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Chondrocyte apoptosis induced by hydrogen peroxide requires caspase activation but not mitochondrial pore transition
ELSEVIER
Journal of Orthopaedic Research
Journal of Orthopaedic Research 22 (2004) 1120-1125
www.elsevier.com/locate/orthres
Chondrocyte apoptosis induced by hydrogen peroxide requires caspase activation but not mitochondrial pore transition Marvin Y. Lo
a,
Hubert T. Kim
b7*
Department of Orthopaedic Surgery, University of California, San Francisco, CA, USA Veterans Affairs Medical Centre, 4150 Clement Street (112), San Francisco, CA 94121, USA Received 21 October 2003; accepted 31 December 2003
Abstract
The primary objective of this study was to test the hypothesis that inhibition of mitochondrial permeability transition andlor inhibition of caspase family enzymes can block chondrocyte apoptosis induced by H202. Primary human chondrocytes were isolated from normal cartilage by enzymatic digestion. Apoptosis was induced by exposure to H202. Chondrocyte apoptosis was quantified using an ELISA for nucleosome formation. Independent confirmation of apoptosis was obtained by TUNEL analysis. H202 induced apoptosis in primary human chondrocytes in a time and dose dependent manner. The effects of candidate apoptosis inhibitors were then tested. Chondrocytes were pretreated with inhibitors of mitochondrial permeability transition, or one of three different caspase inhibitors, and then incubated with H202. Apoptosis was then measured after 16 h of exposure to H202.Pretreatment with inhibitors of mitochondrial permeability transition did not block apoptosis induced by H202. A non-selective caspase inhibitor, a caspase 3-selective inhibitor, and a caspase 1-selective inhibitor, all blocked chondrocyte apoptosis induced by H202. These results show that H 2 0 2triggers chondrocyte apoptosis through caspase activation, independent of mitochondrial membrane permeability transition. 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Cartilage; Apoptosis; Caspase; Mitochondria; Peroxide
Introduction Increased chondrocyte programmed cell death (PCD), or “apoptosis”, has been observed in samples of cartilage obtained from patients with rheumatoid arthritis [l 11. In response to cytokine stimulation, articular chondrocytes have the ability to produce a variety of reactive oxygen species (ROS) including: peroxynitrite, superoxide anion, nitric oxide, and hydrogen peroxide (H202) [7-9,191. Furthermore, inflammatory cells and synoviocytes are major sources of ROS. H202 induces PCD in many cell types and may mediate cartilage degeneration associated with inflammatory joint diseases by inducing chondrocyte apoptosis [ 11. The mechanisms by which H202 induces chondrocyte apoptosis are not well understood; however, in most cell types, H202 alters mitochondrial membrane per-
* Corresponding author. Tel.: +1-415-221-4810x2940; fax: +1-415750-2181. E-maif address: [email protected] (H.T. Kim).
meability that allows translocation of cytochrome C into the cytoplasm and formation of an “apoptosome” complex [24]. This apoptosome complex activates proteolytic enzymes termed caspases that are key mediators of apoptosis. Agents such as cyclosporin A (CsA) and aristolochic acid (ArA) stabilize the mitochondrial membrane by preventing the formation of permeability transition pores (PTPs), and prevent apoptosis induced by H202 in some cell types [24]. The effects of these agents on chondrocyte apoptosis are unknown. The caspase family of proteolytic enzymes has been a frequent target for apoptotic inhibition experiments because these enzymes are critical to many aspects of the apoptotic pathway. Different caspases serve as triggers, regulatory elements, and death effectors [20,23]. Caspases 1 family members (1,4, 5, and 13) are believed to be involved in the initiation of the apoptotic cascade while caspases three family members (2, 3, and 7) take part in the execution phase of programmed cell death [16]. Exposure of cultured bovine chondrocytes to H202has been shown to activate caspase 3 [l]. Furthermore, several recent studies have shown that caspase inhibitors
0736-0266/$ - see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. Ail rights reserved, doi: 10.1016/j.orthres.2003.12.022
M. Y. Lo, H. T. Kim I Journal of Orthopaedic Research 22 (2004) 1120-1125
can block chondrocyte apoptosis induced by camptothecin in cultured cells [ 12,181, and by mechanical injury in cartilage explants [5]. Taken together, these findings suggest that caspase inhibitors may block chondrocyte apoptosis induced by H202. To our knowledge, this hypothesis has not been tested previously.
Materials and methods Primary human chondrocyte culture
Macroscopically normal human cartilage was obtained from patients at the time of surgical procedures such as limb amputation and joint replacement (number of donors = 6; ages = 18-68). The cartilage was finely minced and incubated in DMEM with 0.1% hyaluronidase (Sigma, St. Louis, MO) for 30 min on a 37 "C shaker. The cartilage was then incubated in DMEM with 10% BCS and 0.1% collagenase type IV (Sigma) overnight on a 37 "C shaker. The supernatant was filtered through a 100 pm cell strainer (Falcon, Franklin Lake, New Jersey) and then centrifuged at 500xg for 10 min. The cell pellet was washed twice with PBS. Isolated chondrocytes were re-suspended in DMEM with 10% FCS and antibiotics, and seeded at high density in standard tissue culture flasks (Falcon). Cultures were maintained in DMEM supplemented with 10% FCS and antibiotics for approximately two weeks before further manipulation. H z 0 2 treatment
Primary chondrocytes were re-plated into either 96-well flat bottom plates or 4-well chamber slides (Nunc, Roskilde, Denmark) at a density of los cells/cm2,and placed overnight at 37 "C in a humidified 5% COz incubator. Cells were then treated with media containing H202 at indicated final concentrations and maintained under standard tissue culture conditions. At the stated time points, cells were harvested for subsequent analysis.
