ARTICLE |
Correspondence to: Cheryl B. Knudson, Dept. of Biochemistry, Rush Medical College, 1653 W. Congress Parkway, Chicago, IL 60612. E-mail: cknudson@rush.edu
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Summary |
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The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure. (J Histochem Cytochem 50:13131323, 2002)
Key Words: cartilage, chondrocytes, proteoglycans, hyaluronan, CD44, latrunculin, cytochalasin, matrix assembly
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Introduction |
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Cellmatrix interactions are crucial for maintaining cartilage homeostasis. At the cell surface, matrix receptors link the extracellular matrix (ECM) to the cell interior through elements of the cytoskeleton and other component proteins of signal transduction pathways. Binding of matrix components to cell surface receptors also establishes a pericellular pool of ECM molecules that may stabilize the cell phenotype. One such cell receptor is the glycoprotein CD44, the principal hyaluronan receptor expressed by chondrocytes (
The cortical cytoskeleton of chondrocytes helps to maintain phenotypic stability, organize the distribution of the cell organelles, and interact with the membrane proteins. On ligand binding, the interaction of certain membrane receptors with the cytoskeletal proteins sometimes brings about a change in the organization of the cytoskeleton. The punctate peripheral staining of actin observed in chondrocytes increased in organization with matrix assembly during culture in agarose culture (
The objective of this study was to determine the role of the actin cytoskeleton in CD44hyaluronan-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular chondrocytes cultured in alginate beads and adult bovine articular cartilage tissue slices in explant cultures were treated with latrunculin A or dihydrocytochalasin B. CD44 expression determined by flow cytometry, the detergent solubility of CD44, and hyaluronan binding were examined. Pericellular matrix assembly was monitored and histological assessment of cartilage was performed. Although disruption of the cytoskeleton did not alter cell surface CD44 expression, detergent extraction of CD44 increased. In addition, there was a reduction in hyaluronan binding, pericellular matrix assembly, and cartilage matrix retention. Therefore, disruption of the cytoskeleton is apparently another mechanism by which uncoupling chondrocytes from the matrix results in changes in chondrocyte metabolism and deleterious changes in matrix structure. The direct interaction of chondrocytes with their matrix is vital for maintenance of cartilage homeostasis.
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Materials and Methods |
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Cell Culture
Bovine cartilage was collected from the metacarpophalangeal joints of 18-month-old animals obtained from a local slaughterhouse. For explant cultures, the cartilage was dissected into 1-cm2 slices; 25 mg was the average wet weight per tissue slice. Alternatively, the chondrocytes were isolated with a sequential 1-hr treatment with 0.2% Pronase (Calbiochem; La Jolla, CA) followed by an overnight treatment with 0.025% collagenase P (BoehringerMannheim; Indianapolis, IN) in Ham's F-12 medium (Cellgro; Mediatech, Washington, DC) supplemented with 5% FBS (Summit Biotechnology; Fort Collins, CO) (
Effects of Latrunculin A and Dihydrocytochalasin B Treatment on Cell Surface CD44 Expression
