Objectives: Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis. In advanced osteoarthritis, cartilage shows chondrocyte apoptosis, overexpression of annexin 5 (A5) and BCP crystal deposition within matrix vesicles. We assessed in vitro whether BCP crystals and overexpression of A5 increased chondrocyte apoptosis.
Methods: Apoptosis was induced by BCP crystals, tumour necrosis factor (TNF)-α (20 ng/ml) and Fas ligand (20 ng/ml) in normal articular chondrocytes (control) and in A5 overexpressed chondrocytes, performed by adenovirus infection. Apoptosis was assessed by caspase 3 (Cas3) activity, and DNA fragmentation.
Results: All BCP crystals, TNF-α and Fas ligand induced chondrocyte apoptosis as demonstrated by decreased cell viability and increased Cas3 activity and DNA fragmentation. TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling)-positive staining chondrocytes were increased by OCP (12.4 (5.2)%), CA (9.6 (2.6)%) and HA (9.2 (3.0)%) crystals and TNF-α (9.6 (2.4)%) stimulation compared with control (3.1 (1.9)%). BCP crystals increased Cas3 activity in a dose-dependent fashion. BCP-crystal-induced chondrocyte apoptosis was independent from TNF-α and interleukin-1β pathways but required cell-crystal contact and intralysosomal crystal dissolution. Indeed, preincubation with ammonium chloride, a lysosomal inhibitor of BCP crystal dissolution, significantly decreased BCP-crystal-induced Cas3 activity. Finally, overexpression of A5 enhanced BCP crystal- and TNF-α-induced chondrocyte apoptosis.
Conclusions: Overexpression of A5 and the presence of BCP crystals observed in advanced osteoarthritis contributed to chondrocyte apoptosis. Our results suggest a new pathophysiological mechanism for calcium-containing crystal arthropathies.
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Osteoarthritis (OA) is a disabling disorder characterised by joint cartilage destruction. The multifactorial pathogenesis of OA involves not only multiple genes, but also mechanical overload, ageing and the intra-articular crystals of basic calcium phosphate (BCP). The presence of Ca2+ crystals within the cartilage has recently been associated to ageing.1 In advanced OA the upper zone of the cartilage shows matrix breakdown, chondrocyte apoptosis, overexpression of type X collagen and annexin 5 (A5), and BCP crystal deposition within matrix vesicles.2
BCP crystals include octacalcium phosphate (OCP), tricalcium phosphate, carbonate-substituted apatite (CA) and hydroxyapatite (HA). They are associated with severe forms of OA3 and joint destruction.4 BCP crystals were identified in synovial fluid,3 found in OA cartilage and observed within chondrocytes.5 6 BCP crystals can be released from subchondral bone through cartilage lesions and/or synthesised in situ, in particular in the ageing joint, within matrix vesicles2 7 and apoptotic bodies,8 whose numbers are increased in osteoarthritic cartilage.
The mechanism of cartilage breakdown in BCP crystal-associated OA remains unclear. In vitro, BCP crystals stimulated: the proliferation of human skin fibroblasts;9–11 production of matrix-degrading metalloproteases (MMP);10 12 13 and secretion of inflammatory mediators.14–17 We recently reported that OCP crystals directly stimulated joint chondrocytes to produce nitric oxide (NO) through the JNK and p38 MAPK pathways.17 NO mediates catabolism in OA18 and chondrocyte apoptosis.18 19
Chondrocyte apoptosis is a hallmark of OA.2 8 20 Chondrocytes undergo apoptosis after exposure to NO19 or Fas ligand (Fas-L).21 Apoptotic bodies share similarities with the matrix vesicles that are released by terminally differentiated, mineralisation-competent, hypertrophic chondrocytes.7 8 Both particles are cell-derived membrane-enclosed structures that can precipitate calcium and initiate mineralisation.8 Matrix vesicles also contain MMPs, and phospholipid-binding proteins belonging to the annexin family.22 23
Annexins are ubiquitous proteins that can interact with acid phospholipids, membranes and cytoskeleton constituents in the presence of Ca2+.24 They are involved in regulating intracellular and extracellular activities such as endocytosis and exocytosis and Ca2+ fluxes.24 Chondrocytes produce annexins 2, 5 and 6 (A2, A5 and A6), whose levels are increased in OA cartilage.2 25 A2, A5 and A6 have been identified on matrix vesicles.2 25 A5 can form voltage-gated Ca2+ channels24 and mediates Ca2+ influx into matrix vesicles, which initiates extracellular mineralisation,23 and into cellular cytoplasm, which induces apoptosis of growth-plate chondrocytes.26 27 We showed that A5 promoted the apoptosis of cardiomyocytes.28
We designed an in vitro study to investigate the direct effect of BCP crystals on cartilage chondrocyte apoptosis and the potential role for A5.