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Chondrocyte apoptosis inhibition
Chondrocytes were pre-treated with candidate apoptosis inhibitors one hour before the addition H202 (final concentration 1 mM). Cells were harvested after 16 h for subsequent apoptosis analysis. The caspase inhibitors tested were: Z-VAD(0Me)-FMK (non-selective), Z-D(OMe)E(OMe)VD(OMe)-FMK (caspase 3 selective), and ZYVAD(0Me)-FMK (caspase 1 selective). All caspase inhibitors were obtained from Calbiochem (San Diego, CA). The mitochondria1 membrane stabilizers tested were CsA and ArA. Mitochondria] membrane stabilizers were purchased from Sigma (St. Louis, MO). All candidate inhibitors were reconstituted in DMSO at a concentration of 10 mM, and then diluted to working concentrations in media immediately prior to use. Miiochondrial membrane potential analysis
Primary chondrocytes were re-plated onto 4-well chamber slides (Nunc). After incubation overnight under standard tissue culture conditions, chondrocytes were treated with: (1) medium; (2) carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (100 pM); (3) H202 (1 mM); or (4) H 2 0 2(1 mM) + CsA (2 pM, added 1 h before the addition of H202). After 4 h, cells were labeled with Mitotracker@ Red CMXRos (100 nM final concentration) for 15 min. Cells were washed with fresh media then fixed with 1% paraformaldehyde in PBS for 15 min at room temperature. Slides were mounted with Vectashield mounting media (Vector laboratories) containing 1 pg/ml DAPI, and viewed under fluorescence microscopy using appropriate filters. Images were captured using a digital camera at 9 megapixel resolution. Statistical analysis
Students t-test was used for all analyses, and calculations were performed using SPSS 11.0 software (SPSS, Chicago, IL). Data are presented as mean f SD of triplicate or quadruplicate sample and are representative of multiple independent experiments. H202 dose response experiments were performed using chondrocytes isolated from six different donors. Apoptosis inhibition experiments were performed using chondrocytes isolated from five of the six donors.
Apoptosis analysis using nucleosome ELISA
The presence of nucleosomes in cell lysates is a specific and quantitative measure apoptosis. The nucleosome ELISA technique has been well-validated and used successfully in several previous studies of chondrocyte apotosis [12,18]. The Cell Death Detection ELISA Plus kit from Roche (Indianapolis, IN) was used with minor modifications to the supplied protocol. In brief, 96-well plates containing the treated chondrocytes were centrifuged at 200xg for 15 rnin to pellet any nonadherent cells. Media was carefully aspirated, and 200 p1 of kit lysis buffer was added. After 30 min, the samples were centrifuged at 2500xg for 15 min. 20 p1 of each sample was used for subsequent analysis following the manufacturer's protocol precisely. Results are expressed as absorbance values with a dual wavelength spectrophotometer at 405 nm with 490 nm as a reference. TUNEL (TdT-mediaied dUTP-biotin nicked end labeling) analysis
TUNEL analysis identifies cells with characteristic DNA fragmentation that is a classical finding in late stages of apoptosis. Primary chondrocytes were re-plated onto 4-well plastic chamber slides (Nunc). After overnight incubation, the cells were treated with: (1) medium alone, or (2) medium containing H 2 0 2at the indicated final concentration. After 16 h, the slides were spun at 300xg for 10 min. The media was aspirated and the cells were washed once with PBS. The cells were fixed with 1% paraformaldehyde for 10 min. The cells were post-fixed with ethanokacetic acid (2:l) at -20" for 5 min then washed with PBS. TUNEL analysis was performed using the ApopTag Direct kit (Intergen, Purchase, New York) following the manufacturer's protocol. The slides were subsequently mounted with Vectashield mounting media (Vector laboratories, Burlingame, CA) containing DAPI ( I pg/ml) and viewed with fluorescence microscopy using appropriate filters. Images were captured using a digital camera at 1 megapixel resolution.
Results HZ02 induces apoptosis in human chondrocyte
Primary human chondrocytes isolated from normal cartilage were grown in monolayer culture and then treated with H202 to study its ability to induce apoptosis. Nucleosome ELISA was used as the primary method for measuring chondrocyte apoptosis. H202 induced apoptosis in human primary chondrocytes in a time and dose dependent manner. After 16 h of treatment, chondrocyte apoptosis was observed at final H202 concentrations as low as 0.4 mM. Apoptosis peaked at a concentration of 10 mM (Fig. la). At a final H202 concentration of 4 mM, chondrocyte apoptosis was detectable by nucleosome ELISA as early as 6 h after continuous exposure to H202,and then increased over the course of the 24 h experiment (Fig. lb). To confirm the nucleosome ELISA data, we performed TUNEL analysis of chondrocytes grown on chamber slides and treated with H202 for 16 h (Fig. 2). Chondrocytes treated with media alone demonstrated only a few scattered TUNEL positive cells. In contrast, chondrocytes treated with H202 (4 mM final
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M. Y. Lo, H.T. Kim I Journal of Orthopaedic Research 22 (2004) 1120-1125
In order to confirm that mitochondrial membrane stabilizers effectively prevent MPT under the tested conditions, treated cells were stained with Mitotracker@ Red CMXRos. Mitotracker@ Red CMXRos is a fluorescent dye that is actively concentrated by functional mitochondria, and has been widely used as an indicator of mitochondrial membrane potential integrity [15]. Chondrocytes pre-treated with CsA (2 pM) prior to treatment with H202 (1 mM) stained brightly with Mitotracker@ Red CMXRos, indicating that MPT was inhibited and mitochondrial membrane potential was maintained (Fig. 4). As a positive control, cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a potent uncoupler of oxidative phosphorylation. After treatment with CCCP, chondrocytes completely lost the capacity to maintain mitochondrial membrane potential as measured by Mitotracker@Red CMXRos staining (Fig. 4).
