Disassembly of the vimentin cytoskeleton disrupts articular cartilage chondrocyte homeostasis

Matrix Biology 25 (2006) 398 – 408 www.elsevier.com/locate/matbio

Disassembly of the vimentin cytoskeleton disrupts articular cartilage chondrocyte homeostasis Emma J. Blain ⁎, Sophie J. Gilbert, Anthony J. Hayes, Victor C. Duance Connective Tissue Biology Laboratories, Biomedical Sciences Building, School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, Wales, UK Received 7 February 2006; received in revised form 18 May 2006; accepted 7 June 2006

Abstract Articular cartilage functions in dissipating forces applied across joints. It comprises an extracellular matrix containing primarily collagens, proteoglycans and water to maintain its functional properties, and is interspersed with chondrocytes. The chondrocyte cytoskeleton comprises actin microfilaments, tubulin microtubules and vimentin intermediate filaments. Previous studies have determined the contribution of actin and tubulin in regulating the synthesis of the extracellular matrix components aggrecan and type II collagen. The contribution of vimentin to extracellular matrix biosynthesis in any cell type has not previously been addressed. Therefore the aim of this study was to assess the role of vimentin in cartilage chondrocyte metabolism. Vimentin intermediate filaments were disrupted in high-density monolayer articular chondrocyte cultures using acrylamide for 7 days. De novo protein and collagen synthesis were measured by adding [3H]-proline, and sulphated glycosaminoglycan (sGAG) synthesis measured by adding [35S]-sulphate to cultures. Vimentin disruption resulted in decreased collagen synthesis, whilst sGAG synthesis was unaffected. In addition, there was a significant reduction in type II collagen and aggrecan gene transcription suggesting that the effects observed occur at both the transcriptional and translational levels. A 3-day cold chase demonstrated a significant inhibition of collagen and sGAG degradation; the reduction in collagen degradation was corroborated by the observed reduction in both pro-MMP 2 expression and activation. We have demonstrated that an intact vimentin intermediate filament network contributes to the maintenance of the chondrocyte phenotype and thus an imbalance favouring filament disassembly can disturb the integrity of the articular cartilage, and may ultimately lead to the development of pathologies such as osteoarthritis. © 2006 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Articular chondrocyte; Vimentin; Glycosaminoglycans; Collagen; Matrix metalloproteinases

1. Introduction Articular cartilage is a highly specialised tissue that functions in dissipating applied forces and ensuring easy and frictionless articulation of joints. It is composed of a dense extracellular matrix containing primarily collagens, proteoglycans and water which is interspersed with its only cell type, the chondrocyte. All cells contain a cytoskeleton which is important in orchestrating cellular events such as cell motility, protein trafficking/secretion and mitosis. In chondrocytes, the cytoskeleton contains predominantly actin microfilaments, tubulin microtubules and vimentin and nuclear lamin intermediate filaments (Benjamin et al., 1984). Actin micro-

⁎ Corresponding author. Tel.: +44 29 20875419; fax: +44 29 20874594. E-mail address: [email protected] (E.J. Blain).

filaments are responsible for providing the cell with mechanical integrity to withstand compressive loads (Guilak, 1995), induce chondrogenesis (Archer et al., 1982) and help maintain a chondrocytic phenotype (Brown and Benya, 1988). Microtubules have essential roles in organising the distribution of organelles, protein trafficking and secretion, and in forming the mitotic spindle during cell division (Thyberg and Moskalewski, 1999). Previous studies in several cell types including fibroblasts (Diegelmann and Peterkofsky, 1972; Bauer and Valle, 1982; Newman and Watt, 1988; Wang et al., 1993; Hermann and Aebi, 2000), synoviocytes (Aggeler, 1990; Harris and Krane, 1971), peritubular cells (Thiebot et al., 1999), epiphyseal (Newman and Watt, 1988) and foetal chondrocytes (Jansen and Bornstein, 1974; Lohmander et al., 1979; Bodo et al., 1996) have demonstrated that when either actin filaments or tubulin microtubules were disrupted using cytochalasin B/D or colchicine, respectively, there

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were concomitant decreases in the synthesis and secretion of both collagen and proteoglycan. The third type of cytoskeletal element is the vimentin intermediate filament which is distributed from the nuclear surface to the plasma membrane, whilst nuclear lamins form a concentrated meshwork at the inner surface of the nuclear envelope (Benjamin et al., 1984). Vimentin filaments possess unique viscoelastic properties that allow greater resistance to mechanical stress (Trickey et al., 2004). Although the vimentin knockout mouse displays no obvious phenotype (Colucci-Guyon et al., 1994), a reduction in stiffness, mechanical stability, motility and directional migration of vimentin −/− fibroblasts has been previously reported (Eckes et al., 1998) indicating cellular fragility. The vimentin network is highly dynamic, the mechanisms of which are regulated by phosphorylation and dephosphorylation events (Benjamin et al., 1984), and in vitro has been demonstrated to be a target for a number of kinases including protein kinase A (PKA) and C (PKC) (Inagaki et al., 1996). This has led to the hypothesis that phosphorylation of vimentin and the resulting changes in cytoskeleton architecture may reflect a pathway for mechanical signal transduction (Goldman and Chou, 1999). Very little is known about the specific function of vimentin intermediate filaments in chondrocytes, and to date, there have been no studies on the effect of disrupting the vimentin intermediate

