to CD44 isolated from baby hamster kidney cells (Tsukita et al. 1994). The monoclonal antibody IVd4 (a kind gift from Prof. Bryan Toole, Tufts University, Boston, MA) was raised against an HABP isolated from embryonic chick brain (Banerjee and Toole 1991 ). Antibody R7.2 (a kind gift from Prof. Eva A. Turley, Institute of Cell Biology, Manitoba, Canada) was raised against a 12-peptide sequence of rabbit receptor for HA-mediated migration (RHAMM) (Nagy et al. 1995 ). Antibodies CR22, M11, and M22 (gifts from Dr. S. Tsukita) were raised against baby hamster kidney cell ERM proteins (Tsukita et al. 1989; Sato et al. 1991 ). Finally, polyclonal antibodies 454, 457, and 464 raised against mouse radixin (454)-, ezrin (457)-, and moesin (464)-specific peptides were obtained from Dr. Frank Solomon (Massachusetts Institute of Technology, Boston, MA) (Winkler et al. 1994 ).


 
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Table 1. Antibodies used to localize HABPs and ERM family members in cavitating chick joints

Immunocytochemistry. Immediately before immunolabeling, cryostat sections were dried under a fan for 30 min at room temperature (RT) and rinsed briefly with Tris-buffered saline (TBS, pH 7.2; Sigma, St Louis, MO) for 5 min. Antibodies were then applied after the optimal antibody dilution and incubation times had been determined (see Table 1). Sections were then washed in TBS (three times for 5 min) and appropriate fluoroscein isothiocyanate (FITC)-conjugated secondary antibodies (Sigma) diluted 20% in heat-inactivated chick serum (Gibco; Paisley, UK) in TBS were applied for 1 hr at RT. Subsequently, sections were washed with TBS (three times for 5 min) and mounted in 0.05 M Tris buffer (pH 8.5), containing 33% glycerol, 15% polyvinyl alcohol (Sigma), and 2.5% DABCO (1,4,diazobicyclo[2.2.2.] octane; Sigma) to reduce bleaching of fluorescence. Controls consisted of sections treated with TBS or appropriate nonimmune immunoglobulins instead of primary antibody.

Localization of Actin Filaments
Polymerized actin filaments were localized using 1 µg/ml FITC-conjugated phalloidin (in TBS; Sigma) applied to sections for 30 min at RT and washed in TBS (three times for 5 minutes). Thereafter, nuclei were counterstained by incubating sections with propidium iodide (0.5 µg/ml in TBS; Sigma) for 2 min and were subsequently washed in TBS (three times for 5 minutes) and mounted in DABCO. Dual labeled sections were examined using a Molecular Dynamics (Sunnyvale, CA) Sarastro 2000 laser scanning confocal microscope.

Detection of HA Binding Sites Using Biotinylated HA
Biotinylated HA (bHA) was a kind gift from Dr. Jim Melrose (University of Sydney, Australia) (Melrose et al. 1996 ). After wetting in TBS–Tween for 5 min, total HA binding sites within tissue sections were disclosed by incubation with 20 U/ml Streptomyces hyaluronidase (Sigma) in TBS, pH 6.0, at 37C for 1 hr. Comparison of such hyaluronidase pretreated sections (total HA binding sites) with adjacent serial sections incubated with bHA without hyaluronidase pretreatment (free HA binding sites) allowed the distribution of occupied and unoccupied HA binding sites to be identified. Sections were then washed in TBS (three times for 5 mins) and incubated overnight with 100 µg/ml bHA at RT. Sections were then washed in TBS (three times for 5 min) and incubated with alkaline phosphatase-conjugated streptavidin (DAKO; Buckinghamshire, UK), reacted for alkaline phosphatase activity as described by Pitsillides et al. 1995 , and mounted in Aquamount (Merck; Leicestershire, UK). Controls consisted of sections treated with TBS instead of bHA, both before and after hyaluronidase digestion.

Manipulation of Joint Cavity Formation Using Oligosaccharides of HA
HA oligosaccharides (HAO) with no more than six disaccharides per chain were prepared as previously described (Kumar and West 1987 ) and were used at a concentration of 1 mg/ml. Agarose AG 1 2AX beads (Sigma) were derivatized overnight in 1 N formic acid, washed extensively in distilled water, and stored at 4C until use (Tickle et al. 1982 ).

