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Arthritis Rheum. Author manuscript; available in PMC Sep 1, 2009.
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PMCID: PMC2562294
NIHMSID: NIHMS59048

Rho-Dependent CCL20 Induced by Dynamic Compression of Human Chondrocytes

Dominik R. Haudenschild, Ph.D.,1,2 Bao Nguyen, M.S.,2 Jianfen Chen, B.S.,1 Darryl D. D'Lima, Ph.D., M.D.,2 and Martin K. Lotz, M.D.1

Abstract

Objective

Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. This study examines a role for both actin and Rho in mechanotransduction in chondrocytes.

Methods

We embedded human articular chondrocytes in 2mm × 6mm agarose discs at 5×106 cells/ml and subjected the discs to unconfined dynamic compression at 0.5Hz. By comparing samples with and without dynamic compression, we identified Rho activation by measured GTP-bound active RhoA in cell lysates. We identified rearrangements in filamentous actin structures using fluorescently labeled phalloidin and confocal microscopy on fixed samples. We identified altered gene expression using TaqMan quantitative RT-PCR. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho Kinase inhibitors.

Results

RhoA activation occurred within 5 to 10 minutes of dynamic compression. Rho Kinase-dependent actin reorganization occurs upon dynamic compression within 20 minutes, apparent as “punctate” F-actin structures visible in confocal microscopy. We identify early-phase mechanoresponsive genes CCL20 and iNOS that are highly upregulated within one hour of dynamic compression in a Rho kinase and actin-dependent manner.

Conclusion

Together, these results demonstrate for the first time that the Rho-ROCK pathway and actin cytoskeletal reorganization are required for changes in gene expression involved in human chondrocyte mechanotransduction.

INTRODUCTION

Mechanical forces regulate chondrocyte proliferation, survival, differentiation, gene expression, and biosynthetic responses. The type and duration of mechanical stimulation determine the outcome of the cellular responses that in their extreme manifestations can range from cell proliferation to cell death and from matrix formation to matrix destruction. Although it is accepted that an abnormal response to mechanical stimuli can be a contributing factor to osteoarthritis (1), the signal transduction mechanisms in chondrocytes that recognize the applied forces and elicit the appropriate biochemical cellular responses are not well characterized.

In chondrocytes, there is abundant evidence of a link between cell shape and phenotype as defined by gene expression (2-4). Drastic changes in cell size and shape are hallmarks of chondrocyte differentiation (5, 6), and the changes in cell shape are closely related to changes in chondrocyte gene expression (2, 5, 7).

The chondrocyte cell shape is dependent on the cytoskeleton, which is largely determined by the organization of filamentous actin. The actin cytoskeleton is important in the control of chondrogenic phenotype and differentiation (5, 8-10). Actin microfilaments are a dynamic structure of actin polymers and associated actin-binding proteins (11, 12). The actin cytoskeleton is important in regulating cell shape changes during mitosis and in response to extracellular stimuli (13), migration (14), adhesion (15), signaling (16), organelle movements (17), and endocytosis (18). While all microfilaments and microtubules also contribute to mechanical properties, actin contributes the most to the generation of mechanical forces within the cell (19, 20). Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response (21, 22).

Rho GTPases play a central role in the dynamics of the actin cytoskeleton (reviewed in (23-26)). They affect actin dynamics through many pathways, including the activation of effector kinases PAK, ROCK, and MRCK, which all phosphorylate Lim Kinase, and downstream Cofilin, an actin filament severing protein. Rho activity can also affect microtubule dynamics. Recent studies directly link Rho signaling and the actin cytoskeleton to chondrocyte differentiation from pluripotent mesenchymal cells (27, 28).

Rho GTPases and actin cytoskeleton reorganization have been examined in mechanotransduction pathways using vascular endothelium and smooth muscle cell cultures (29-32), but their contribution in chondrocyte mechanotransduction remains unknown. Most of the studies in vascular cells were conducted using monolayer cell culture systems subjected to stretch or shear forces. Monolayer culture conditions have only limited relevance to the biology of native chondrocytes, which exist embedded in 3-dimensional cartilage matrix with limited or no direct cell-cell contact. In this manuscript we present evidence for the first time that mechanotransduction in 3D cultured human articular chondrocytes causes activation of Rho GTPase and is further mediated by the activity of downstream Rho Kinase and a subsequent reorganization of the actin cytoskeleton.