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filaments in chondrocytes. However, a significant, 20% reduction in vimentin expression was recently reported in the chondrocytes of a rat model of osteoarthritis (OA) (Capin-Gutierrez et al., 2004), and a disorganised vimentin cytoskeleton was also observed in human OA articular cartilage chondrocytes (Fioravanti et al., 2003; Holloway et al., 2004), indicating that changes in the chondrocyte vimentin cytoskeleton may be involved in OA pathogenesis. OA, the most common joint disorder worldwide (Cantatore et al., 2001), is characterised by joint space narrowing and focal areas of articular cartilage damage (Dieppe, 1998). Degradative events are initiated by an imbalance of matrix catabolism over matrix synthesis, which is due in part to the activity of the matrix-degrading enzymes — the matrix metalloproteinases (MMPs) (Dean et al., 1989). Previous studies, conducted in rabbit synovial fibroblasts, indicated that upon actin disruption using cytochalasin D, expression and activation of several MMPs were induced (Unemori and Werb, 1986; Werb et al., 1986; Tomasek et al., 1997; Lambert et al., 2001). Despite there being extensive evidence for the importance of both the actin microfilaments and the tubulin microtubules in modulating matrix biosynthesis and degradation, there are no reports on the contribution of the vimentin intermediate filaments in maintaining the chondrocyte phenotype. Therefore the aim of this study was to determine the importance of vimentin on anabolic and catabolic events in cartilage chondrocyte metabolism.

Fig. 1. The cytoskeleton of chondrocytes depicting (A, C, E) intact vimentin intermediate filaments, and (B, D, F) vimentin filaments disrupted using 5 mM acrylamide after (A–B) 1, (C–D) 3 or (E–F) 7 days of treatment (arrows indicate regions of vimentin disassembly as evidenced by collapse of filaments over the nucleus). The organisation of (G) actin microfilaments or (I) tubulin microtubules in untreated cells was compared to (H, J) cells after 7 days of 5 mM acrylamide treatment respectively. Cells were visualised using FITC-conjugated antibodies (counterstained with propidium iodide in conjunction with scanning confocal microscopy (scale bar = 5 μm).

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2. Results 2.1. Acrylamide treatment disassembles vimentin intermediate filaments in chondrocytes Disruption of the vimentin elements was confirmed using scanning confocal microscopy (Fig. 1). In intact cells, vimentin is evident as a dense network of filaments throughout the cytoplasm (Fig. 1A, C and E), which upon the addition of 5 mM acrylamide causes the collapse of the filaments over the nuclei (Fig. 1B, D and F). Clearly there is a temporal disassembly of the vimentin filaments, as a partial collapse of vimentin at the nucleus is evident as early as 24 h after acrylamide treatment (Fig. 1B). This aggregation of vimentin at the nucleus is further exemplified at day 3 (Fig. 1D) and a complete collapse is evident at day 7 (Fig. 1F). There is a distinct “shrinkage” of the acrylamide treated cells at all time points examined, and by day 7 there was an approximate 50% reduction in cell diameter that was consistent across the experiments. However, at day 7, acrylamide treatment does not affect the organisation of the other cytoskeletal elements actin (Fig. 1H) and tubulin (Fig. 1J) which have similar arrangements to the untreated cells (Fig. 1G and I respectively). Although the cells have “shrunk” with acrylamide treatment, overall the tubulin and actin networks retain their cytoskeletal organisation.

Fig. 2. Caspase 3 activation in chondrocytes exposed to 5 mM acrylamide. Caspase 3 activity (17 kDa) was detected in cell extracts by Western blotting using a rabbit anti-caspase 3 antibody. Strong immunoreactivity was observed in the acrylamide treated chondrocytes but there was no activity in untreated cells.

chondrocytes after exposure to acrylamide for 7 days, but was completely absent in untreated control cells (Fig. 2). All of the biochemical data is thus presented as mean ± S.E.M. after normalisation to cell number.

2.2. Decreased cell number in chondrocytes after acrylamide treatment Cell death over the culture period was measured using the CytoTox 96® assay. Exposure to 5 mM acrylamide caused no significant release of lactate dehydrogenase in chondrocyte cultures (data not shown). Total cell number with and without acrylamide treatment was also determined using the CytoTox 96™ assay (Table 1). Untreated cells showed proliferation with the total absorbance approximately doubling over the 10-day culture period. Significantly fewer cells were observed following treatment with acrylamide at both 7 days, and after replenishment of treatment for a further 3 days ( p < 0.005). The vimentin filaments are believed to be a substrate for caspases (Niu and Nachmias, 2000; Byun et al., 2001; Dinsdale et al., 2004), so disassembly of the vimentin networks concomitant with cleavage of vimentin by active caspases result in death by apoptosis. Indeed, active caspase 3 enzyme (17 kDa) was detected, by Western blotting, in Table 1 Effect of vimentin disruption on cell number as assayed using the CytoTox 96® assay

Control (untreated) Acrylamide (vimentin)

Day 1

Day 7

Day 10

0.818 ± 0.041 0.861 ± 0.034

1.0094 ± 0.0262 0.522 ± 0.077***

1.487 ± 0.096 0.190 ± 0.010***

The cell number is expressed as absolute absorbance (at a wavelength of 490 nm) at days 1 and 7, and after re-exposure of the cells to 5 mM acrylamide for a further 3 days (day 10). There was a significant decrease in cell number after acrylamide treatment for 7 and 10 days when compared with the untreated cultures. Data are presented as mean ± S.E.M. (n = 4) (***p < 0.001).