White Leghorn chick embryos were windowed at Stage 24, the windows resealed using transparent adhesive tape, and replaced in the incubator at 39C. At Stage 30, windows were reopened and agarose beads, previously soaked for 1 hr in 50 µl of 1 mg/ml HAO and washed in PBS containing 0.1% phenol red for 15 min, were applied to wounds cut in the presumptive knee joints of the right leg after exposure of the limb by careful dissection of the extraembryonic membranes. Control experiments consisted of contralateral limbs, limbs into which a PBS-soaked bead were implanted, sham-operated limbs, and unoperated (i.e., normal) limbs. After application of the bead, the extraembryonic membranes were carefully closed over the embryo and the window resealed with transparent adhesive tape. Embryos were replaced in the incubator at 39C and allowed to develop for a further 3 days (up to Stage 36), after which knee joints were removed, chilled, and sectioned as described above.

Sagittal cryosections were stained with 0.1% toluidine blue in 0.1 M acetate buffer (pH 6.0) and examined by routine microscopy to histologically assess the effect of HAO bead application on joint cavitation compared with controls. Sections were also labeled with antibody (30189) to CD44, and actin filament expression was observed using FITC-conjugated phalloidin as described above.

Assessment of UDPGD Activity in HAO Beaded Limbs and Controls
Limbs were reacted and assesssed microdensitometrically for UDPGD activity as described by Pitsillides et al. 1995 . To assess alterations in UDPGD activity associated with joint cavity manipulation, microdensitometric measurements were made in 20 cells within joint fibrocartilage (controls), epiphyseal chondrocytes (controls and experimental), and within the site of fusion in experimental limbs. These measurements were converted into units of mean integrated extinction (MIE) x 100/cell/45 min reaction time. Results were expressed as mean UDPGD activity/cell ± SEM and compared using Students t-test.


  Results
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Summary
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Materials and Methods
Results
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Distribution of Cell Surface HABPs
All joints examined (knee, MTP, and interphalangeal joints) exhibited the same distribution of CD44 (as shown by immunolabeling with antibody 30189) during the process of joint cavitation. Before and during joint cavitation, intense labeling for CD44 was observed on cells of the interzone and developing articular fibrocartilages, with the most intense label associated with the developing articular fibrocartilages, whereas the epiphyseal chondrocytes were weakly labeled (Figure 1A). On completion of the cavitation process, a decreasing gradient of labeling intensity was evident, from the most intensely labeled surface cells of the articular fibrocartilage to less intense labeling in the deeper regions of articular fibrocartilage and finally weak labeling in epiphyseal chondrocytes (Figure 1D).



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Figure 1. Immunocytochemical labeling of HABPs before (St. 40; A–C) and after (St. 44; D–F) cavitation of the MTP joint. Label for CD44 (A, D) is present in the interzone (iz), the cells of the developing articular fibrocartilage (af), and weak label is present in the chondrocytes of the epiphysis (e). RHAMM (B,E) is present in the cells of the interzone, articular fibrocartilage, and chondrocytes of the epiphysis. There is less differential labeling intensity between the cells of both the interzone and articular fibrocartilage and the epiphyseal chondrocytes compared with CD44. The IVd4 epitope (C,F) is also present within the cells of the interzone, articular fibrocartilage, and epiphyseal chondrocytes. Labeling for the IVd4 epitope becomes more restricted to the articular fibrocartilage after cavitation (F). The proximal element (metatarsus) is towards the bottom and the distal element (first phalange) is towards the top in all figures. Bars = 50 µm.

Figure 2. Immunocytochemical labeling of the MTP joint using monoclonal antibodies to ezrin (A) and radixin (B), showing no immunoreactivity in the joint region. Immunolabeling with antibody M22 reveals that moesin is expressed in the cells of the interzone (iz) and developing articular fibrocartilage before cavitation (St. 40; C) and in the articular fibrocartilage after cavitation (St. 44; D) of the MTP joint. Bars = 50 µm.

Before, during, and after formation of the joint cavity, label for the RHAMM epitope (antibody R7.2) was also intense in the cells of the interzone and developing articular surfaces (Figure 1B and Figure 1E). However, in contrast to the marked differential labeling intensity evident for CD44, labeling for RHAMM in the epiphyseal chondrocytes was only slightly less intense than in the articular fibrocartilage (Figure 1E).