Bovine chondrocytes in alginate beads were cultured either with 10 µg/ml dihydrocytochalasin B (DHCB; Sigma, St Louis MO) or 0.2 µg/ml latrunculin A (Calbiochem) or the vehicle buffer for various time periods: 3 hr, 1, 2, 5, 6, and 7 days. After the respective incubation period, the cells were fixed with 1% paraformaldehyde (Fisher; Pittsburgh, PA) in PBS, pH 7.4, for 1 hr at 4C. The alginate beads were then depolymerized with 55 mM sodium citrate and the cells were rinsed twice with PBS, incubated with 0.2 M glycinePBS for 5 min, and blocked with 4% non-fat dry milk in PBS for 1 hr. The cells were then probed for CD44 with biotinylated monoclonal antibody IM7.8.1 (Pharmingen; San Diego, CA) 2.5 µg/ml per 106 cells, for 1 hr at 4C, which was detected with 2.5 µg/ml streptavidinphycoerythrin (Gibco BRL; Life Technologies, Rockville, MD). The cells were then analyzed by flow cytometry as described (
Detergent Extraction of CD44 on Cytoskeletal Disruption
Bovine chondrocytes were plated into 24-well plates (Falcon 3047) at 2 x 105cells per well. The cells were then incubated with either 0.5 µg/ml latrunculin A, 10 µg/ml DHCB, or the vehicle, for 1 hr at 4C. After the incubation period, the cells were extracted with 0.1% Nonidet P-40 (NP-40; Calbiochem) for 30 min on ice. The NP-40 buffer consisted of 50 mM Tris-HCl (Bio-Rad; Hercules, CA), 150 mM NaCl, 5 mM EDTA, 100 mM -amino-N-caproic acid, 2 ng/ml pepstatin A, 10 µg/ml leupeptin, 10µg/ml aprotinin, and 5 mM benzamidine (all from Sigma) at a pH of 7.4 (
Hyaluronan Binding by Chondrocytes
Fluorescein hyaluronan (fl-HA) was prepared as previously reported (
Bovine chondrocytes were released from the alginate beads, suspended 5 x 105 cells/ml, and treated with 5 U/ml Streptomyces hyaluronidase (Sigma) in DMEM supplemented with 10% FBS for 60 min at 37C to remove endogenous ligand, hyaluronan, and expose total CD44 receptors. After the enzyme treatment, the cells were rinsed and then incubated for 3 hr at 37C with 10 µg/ml DHCB, 1 µg/ml latrunculin A, or vehicle buffer. At the end of this pre-incubation period, the cells were incubated for 80 min at 4C with 200 µg/ml fl-HA cells in the continued presence of the above agents. The cells were rinsed twice, counted, and resuspended in DMEM to give a final cell concentration of 3.3 x 105 cells/ml. These cells were then transferred into a 96-well plate at a volume of 200 µl per condition in triplicate for analysis on a fluorescent plate reader (Bio-Tek FL500; Winooski, VT). In parallel, cell surface CD44 expression on pretreated chondrocytes was determined by flow cytometry as described above.
Pericellular Matrix Retention or Assembly on Cytoskeletal Disruption
Chondrocytes were cultured in alginate for 5 days and then treated either with 0.2 µg/ml latrunculin A, 10 µg/ml DHCB, or vehicle buffer for 15 hr. After release from the alginate beads, the cells in suspension were "splatted" onto a six-well plate (Falcon 3046) at a density of 1 x 105 cells per well by centrifugation at 550 x g for 10 min (
Maintenance of the Extracellular Matrix in Cartilage Explants on Cytoskeletal Disruption
Safranin O Staining of Cartilage Tissue.
Bovine cartilage slices were allowed to equilibrate for 24 hr in DMEM with 10% FBS, transferred into individual wells of a 96-well plate (Falcon 3072), and cultured in the presence or absence of 0.2 µg/ml latrunculin A or 10 µg/ml DHCB. Each well contained the explant cartilage and 200 µl of the respective medium condition, which was changed daily. Explants were collected after 1, 2, 4, 5, 7, 8, 12, or 13 days in culture. Some of the cultures after 7 days of treatment were cultured for an additional 3 days in the absence of latrunculin A or DHCB. The cartilage was fixed in Perfix histological fixative (Fisher) containing 0.1% safranin O (Fisher) for 2 hr at 4C. After fixation the tissue was rinsed twice with PBS and quenched in PBS containing 2 mM glycine, 5% glycerol, and 1% BSA. Explants were then embedded in paraffin. Ten-µm sections were stained with fast green and safranin O (
Detection of the NITEGE Neoepitope in Cartilage Tissue.