Fig. 3. De novo synthesis of sGAG in chondrocytes. (A) Amount of sGAG released from chondrocytes treated with 5 mM acrylamide for 7 days as measured using the DMMB assay. [35S]-sulphate (10 μCi/ml) was added to the media in the presence of 5 mM acrylamide for 7 days, unincorporated label removed and counts (cpm) measured in the (B) media and (C) cell extract. There was no significant effect on sGAG amounts after acrylamide disruption of the vimentin filaments. Normalised data are presented as mean ± S.E.M. (n = 4).

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2.3. Disassembled vimentin filaments do not affect de novo sGAG synthesis Analyses using the dimethylmethylene blue assay indicated that acrylamide treatment did not significantly affect sGAG release into the media compared to untreated cells ( p = 0.114) (Fig. 3A). To determine whether disruption of the vimentin networks by acrylamide treatment affected de novo synthesis, radiolabelling experiments were performed. Cells were treated with 5 mM acrylamide in the presence of [35S]-sulphate, and rates of biosynthesis measured at day 7 in both media and cell extract (Fig. 3B and C). No significant differences in the level of [35S]-sGAG were detected in the acrylamide and untreated media ( p = 0.071) and cell extracts ( p = 0.758). 2.4. An abrogated vimentin cytoskeleton reduces degradation of newly-synthesised sGAG To determine the amount of degraded de novo sGAGs over the 7-day period, 3-day cold chase experiments were performed. After 7 days of [35S]-radiolabelling, the labelled media was removed and replaced with “cold” media to chase out any remaining [35S]-sulphate. The amount of label remaining at the end of the 3-day chase, which was removed through the low molecular weight cut-off filter units, provide an indication of degraded sGAGs. A significant reduction in sGAG degradation was observed over the 7 days of acrylamide culture, accounting for 8.2% of the newly synthesised [35S]-sGAGs in the treated cultures and 17.1% in the untreated controls (p = 0.002) (Fig. 4). 2.5. Decreased de novo collagen protein synthesis in vimentin disrupted chondrocytes Using the hydroxyproline assay there was a significant reduction in collagen release from chondrocytes treated with acrylamide compared with levels released from untreated cells ( p< 0.001) (Fig. 5A). Levels of de novo collagen synthesis were therefore determined by labelling chondrocytes with [3H]-proline and ditgesing newly synthesised [3H]-protein with collagenase to give an

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indication of de novo collagen synthesis (collagen (cpm)= total protein (counts prior to digestion)− non-collagenous protein (counts after digestion)). As predicted, there was a steady increase in collagen synthesis in untreated chondrocytes over the 7 days of culture (data not shown). There was no difference in the [3H]collagen levels measured in the media of chondrocytes treated with acrylamide (p= 0.068) (Fig. 5B). However, a significant decrease in radiolabelled collagen was detected in the cells ( p < 0.001) (Fig. 5C). This decrease in collagen synthesis is clearly evident when immunoblotting for type II collagen. Type II pro-collagen synthesis was hardly detectable in acrylamide treated chondrocytes (Fig. 5D), whilst strong immunolabelling was detected in the untreated cells. Type I collagen was not observed in either cultures over the 7-day period (data not shown). 2.6. A disrupted vimentin network decreases collagen degradation in chondrocytes concomitant with a reduction in MMP synthesis To determine whether a reduction in de novo collagen was due to collagen degradation, a 3-day cold chase was performed (Fig. 6A) as outlined for [35S]-sGAG degradation. There was a significant reduction in collagen degradation after acrylamide treatment (12.3%) when compared with untreated chondrocytes (18.37%) ( p = 0.023). Media from chondrocytes treated with 5 mM acrylamide were analysed for MMPs 2 and 9 expression and activation by gelatin zymography (Fig. 6B). There was a significant reduction in pro-MMP 2 synthesis ( p = 0.003) (Fig. 6B) concomitant with a decrease in active MMP 2 ( p = 0.027) compared to untreated controls. Levels of pro-MMP 9 were below the limit of detection and could not be quantified. Media was also analysed for TIMP expression by gelatin reverse zymography (Fig. 6C). There was a significant decrease in the expression of TIMP 1 in acrylamide treated chondrocytes ( p = 0.013). However TIMP 2 expression was unaffected (data not shown). 2.7. Decreased aggrecan and type II collagen gene expression in vimentin disrupted chondrocytes Aggrecan and type II collagen gene expression was determined by quantitative PCR following normalisation to the housekeeping gene GAPDH. There was a significant reduction in both aggrecan ( p = 0.049) and type II collagen gene expression ( p = 0.012) in acrylamide treated cells over the duration of culture (Table 2). 2.8. Decreased matrix synthesis is independent of cell death

Fig. 4. Amount of sGAG degradation induced by acrylamide treatment. Degradation of de novo sGAGs was measured by performing a 3-day cold chase after 7 days of radiolabelling/treatment. Data is presented as a % degradation of newly synthesised sGAG. Disruption of vimentin caused a significant reduction in sGAG degradation (8.2%) compared with 17.1% in untreated cells. Normalised data are presented as mean ± S.E.M. (n = 4) (*p < 0.05).