The most commonly studied mechanoresponsive genes in cartilage are aggrecan and type II collagen, and these genes represent the most abundant components of the cartilage extracellular matrix. However, the magnitude of induction of these genes is relatively small, on the order of 25% to 100% increases compared to baseline. Using whole-genome microarray as a screen to pick highly induced genes, we identify CCL20 and iNOS as rapidly induced mechanoresponsive genes in chondrocytes. Furthermore, we demonstrate that both Rho kinase activity and actin reorganization are required for their induction by dynamic compression.

MATERIALS AND METHODS

Culture of chondrocytes in 3D

Primary human articular chondrocytes were obtained from the femoral and tibial chondyles as previously described (33). This study used chondrocytes from a total of 14 donors of ages ranging from 17 to 58 (average age was 42), both male and female, and OA grades I-II. Each experiment was repeated with chondrocytes from at least three different donors. Chondrocytes were expanded in monolayer culture until nearly confluent, then passaged at most three times. Chondrocytes were released from monolayer culture with trypsin digestion, concentrated by centrifugation to 10×106 cells/ml, mixed with an equal volume of 6% low gelling point agarose at 42°C, and immediately pipetted into a custom casting chamber exactly 2mm thick. The agarose was allowed to gel at 4°C for 30 minutes, and then a dermal punch was used to make agarose discs measuring 6mm in diameter and 2mm height. The final cell density was 5×106 cells/ml in 3% agarose. Cells were cultured with one gel per well in 24-well plates, in DMEM supplemented with 10% fetal calf serum (FCS) and 25μM ascorbic acid. Media was changed every 2 to 3 days. Aggrecan and type II collagen expression were monitored in the agarose-embedded chondrocytes to verify the chondrocyte phenotype.

Dynamic compression

To apply physiological loading to the cells, the agarose-embedded chondrocytes were placed in a custom designed polysulfone chamber and subjected to continuous sinusoidal cyclic compression of 5% amplitude above and below a 10% offset (5%−15% strain) at a frequency of 0.5Hz. Strain was applied with a computer-controlled actuator with 5μm resolution, the entire apparatus was housed in an incubator at 37 degrees with a 5% CO2 humidified atmosphere. Cell number and viability were unaffected by this treatment and we did not observe evidence of apoptosis.

Rho activation assay

To determine whether dynamic compression activates RhoA, agarose-embedded chondrocytes were first serum-starved for 48 hours to reduce basal Rho activation. Cells were then subjected to 2, 5, or 10 minutes of dynamic compression from 5% to 15% cyclic strain at 0.5Hz frequency. Immediately at the end of dynamic compression, the agarose gels and cells were homogenized in lysis buffer and RhoA activation measured using the RhoA G-LISA kit (Cytoskeleton, Denver, CO). Results represent the average increase of RhoA activation in three chondrocyte preparations from different donor individuals. Statistical significance was calculated using one-way ANOVA from duplicate measurements of at least two gels for each condition using JMP5.1 (SAS Institute, Cary, NC).

Quantification of changes in actin cytoskeleton

To determine whether actin rearrangement occurs upon dynamic compression, confocal microscopy and image analysis of phalloidin-stained chondrocytes was performed. Agarose-embedded chondrocytes were subjected to dynamic compression from 0% to 10% strain at a frequency of 0.5Hz for 20 minutes. Immediately after dynamic compression, gels were fixed in 3.7% neutral buffered formaldehyde for 1 hour at room temperature. The actin cytoskeleton was stained with phalloidin-Alexa-488 (Molecular Probes, Portland, OR). Confocal z-stacks were taken using an inverted Zeiss LSM510 microscope with a 63× water-immersion objective. Images were exported as 512 × 512 tiff files and imported into ImageJ (http://rsb.info.nih.gov/ij/). The extent of cytosolic punctate actin staining was quantified with an ImageJ macro command. Briefly, the macro reduces the graininess with a median-blur set to 3.5 pixel radius, highlights edge features with a convolution algorithm, thresholds the resulting image into black/white data, and then counts the number of spots of a given size range between 10 and 50 pixels in diameter.