MATERIALS AND METHODS
Chondrocyte isolation and culture
Chondrocytes were isolated from 3-year-old calf metatarsophalangeal joints, as previously described.17 Briefly, joint cartilage was cut into small pieces, and chondrocytes were released by digestion with bacterial collagenase type II (Sigma-Aldrich) (0.2% in Dulbecco modified Eagle’s medium; Invitrogen). Chondrocytes were collected through a 100 μm nylon cell strainer (Falcon, VWR), washed, and plated at high density (107 cells/ml) in complete medium (high glucose Dulbecco modified Eagle’s medium, 10% fetal calf serum (Dominique Dutscher) with antibiotics). At subconfluence, cells were harvested and replated at 104 cells/well in 96-well plates (for viability tests), 2×105 cells/well in 24-well plates (for caspase 3 activity studies), and 5×105 cells/well in six-well plates (for TUNEL studies (terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling; Roche)).
Basic calcium phosphate and urate monosodium crystal preparation
Sterile, pyrogen-free BCP and urate monosodium (MSU) crystals were synthesised as previously described.29 BCP crystal sizes and shapes were determined using a Philips EM 300 transmission electron microscope and their nature was confirmed by x-ray diffraction and infrared spectroscopy. BCP crystals were sterilised by exposure to 60Co γ-radiation by CisBio International (Laboratoire des Produits d’Irradiation at the Commissariat à l’Energie Atomique, Saclay, France). MSU crystals were sterilised by heating at 180°C for 30 min. MSU and BCP crystals were confirmed to be pyrogen-free, as previously reported.29 Crystals were suspended in serum-free culture medium and dispersed by brief sonication.
Subconfluent chondrocytes were harvested and seeded on 96-well plates (104 cells/well), 24-well plates (2×105 cells/well), or six-well plates (5×105 cells/well). After being cultured overnight in complete medium, chondrocytes were washed, starved overnight in serum-free medium and then washed again before apoptosis induction with BCP crystals, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, Fas-L (R&D Systems Inc, Lilles, France) or sodium nitroprusside (SNP) (Sigma-Aldrich).
Caspase-3 activity measurement
After apoptosis induction, cells were incubated on ice for 15 min in lysis buffer (10 mM Tris pH 7.4, 200 mM NaCl, 5 mM EDTA pH 7.4, 10% glycerol and 1% NP40). Lysates were centrifuged at 12 000 g for 10 min, and supernatants were collected and stored at −20°C until used. Lysate, 100 μl, was incubated for 2 h at 37°C with 200 μl of reaction buffer (0.1 mM phenylmethylsulphonyl fluoride, 10 mM dithiothreitol, 10 mM Hepes/NaOH pH 7.4) and 200 μM of fluorophore-coupled Cas3 substrate (DEVD coupled with 7-amino-4-trifluoromethyl coumarin (DEVD-AFC) (Interchim)). Cas3 cleavage led to the emission of free AFC fluorescence (505 nm), which was measured after excitation at 400 nm. Cas3 activity was normalised for the protein content. Cas3 activity was expressed as arbitrary units (AU) with Cas3 activity of the untreated cells taken as 1.
Viability was assessed in 96-well plates (replicates of six) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) test. MTT, 10 μl of a 5 mg/ml solution, was incubated with cells 4 h before the end of the experiments. Supernatants were then discarded and the formazan formed by living cells was dissolved with dimethyl sulphoxide, which yielded a purple colour. Colour intensity was measured at 560 nm.