Due to the decrease in cell number following acrylamide treatment (Table 1) and the presence of caspase 3 (Fig. 2), the contribution of apoptosis to the observed decrease in matrix synthesis was assessed by addition of the pan-caspase inhibitor Z-VAD FMK to the acrylamide treated cultures. The addition of 20 μM Z-VAD FMK rescued 50% of the cells with respect to those treated with acrylamide alone ( p = 0.01). The number of cells detected after acrylamide/Z-VAD FMK treatment corresponded to approximately 70% of the untreated cell population (Fig. 7A). However, the increased survival of cells treated with acrylamide/Z-VAD FMK

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Fig. 5. De novo synthesis of collagen in chondrocytes. (A) Amount of hydroxyproline from chondrocytes treated with 5 mM acrylamide for 7 days as measured using the hydroxyproline assay. [3H]-proline was added to the media in the presence of 5 mM acrylamide for 7 days, unincorporated label removed, subjected to bacterial collagenase digestion, re-purified and counts (cpm) measured in the (B) media and (C) cell extract. Acrylamide disruption of vimentin decreased collagen amounts in the media and significantly inhibited collagen synthesis in the cell extracts ( p < 0.001). (D) Expression of type II procollagen in cell extracts was detected by Western blotting using a monoclonal antibody AVT-6E3. Strong immunoreactivity was observed in the untreated chondrocytes but there was an almost complete abrogation of type II pro-collagen in acrylamide treated cells. Normalised data are presented as mean ± S.E.M. (n = 4) (***p < 0.001).

did not significantly affect the levels of sGAG and type II collagen synthesis. Similar amounts of sGAG were detected in the media of acrylamide treated cells irrespective of the presence of caspase inhibitor ( p= 0.621), as measured using the dimethylmethylene blue assay (Fig. 7B). Likewise, very little type II pro-collagen was observed in acrylamide treated cells in the presence or absence of inhibitor (Fig. 7C), whereas strong immunolabelling was detected in the untreated cells (+/− inhibitor). 3. Discussion Previous studies have demonstrated the importance of an intact cytoskeleton on cellular function (Bauer and Valle, 1982; Newman

and Watt, 1988; Wang et al., 1993; Hermann and Aebi, 2000) implicating both actin microfilaments and tubulin microtubles in diverse fundamental events such as mitosis, cell locomotion, protein synthesis and trafficking. Importantly, both actin microfilaments and the tubulin microtubules were shown to be crucial in maintaining the phenotype of cartilage chondrocytes. Several studies in epiphyseal (Newman and Watt, 1988) and foetal articular cartilage chondrocytes (Jansen and Bornstein, 1974; Lohmander et al., 1979; Bodo et al., 1996) have demonstrated that when either actin filaments or tubulin microtubules were disrupted using cytochalasin B/D or colchicine, respectively, there were concomitant decreases in the synthesis and secretion of both collagen and proteoglycan. We also find that in articular cartilage chondrocytes

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Table 2 Expression levels of aggrecan and type II collagen mRNA in chondrocytes treated with 5 mM acrylamide for 7 days, as assessed using quantitative “realtime” PCR

Control (untreated) Acrylamide (vimentin)

Aggrecan mRNA

Col2A1 mRNA

100% 35 ± 15%*

100% 1 ± 0.1%*

There was a significant reduction in both aggrecan ( p = 0.049) and type II collagen mRNA levels ( p = 0.012). Gene expression was normalised to the housekeeping gene GAPDH, and normalised data are presented as mean ± S.E.M. (n = 4) (*p < 0.05).

previously reported in glial cells (Eckert, 1985). A clear reduction in cell diameter was observed in chondrocytes treated with acrylamide in a time-dependent fashion with a reduction of approximately 50% by day 7. However, there appeared to be no overt effect of acrylamide on either the actin microfilaments or the tubulin

Fig. 6. Proteolytic profile of chondrocyte cultures treated with acrylamide for 7 days. (A) Amount of collagen degradation induced by vimentin disruption. Degradation of de novo collagen was measured by performing a 3-day cold chase after 7 days of radiolabelling/treatments. Data is presented as a % degradation of newly synthesised collagen. There was a significant reduction in collagen degradation after disruption of vimentin (12.3%) ( p = 0.013) when compared to control cells (18.4%). Normalised data are presented as mean ± S.E.M. (n = 4). (B) A representative gelatin zymogram depicting pro- and active-MMP 2 levels. Addition of acrylamide significantly inhibited pro- and active-MMP 2 amounts. Levels of pro-MMP 9 were below the limit of detection. (C) A representative reverse zymogram depicting TIMP 1 expression. There was a significant decrease in TIMP 1 expression. TIMP 2 was unaffected by acrylamide disruption of vimentin (data not shown).

both collagen and sGAG synthesis are decreased in the absence of a functional actin or tubulin network (data not shown). Such a sustained reduction in collagen and proteoglycan synthesis is detrimental to the cartilage tissue, and there are three recent studies suggesting that a disorganised cytoskeleton exists in articular cartilage chondrocytes from human OA (Fioravanti et al., 2003; Holloway et al., 2004) and a rat model of OA (CapinGutierrez et al., 2004.). In the latter case, a significant 20% reduction in vimentin protein was observed. We therefore undertook the current study to specifically investigate the role of vimentin intermediate filaments in articular cartilage chondrocyte matrix turnover and its potential implication in OA pathogenesis. Acrylamide, widely used to disassemble intermediate filaments, completely disrupted the normal organisation of the vimentin networks in the articular chondrocytes. Aggregation of the vimentin filaments onto the nucleus was a temporal event noticeable after 24 h, becoming more pronounced at days 3 and 7. The characteristic collapse of the filaments over the nuclei has also been

Fig. 7. Effect of the pan-caspase inhibitor Z-VAD FMK on acrylamide treated chondrocytes. Chondrocytes were treated for 7 days with acrylamide in the absence or presence of 20 μM Z-VAD FMK. (A) Cell number was determined using the CytoTox 96™ assay. Data are presented relative to the control cells. Addition of ZVAD FMK to the acrylamide treatment rescued approximately 50% of the cells (p = 0.01). (B) In the presence of Z-VAD FMK there was no difference in sGAG levels as measured using the DMMB assay. Data is presented as μg sGAG per cell (mean ± S.E.M.). (C) Type II pro-collagen was detected by immunoblotting with AVT-6E3 antibody. An equivalent number of cells were loaded. The addition of ZVAD FMK did not rescue type II collagen production. Data are presented as mean ± S.E.M. (n = 4) (**p < 0.01 ***p < 0.001).