RNA isolation and real time quantitative reverse transcription-PCR

To determine changes in gene expression, total RNA was isolated from agarose-embedded chondrocytes using an RNA isolation protocol described by Li and Trick (34). First strand cDNA synthesis was performed with Superscript III using total RNA as a template according to the manufacturer's protocols (Invitrogen, Carlsbad, CA). Quantitative RT-PCR was performed using TaqMan RT-PCR reagents. GAPDH and iNOS were detected using Assays-on-Demand primer/probe sets (Applied Biosystems, Foster City, CA). The CCL20 primer/probe set (Fwd-TCTGTGTGCGCAAATCCAA, Rev-CCATTCCAGAAAAGCCACAGT, probe-TGTGCGTCTCCTCAGTAA) was designed to identify a 100bp sequence spanning exons 3 to 4 of human CCL20 (locus NM_004591). Expression levels were normalized to GAPDH using the recommended ΔCt method, and fold-change calculated using the 2ΔΔCt formula (35). RT-PCR was performed in triplicate for each RNA, and Student's t-test was used to assess statistical significance.

Cell culture treatments

In certain experiments, IL-1β (Peprotech, Rocky Hill, NJ) was added at 10ng/ml for 2 hours. Rho kinase inhibitors hydroxyfasudil (5μM) or Y27632 (20μM) were added for 2 hours prior to any other treatments (Calbiochem, San Diego, CA). Cytochalasin-D (Calbiochem) was added at 10μM for 2 hours prior to any other treatments.

RESULTS

RhoA is activated by Dynamic Compression

To test whether RhoA activation mediates mechanotransduction during dynamic compression of agarose-embedded chondrocytes, a timecourse of RhoA activation was performed. Dynamic compression activated RhoA within 5 minutes and for at least 10 minutes (Fig. 1). A significant 85% increase in active GTP-bound RhoA was detectable after 5 minutes of dynamic compression (p<0.025) and an even greater increase after 10 minutes (p<0.002). Two minutes of dynamic compression caused an increase of RhoA activation in cells from some but not all donors and was thus not statistically significant (p=0.59).

Figure 1
Dynamic Compression Causes RhoA Activation in Chondrocytes

Cytoskeletal changes are induced by Dynamic Compression

A major role for Rho proteins is to coordinate the reorganization of the actin cytoskeleton (36). To test whether dynamic compression induces actin rearrangements, agarose-embedded chondrocytes were subjected to 20 minutes of dynamic compression then formaldehyde-fixed, and the phalloidin-stained F-actin structures examined by confocal fluorescence microscopy. While several aspects of the actin cytoskeleton were altered in cells subjected to dynamic compression, the most readily quantifiable and reproducible change was in the number of punctate F-actin features observed in the chondrocyte cytosol (Fig. 2). Thus, quantification of the punctate F-actin was taken as a reliable indicator of cytoskeletal rearrangements in 3D cultured chondrocytes (Fig. 3).

Figure 2
Dynamic Compression induces rearrangements in the actin cytoskeleton
Figure 3
Dynamic Compression increases punctate cytosolic actin

Cytoskeletal rearrangements require Rho kinase activity

Rho kinase (ROCK) is a major effector protein downstream of Rho activation, providing a link to actin remodeling (37, 38). To test for a requirement of ROCK activity for the cytoskeletal rearrangements observed upon dynamic compression, agarose-embedded chondrocytes were pretreated with ROCK inhibitor Y27632 for 2 hours to reduce baseline ROCK activity, followed by 20 minutes of dynamic compression in the continued presence of Y27632. When dynamic compression was applied with ROCK inhibitor, the number of punctate F-actin features was similar to the baseline levels in unstimulated chondrocytes (Figs. 2 and and3).3). This demonstrates that the dominant actin rearrangements induced by dynamic compression require active Rho kinase.

Changes in gene expression are induced by Dynamic Compression

We used a whole-genome microarray approach to determine the genes regulated by dynamic compression. This identified iNOS and CCL20 as two genes highly upregulated by dynamic compression in our system. TaqMan quantitative RT-PCR on dynamically compressed chondrocytes from several donors showed consistent increases of approximately 50-fold and 200-fold in iNOS and CCL20 expression, respectively (Fig. 4a). This response was independent of the sex of the tissue donor and did not correlate strongly with either the age of the tissue donor or the grade of osteoarthritis of the cartilage from which the chondrocytes were isolated. The age of the tissue donors analyzed in figure 4 ranged from 17 to 55 years (average 39.4 years) with an osteoarthritis grade of I or II, representing completely normal tissue or only mild fibrillations.