The IVd4 epitope was also detected within the cells of the interzone and developing articular fibrocartilage before, during, and after cavitation (Figure 1C and Figure 1F). As with the RHAMM antibody, labeling in the epihyseal chondrocytes was only slightly less intense than that seen in the articular fibrocartilage (Figure 1F). Therefore, the differential labeling intensity observed between the cells bordering the developing joint cavity and their neighboring epiphyseal chondrocytes was most marked for CD44. Control sections treated with the appropriate nonimmune serum or without primary antibody in the initial incubation and examined using routine microscopy showed no labeling (data not shown).

Immunocytochemical Labeling for ERM Family Members
Within the developing anlagen, immunocytochemical labeling using a pan-specific antibody (CR22) recognizing all members of the ERM family was concentrated at the presumptive joint line in the interzone and developing fibrocartilage before cavitation, and this differential distribution was maintained in articular fibrocartilage during subsequent stages of joint morphogenesis (data not shown). Using antibodies specific to individual members of the ERM family of proteins, we found that moesin (antibodies M22 and 454) was preferentially expressed by cells of the interzone and articular fibrocartilage before cavitation (Figure 2C) and that expression of moesin was maintained in the articular fibrocartilage after cavitation (Figure 2D). At all stages during cavitation, weak labeling was evident for moesin in neighboring epiphyseal chondrocytes (Figure 2C and Figure 2D). Although failure to detect specific proteins immunocytochemically is not necessarily absolute evidence of their absence, label for the other members of the ERM family, i.e., ezrin and radixin (antibodies 464 and 457, respectively), was not present at the joint line or within epiphyseal chondrocytes (Figure 2A and Figure 2B).

Localization of Hyaluronan Binding Sites
Disclosure of occupied HA binding sites after hyaluronidase pretreatment of sections (total HA binding sites) showed intense cell-associated pericellular labeling within the developing fibrocartilaginous articular surfaces and, to a lesser extent, in the interzone before cavitation (Figure 3A). There was very weak labeling with bHA in the epiphyseal chondrocytes after hyaluronidase pretreatment (Figure 3A). After cavitation, binding of bHA in hyaluronidase-pretreated sections was most prominent in chondrocytes of the epiphysis, which showed progressively weaker labeling intensity in the articular fibrocartilage, although some label was apparent on the surface cells of the fibrocartilage (Figure 3B). In contrast, sections treated with bHA without prior hyaluronidase treatment showed very little if any staining for freely available (unoccupied) HA binding sites within the interzone or the fibrocartilage at any stage examined before, during, or after cavitation. Similarly, under these conditions extremely low levels of bHA binding were observed in the cartilage matrix (data not shown). Control sections treated with TBS instead of bHA showed no staining (data not shown).



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Figure 3. Binding of bHA to tissue sections after hyaluronidase treatment reveals strong binding at the developing articular fibrocartilage (af) and interzone (iz) before cavitation (St. 40; A) of the MTP joint. There is relatively weak pericellular and matrix staining in the epiphysis (e). After cavitation of the MTP joint (St. 44; B), bHA binds weakly to the surface of the articular fibrocartilage (arrows) and very little label is present in the deeper fibrocartilage (arrowheads). Note the strong binding of bHA to epiphyseal chondrocytes and the territorial matrix of the epiphysis. Bars = 50 µm.

Actin Filament Distribution in Cavitating Joints
Using dual labeled sections and confocal microscopy, intense staining for filamentous actin was observed in the cells of the developing interzone and fibrocartilaginous articular surfaces before, during, and after cavitation (Figure 4A and Figure 4B). Epiphyseal chondrocytes also stained for actin, but the staining intensity in these cells was much weaker than in the cells at the joint line (Figure 4A and Figure 4B). Label within interzone and fibrocartilage cells appeared linear, extending away from the nuclei and following the plane of joint cavitation, whereas the weak labeling in chondrocytes extended away from the nuclei but did not appear to follow the plane of cavitation (Figure 4A and Figure 4B).