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microtubules, although there is compaction of the organisations due to cell shrinkage. It has been previously reported that acrylamide does not affect the organisation of either actin or tubulin in chondrocytes (Trickey et al., 2004). This suggests that vimentin disassembly contributes directly to this cell “shrinkage”. It is widely accepted that the intermediate filaments play an important role in withstanding tensile stress (Wang et al., 1993) and contribute to the viscoelastic properties of the chondrocyte (Trickey et al., 2004). A reduction in stiffness, mechanical stability, motility and directional migration of vimentin −/− fibroblasts has been previously reported (Eckes et al., 1998) indicating a cellular fragility, a characteristic inherent in several disease states where loss of function mutations in the vimentin gene have been observed (Fuchs et al., 1994). Therefore these cells are incapable of resisting deformation at the cell surface (Eckes et al., 1998; Trickey et al., 2004). Since vimentin filaments regulate the swelling pressure of articular chondrocytes in situ (Durrant et al., 1999), an absence of organised vimentin architecture may reduce cell size due to an alteration in the swelling pressure. Such a profound alteration to the morphology of the chondrocyte (containing a disorganised vimentin architecture) would have severe implications on multiple functions including mechanotransduction and hence matrix turnover, as described in this paper. It is therefore likely that similar but less dramatic changes that have been observed in osteoarthritic cartilage may contribute to the phenotype observed in osteoarthritis. A dose response for acrylamide has previously been determined and 5 mM acrylamide was deemed to be non-toxic to glial cells over a 24-h period (Eckert, 1985). In order to measure collagen synthesis in our study, the period of acrylamide treatment was extended to 7 days and over this period a significant reduction in cell number was observed. This reduction in cell number can be attributed to either a reduction in cell proliferation or an increase in cell death, and we have evidence to suggest the latter occurs. As no lactate dehydrogenase was detected in the media, thus implying an intact cell membrane, we believe that the acrylamide treated chondrocytes are undergoing apoptosis. Untreated cells continue to proliferate over the 10-day culture period. Cell proliferation would not have been expected in the acrylamide treated cells as vimentin-deficient cells are slow to proliferate due to decreased DNA synthesis (Wang and Stamenovic, 2000). Vimentin filaments are a substrate for caspases (Niu and Nachmias, 2000; Byun et al., 2001; Dinsdale et al., 2004), so disassembly of the vimentin networks followed by caspase cleavage could induce apoptosis. Indeed, active caspase 3 (17 kDa) was detected in the acrylamide treated chondrocytes whereas it was undetectable in the untreated cultures, suggesting an apoptotic response to vimentin disassembly. Notably, addition of the pan-caspase inhibitor, Z-VAD FMK, reversed the effect of acrylamide on cell number, rescuing approximately 50% of the cells after treatment with acrylamide, indicating a role for the caspases in this cell death. Intermediate filaments are known to sequester pro-apoptotic molecules preventing cell death (Tzivion et al., 2000; DePianto and Coulombe, 2004), therefore, the absence of a functional vimentin network could promote a pro-apoptotic response. Interestingly, sGAG synthesis did not seem to be affected by disruption of the vimentin filaments as measured by [35S]-sulphate radiolabelling, but there was a significant decrease in aggrecan