8
Efsects of selective and non-selective caspase inhibition on chondrocyte apoptosis induced by H ~ 0 2
8
(b)
hours
Fig. 1. Dose response and time course of human chondrocyte apoptosis induced by H202. Primary human chondrocytes were re-plated into 96-well plates. The following day, the chondrocytes were treated with (a) H202 at the various concentrations for 16 h; or (b) Hl02 at 4 mM concentration for various lengths of time. The amount of apoptosis was quantified using a nucleosome ELISA assay. The graphs show data from representative experiments. Each bar represents the mean f SD of triplicate or quadruplicate samples.
concentration) were TUNEL positive at a rate of 83 2 12% (p = 0.006). Examination of DAPI stained cells showed that the TUNEL positive cells consistently exhibited nuclear condensation with a subset also exhibiting nuclear “blebbing”, both hallmarks of apoptosis (Fig. 2).
In order to investigate the role of caspases in chondrocyte apoptosis induced by H202,we tested the effects of selective and non-selective caspase inhibitors. Three caspase inhibitors were tested: Z-VAD, a non-selective irreversible caspase inhibitor; Z-DEVD, a selective inhibitor of caspase 3; and Z-YVAD, a selectiveinhibitor of caspase 1. The non-selective inhibitor Z-VAD consistently had the most dramatic effect, essentially blocking H202-induced apoptosis to background levels (Fig. 3). The caspase 3 and caspase 1-selective inhibitors also reduced H202-induced apoptosis to a significant extent (Fig. 3), but not as completely as the non-selective inhibitor. Combination of caspase 3 selective and caspase 1 selective inhibitors blocked chondrocyte apoptosis to the same degree as the non-selective inhibitor Z-VAD. Higher concentrations of caspase inhibitors (up to 50 pM final concentration) did not cause higher levels of inhibition (not shown). Therefore, a concentration of 10 pM was selected to minimize potential non-specific caspase inhibitory effects.
Discussion Mitochondria1 membrane stabilization does not prevent chondrocyte apoptosis induced by Hz02 In order to determine whether or not mitochondrial permeability transition (MPT) is a requirement for H202 induced chondrocyte apoptosis, we studied the effects of the prototypical MTP inhibitors CsA and ArA. Pre-treatment of chondrocytes with the CsA (2 pM) failed to inhibit apoptosis induced by H202 (1 mM) (Fig. 3). Similarly, pre-treatment with CsA (up to 10 pM) in combination with ArA (up to 50 pM)also failed to inhibit chondrocyte induced by H202 (not shown).
Chondrocyte apoptosis has been implicated in the pathogenesis of degenerative joint diseases including osteoarthritis and rheumatoid arthritis [3,10,11]. The specific role that H202 plays in cartilage degeneration remains unclear. In rheumatoid arthritis, there is evidence that H202 is both exogenously supplied and endogenously produced. Increased numbers of polymorphonuclear leukocytes and macrophages release exogenous H202, while chondrocytes produce H 2 0 2in response to a number of chemical signals thought to be present in inflammatory joint diseases. H202 may have
M . Y. Lo, H. 7: Kim i Journal OJ Orthopaedic Research 22 (2004) 1120-1125
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Fig. 2. TUNEL analysis of human chondrocytes treated with H202. Primary human chondrocytes were re-plated onto chamber slides. The following day, chondrocytes were treated with H102 (2 mM) for 16 h. T U N E L analysis was performed as described. Apoptotic chondrocytes are identified by bright green fluorescence. The upper right panel shows TUNEL analysis of treated chondrocytes. The lower right panel shows an image of the same microscope field captured with filters for DAPI to show all nuclei. The upper left and lower left panels show TUNEL and DAPI staining of untreated chondrocytes. Original magnification 5x.
Fig. 4. Mitochondria1 membrane potential analysis of chondrocytes exposed to H202 with and without CsA pre-treatment. Primary human chondrocytes were re-plated onto chamber slides. The following day, chondrocytes were treated with media alone (negative control), CCCP (positive control), H202 (1 mM], or HzOz(1 mM) after pre-treatment with CsA (2 pM). The chondrocytes were then labeled with the potential sensitive dye Mitotracker@ Red CMXRos. Cells with active mitochondria show bright red cytoplasmic staining, Nuclei are stained blue with DAPI. Original magnification 40x.
direct deleterious effects on cartilage matrix by inhibiting proteoglycan synthesis by chondrocytes [ 141. The data presented in this report indicate that H202 may
also cause cartilage degeneration by inducing chondrocyte apoptosis. This finding is consistent with results from a similar study in bovine chondrocytes [2].
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M. Y. Lo, H . T. Kim I Journal of Orlhopaedic Research 22 (2004) 1120-1125
n
0
Fig. 3. Effects of caspase inhibitors and CsA on human chondrocyte apoptosis induced by H20z. Primary human chondrocytes were replated onto 96-well plates. The following day, the chondrocytes were treated with caspase inhibitors (Pan C = Z-VAD, non-selective; C3 = Z-DEVD, caspase 3 selective; C1= Z-YVAD, caspase 1 selective), or with CsA, a prototypical inhibitor of MPT. The amount of apoptosis was quantified using a nucleosome ELISA assay. The graphs show data from representative experiments. Each bar represents the mean 2 SD of triplicate or quadruplicate samples. * indicates P < 0.05 compared to H 2 0 2 treated control.
