1 Department of Biochemistry, School of Dental Medicine, Philadelphia, and 2 Stokes Research Institute, Children's Hospital at Philadelphia and Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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An elevation in inorganic phosphate
(Pi) concentration activates epiphyseal chondrocyte
apoptosis. To determine the mechanism of apoptosis,
tibial chondrocytes were treated with Pi, and
nitrate/nitrite (NO
epiphyseal chondrocyte; nitric oxide synthase; caspase
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INTRODUCTION |
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CHONDROCYTES CONTAINED within the epiphyseal growth plate facilitate rapid growth of tubular bones. After rounds of rapid proliferation, these cells initiate a developmental program that leads to terminal differentiation. Terminally differentiated chondrocytes exhibit well-documented changes in phenotype, including increased alkaline phosphatase activity, synthesis of type X collagen and downregulation of type II collagen, expression of vitamin D receptors, and release and mineralization of matrix vesicles. Maturing cartilage cells also exhibit an extraordinary profile of metabolic changes. For example, Rajpurohit et al. (29) have shown that there is a maturation-dependent loss of mitochondrial function in growth plate chondrocytes. Although these cells are very active biosynthetically, they generate almost all of their metabolic energy through glycolysis.
Considerable debate still exists concerning the fate of the postmitotic chondrocyte. Work from a number of laboratories provides strong support for the hypothesis that in the growth plate, the terminally differentiated cells undergo apoptosis or programmed cell death (7, 9, 10, 13). Recently, it was shown that inorganic phosphate (Pi) ions can induce apoptosis of hypertrophic chondrocytes (22, 23). Because a rise in Pi levels occurs at sites of cartilage mineralization, these workers have linked a late stage extracellular change with an intracellular event that may lead to deletion of terminally differentiated cells from the tissue. Aside from Pi, factors that may serve to sensitize mature chondrocytes to apoptosis include expression of the hypertrophic phenotype (8), loss of mitochondrial oxidative activity, and a low level of Bcl-2 expression (29). It has also been observed that Pi treatment of isolated postmitotic sternal chondrocytes results in a decrease in the levels of reduced thiols (35). Whether the loss of thiol reserve activates the apoptotic process has not been determined.
Generation of nitric oxide (NO) by NO synthase (NOS) and formation of other nitrogen species have been associated with depletion of intracellular glutathione and inhibition of complexes I to IV of the mitochondrial respiratory chain (4, 6, 12, 40, 43). Relevant to the pathophysiology of skeletal tissues, it has been observed that when articular cartilage is treated with inflammatory mediators, there is an increase in NO generation (1, 2, 17). Whether a similar pathway is activated in epiphyseal chondrocytes has yet to be determined. In this report, we investigate the possibility that Pi induction of chondrocyte apoptosis is linked to NO generation. Results of the study show that chondrocyte apoptosis is mediated by NO, and inhibition of NOS protects cells from the anionic apoptogen.
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MATERIALS AND METHODS |
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Design of the study. The overall goal of the study was to learn whether Pi-dependent chondrocyte apoptosis is linked to the generation of NO. The first series of experiments was designed to establish that NO release is directly related to Pi-induced cell death. Thus hypertrophic chondrocytes were treated with increasing concentrations of Pi, and cell death [measured by the modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay] and NO levels were determined. To confirm that NO generation is linked to Pi-induced apoptosis, chondrocyte viability was measured in the presence of inhibitors that block NO generation [NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA)], Pi transport (phosphonoformic acid or PFA), and an agent that generates exogenous NO (S-nitrosoglutathione or SNOG). In a parallel experiment, we determined whether NOS is associated with changes in the functional state of the Pi-treated cell. Thus we examined mitochondrial activity using a fluorescent voltage-sensitive dye (rhodamine 123) in the presence of NMMA and PFA. Because Pi caused a change in mitochondrial oxidative activity and mediated a decrease in the thiol status of the cell, we explored the possibility that the apoptogen stimulates apoptosis by lowering the thiol reserve of the cell. To test this hypothesis, we determined glutathione levels in chondrocytes that had been treated with a concentration of the anion that was sufficient to cause apoptosis. Finally, we probed downstream events linked to the generation of NO. We measured caspase activity in cells that had been treated with the apoptogen; in concert with this experiment, we determined whether cell death could be blocked by treating chondrocytes with caspase inhibitors.