mRNA transcription. As our data suggests that the cells could be entering apoptosis we hypothesise that the sGAG detected in the cell extract and media may be due to accumulation over the duration of culture, and may not reflect continuing biosynthetic activity. This accumulation of sGAG in the media of acrylamide treated cells may, in part, be attributed to the significant reduction in degradation of newly-synthesised [35S]-sGAGs. As yet we have no evidence to explain why degradation is reduced in the chondrocytes with disrupted vimentin filaments, but investigation into the levels of proteoglycan degrading enzymes may offer some insight. Unlike sGAG biosynthesis, de novo collagen synthesis was significantly affected by disruption of the chondrocyte vimentin networks. A 75% reduction in [3H]-collagen was observed in the cell extract compared with untreated cells. As [3H]-proline is preferentially incorporated into collagen (and accounted for over 90% of total newly synthesised protein in our study (data not shown)), this correlates closely with the 70–80% inhibition of protein synthesis observed in other cell types treated with 5 mM acrylamide (Klymkowsky, 1988; Aggeler and Seely, 1990). The reduction in collagen biosynthesis was confirmed by immunoblotting for type II collagen, the predominant collagen type in cartilage. In the untreated cells, high levels of type II pro-collagen were observed, as would be expected in high-density monolayer cultures where it has been documented that a chondrocytic phenotype is maintained (Goldring, 2004). However, there was almost complete abrogation of type II collagen in the acrylamide treated cells. This decrease in type II collagen could not be attributed to a de-differentiated, fibroblast-like phenotype as type I collagen was not observed (data not shown). Over the duration of culture, acrylamide treated cells also resulted in a significant inhibition of type II collagen gene transcription. We attribute this to a potential loss of intracellular signalling, perhaps mediated by the MAP kinases, and a shutting down of cellular processes marking the cells' entry into apoptosis. This is currently under investigation. Although there is an increase in cell death upon exposure to acrylamide, this does not account for the observed reduction in matrix synthesis. All data has been normalised to cell number, therefore accounting for the effect of cell death. Furthermore, the addition of the pan-caspase inhibitor Z-VAD FMK rescued approximately 50% of the cells, but this did not alter the biosynthetic activity of the cell. A significant decrease in both sGAG and type II collagen, as detected by DMMB assay and immunoblotting respectively, was still observed in the presence of the inhibitor indicating that acrylamide's effect on cell death and matrix turnover are by two separate mechanisms. Analysis of degraded [3H]-collagen fragments indicated that there was also an inhibition of collagen degradation in the acrylamide treated chondrocytes. This reduction in collagen degradation correlates with lower levels of pro- and active-MMP 2 as observed by gelatin zymography. Recent reports have implicated vimentin in several carcinomas (Hu et al., 2004; Taki et al., 2006). Increased vimentin was observed in highly motile tumour cells, and it is hypothesised that vimentin, by an as yet unidentified mechanism, may induce pro-MMP 2 synthesis and activation which enhances the invasiveness of these cells in promoting tumour progression. Thus, the loss of an intact vimentin network may promote the converse effect of down-regulating pro-MMP 2 synthesis and activation as observed in our study.

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As the vimentin network extends from the plasma to the nuclear membrane, and disruption by acrylamide leads to collapse of the filaments around the perinuclear region, it is not surprising that these cytoskeletal elements modulate cellular responses. Interestingly, recent studies have shown a similar perinuclear collapse of vimentin filaments in cells in which the GTPases have been manipulated. Cells transfected with active cdc42Hs or Rac1 showed a perinuclear collapse of the vimentin filaments initiated by activation of specific tyrosine kinase(s) (Meriane et al., 2000). It has been previously documented that the RhoA-binding kinase α (ROKα) facilitates collapse of the vimentin network (Sin et al., 1998), and it has since been reported that ROKα activation, by Rho, results in juxtanuclear aggregation of vimentin via ROKα phosphorylation of the filaments (Ehrenreiter et al., 2005). Downstream targets of Rho include proteins which localise at focal contacts i.e. erzin, FAK and paxillin (Meriane et al., 2000). Recently, it has been shown that vimentin filament assembly colocalises with sites of focal contact assembly (Tsuruta and Jones, 2003). Down-regulation of vimentin expression in chondrocytes, utilising specific phosphorothioate oligonucleotides, results in disparities in paxillin localisation in these antisense treated cells when compared to untreated control cells (unpublished observations). Interestingly, abrogation of vimentin in endothelial cells using siRNA resulted in smaller focal contacts with dramatically reduced adhesion properties (Tsuruta and Jones, 2003). Therefore we postulate that a loss of cell contact with the surrounding matrix, due to disruption of the vimentin filaments/associated focal contacts, could alter the signalling dynamics of the cell, and in effect, shut down transcriptional events. Studies are ongoing to determine the effect of vimentin filament disruption on formation of focal contacts and the possible involvement of the GTPases in the signalling mechanisms that perpetuate decreased matrix synthesis. We endeavour to substantiate a link between the effects observed in our in vitro model of vimentin disassembly and the altered vimentin architecture observed in OA chondrocytes (Fioravanti et al., 2003; Capin-Gutierrez et al., 2004; Holloway et al., 2004), as we believe that disruption of the vimentin network could promote an imbalance in cartilage homeostasis due to reduced anabolism. This would ultimately disturb the integrity of the tissue as observed in the pathology of OA. Thus this study reports, for the first time, the involvement of the vimentin cytoskeletal architecture in modulating matrix biosynthesis, as characterised by changes at both the transcriptional and translational level, and its role in mediating catabolic events in the chondrocyte. Further, we have demonstrated that an intact vimentin intermediate filament network is necessary in maintaining the chondrocyte phenotype and thus an imbalance favouring filament disassembly has the potential to disturb the integrity of the articular cartilage matrix.

streptomycin, 50 μg/ml L-ascorbate-2-phosphate and 1× Insulin– Transferrin–Sodium selenite (ITS). For radiolabelling experiments, DMEM-Glutamax I™ was replaced with DMEM glutamax I™/HAMS F12 media (1:1).

4. Experimental procedures

The effect of vimentin filament disruption on cell viability was assessed using the CytoTox 96® non-radioactive cytotoxicity assay (Promega) according to the manufacturer's instructions. The enzymatic assay quantitatively measures lactate dehydrogenase levels in culture medium as a consequence of cell lysis i.e. through cell death.