Mitochondria are felt to play a pivotal role in apoptosis. In other cell types, exposure to H202 induces apoptosis through pathways that involve disruption of normal mitochondria function. One key perturbation appears to be the opening of mitochondrial permeability transition pores that allow passage of small pro-apoptotic molecules into the cytoplasm [6,13,22]. Mitochondria1 membrane stabilizers such as CsA and ArA have been used to prevent apoptosis induced by H202 in cell types such as fibroblasts and endothelial cells [21,24]. However, these inhibitors of mitochondrial permeability transition (MPT) failed to block apoptosis in our experiments using human chondrocytes, even at concentrations much higher than levels found to be effective in other cell types. One explanation is that chondrocytes may undergo apoptosis through both mitochondrial dependent and independent pathways as has been demonstrated in other cell types such as cultured neurons [4]. Our experiments show that MPT is not essential for H202 induced chondrocyte apoptosis; however, they do not exclude the possibility that MTP contributes to the execution of chondrocyte PCD in a non-critical fashion. Furthermore, our findings are only applicable to chondrocyte apoptosis induced by H202; apoptosis induced by other stimuli may involve pathways with an absolute requirement MPT. Recent studies have shown the ability to caspase inhibitors to decrease chondrocyte apoptosis and retain cell functionality [ 12,181. Although apoptosis can occur independently of caspase involvement in some cell types [13], all existing data indicates that caspase activation is a requirement for chondrocyte apoptosis. Caspase 3, in particular, is a critical mediator of apoptosis in many cell types including chondrocytes. Caspase 3 activity has been shown to increase after H202 exposure in cultured
bovine chondrocytes [ 11. Our data shows that Z-DEVD, a caspase 3 selective inhibitor, blocks H202-induced apoptosis in human chondrocytes. Together, these findings clearly demonstrate that activation of caspase 3 is a necessary step in the cascade of cellular events that leads to chondrocyte apoptosis after exposure to H202. Our finding that the caspase-1 inhibitor Z-YVAD blocks chondrocyte apoptosis induced by H202 is especially interesting. In other cell types, caspase 1 has been shown to be involved in many aspects of programmed cell death including TNF-mediated apoptosis. Caspase 1 is involved in the processing of interleukin (1L)-1 and IL-18. Pelletier and colleagues noted increased caspase 1 expression in samples of osteoarthritic cartilage; however a direct association with chondrocyte apoptosis was not established [17]. Furthermore, previous studies found that caspase 1 inhibition does not block camptothecin-induced apoptosis in cultured chondrocytes [18], a finding we have also confirmed (data not shown). Our data show a direct link between caspase 1 activity and chondrocyte apoptosis induced by H202 suggesting that the contribution of caspase 1 to chondrocyte apoptosis is stimulus dependent. In summary, human articular chondrocytes undergo apoptosis when treated with H202. In contrast to what has been observed in many other cell types, mitochondrial membrane permeability transition is not a requirement for chondrocyte apoptosis to occur following exposure to H202. Caspase activation, however, is a required step in the chondrocyte apoptotic pathway triggered by H202 exposure. Of particular significance is the finding that both caspase 1 and caspase 3 play direct roles in chondrocyte apoptosis induced by H202. These results suggest that multiple caspases are possible targets for inhibition of chondrocyte apoptosis that may occur in inflammatory joint diseases where ROS such as H202 are up-regulated. Potentially, these findings may lead to therapeutic approaches that decrease cartilage degeneration by minimizing chondrocyte loss. However, both the current study and previous related studies used short-term exposure to supra-physiologic concentrations of H202 in order to demonstrate large changes in chondrocyte function and survival, and to dissect out the pathways involved. The clinical significance of our findings and the potential utility of apoptosis inhibitor therapy depend upon the extent to which the observed responses take place at physiologic concentrations. Therefore, additional studies that examine the effects of long-term exposure to lower levels of ROS are merited.
Acknowledgement This research was supported by a MREP award from VA Medical Research.
M. Y. Lo, H . T. Kim I Journal of Orthopaedic Research 22 (2004) 1120-1125
References [l] Asada S, Fukuda K, Nishisaka F, Matsukawa M, Hamanisi C. Hydrogen peroxide induces apoptosis of chondrocytes; involvement of calcium ion and extracellular signal-regulated protein kinase. Inflamm Res 2001;50:19-23. [2] Asada S, Fukuda K, Oh M, Hamanishi C, Tanaka S. Effect of hydrogen peroxide on the metabolism of articular chondrocytes. lnflamm Res 1999;48:399403. [3] Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis a possible pathway for osteoarthritis pathology. Arthritis Rheum 1998;41:284-9. [4] Budd SL, Tenneti L, Lishnak T , Lipton SA. Mitochondria1 and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons. Proc Natl Acad Sci USA 2000;97:6161-6. [S] D’Lima DD, Hashimoto S, Chen PC, Lotz MK, Colwell Jr CW. Prevention of chondrocyte apoptosis. J Bone Jnt Surg Am 2001; 83-A(Suppl 2):25-6. [6] Daugus E, Nochy D, Ravagnan L, Loeffler M, Susin SA, Zamzani N, et al. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett 2000; 476:118-23. [7] Fukuda K, Dan H, Saitoh M, Takayama M, Tanaka S. Superoxide dismutase inhibits interleukin-I-induced degradation of human cartilage. Agents Actions 1994;42:71-3. [8] Fukuda K, Kumano F, Takayama M, Saito M, Otani K, Tanaka S. Zonal differences in nitric oxide synthesis by bovine chondrocytes exposed to interleukin-1. Inflamm Res 1995;44:434-7. [9] Fukuda K, Takayama M, Ueno M, Oh M, Asada S, Kumano F, et al. Hyaluronic acid inhibits interleukin-1-induced superoxide anion in bovine chondrocytes. Inflamm Res 1997;46:114-7. Hashimoto S, Ochs RL, Komiya S, Lotz M. Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum 1998;41:1632-8. Kim HA, Song YW. Apoptotic chondrocyte death in rheumatoid arthritis. Arthritis Rheum 1999;42:1528-37. Lee D, Long SA, Adams JL, Chan G, Vaidya KS, Francis TA, et al. Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality. J Biol Chem 2000275: 16007-14.