Apoptosis was induced as described above. DNA fragmentation was assessed using an enzyme-linked immunosorbent assay kit (Roche).
Terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling assay
TUNEL (Roche) was performed according to the manufacturer’s instructions. Briefly, after induction of apoptosis, chondrocytes were fixed with acetone/methanol (volume/volume) and counterstained with Hoechst 33342 (1 μg/ml) (Sigma-Aldrich) for cell counting. TUNEL staining was then performed. Immunofluorescent images were obtained and were counted by coupling to an imaging analysis system (Histolab software, Microvision). Ten fields were counted for each dish.
Annexin 5 overexpression
Human full-length A5 cDNA in pRC-CMV vector (kindly donated by Professor Moss, London, UK) was cloned and ligated downstream from the cytomegalovirus promoter into vector pDC-515. Replication-defective recombinant adenovirus vectors were used. A5 or LacZ pDC-515 vectors were recombined with pBHGfrtΔE1.3flp plasmid (Microbix Bioreagents) according to standard procedures.30 Monolayer chondrocyte cultures were infected with adenovirus expressing either A5 or LacZ at different multiplicities of infection (MOI) for 6 h. Culture media were then changed and the adenovirus was allowed to replicate for 48 h before apoptosis induction using different stimuli. A5 overexpression was checked by immunoblotting for each experiment.
Preparation of cytoplasmic extract
Monolayer chondrocyte cultures were placed in lysis buffer. After sonication, lysates were incubated on ice for 15 min and centrifuged at 14 000 rpm for 10 min at 4°C. The supernatants containing cell lysates were collected, and the protein concentrations were measured using the method of Bradford.
Crystal dissolution to detect bound annexin 5
After 48 h stimulation with BCP and MSU crystals, chondrocytes were incubated in lysis buffer for 15 min on ice. Cells and crystals were collected. Crystals were settled by slow centrifugation (2 g) for 1 min and dissolved with 0.1 mol/l HCl. Dissolved samples containing proteins bound to crystals were subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and immunoblotting with anti-A5 antibody (kindly donated by Dr Françoise Russo-Marie, Institut Cochin, Paris) was performed.
Cytoplasmic and supernatant proteins (15 μg) were diluted in Laemmli buffer, separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred on to PVDF membranes. Membranes were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline-Tween (TBS-T), washed three times with TBS-T, and incubated overnight at 4°C with anti-A5 antibody (1/1000) in TBS-T 5% milk. After washing with TBS-T, blots were incubated with a horseradish peroxidase-conjugated secondary antibody. The protein complexes were visualised by chemiluminescence Luminol reagent (Pierce, Brebieres, France). The membranes were subsequently stripped and re-probed with anti-actin (Sigma-Aldrich) antibody.
RNA isolation and reverse transcription–polymerase chain reaction
Cultured chondrocyte RNA were isolated with TRIzol reagent according to manufacturer’s instructions. Then, 2 μg of each sample was reverse-transcribed using the M-MLV reverse transcription–polymerase chain reaction (reverse transcription–PCR) system. The resulting cDNA samples were amplified by PCR. PCR primers for bovine collagen IIα1 primers, which generated a 527 bp product, were as follows: sense, 5′-GAT CCG CAA CAT GGA GAC TGG CGA-3; and anti-sense, 5′-CAA GAA GCA GAC AGG CCC TAT GTC CAC-3′. Primers for the housekeeping gene GAPDH amplified a 579 bp product and were as follows: sense, 5′-ATC ACC ATC TTC CAG GAG CG-3; and anti-sense, 5′-CCT GCT TCA CCA CCT TCT TG-3′. The PCR products were analysed by electrophoresis on 2% agarose gel containing ethidium bromide.
Values are the mean±SEM of at least four independent experiments performed in four replicates, unless stated otherwise. Groups were compared using ANOVA and Bonferroni tests. p<0.05 was considered statistically significant.