The same cartilage explants were also used to stain for the NITEGE neoepitope of aggrecan (
Cell Viability Assay
Cell viability was determined by co-incubation of chondrocytes in alginate beads or cartilage slices in 2 µM calcein AM and 4 µM ethidium homodimer-1 (EthD-1) in PBS (LIVE/DEAD Kit, L-3224; Molecular Probes, Eugene, OR) for 40 min, followed by rinsing in Hanks' balanced salt solution (GIBCO BRL.) Chondrocytes were released from the alginate beads and resuspended in a small volume of 10% glycerol in PBS, which was placed on a glass slide and coverslipped. Cartilage was embedded in OCT (EM Sciences; Ft Washington, PA), and frozen sections placed on glass slides and warmed before viewing with a Nikon Eclipse E600. Uncultured cartilage slices were examined as a control. Dead cells (red nuclear fluorescence) were evaluated by the uptake of EthD-1 (ex/em = 495 nm/635 nm). Living cells were visualized by the fluorescence (green) of the calcein (ex/em = 495 nm/515 nm) as a result of endogenous esterase activity.
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Results |
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Chondrocyte Pericellular Matrix Assembly and Retention
Bovine articular cartilage chondrocytes were cultured for 5 days in alginate beads, in which the cells maintain a round morphology and synthesize an ECM. The pericellular matrix is dependent on a scaffold of hyaluronan and is removed by treatment with Streptomyces hyaluronidase (
The ability of the cells after disruption of the cytoskeleton to re-assemble a pericellular matrix following Streptomyces hyaluronidase treatment was examined with the particle exclusion assay. Fig 1 shows the pattern of matrix assembly monitored at day 1 (Fig 1A1C), day 2 (Fig 1D1F), and day 3 (Fig 1G1I) after Streptomyces hyaluronidase treatment. The progressive increase in pericellular matrix seen in control cells (Fig 1A, Fig 1D, and Fig 1G) was reduced in the presence of either cytochalasin (Fig 1B, Fig 1E, and Fig 1H) or latrunculin (Fig 1C, Fig 1F, and Fig 1I). After washout of the treatment reagents, matrix assembly progressed to that exhibited by controls (data not shown.) A morphometric analysis of the matrix:cell area ratio was determined at day 3 of matrix assembly (Table 1). Whereas only 7% of the control chondrocytes had a smaller pericellular matrix (ratio <1.5), 50% of the chondrocytes in the DHCB- or latrunculin-treated cultures exhibited a matrix:cell area ratio of <1.5. Conversely, whereas nearly 60% of the control chondrocytes exhibited a matrix:cell area ratio of >2.0, less than 10% of the chondrocytes in either experimental group exhibited these larger matrices. Chondrocyte viability after 3 days of treatment (Fig 2) was similar between DHCB-treated (Fig 2A), latrunculin-treated (Fig 2B), and untreated (Fig 2C) chondrocytes or after 5 days (data not shown) as revealed by the LIVE/DEAD cell assay.
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The ability of chondrocytes to maintain the existing pericellular matrix after treatment to disrupt the cytoskeleton was analyzed. Chondrocytes released from the beads after depolymerization of the alginate exhibit a matrix that can be visualized using the particle exclusion assay (
Cytoskeletal Disruption of Cells in Articular Cartilage
The results with isolated chondrocytes suggested that an intact actin cytoskeleton was critical to maintain chondrocytematrix interactions. To expand these studies to intact cartilage, latrunculin and cytochalasin treatments of explant cultures were evaluated. The chondrocytes in the latrunculin- or cytochalasin-treated cartilage appeared more compact. There was a decrease in the safranin O staining of both cytochalasin-treated (Fig 4B; 2 µg/ml, 2 days) and latrunculin-treated (Fig 4C; 10 µg/ml, 5 days) cartilage, indicating the loss of proteoglycan from these tissues compared to cultured controls (Fig 4A, Fig 5 days). When the explants were allowed to recover for 3 days after a 5-day treatment, there appeared to be an increase in safranin O staining, especially in the area right around the cells (insets in Fig 4B and Fig 4C shown at higher magnification, but from the middle zone of the cartilage indicated by the rectangles).