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[I31 Lorenzo H, Susin SA, Penninger J, Kroemer G. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 1999;6: 51624. [I41 Lowther D, Sandy JD, Santer VB, Brown HLG. Antigen-induced arthritis: decreased proteoglycan content and inhibition of proteoglycan synthesis in articular cartilage. Arthritis Rheum 1978;21:675-80. [IS] Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, et al. Bax and Adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 1998;281:2027-31. [16] Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999;6: 102842. [I71 Nuki G. Osteoarthritis: a problem ofjoint failure. Z Rheum 1999; 58:142-7. [I81 Nuttall ME, Nadeau DP, Fisher PW, Wang F, Keller PM, DeWolf Jr WE, et al. Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro. J Orthop Res 2000;18:35663. [I91 Rathakrishnan C, Tiku K, Raghavan A, Tiku M. Release of oxygen radicals by articular chondrocytes: a study of luminoldependent chemiluminescence and hydrogen peroxide secretion. Miner Res 1992;7:113948. [20] Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ 1999;6: 1067-74. [21] Sugano N, Ito K, Murai S. Cyclosporine A inhibits H202 induced apoptosis of human fibroblasts. FEBS Lett 1999;447:274-6. [22] Susin Z, Zanzani N, Castedo M, Hirsch T, Marchetti P, Macho A, et al. Bcl-2 inhibits the mitochondria1 release of an apoptogenic protease. J Exp Med 1996;1:133141. [23] Utz PJ, Anderson P. Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules. Cell Death Differ 2000;7:589-602. [24] Walter DH, Haendeler J, Galle J, Zeiher AM, Dimmeler S. Cyclosporine A inhibits apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria. Circulation 1998;98:1 153-7.
Journal of Orthopaedic Research
Journal of Orthopaedic Research 22 (2004) 1120-1125
www.elsevier.com/locate/orthres
Chondrocyte apoptosis induced by hydrogen peroxide requires caspase activation but not mitochondrial pore transition Marvin Y. Lo
a,
Hubert T. Kim
b7*
Department of Orthopaedic Surgery, University of California, San Francisco, CA, USA Veterans Affairs Medical Centre, 4150 Clement Street (112), San Francisco, CA 94121, USA Received 21 October 2003; accepted 31 December 2003
Abstract
The primary objective of this study was to test the hypothesis that inhibition of mitochondrial permeability transition andlor inhibition of caspase family enzymes can block chondrocyte apoptosis induced by H202. Primary human chondrocytes were isolated from normal cartilage by enzymatic digestion. Apoptosis was induced by exposure to H202. Chondrocyte apoptosis was quantified using an ELISA for nucleosome formation. Independent confirmation of apoptosis was obtained by TUNEL analysis. H202 induced apoptosis in primary human chondrocytes in a time and dose dependent manner. The effects of candidate apoptosis inhibitors were then tested. Chondrocytes were pretreated with inhibitors of mitochondrial permeability transition, or one of three different caspase inhibitors, and then incubated with H202. Apoptosis was then measured after 16 h of exposure to H202.Pretreatment with inhibitors of mitochondrial permeability transition did not block apoptosis induced by H202. A non-selective caspase inhibitor, a caspase 3-selective inhibitor, and a caspase 1-selective inhibitor, all blocked chondrocyte apoptosis induced by H202. These results show that H 2 0 2triggers chondrocyte apoptosis through caspase activation, independent of mitochondrial membrane permeability transition. 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Cartilage; Apoptosis; Caspase; Mitochondria; Peroxide
Introduction Increased chondrocyte programmed cell death (PCD), or “apoptosis”, has been observed in samples of cartilage obtained from patients with rheumatoid arthritis [l 11. In response to cytokine stimulation, articular chondrocytes have the ability to produce a variety of reactive oxygen species (ROS) including: peroxynitrite, superoxide anion, nitric oxide, and hydrogen peroxide (H202) [7-9,191. Furthermore, inflammatory cells and synoviocytes are major sources of ROS. H202 induces PCD in many cell types and may mediate cartilage degeneration associated with inflammatory joint diseases by inducing chondrocyte apoptosis [ 11. The mechanisms by which H202 induces chondrocyte apoptosis are not well understood; however, in most cell types, H202 alters mitochondrial membrane per-
* Corresponding author. Tel.: +1-415-221-4810x2940; fax: +1-415750-2181. E-maif address: [email protected] (H.T. Kim).
meability that allows translocation of cytochrome C into the cytoplasm and formation of an “apoptosome” complex [24]. This apoptosome complex activates proteolytic enzymes termed caspases that are key mediators of apoptosis. Agents such as cyclosporin A (CsA) and aristolochic acid (ArA) stabilize the mitochondrial membrane by preventing the formation of permeability transition pores (PTPs), and prevent apoptosis induced by H202 in some cell types [24]. The effects of these agents on chondrocyte apoptosis are unknown. The caspase family of proteolytic enzymes has been a frequent target for apoptotic inhibition experiments because these enzymes are critical to many aspects of the apoptotic pathway. Different caspases serve as triggers, regulatory elements, and death effectors [20,23]. Caspases 1 family members (1,4, 5, and 13) are believed to be involved in the initiation of the apoptotic cascade while caspases three family members (2, 3, and 7) take part in the execution phase of programmed cell death [16]. Exposure of cultured bovine chondrocytes to H202has been shown to activate caspase 3 [l]. Furthermore, several recent studies have shown that caspase inhibitors
0736-0266/$ - see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. Ail rights reserved, doi: 10.1016/j.orthres.2003.12.022
M. Y. Lo, H. T. Kim I Journal of Orthopaedic Research 22 (2004) 1120-1125
can block chondrocyte apoptosis induced by camptothecin in cultured cells [ 12,181, and by mechanical injury in cartilage explants [5]. Taken together, these findings suggest that caspase inhibitors may block chondrocyte apoptosis induced by H202. To our knowledge, this hypothesis has not been tested previously.