Basic calcium phosphate crystals induced chondrocyte apoptosis
Collagen type II expression remained unchanged during the experiments, which lasted 4 days at the most (fig 1A). Compared with the control condition, exposure to BCP crystals for 1 day led to significant increases in Cas3 activity 15.9 (4.2) arbitrary units (AU)/μg proteins with OCP, 14.3 (1.2) AU/μg with CA, and 13.9 (4.1) AU/μg with HA, compared with 7.0 (0.1) AU/μg with the control; p<0.0001 for all three comparisons) (fig 1B). After 2 days of exposure to the crystals, Cas3 activity reached a plateau, at which the increases compared with control cells were 3.7-fold (±0.8) with OCP, 3.5-fold (±0.5) with CA, and 4.0-fold (±1.1) with HA (p<0.0001 for all three comparisons) (fig 1B). The effect of BCP crystals on Cas3 activity was dose-dependent (fig 1C).
To establish that increased Cas3 activity led to chondrocyte apoptosis, we assessed cell viability and DNA fragmentation. All three BCP crystals decreased cell viability, as evaluated by the MTT test, in a dose-dependent manner (fig 2A). Furthermore, DNA fragmentation increased, denoting increased apoptosis; the increases were 4.7-fold (±0.9) with OCP, 2.7-fold (±0.2) with CA and 3.0-fold (±0.7) with HA, compared with untreated cells (fig 2B). TUNEL staining confirmed the increase in apoptosis: percentages of TUNEL-positive cells after 48 h of crystal exposure were 12.4 (5.2)% with OCP, 9.6 (2.6)% with CA and 9.2 (3.0)% with HA, compared with 3.1 (1.9)% without crystals (p<0.001 for all three comparisons) (fig 2C,D). Other apoptotic factors, such as the NO donor SNP, Fas-L and etoposide, dramatically increased Cas3 activity (fig 3A). TNF-α also increased Cas3 activity and apoptosis as demonstrated by TUNEL staining (fig 3A,B). In contrast, IL-1β did not induce chondrocyte apoptosis (fig 3A), in agreement with previous studies.19
Basic calcium phosphate crystal-induced chondrocyte apoptosis was independent from the tumour necrosis factor-α pathway but required cell-crystal contact and intracellular crystal dissolution
As both SNP and TNF-α induced chondrocyte apoptosis, we investigated their role in BCP-crystal-induced apoptosis. As monolayer-cultured chondrocytes did not produce NO, even after stimulation with IL-1β (data not shown), BCP-crystal-induced apoptosis was NO independent, at least for the 3-year-old animals. We used the anti-TNF-α monoclonal antibody infliximab (10 μg/ml, Schering-Plough, Puteaux, France) to investigate whether the effects of BCP crystals were dependent on TNF-α. Infliximab pre-treatment did not modify BCP-crystal-induced chondrocyte apoptosis but completely inhibited TNF-α effect (fig 3D). Thus, BCP-crystal-induced chondrocyte apoptosis was independent from the TNF-α pathway.
Next, we evaluated whether chondrocyte apoptosis induced by BCP crystals was related to crystal dissolution causing calcium and/or phosphate concentrations to rise in the culture medium. To test this hypothesis, we stimulated cells with supernatants of crystals that were suspended and maintained in serum-free medium for 48 h at 37°C. As shown in fig 4A, Cas3 activity was unchanged by exposure to crystal supernatant. In contrast, when we prevented cell-crystal contact by using a Transwell chamber (Costar, ATGC, Noisy-Le Grand, France) whose 1 μm pore membrane allowed diffusion of ions, but not crystals, BCP crystals failed to increase Cas3 activity (fig 4A). Thus, chondrocyte apoptosis induced by BCP crystals was independent from elevations in extracellular calcium and/or phosphate concentrations but required direct cell-crystal contact. To study the role of intracellular calcium variations in BCP-crystal-induced apoptosis we used ammonium chloride (NH4Cl2) (10 mM) to inhibit intracellular BCP crystal dissolution by increasing the intralysosomal pH.13 We showed that pre-treatment with NH4Cl2 significantly decreased Cas3 activity induced by BCP crystals (OCP, 4.7 (0.3) vs 15.1 (1.0); CA, 4.2 (0.1) vs 8.7 (0.6); and HA, 2.9 (0.5) vs 8.1 (0.3); p<0.0001) (fig 4B). Of interest, when chondrocytes were treated with the endocytosis inhibitor cytochalasin B, Cas3 activity induced by BCP crystals was significantly reduced (OCP, 5.0±0.4 vs 13.2±1.0; CA, 4.4±0.1 vs 7.6±0.7; and HA, 3.8±0.2 vs 7.1±0.3; all p<0.0001) (fig 4C), whereas TNF-α effect was not modified (fig 4C). Taken together, these results demonstrated that BCP-crystal-induced chondrocyte apoptosis involved cell-crystal contact, crystal endocytosis and intralysosomal crystal dissolution.