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Figure 4. Confocal microscopy in conjunction with fluoroscein-conjugated phalloidin reveals bundles of actin filaments (arrows) in the interzone (iz) and at the developing articular fibrocartilage (af) before (St 40; A) and after (St. 44; B) cavitation of the MTP joint. Note that actin filament bundles extend from the nuclei (stained red with propidium iodide) and follow the plane of cavitation across the middle of the field of view. The deeper fibrocartilage cells and the chondrocytes of the epiphysis (e) label less intensely for actin and the filaments do not follow the plane of cavitation (arrowheads) Bars = 10 µm.

Effect of Oligosaccharides of HA on Joint Cavitation
From a total of 24 embryos implanted with HAO-soaked beads, seven embryos survived to Stage 36/37 (29% survival). This low survival rate is probably due to the late stage at which the manipulations were performed because PBS-beaded and sham-operated embryos both had similarly low survival rates (30 and 31%, respectively). Histological examination of the seven HAO-treated embryos revealed disruption of cavitation in six embryos and no effect in the other one (85% joint disruption). This disruption was noted histologically as a loss of the demarcation between the developing articular fibrocartilage and the interzone and the lack of an obvious cavity between the femur and the intervening menisci (Figure 5A and Figure 5B). Compared with control limbs (PBS-beaded, sham-operated, and contralaterals), the cartilage anlagen of the femur and tibia of manipulated limbs were separated by a loose mass of "mesenchymal cells" with no distinct developing articular fibrocartilage and no development of the joint cavity. There was little evidence of differentiation of the menisci in manipulated limbs.



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Figure 5. Toluidine blue-stained sections from control (A) and experimental (B) knees show the differences in the development of the knee joint. Cavitation (arrows) has commenced between the developing menisci (m), femoral condyle (f), and tibial plateau (t) in the control animal, whereas the "cavity" in HAO-treated limbs contains a loose mass of mesenchymal cells (asterisks). Bars = 100 µm. When control (C) and experimental (F) animals are immunolabeled for CD44, there is intense labeling in the articular fibrocartilage (af) of controls compared with the fused region (fr) of experimental animals. Actin filaments label intensely at the joint line of control animals (D), whereas experimental animals are much less intensely labeled in the fused region (G). Sections of developing knee joints from control (E) and HAO-treated (H) limbs reacted for UDPGD activity. Note the intense activity in the cells of the fibrocartilaginous articular surface (af) of controls and the decreased activity in the fused region (fr) of HAO-treated limbs at comparable sites. Bars = 50 µm.

With the anti-CD44 antibody 30189, there was less intense immunolabeling for CD44 in manipulated limbs compared with control limbs (Figure 5C and Figure 5F), and there was decreased labeling of actin filaments with FITC–phalloidin in manipulated limbs (Figure 5D and Figure 5G).

Assessment of UDPGD Activity in Manipulated and Control Limbs
The UDPGD activity/cell at various sites in experimental and control limbs is shown in Figure 6. Control limbs showed significantly (p=0.01) higher UDPGD activity in articular fibrocartilage cells than in the femoral or tibial chondrocytes (Figure 5E and Figure 5H), whereas UDPGD activity in these control epiphyseal chondrocytes was not significantly different from that in epiphyseal chondrocytes of HAO-treated limbs (p>0.05). However, although UDPGD activity in sites of joint fusion in HAO-treated limbs was significantly higher than the UDPGD activity in the epiphyseal chondrocytes of the same limbs (p=0.001), it was also significantly decreased compared with UDPGD activity found in control articular fibrocartilage cells (p=0.01).



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Figure 6. Effect of HAO application on UDPGD per cell (± SEM) at various sites in the femur and tibia of control and HAO-treated limbs at stage 36. *p = 0.01 compared with activity in epiphyseal chondrocytes of the same limb; NS, p>0.05 relative to epiphyseal chondrocytes in control limbs; **p = 0.001 compared with activity in epiphyseal chondrocytes in HAO-treated limbs; and ***p<0.01 compared with activity in cells of articular fibrocartilage in control limbs.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Our findings indicate that HABPs play a central role during synovial joint formation and that their interaction with HA may exert a primary influence coordinating tissue separation at these sites. We have demonstrated (a) that three cell surface HABPs, i.e., CD44, RHAMM, and the IVd4 epitope, are present at sites of joint formation in embryonic chick limbs, (b) that CD44 is co-expressed with moesin and linear bundles of filamentous actin in a joint line-selective distribution that is closely associated with cavitation, (c) that occupied HA binding sites are present at these locations during but not after cavitation, and finally (d) that application of HAOs close to sites of presump-tive cavitation disrupts joint cavity formation and is associated with local decreases in UDPGD activity/cell and with the expression of CD44 and filamentous actin.