Materials and methods Primary human chondrocyte culture
Macroscopically normal human cartilage was obtained from patients at the time of surgical procedures such as limb amputation and joint replacement (number of donors = 6; ages = 18-68). The cartilage was finely minced and incubated in DMEM with 0.1% hyaluronidase (Sigma, St. Louis, MO) for 30 min on a 37 "C shaker. The cartilage was then incubated in DMEM with 10% BCS and 0.1% collagenase type IV (Sigma) overnight on a 37 "C shaker. The supernatant was filtered through a 100 pm cell strainer (Falcon, Franklin Lake, New Jersey) and then centrifuged at 500xg for 10 min. The cell pellet was washed twice with PBS. Isolated chondrocytes were re-suspended in DMEM with 10% FCS and antibiotics, and seeded at high density in standard tissue culture flasks (Falcon). Cultures were maintained in DMEM supplemented with 10% FCS and antibiotics for approximately two weeks before further manipulation. H z 0 2 treatment
Primary chondrocytes were re-plated into either 96-well flat bottom plates or 4-well chamber slides (Nunc, Roskilde, Denmark) at a density of los cells/cm2,and placed overnight at 37 "C in a humidified 5% COz incubator. Cells were then treated with media containing H202 at indicated final concentrations and maintained under standard tissue culture conditions. At the stated time points, cells were harvested for subsequent analysis.
1121
Chondrocyte apoptosis inhibition
Chondrocytes were pre-treated with candidate apoptosis inhibitors one hour before the addition H202 (final concentration 1 mM). Cells were harvested after 16 h for subsequent apoptosis analysis. The caspase inhibitors tested were: Z-VAD(0Me)-FMK (non-selective), Z-D(OMe)E(OMe)VD(OMe)-FMK (caspase 3 selective), and ZYVAD(0Me)-FMK (caspase 1 selective). All caspase inhibitors were obtained from Calbiochem (San Diego, CA). The mitochondria1 membrane stabilizers tested were CsA and ArA. Mitochondria] membrane stabilizers were purchased from Sigma (St. Louis, MO). All candidate inhibitors were reconstituted in DMSO at a concentration of 10 mM, and then diluted to working concentrations in media immediately prior to use. Miiochondrial membrane potential analysis
Primary chondrocytes were re-plated onto 4-well chamber slides (Nunc). After incubation overnight under standard tissue culture conditions, chondrocytes were treated with: (1) medium; (2) carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (100 pM); (3) H202 (1 mM); or (4) H 2 0 2(1 mM) + CsA (2 pM, added 1 h before the addition of H202). After 4 h, cells were labeled with Mitotracker@ Red CMXRos (100 nM final concentration) for 15 min. Cells were washed with fresh media then fixed with 1% paraformaldehyde in PBS for 15 min at room temperature. Slides were mounted with Vectashield mounting media (Vector laboratories) containing 1 pg/ml DAPI, and viewed under fluorescence microscopy using appropriate filters. Images were captured using a digital camera at 9 megapixel resolution. Statistical analysis
Students t-test was used for all analyses, and calculations were performed using SPSS 11.0 software (SPSS, Chicago, IL). Data are presented as mean f SD of triplicate or quadruplicate sample and are representative of multiple independent experiments. H202 dose response experiments were performed using chondrocytes isolated from six different donors. Apoptosis inhibition experiments were performed using chondrocytes isolated from five of the six donors.
Apoptosis analysis using nucleosome ELISA
The presence of nucleosomes in cell lysates is a specific and quantitative measure apoptosis. The nucleosome ELISA technique has been well-validated and used successfully in several previous studies of chondrocyte apotosis [12,18]. The Cell Death Detection ELISA Plus kit from Roche (Indianapolis, IN) was used with minor modifications to the supplied protocol. In brief, 96-well plates containing the treated chondrocytes were centrifuged at 200xg for 15 rnin to pellet any nonadherent cells. Media was carefully aspirated, and 200 p1 of kit lysis buffer was added. After 30 min, the samples were centrifuged at 2500xg for 15 min. 20 p1 of each sample was used for subsequent analysis following the manufacturer's protocol precisely. Results are expressed as absorbance values with a dual wavelength spectrophotometer at 405 nm with 490 nm as a reference. TUNEL (TdT-mediaied dUTP-biotin nicked end labeling) analysis
TUNEL analysis identifies cells with characteristic DNA fragmentation that is a classical finding in late stages of apoptosis. Primary chondrocytes were re-plated onto 4-well plastic chamber slides (Nunc). After overnight incubation, the cells were treated with: (1) medium alone, or (2) medium containing H 2 0 2at the indicated final concentration. After 16 h, the slides were spun at 300xg for 10 min. The media was aspirated and the cells were washed once with PBS. The cells were fixed with 1% paraformaldehyde for 10 min. The cells were post-fixed with ethanokacetic acid (2:l) at -20" for 5 min then washed with PBS. TUNEL analysis was performed using the ApopTag Direct kit (Intergen, Purchase, New York) following the manufacturer's protocol. The slides were subsequently mounted with Vectashield mounting media (Vector laboratories, Burlingame, CA) containing DAPI ( I pg/ml) and viewed with fluorescence microscopy using appropriate filters. Images were captured using a digital camera at 1 megapixel resolution.