Annexin 5 overexpression increased chondrocyte apoptosis induced by basic calcium phosphate crystals
Our results suggested that variations in intracellular calcium levels secondary to intralysosomal BCP crystal dissolution contributed to BCP-crystal-induced apoptosis. BCP crystals can induce Ca2+ influx within minutes.13 A5 can induce Ca2+ channel formation and is increased and associated with BCP crystal presence in advanced OA cartilage.31 Therefore, we investigated whether A5 overexpression, performed by adenovirus infection, modulated chondrocyte apoptosis induced by BCP crystals. A5 overexpression was confirmed by immunostaining (fig 5A) and immunoblotting (fig 5B). Compared with normal cells, chondrocytes that overexpressed A5 showed enhanced Cas3 activity after stimulation by either BCP crystals (OCP, 8.5 (2.6) vs 5.1 (1.0), p<0.0001; CA, 1.5 (0.3) vs 1.3 (0.2), p<0.05; and HA, 3.6 (0.9) vs 2.6 (0.5), p<0.005) (fig 5C,D) or TNF-α (fig 5E). A5 and actin (fig 5C–E) proteins were assayed in each experiment by immunoblotting. Furthermore, BCP-crystal-induced Cas3 activity increased with the level of A5 overexpression (fig 5D).
Annexin 5 can bind specifically to basic calcium phosphate crystals but not to urate monosodium crystals
We investigated whether BCP crystals modulated, and interacted with, A5 expression. A5 coating was evidenced by immunoblotting after crystal dissolution in 0.1 mol/l HCl (fig 6A). This result was confirmed by immunofluorescence, in which synthetic BCP crystals incubated with fluorescein isothiocyanate-coupled A5 displayed green fluorescence (fig 6B), whereas MSU crystals and calcium-free crystals were not stained.
BCP crystals are found in OA cartilage and observed in chondrocytes and are associated with severe forms of OA.3 5 6 32 Previous studies showed that the loss of cells in OA cartilage was chiefly attributed to chondrocyte apoptosis,2 20 33 which might generate apoptotic bodies, thereby promoting BCP crystal formation. There is no evidence yet for chondrocyte apoptosis in early OA cartilage. However, the effect of BCP crystals on chondrocyte apoptosis had not been assessed previously. In contrast to Mitchell et al’s study who showed that BCP crystals induced porcine chondrocyte proliferation,34 we demonstrated that BCP crystals consistently induced bovine chondrocyte apoptosis. Chondrocyte apoptosis was evidenced after 1 day of exposure to BCP crystals or other known apoptotic stimuli such as Fas-L, NO and TNF-α. These contradictory results can be secondary to cell origin as BCP crystals had different effects according to cell type. Thus, BCP-crystal-induced chondrocyte inducible nitric oxide synthase production involved the p38 and JNK MAPK pathways,17 whereas fibroblast MMP-1 induction was secondary to Erk 1/2 MAPK pathway activation.35 However, we hypothesise that BCP-crystal-induced chondrocyte apoptosis contributes to cartilage damage observed in destructive OA related to calcium-containing crystals.