It has been shown that low HA concentrations may promote adhesive interactions between adjacent HABP-expressing cells. Conversely, increased HA concentrations may facilitate cell–cell separation by a process involving saturation of their cell surface-associated HABPs (Toole 1981 ). Our findings demonstrate that several HABPs, i.e., CD44, RHAMM, and the IVd4 epitope, appear to be present at sites of joint cavity formation in chick limbs. Of these, CD44 shows the most marked joint line-selective distribution, with the most intense labeling evident in cells at the joint interzone before cavitation and at the developing articular surfaces during and after cavitation. In comparison, neighboring epiphyseal chondrocytes at all stages contained relatively little CD44 labeling. The HA binding role of CD44 is well established (Sherman et al. 1994 ) and its localization at the joint line, along with RHAMM and IVd4 epitopes, supports the hypothesis that HA–HABP interactions play an important role in the formation and subsequent maintenance of synovial joint cavities.

Of these HABPs, only CD44 contains an aggrecan-like link module, HA binding domain (Neame and Barry 1993 ), and although our results do not exclude the involvement of RHAMM and IVd4 binding epitopes, they do suggest that CD44 exerts a dominant role in the events that culminate in loss of cohesion at this site. Nevertheless, RHAMM and IVd4 epitopes have been ascribed additional roles, raising the possibility that they may contribute to, or indeed control, wider aspects of joint formation. Recently, the IVd4 epitope was found to possess mRNA sequence homology to the yeast cell cycle control protein cdc37 (Grammatikas et al. 1995), suggesting that interaction between locally synthesized HA and these IVd4 epitopes may regulate proliferation at the presumptive joint line which, by affecting local growth rates, may contribute indirectly to the cavitation process. A role for RHAMM in HA-mediated motility has been clearly established in vitro (Turley et al. 1990 ), and its expression in the joint interzone suggests that it may direct cell migration from this site to either, or both, developing articular surfaces and synovium. This is consistent with studies indicating that, during blastocoel formation, HA not only provides a substrate for migration but also fills the remaining space after migration is completed (Fisher and Solursh 1977 ).

CD44 can exist in one of three distinct HA binding states: inactive, activatable, and constitutively active (Perschl et al. 1995 ). Therefore, it is evident that although immunolabeling may describe HABP distribution, it provides no information on functional HA binding status. Using biotin-labeled HA, we have shown that cells in joint interzones, but not those in fibrocartilaginous articular surfaces of fully cavitated joints, do indeed exhibit HA binding ability. This supports our earlier studies which indicated that labeling for free HA, using a biotinylated aggrecan HA binding region, was negligible in fibrocartilaginous articular surfaces, whereas all other tissues lining joint cavities were intensely labeled (Pitsillides et al. 1995 ). Therefore, cells associated with a loss of cohesion at developing joint lines show abundant CD44 expression, HA binding capacity, and an ECM containing abundant free HA. In contrast, cells in the structurally coherent fibrocartilaginous articular surfaces of established joints show less CD44 expression, little HA binding capacity, and an ECM with low levels of free HA, suggesting that the initial cell separation which occurs during joint cavity formation may involve interaction between CD44 and HA. This differs from the markedly enhanced hyaluronidase-induced disclosure of HA binding sites within the territorial matrix of epiphyseal chondrocytes after joint formation (see Figure 3B), which may reflect the presence of link protein- or aggrecan-associated bHA binding within the cartilage matrix.

The "active" HA binding role for CD44 at sites of initial tissue separation is also strengthened by our findings regarding the distribution of the ERM protein family members and filamentous actin. We have shown that interzone cells label strongly with a pan-specific antibody to these actin capping ERMs, and that one ERM family member in particular, i.e., moesin, exhibits a marked differential expression at presumptive and forming joint lines. Moreover, our confocal microscopic analyses using FITC-conjugated phalloidin also revealed a specific alignment of polymerized filamentous actin parallel to developing joint surfaces, and indicate that the intensity of staining as well as the degree of specific alignment is most obvious in cells directly bordering sites of presumptive and recently formed cavities. In contrast, the neighboring epiphyseal chondrocytes show reduced levels of staining that lacks such regular or specific alignment.