Results HZ02 induces apoptosis in human chondrocyte
Primary human chondrocytes isolated from normal cartilage were grown in monolayer culture and then treated with H202 to study its ability to induce apoptosis. Nucleosome ELISA was used as the primary method for measuring chondrocyte apoptosis. H202 induced apoptosis in human primary chondrocytes in a time and dose dependent manner. After 16 h of treatment, chondrocyte apoptosis was observed at final H202 concentrations as low as 0.4 mM. Apoptosis peaked at a concentration of 10 mM (Fig. la). At a final H202 concentration of 4 mM, chondrocyte apoptosis was detectable by nucleosome ELISA as early as 6 h after continuous exposure to H202,and then increased over the course of the 24 h experiment (Fig. lb). To confirm the nucleosome ELISA data, we performed TUNEL analysis of chondrocytes grown on chamber slides and treated with H202 for 16 h (Fig. 2). Chondrocytes treated with media alone demonstrated only a few scattered TUNEL positive cells. In contrast, chondrocytes treated with H202 (4 mM final
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In order to confirm that mitochondrial membrane stabilizers effectively prevent MPT under the tested conditions, treated cells were stained with Mitotracker@ Red CMXRos. Mitotracker@ Red CMXRos is a fluorescent dye that is actively concentrated by functional mitochondria, and has been widely used as an indicator of mitochondrial membrane potential integrity [15]. Chondrocytes pre-treated with CsA (2 pM) prior to treatment with H202 (1 mM) stained brightly with Mitotracker@ Red CMXRos, indicating that MPT was inhibited and mitochondrial membrane potential was maintained (Fig. 4). As a positive control, cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a potent uncoupler of oxidative phosphorylation. After treatment with CCCP, chondrocytes completely lost the capacity to maintain mitochondrial membrane potential as measured by Mitotracker@Red CMXRos staining (Fig. 4).
8
Efsects of selective and non-selective caspase inhibition on chondrocyte apoptosis induced by H ~ 0 2
8
(b)
hours
Fig. 1. Dose response and time course of human chondrocyte apoptosis induced by H202. Primary human chondrocytes were re-plated into 96-well plates. The following day, the chondrocytes were treated with (a) H202 at the various concentrations for 16 h; or (b) Hl02 at 4 mM concentration for various lengths of time. The amount of apoptosis was quantified using a nucleosome ELISA assay. The graphs show data from representative experiments. Each bar represents the mean f SD of triplicate or quadruplicate samples.
concentration) were TUNEL positive at a rate of 83 2 12% (p = 0.006). Examination of DAPI stained cells showed that the TUNEL positive cells consistently exhibited nuclear condensation with a subset also exhibiting nuclear “blebbing”, both hallmarks of apoptosis (Fig. 2).
In order to investigate the role of caspases in chondrocyte apoptosis induced by H202,we tested the effects of selective and non-selective caspase inhibitors. Three caspase inhibitors were tested: Z-VAD, a non-selective irreversible caspase inhibitor; Z-DEVD, a selective inhibitor of caspase 3; and Z-YVAD, a selectiveinhibitor of caspase 1. The non-selective inhibitor Z-VAD consistently had the most dramatic effect, essentially blocking H202-induced apoptosis to background levels (Fig. 3). The caspase 3 and caspase 1-selective inhibitors also reduced H202-induced apoptosis to a significant extent (Fig. 3), but not as completely as the non-selective inhibitor. Combination of caspase 3 selective and caspase 1 selective inhibitors blocked chondrocyte apoptosis to the same degree as the non-selective inhibitor Z-VAD. Higher concentrations of caspase inhibitors (up to 50 pM final concentration) did not cause higher levels of inhibition (not shown). Therefore, a concentration of 10 pM was selected to minimize potential non-specific caspase inhibitory effects.
Discussion Mitochondria1 membrane stabilization does not prevent chondrocyte apoptosis induced by Hz02 In order to determine whether or not mitochondrial permeability transition (MPT) is a requirement for H202 induced chondrocyte apoptosis, we studied the effects of the prototypical MTP inhibitors CsA and ArA. Pre-treatment of chondrocytes with the CsA (2 pM) failed to inhibit apoptosis induced by H202 (1 mM) (Fig. 3). Similarly, pre-treatment with CsA (up to 10 pM) in combination with ArA (up to 50 pM)also failed to inhibit chondrocyte induced by H202 (not shown).
Chondrocyte apoptosis has been implicated in the pathogenesis of degenerative joint diseases including osteoarthritis and rheumatoid arthritis [3,10,11]. The specific role that H202 plays in cartilage degeneration remains unclear. In rheumatoid arthritis, there is evidence that H202 is both exogenously supplied and endogenously produced. Increased numbers of polymorphonuclear leukocytes and macrophages release exogenous H202, while chondrocytes produce H 2 0 2in response to a number of chemical signals thought to be present in inflammatory joint diseases. H202 may have
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Fig. 2. TUNEL analysis of human chondrocytes treated with H202. Primary human chondrocytes were re-plated onto chamber slides. The following day, chondrocytes were treated with H102 (2 mM) for 16 h. T U N E L analysis was performed as described. Apoptotic chondrocytes are identified by bright green fluorescence. The upper right panel shows TUNEL analysis of treated chondrocytes. The lower right panel shows an image of the same microscope field captured with filters for DAPI to show all nuclei. The upper left and lower left panels show TUNEL and DAPI staining of untreated chondrocytes. Original magnification 5x.