Figure 4
CCL20 and iNOS are early-response genes induced by dynamic compression

Gene expression changes require Rho Kinase activity and actin remodeling

To determine whether the induction of iNOS and CCL20 expression requires Rho kinase activity and actin remodeling, agarose-embedded chondrocytes were subjected to dynamic compression in the presence of either ROCK inhibitor or cytochalasin-D, which inhibits actin filament growth. The presence of either inhibitor significantly attenuated the gene-expression response (Fig. 5) for both iNOS and CCL20 (p<0.05 by Student's t-test). This indicates a requirement for both Rho kinase activity and actin cytoskeletal remodeling during mechanotransduction in 3D cultured human articular chondrocytes.

Figure 5
Effect of dynamic compression on gene expression requires both Rho Kinase activity and Actin Cytoskeletal remodeling

IL-1β response is not ROCK-dependent

Both CCL20 and iNOS can be induced by IL-1β (39, 40). To test whether the gene expression response seen upon dynamic compression could be mimicked simply by addition of IL-1β, we tested for a requirement of Rho kinase in IL-1β signaling. Expression of both iNOS and CCL20 were greatly increased by IL-1β treatment for 4 hours, but this increase was not affected by the presence of Rho kinase inhibitor hydroxyfasudil (Fig. 6). Hydroxyfasudil alone did not significantly induce or repress the expression of either iNOS or CCL20 mRNA.

Figure 6
Induction of CCL20 and iNOS by IL-1 is not dependent on Rho Kinase activity

DISCUSSION

This study addressed the hypothesis that mechanotransduction in 3D cultured human articular chondrocytes is mediated by the activation of Rho GTPases and a subsequent reorganization of the actin cytoskeleton. Our findings demonstrate for the first time that dynamic compression causes RhoA activation in chondrocytes. RhoA activation occurred within 10 minutes, followed within 20 minutes by changes in the actin cytoskeleton, and subsequently by changes in gene expression within an hour of dynamic compression. We demonstrated a requirement for Rho kinase activity in the actin cytoskeletal rearrangements and further demonstrated that both Rho kinase activity and actin rearrangements are required for the changes in gene expression induced by dynamic compression.

The immediate regulatory mechanism of Rho GTPase signaling is activation via binding to GTP or inactivation via hydrolysis of GTP to GDP, and is regulated by a balance between the activities of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPS). In experiments on vascular endothelium exposed to stretch or shear stress, activation of Rho GTPases via GTP-binding often occurs within seconds or minutes of applied stimuli (29, 41). The duration of the activated state is usually measured in minutes and generally returns to baseline within an hour. We observed that dynamic compression of 3D cultured chondrocytes does activate RhoA and that the timecourse of activation is similar to that observed in vascular cells. Future work will identify the GEFs and GAPs responsible for RhoA activation in chondrocytes during mechanotransduction.

The Rho family of GTPases exert profound effects on the actin cytoskeleton. One of the most common features of Rho activation in monolayer cultures is the formation of stress-fibrils. However, 3D cultured chondrocytes do not contain readily identifiable stress fibrils, nor do they contain the lamellipodia or filopodia structures traditionally ascribed (in monolayer cultures) to the activities of closely related GTPases Rac or Cdc42 (5, 42-45). While descriptions of actin structures in monolayer cell cultures are abundant, we were unable to find categorical descriptions of the types of actin structures induced by the Rho GTPases RhoA, Rac or Cdc42 in 3D chondrocyte cultures.

The actin cytoskeleton of 3D chondrocytes is traditionally described as a “cortical shell” (46, 47). High-resolution confocal microscopy revealed (from our unpublished observations) that this shell might be more accurately described as a tightly interwoven mesh of filaments, which only appears to form a continuous “shell” due to the limited resolution of the microscopes in the z-axis. The apparent thickness of this shell is determined by the number of overlapping filaments and varies considerably around any particular circumference, making precise measurements difficult.