Many studies suggest that interactions between actin cytoskeletal elements and cell surface HABPs are a prerequisite for effective ligand binding (Lacy and Underhill 1987 ; Isacke 1994 ; Perschl et al. 1995 ). Furthermore, HA binding to CD44 also appears to be regulated by ERM proteins (Tsukita et al. 1994), which co-localize with barbed ends of actin filaments in a manner suggesting that they ensure the association of actin with cell surface proteins (Sato et al. 1992 ). Therefore, the presence of polymerized actin and concomitant co-distribution of moesin at sites of joint cavity formation suggest that cells at these sites are functionally engaged in binding to HA. In addition, the presence of linear arrays of actin filament bundles at the presumptive site of cavitation suggests that initial tissue separation occurs at predefined sites within the apparently structurally homogenous interzone. Although we cannot necessarily exclude the possibility that cavitation sites are acquired in response to mechanically engendered stimuli, our results nevertheless suggest that "potential cleavage planes" may be predetermined within a designated population of interzone cells possessing a specific actin filament alignment.

Using chondrocytes, fibrosarcoma and endothelial cells (Banerjee and Toole 1992 ; Yu et al. 1992 ) as well as 3T3 cells (Underhill 1982 ), it has been shown that HAO (hexasaccharides) can block the association of nascent pericellular HA with its cell surface-associated HABPs. We found that joints close to sites of HAO-soaked bead implantation failed to form an overt cavity and that cells in these "fused" areas adopted a rounded phenotype with reduced CD44 and actin expression, and, most markedly, decreases in UDPGD activity. It has also been shown that high exogenous HA concentrations decrease its synthesis by chondrocytes, suggesting that HABP occupancy or, more precisely, their saturation inhibits HA synthesis (Mason et al. 1989 ). Therefore, HAOs may not only disrupt nascent HA–HABP interactions but may also decrease HA synthesis by a process involving cell surface HABP saturation. This is supported by the marked local decreases in the activity of UDPGD at these sites.

Our results indicate that HAO application also results in structural loss of coherence in developing fibrocartilaginous articular surfaces. However, because these cells have neither HA binding capacity (even after hyaluronidase digestion) nor an association with an ECM containing high HA levels, it is apparent that HAOs produce loss of fibrocartilage structural integrity by a mechanism distinct from that apparent at the site of cavity formation. Interestingly, HAO beading failed to disrupt the integrity of epiphyseal cartilage, suggesting that HAOs either failed to reach the epiphyseal region or that HA–HABP interactions in these epiphyses are less sensitive to HAO-mediated disruption. Indeed, the size of the HAOs used in this study (less than 12 disaccharides) is insufficient to disrupt HA–aggrecan interactions (Kimura et al. 1980 ).

In conclusion, on the basis of a close temporospatial association between increased local HA synthesis (Pitsillides et al. 1995 ) and its cell surface binding proteins, particularly CD44, these findings suggest that HABP interactions are involved in joint cavitation in the developing limb. The arrays of actin filaments, spatially coordinated expression of the actin capping ERM protein moesin, and increased availability of HA binding sites at the presumptive joint line also indicate an active HA binding role for HABPs at the site of initial cavity formation. Moreover, the presence of arrays of actin filament bundles within the interzone provides evidence for the establishment of distinct and structurally defined planes of potential separation before overt cavity formation. Finally, HAO-mediated disruption of the cavitation process suggests that HA–HABP interactions have an essential role during the initial loss of cohesion at the forming diarthrodial space in developing joints.


  Acknowledgments

We wish to thank Prof Bryan Toole, Prof Eva Turley, Dr Sochiro Tsukita, and Dr Frank Solomon for kindly supplying primary antibodies, Dr Jim Melrose for supplying biotinylated hyaluronan, and Dr Mike Bayliss for the supply of HAOs. GPD was supported by the Arthritis and Rheumatism Council of Great Britain. For the use of the confocal microscopy facilities at the CTBL, University of Wales, Cardiff, we wish to thank Dr Jim Ralphs.

Received for publication June 11, 1997; accepted December 10, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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