Fig. 4. Mitochondria1 membrane potential analysis of chondrocytes exposed to H202 with and without CsA pre-treatment. Primary human chondrocytes were re-plated onto chamber slides. The following day, chondrocytes were treated with media alone (negative control), CCCP (positive control), H202 (1 mM], or HzOz(1 mM) after pre-treatment with CsA (2 pM). The chondrocytes were then labeled with the potential sensitive dye Mitotracker@ Red CMXRos. Cells with active mitochondria show bright red cytoplasmic staining, Nuclei are stained blue with DAPI. Original magnification 40x.
direct deleterious effects on cartilage matrix by inhibiting proteoglycan synthesis by chondrocytes [ 141. The data presented in this report indicate that H202 may
also cause cartilage degeneration by inducing chondrocyte apoptosis. This finding is consistent with results from a similar study in bovine chondrocytes [2].
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n
0
Fig. 3. Effects of caspase inhibitors and CsA on human chondrocyte apoptosis induced by H20z. Primary human chondrocytes were replated onto 96-well plates. The following day, the chondrocytes were treated with caspase inhibitors (Pan C = Z-VAD, non-selective; C3 = Z-DEVD, caspase 3 selective; C1= Z-YVAD, caspase 1 selective), or with CsA, a prototypical inhibitor of MPT. The amount of apoptosis was quantified using a nucleosome ELISA assay. The graphs show data from representative experiments. Each bar represents the mean 2 SD of triplicate or quadruplicate samples. * indicates P < 0.05 compared to H 2 0 2 treated control.
Mitochondria are felt to play a pivotal role in apoptosis. In other cell types, exposure to H202 induces apoptosis through pathways that involve disruption of normal mitochondria function. One key perturbation appears to be the opening of mitochondrial permeability transition pores that allow passage of small pro-apoptotic molecules into the cytoplasm [6,13,22]. Mitochondria1 membrane stabilizers such as CsA and ArA have been used to prevent apoptosis induced by H202 in cell types such as fibroblasts and endothelial cells [21,24]. However, these inhibitors of mitochondrial permeability transition (MPT) failed to block apoptosis in our experiments using human chondrocytes, even at concentrations much higher than levels found to be effective in other cell types. One explanation is that chondrocytes may undergo apoptosis through both mitochondrial dependent and independent pathways as has been demonstrated in other cell types such as cultured neurons [4]. Our experiments show that MPT is not essential for H202 induced chondrocyte apoptosis; however, they do not exclude the possibility that MTP contributes to the execution of chondrocyte PCD in a non-critical fashion. Furthermore, our findings are only applicable to chondrocyte apoptosis induced by H202; apoptosis induced by other stimuli may involve pathways with an absolute requirement MPT. Recent studies have shown the ability to caspase inhibitors to decrease chondrocyte apoptosis and retain cell functionality [ 12,181. Although apoptosis can occur independently of caspase involvement in some cell types [13], all existing data indicates that caspase activation is a requirement for chondrocyte apoptosis. Caspase 3, in particular, is a critical mediator of apoptosis in many cell types including chondrocytes. Caspase 3 activity has been shown to increase after H202 exposure in cultured
bovine chondrocytes [ 11. Our data shows that Z-DEVD, a caspase 3 selective inhibitor, blocks H202-induced apoptosis in human chondrocytes. Together, these findings clearly demonstrate that activation of caspase 3 is a necessary step in the cascade of cellular events that leads to chondrocyte apoptosis after exposure to H202. Our finding that the caspase-1 inhibitor Z-YVAD blocks chondrocyte apoptosis induced by H202 is especially interesting. In other cell types, caspase 1 has been shown to be involved in many aspects of programmed cell death including TNF-mediated apoptosis. Caspase 1 is involved in the processing of interleukin (1L)-1 and IL-18. Pelletier and colleagues noted increased caspase 1 expression in samples of osteoarthritic cartilage; however a direct association with chondrocyte apoptosis was not established [17]. Furthermore, previous studies found that caspase 1 inhibition does not block camptothecin-induced apoptosis in cultured chondrocytes [18], a finding we have also confirmed (data not shown). Our data show a direct link between caspase 1 activity and chondrocyte apoptosis induced by H202 suggesting that the contribution of caspase 1 to chondrocyte apoptosis is stimulus dependent. In summary, human articular chondrocytes undergo apoptosis when treated with H202. In contrast to what has been observed in many other cell types, mitochondrial membrane permeability transition is not a requirement for chondrocyte apoptosis to occur following exposure to H202. Caspase activation, however, is a required step in the chondrocyte apoptotic pathway triggered by H202 exposure. Of particular significance is the finding that both caspase 1 and caspase 3 play direct roles in chondrocyte apoptosis induced by H202. These results suggest that multiple caspases are possible targets for inhibition of chondrocyte apoptosis that may occur in inflammatory joint diseases where ROS such as H202 are up-regulated. Potentially, these findings may lead to therapeutic approaches that decrease cartilage degeneration by minimizing chondrocyte loss. However, both the current study and previous related studies used short-term exposure to supra-physiologic concentrations of H202 in order to demonstrate large changes in chondrocyte function and survival, and to dissect out the pathways involved. The clinical significance of our findings and the potential utility of apoptosis inhibitor therapy depend upon the extent to which the observed responses take place at physiologic concentrations. Therefore, additional studies that examine the effects of long-term exposure to lower levels of ROS are merited.
Acknowledgement This research was supported by a MREP award from VA Medical Research.
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