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A decrease in safranin O staining could be due to decreased proteoglycan synthesis or increased turnover owing to the activity of matrix metalloproteinases. To investigate this later possibility, sections from the treated and untreated control explants were stained for the NITEGE neoepitope of aggrecan. Fig 5A and Fig 5B are the untreated control tissue, with Fig 5A showing negative control for the immunohistochemistry and Fig 5B the NITEGE epitope. Fig 5C shows the DHCB-treated tissue and Fig 5D shows the latrunculin-treated tissue. There was a detectable level of NITEGE staining in all of the tissue sections, indicative of endogenous "aggrecanase" activity (
Cell viability in the freshly isolated, uncultured cartilage slices (Fig 6A) was quite high, as detected by green cytosolic fluorescence. Cartilage explants cultured for 3 or 5 days were also evaluated for chondrocyte viability. Although some dead cells (red nuclear fluorescence) could be detected in all sections, no increase in the number of dead cells was detected at day 3 (data not shown) or day 5 of culture in the absence (Fig 6B) or presence of DHCB (Fig 6C) or latrunculin (Fig 6D).
CD44 Expression, Association with the Cytoskeleton, and Hyaluronan Binding
Because CD44 is a critical receptor in chondrocytematrix interactions, cell surface expression of CD44 was monitored by flow cytometry. Five-day alginate bead cultures of chondrocytes were cultured in the presence or absence of either DHCB or latrunculin A for an additional 3 hr to 7 days. It appears that both of the agents used had similar minimal effects on the level of cell surface CD44 (Fig 7). The treated cells gave, at almost any given incubation time, less then a 25% decrease in the levels of cell surface CD44 expression. For DHCB the average decrease in cell surface CD44 was 9.2% and for latrunculin the average decrease in cell surface CD44 was 19.4%.
Immunocytochemistry combined with the differential detergent extraction protocol was used to visualize the cytoskeleton-associated pool of CD44 after disruption of the cytoskeleton. Fig 8A shows representative results. Fig 8A shows staining of total cell surface CD44 on untreated control chondrocytes. The inset in Fig 8A shows the negligible background staining with the omission of the primary antibody and using the secondary reagents only. No discernible differences in total cell surface staining between untreated control, cytochalasin-treated, or latrunculin-treated chondrocytes were noted (data not shown.) Fig 8B shows the residual CD44 on untreated control cells after extraction with 0.1% NP-40. There is a decrease in epitope staining due to the extraction of CD44 by the detergent, and the residual staining represents the cytoskeleton-associated pool of CD44. Chondrocytes treated with DHCB (Fig 8C) or latrunculin (Fig 8D) for 3 hr were then extracted with 0.1% NP-40 before CD44 detection. The staining intensity for the cytoskeleton-associated pool of CD44 was reduced after treatments with these agents, suggesting that in the absence of an intact actin cytoskeleton, CD44 is more readily extracted from the plasma membrane with detergent. These results help to verify that there are in fact two pools of CD44 in the chondrocytes and that the cytoskeleton-associated pool of CD44 is dependent on the stability of the cytoskeleton.
The relationship between CD44cytoskeletal interactions and the functional aspect of hyaluronan binding by CD44 was explored. After the cytoskeleton of the chondrocytes was disrupted for 3 hr, the cells were incubated with fl-HA and the amount of binding to these chondrocytes was determined in comparison to untreated controls. As shown in Fig 9, there was a decrease in the amount of fl-HA that was bound after cytoskeletal disruption compared to the control cells. The relative fluorescence values reflect a 54% decrease in the amount of bound fl-HA in the DHCB-treated cells and a 58% decrease with the latrunculin-treated cells. When cells from the same three populations were analyzed by flow cytometry, there was little difference in CD44 detected after cytoskeleton disruption compared to untreated control (Fig 9). These results indicate that the association between CD44 and the cytoskeleton plays a role in the capacity for the interaction between CD44 and its extracellular ligand hyaluronan.