All reagents were purchased from Sigma (Poole, UK) unless otherwise specified. Culture medium consisted of Dulbecco's Modified Eagle's Medium (DMEM-Glutamax I™, Invitrogen, UK) supplemented with 100 Units ml− 1 penicillin, 100 μg ml− 1

4.1. Primary bovine articular cartilage chondrocyte isolation Primary chondrocytes were isolated from the metacarpalphalyngeal joint of 7-day-old bovine calves as described previously (Vaughan-Thomas et al., 2001). Chondrocytes were plated at a density of 1 × 106 cells/ml per well of a 24-well plate or at 0.4 × 106 cells/ml per well of a 48-well plate for radiolabelling experiments. 4.2. Vimentin intermediate filament disruption Cells were stabilised for 24 h after isolation and the media replaced with culture medium containing 5 mM acrylamide to promote vimentin intermediate filament disruption (Eckert, 1985) for 7 days. Cells were also incubated, for 7 days, with acrylamide in the presence or absence of 20 μM Z-VAD FMK (Promega) — a broad spectrum pan-caspase inhibitor. 4.3. Immunofluorescence and confocal microscopy Chondrocytes (0.5 × 106 cells/ml) were seeded onto glass slides and incubated in the presence or absence of treatment for 1, 3 or 7 days to determine whether there were temporal changes in vimentin organisation over the duration of culture. Cells were fixed in 2% (w/v) paraformaldehyde and permeabilised in icecold 100% methanol. Cells were labelled for vimentin (1:25 dilution of clone V9, Sigma), actin (1:100 dilution of clone AC15, Abcam) or tubulin (neat clone E7, Hybridoma Bank, NIH) for 2 h in a humidified chamber. After removal of primary antibody by repeated washes with 1xPBS, an anti-mouse secondary antibody conjugated to FITC was added (1:64 dilution) for 1 h. After repeated washes in 1× PBS to remove residual antibody, the cells were mounted in VectaShield™ containing propidium iodide (VectorLabs) and visualised using a confocal microscope (Leica TCS SP2 AOBS). Representative cells were scanned using a 63× oil immersion objective (× 8.85 zoom) with appropriate excitation and emission settings for fluoroscein isothiocyanate. Stacks of optical sections through the full depth of chondrocytes were taken at a spacing of 0.3–0.5 μm. Maximum intensity type reconstructions were prepared and images sharpened using Erosion software (Leica Confocal Software). Negative controls, omitting the primary antibody, conducted in parallel were devoid of fluorescent signal (data not shown). 4.4. CytoTox 96® assay: measurement of cytotoxicity

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4.5. CytoTox 96® assay: measurement of cell number The effect of vimentin filament disruption on cell number was also assessed using the CytoTox 96® non-radioactive cytotoxicity assay (Promega). To calculate the relative cell number the cells were lysed in cell extract buffer (0.9% triton X-100 containing 1 μM leupepstatin hemisulphate, 150 nM aproptin, 0.5 mM EDTA disodium salt, 500 μM AEBSF HCl, 1 μM E-64 (Calbiochem)) and the total amount of LDH measured. 4.6. Western blot analysis Cell extract protein from an equivalent number of cells (as determined using the CytoTox 96® assay) was separated on a 10% (v/v) SDS-PAGE gel, and transferred onto PVDF membrane (Millipore) (100 V for 1 h in Laemmli buffer containing 20% methanol). After blocking with TBS (Tris buffered saline, pH 8.3) containing 3% non-fat milk powder, the membranes were probed for 2 h with either a monoclonal antibody, AVT-6E3, which recognises type II collagen (Young et al., 2002) or a rabbit anti-caspase 3 antibody (Stressgen). Membranes were washed 3 times in TBS containing Tween 20® (0.05%) followed by incubation with an affinity purified HRP-conjugated anti-mouse or rabbit antibody for 60 min. After extensive washing to remove residual antibody, the bound antibody was visualised using ECL™ reagent kit (Amersham), and Hyperfilm (Amersham) developed after overnight exposure. 4.7. DMMB assay The amount of sulphated glycosaminoglycans (sGAG) released from the cells was determined using the DMMB assay as described previously (Little et al., 1999) using chondroitin-4-sulphate (sodium salt from shark cartilage) as a standard. 4.8. Hydroxyproline assay The amount of collagen released from the chondrocytes was determined by an adaptation of the method by Woessner (1976). Briefly, media samples were hydrolysed in 6 N HCl at 110 °C for 24 h and samples freeze-dried. A standard curve was prepared by dissolving 4-hydroxyproline in DMEM-glutamax I™ to give a range of 1–10 μg/ml. Hydrolysates (30 μl) and standards were added in triplicate to a 96-well plate followed by 70 μl diluent (66% v/v isopropanol) and 50 μl oxidant (18 mM chloramine T, 10% v/v water, 50% v/v stock buffer) before incubation at room temperature for 5 min. Colour reagent (3.7 mM dimethylamino benzaldehyde, 15% perchloric acid, 85% isopropanol) was added (125 μl) and incubated at 70 °C for 40 min. The absorbance was detected (540 nm) and the hydroxyproline content calculated from the standard curve. 4.9. Metabolic radiolabelling using [35S]-sulphate and [3H]-proline To ascertain whether changes in sGAG or collagen levels, induced by disruption of vimentin were a consequence of de novo