Several aspects of the actin cytoskeleton appeared altered by dynamic compression, including the density of the fibril meshwork comprising the “cortical shell”, the extent of the actin projections beyond this “shell”, and the number of bright punctate F-actin features within the cytosol. None of these structures have previously been attributed to Rho GTPase activity, and to our knowledge, these structures do not have defined functions in generating or preserving the chondrocyte phenotype. We chose to measure the number of central punctate F-actin structures as a quantifiable change induced by dynamic compression of the agarose-embedded chondrocytes, since this type of actin structure was dependent on the Rho Kinase activity and easily quantified by an automated macro in ImageJ. A similar approach was recently published to quantify actin changes after dynamic compression, although these authors did not examine Rho signaling (44). We observed additional actin changes that did not lend themselves as easily to quantification. These included increased thickness of “cortical actin” and increased peripheral extensions.

Many lines of research indicate that dynamic compression of 3D cultured chondrocytes causes increased synthesis of extracellular matrix components aggrecan and type II collagen (48). The magnitudes of the changes in matrix synthesis are minor (approximately 1.3-fold to 2-fold increase over baseline) and require dynamic compression over extended lengths of time (often several hours to days). This makes them inconvenient for studying signal transduction events that occur within seconds or minutes. We therefore used a whole-genome microarray approach to identify early-response genes that were highly upregulated within an hour of dynamic compression. We identified CCL20 and iNOS as early response genes from the microarray analysis and confirmed their induction by dynamic compression with quantitative RT-PCR. The large magnitude (50-fold to 200-fold) of induction of these genes enables good measurements of stimuli that cause their induction, as well as inhibition of those pathways with inhibitors such as Y27632 and cytochalasin. Our results show that the Rho kinase inhibitors Y277632 or hydroxyfasudil and cytochalasin D interfere with the induction of iNOS and CCL20 by dynamic compression. Thus, the activation of Rho and the reorganization of actin appear to be functionally linked to the gene expression response.

There are numerous reports of interactions between the IL-1 pathway and Rho GTPase activation. Most of the evidence suggests that inhibition of Rho kinase potentiates the IL-1-induced gene expression (49). The nature of these interactions is likely to be cell-type specific and has not been studied in chondrocytes. Our observations indicate that inhibition of Rho kinase with either Y277632 or hydroxyfasudil has no effect on IL-1 induced iNOS and CCL20 gene expression in 3D cultures of human articular chondrocytes. This result supports the notion that Rho kinase activation is not a general requirement for the induction of these genes and implies at least some selectivity of this pathway in mechanotransduction.

We have identified iNOS and CCL20 as early and highly responsive genes in chondrocyte mechanotransduction. In the context of this manuscript, they serve as consistent and sensitive outcomes of mechanotransduction. The expression of the CCL20 chemokine by chondrocytes is a novel finding, and to our knowledge its actions on chondrocytes and the chondrocyte actin cytoskeleton are unknown. CCL20 is overexpressed in the synovium of rheumatoid arthritis, and mediates the inflammatory response associated with that disease (reviewed in (50)). CCL20 and CCL6 (the CCL20 receptor) have also been studied in osteoblast proliferation and osteoclast differentiation in the homeostasis of subchondral bone. In hepatic carcinoma, CCL20 induces rapid reorganization of the actin cytoskeleton and actin-based migration. Together, these indirect lines of evidence indicate that CCL20 might influence the chondrocyte cytoskeleton and perhaps the chondrocyte phenotype, but this remains to be determined.

iNOS has been extensively studied in the context of cartilage and arthritis. There are reports that iNOS and NO are up-regulated by shear stresses in chondrocytes and by injury. Others report that iNOS and NO are down-regulated by application of dynamic compressive forces, and that dynamic compression reduces the induction of iNOS by IL-1. In our system, iNOS was consistently up-regulated by dynamic compression in a Rho Kinase dependent manner.

In summary, we have provided evidence for the first time in 3D cultures of human articular chondrocytes that mechanotransduction pathways activated Rho GTPase. Rho activation was followed by Rho kinase-dependent rearrangements of the actin cytoskeleton and by Rho kinase dependent changes in gene expression. We identified two early-response genes, iNOS and CCL20, which were significantly induced by dynamic compression within 1 hour of stimulation. Further studies are needed to determine a more comprehensive set of Rho-dependent genes induced by mechanical stimulation and to determine whether the Rho-dependent mechanotransduction pathway is altered in injured or diseased cartilage.

Grant Supporters

This work was funded through NIH grant AG07996 (MKL) and a generous donation from Donald P. and Darlene V. Shiley.

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