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Discussion |
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Chondrocytematrix interactions and the regulation of ECM assembly are key to cartilage matrix homeostasis. Initial matrix assembly occurs in a zone immediately adjacent to the chondrocyte cell surface. Cellmatrix interactions, mediated by receptors, promote optimal assembly and signal feedback regulation for biosynthesis and/or turnover. This cell-associated matrix can be visualized as a distinct structure within sections of cartilage (
Hyaluronan hexasaccharides (HA6) are a useful tool for probing cell surface hyaluronan receptor function. HA6 do not mediate or disrupt proteoglycan aggregate formation (
Although disruption of the cytoskeleton did not alter cell surface CD44 expression, there was a reduction in hyaluronan binding, pericellular matrix assembly, and cartilage matrix retention. Even at the tissue level, the ability of the chondrocytes to retain the ECM was hindered when the cytoskeleton was disrupted. Therefore, change in the cytoskeleton is apparently another mechanism to uncouple chondrocytes from the matrix, resulting in modulation of chondrocyte metabolism and deleterious changes in matrix structure. However, after removal of the latrunculin or cytochalasin, the cells, both isolated and in cartilage, were able to reconstruct the matrix.
Maintenance of the chondrocyte phenotype, as well as initial cartilage differentiation, may depend on the close association of the cell with its matrix and on cell shape as modulated by the actin cytoskeleton (
Our previous studies demonstrated an increased turnover of CD44 from the chondrocyte cell surface after depletion of the hyaluronan-rich matrix using the enzyme Streptomyces hyaluronidase. In the reverse experiment, when high molecular weight hyaluronan was added back to the matrix-depleted chondrocytes, a partial inhibition of CD44 internalization resulted, returning the CD44 half-life to near baseline levels as seen on matrix-intact chondrocytes (5ß1 on fibroblasts. This integrin was internalized more rapidly in the absence of the fibronectin, while subsequent attachment to fibronectin substrate was found to stabilize the expression of cell surface
5ß1 integrin (
Our results show that destabilization of the actin network in chondrocytes results in decreased matrix assembly or retention. The spatial organization of CD44 at the cell surface, controlled via cytoskeletal interactions, may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffolding for aggrecan. On cytoskeletal disruption, the stability of the CD44 molecules at the cell surface and/or the pattern of distribution on the cell surface may be altered. These results are in accord with previous studies in which cytochalasin treatment of bovine endothelial cells (which express CD44v10) reduced the capacity of these cells to bind hyaluronan (
It becomes apparent that disruption of cytoskeleton of chondrocytes brings about a metabolic response. A preliminary survey of the effects of treatment with DHCB and latrunculin A on the message levels for hyaluronan synthase 2 (HAS-2) and aggrecan indicate that the decrease in matrix assembly and retention on cytoskeletal disruption is not due to a decrease in aggrecan or hyaluronan synthesis because there was little difference in their message levels between the control and treated cells (data not shown). These observations lead us to speculate that signaling events are initiated by the disruption of the cytoskeleton.
Our results suggest that the integrity of microfilament-dependent cytoskeletal structures can be a source of regulatory signals, as suggested previously by others (
Other reports indicate that the use of cytoskeleton-disrupting agents, specifically DHCB, leads to an increase in matrix metalloproteinases (
The interaction of CD44 with the cytoskeleton and the matrix enables us to speculate that both inside-out and outside-in communication patterns are occurring within the chondrocytes via CD44. Chondrocytes may exhibit metabolic and physical changes through the interaction with the matrix, which might induce matrix degeneration, remodeling, or repair. Our results indicate that destabilization of the cytoskeleton brings about a change in the matrix. In addition to artificial cytoskeleton-disrupting agents, such as cytochalasins and latrunculins, natural agents such as NO, which is elevated during degenerative cartilage diseases (
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Acknowledgments |
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Supported by NIH grants AR39507 and AR39239 and by a grant from the National Arthritis Foundation.
This work was submitted by G.A.N. in partial fulfillment of the requirements for the PhD from Rush University, Chicago, IL. We thank Roma A. Andhare for her assistance in obtaining the images in Fig 2 and Fig 6.
Received for publication October 8, 2001; accepted April 24, 2002.
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