synthesis, radiolabelling experiments were performed. Acrylamide was added to cultures (as above) with 20 μCi/ml of [3H]-proline and 10 μCi/ml of [35S]-sulphate (Amersham Biosciences, UK) (Lohmander et al., 1979). At the end of each experiment, protease inhibitors (1 μM leupepstatin hemisulphate, 150 nM aproptin, 0.5 mM EDTA disodium salt, 500 μM AEBSF HCl, 1 μM E-64, Calbiochem) were added to both media and cell extracts to prevent further proteolytic degradation. Protein and sGAG biosynthetic levels were measured by removing unincorporated label using Ultrafree®-MC centrifugal filter units (10 kDa cut-off) according to the manufacturer's instructions (Millipore, UK) and both [3H] and [35S] counts recorded (Beckman scintillation counter). De novo collagen synthesis was determined by digesting collagen in the labelled media and cell extracts with 8U bacterial collagenase (Type III collagenase, Worthington Enzymes, UK) at 37 °C overnight (Scott et al., 1982). Digested collagen fragments were removed (Ultrafree®-MC centrifugal filter units — 10 kDa cut-off) and [3H] counts performed as a measure of non-collagenous proteins. The loss of [3H] counts during this procedure provided a value for collagen produced by the chondrocytes (collagen (cpm)= total protein (counts prior to collagenase digestion) − non-collagenous protein (counts after collagenase digestion)). To assess the level of sGAG and collagen degradation induced by vimentin disruption, a 3-day cold chase experiment was performed. Identical experiments were set up and after 7 days of treatment, media (containing radiolabel) was removed and replaced with non-radioactive media for 3 days. After the 3-day cold chase, counts (from media plus cell extract) were recorded prior to and after removal of unincorporated label to assess degradation of newly synthesised [3H]-collagen and [35S]-sGAGs. 4.10. Quantitative PCR Chondrocytes were lysed in 1 ml of Trizol™ (Invitrogen), and total RNA extracted with chloroform and precipitated with isopropanol according to manufacturer's protocol, and as previously described (Blain et al., 2003). Total RNA was dissolved in 43 μl of RNase free dH2O and treated with 1 unit DNase in the presence of 40 units RNase inhibitor (Promega, UK) at 37 °C for 15 minutes. RNA was purified (RNeasy™, Qiagen, UK), eluted in 50 μl dH2O and the integrity determined by agarose gel electrophoresis. cDNA was produced by priming 11 μl RNA with 0.5 μg/ μl oligo (dT)15 (Promega) followed by adding 200 units Superscript II™ reverse transcriptase in 1× buffer (Gibco BRL) at 42 °C for 50 min. Real-time PCR was carried out on an ABI 7700 Sequence Detection System using the 5′-nuclease assay. Fluorescently labelled probes (5′ 6-carboxyfluorescein and 3′ 6carboxytetramethylrhodamine) were designed to GAPDH (forward primer: 5′-GGCATCGTGGAGGGACTTATGA-3′, reverse primer: 5′-CAGAAGACTGTGGATGGCCC-3′, probe: 5′CACTGTCCACGCCATCACTGC-3′), type II collagen and aggrecan (Darling and Athanasiou, 2005), and obtained from Applied Biosystems. Quantitative PCR was carried out as described previously (Stephens et al., 2004) using 300 nM forward and reverse primers and 200 nM probe (Table 1). Calculation of starting concentration was based on standard curves for each target DNA run in parallel. GAPDH was used as an internal reference of

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housekeeping gene transcription for normalisation between different cDNA samples. 4.11. Gelatin zymography and reverse zymography The expression and activation of MMPs 2 and 9, and the expression of TIMPs 1 and 2 were evaluated in experimental media samples by gelatin zymography and gelatin reverse zymography, respectively (Blain et al., 2001). Their relative quantities were analysed by scanning densitometry (UMAX magic scan) and NIH image software (NIH, Bethesda, MD). 4.12. Statistical analysis Data were normalised to cell number and presented as mean + standard error mean (n = 4). Each experiment was repeated and representative data are presented. Differences (paired t-test; Minitab) were considered significant at P values less than 0.05. Where data was not normally distributed or of unequal variance the Mann–Whitney test was performed. Acknowledgements The authors would like to acknowledge funding from the European Union 5th Framework (ECM Ageing) and the Arthritis Research Campaign. The E7 monoclonal antibody developed by Michael Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. References Aggeler, J., 1990. Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenaseinducing agents. Cell Motil. Cytoskelet. 16, 121–132. Aggeler, J., Seely, K., 1990. Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments. Cell Motil. Cytoskelet. 16, 110–120. Archer, C.W., Rooney, P., Wolpert, L., 1982. Cell shape and cartilage differentiation of early chick limb bud cells in culture. Cell Differ. 11, 245–251. Bauer, E.A., Valle, K.-J., 1982. Colchicine-induced modulation of collagenase in human skin fibroblast cultures: I. Stimulation of enzyme synthesis in normal cells. J. Invest. Dermatol. 79, 398–402. Benjamin, M., Archer, C.W., Ralphs, J.R., 1984. Cytoskeleton of cartilage cells. Microsc. Res. Tech. 28, 372–377. Blain, E.J., Gilbert, S.J., Wardale, R.J., Capper, S.J., Mason, D.J., Duance, V.C., 2001. Up-regulation of matrix metalloproteinase expression and activation following cyclical compressive loading of articular cartilage in vitro. Arch. Biochem. Biophys. 396, 49–55. Blain, E.J., Mason, D.J., Duance, V.C., 2003. The effect of cyclical compressive loading on gene expression in articular cartilage. Biorheology 40, 111–117. Bodo, M., Carinci, P., Baroni, T., Becchetti, E., Bellucci, C., Pezzetti, F., Giammarioli, M., Stabellini, G., Arena, N., 1996. Collagen synthesis and cell growth in chick embryo fibroblasts: influence of colchicines, cytochalasin B and concanovalin A. Cell Biol. Int. 20, 177–185. Brown, P.D., Benya, P.D., 1988. Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression. J. Cell Biol. 106, 171–179. Byun, Y., Chen, F., Chang, R., Trivedi, M., Green, K.J., Cryns, V.L., 2001. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ. 8, 443–450.

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