Calcium-containing crystals and MSU crystals may stimulate cells via several mechanisms: (a) changes in cytoplasmic membrane permeability, which may facilitate the entry of some molecules;36 (b) mechanoreceptor or integrin stimulation;37 (c) endocytosis and intralysosomal crystal dissolution with subsequent elevation of intracellular Ca2+ levels;9 13 and (d) binding to specific membrane receptors related to surface proteins such as Toll-like receptor 2 and 4,38 39 CD14 with CPPD crystals,40 or immunoglobulin Fc-fragment receptor.41
Although the mechanism of BCP-crystal-induced apoptosis was not completely elucidated in our study, we showed it was independent from the IL-1β and TNF-α pathways and found evidence that crystal endocytosis and intralysosomal dissolution were involved, in keeping with reports of BCP crystal effects on fibroblast proliferation.9 10 12 That chondrocytes are capable of phagocytosis was first established by Cheung et al, who used transmission electron microscopy to visualise HA crystals engulfed by chondrocytes.42 Subsequently, a confocal microscopy study showed that chondrocytes were able to phagocytise gold-labelled particles.43
BCP-crystal-induced changes in intracellular Ca2+ levels were reported many years ago in fibroblast-like synoviocytes. BCP crystals induced biphasic variations in intracellular Ca2+ levels in human foreskin fibroblasts.13 The first phase was elevation of intracellular Ca2+ levels due to entry of extracellular Ca2+, and the second phase was associated with dissolution of BCP crystals.13 Increases in intracellular Ca2+ within growth-plate chondrocytes stimulated the induction of terminal differentiation, release of mineralisation-competent matrix vesicles, and apoptosis.26 Intracellular Ca2+ elevation was secondary to Ca2+ influx through Ca2+ channels formed by A2, A5 and A6.27
A2, A5 and A6 are highly expressed in chondrocytes from OA cartilage.2 A5 is the key factor in Ca2+ channel formation,27 and previous studies showed that it actively regulated cell apoptosis. Thus, DT 40 B lymphocytes lacking A5 were resistant to apoptosis induced by reactive oxygen species, as a result of Ca2+ influx inhibition.44 Also, we showed previously that A5 translocation to the plasma membrane or A5 externalisation induced cardiomyocyte apoptosis.28 Here, we demonstrated that A5 overexpression, as described in OA cartilage2 45 contributed to reduce joint cartilage cellularity by increasing BCP crystal- and TNF-α-induced apoptosis.
Although the precise mechanism by which A5 modulates apoptosis is still under investigation, several hypotheses can be put forward. First, A5 may increase cell-crystal contact as it binds to collagen type II45 and collagen type X2 and to BCP crystals. Second, A5 may increase the variations in intracellular Ca2+ levels by forming Ca2+ channels and/or by increasing BCP crystal endocytosis and intralysosomal dissolution. Third, A5 inhibits the phagocytosis of apoptotic bodies46 and then may increase the production of BCP crystals as apoptotic bodies are associated with BCP crystal formation.8 Finally, A5 may inhibit protein kinase C,47 which leads to cell apoptosis.
In summary, BCP crystals directly induced joint chondrocyte apoptosis. This in vitro process was independent from IL-1β, TNF-α and NO pathways. It required cell-crystal contact, crystal endocytosis and intralysosomal crystal dissolution responsible for intracellular Ca2+ elevation. BCP-crystal-induced chondrocyte apoptosis exacerbated by A5 overexpression may involve several mechanisms. Our results suggest a new pathophysiological mechanism for calcium-containing crystal arthropathies.
We thank Mr Daniel Oudot, the slaughterhouse technician, for providing the calf metatarsophalageal joints. We are grateful to Professor Moss (London, UK) for donating the pDC515 vector containing complete human A5 sequence, to Dr Russo-Marie (Institut Cochin, Paris, France) for donating the anti-A5 antibody, and to Professor Rey (ENSIACET, Toulouse, France) for BCP crystal synthesis.
Funding: This work was funded by grants from four non-profit organisations: the Société Française de Rhumatologie (French Society for Rheumatology, Grant 2005), the Association Rhumatisme et Travail, the Association pour la Recherche en Pathologie Synoviale and the Fonds d’Etude et de Recherche des Hôpitaux de Paris et Assistance-Publique. H-K E was supported by the last three organisations.
Competing interests: None.
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