Cell culture. Growth plate chondrocytes from the proximal heads of 19-day-old chick embryo tibia were isolated as previously reported (28). The cells were grown in primary culture for 4 days in Dulbecco's modified high-glucose Eagle's medium (Sigma Chemical, St. Louis, MO) containing 10% defined fetal calf serum (NUSERUM IV; Becton Dickinson, Bedford, MA), 2 mM L-glutamine, and 50 U/ml each of penicillin and streptomycin. Nonadherent chondrocytes were gently washed from the plate and replated at a density of 25,000 cells/cm2. To facilitate cell attachment, cultures were treated with testicular hyaluronidase (4 U/ml of medium). After 3 days, the hypertrophic cells were exposed to agents described below in serum-free medium for 6, 12, or 24 h. The phenotype of these cells was characterized using techniques described earlier (collagen I, II, and X; osteocalcin expression, alkaline phosphatase activity, and Fourier transform infrared spectroscopy) (15, 16, 45).
Treatment protocols. The actual concentration of Pi in the serum-free medium was 1 mM. This value was set as the control Pi concentration. To induce apoptosis, the medium was supplemented with Pi as NaH2PO4. This salt was added to the bulk medium and rapidly mixed; in this way, mineral precipitation was prevented. To investigate the mechanism of Pi-induced cell death, the following agents were used: caspase 3 inhibitor (cat. no. 235423) and caspase 1 inhibitor (cat. no. 400011); fluorogenic caspase 3 substrate Z-DEVD-AFC (cat. no. 264150) and caspase 1 substrate Z-YVAD-AFC (cat. no. 88225; both the caspase inhibitors and fluorogenic substrates were purchase from Calbiochem, San Diego, CA); NO donor SNOG; the NOS inhibitors L-NMMA (acetate salt) and L-NAME; staurosporine, a generalized activator of apoptosis; and the NaPi transporter inhibitor PFA (Sigma).
NO measurements.
NO metabolites nitrite (NO
Caspase activity assay. To measure caspase activity, cells were incubated with specific fluorogenic peptides that serve as caspase substrates. After proteolytic cleavage of the substrate, the release of the fluorescent tag was measured and related to the activity of the caspase. Specifically, the activated chondrocytes were collected in 200 µl of a reaction buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 10 mM Tris · HCl, pH 8.0, and 10 µg/ml leupeptin), sonicated three times for 20 s, and centrifuged for 15 min at 20,000 g. Eighty microliters of lysate were combined with 10 µM caspase substrate in a total volume of 1 ml of reaction buffer. The substrate for caspase 3 was the fluorogenic CPP32/apopain substrate, Z-DEVD-AFC; the substrate for caspase 1 enzyme was the fluorogenic substrate, Z-YVAD-AFC. In both cases, activity was monitored for >60 min by following the blue to green shift in fluorescence upon release of the 7-amino-4-trifluoromethyl-coumarin (AFC) fluorophore. The excitation/emission maxima for AFC is 400/505 nm.
MTT assay. Because dying cells exhibit a sharp decrease in MTT reductive activity, the method is widely used to measure cell death (25, 39). Briefly, cells were grown in 24-well plates in medium with or without the test agent. At the end of the treatment period, they were incubated with MTT (120 µg/ml) in serum-free medium at 37°C. After 3 h, the supernatant was removed, and the formazan crystals were solubilized in 0.04 M HCl in isopropanol and stirred for 10 min at room temperature. The optical density was read at 595 nm using an ELISA plate reader.
Assessment of mitochondrial function. We used the mitochondrial membrane voltage-sensitive dye, rhodamine 123, to evaluate mitochondrial function in control and treated chondrocytes. Cells were treated with 5 mM Pi, 5 mM Pi plus 5 mM L-NMMA, or 5 mM Pi plus 5 mM PFA for 24 h in six-well Falcon plates. Approximately 20 min before termination of the study, the media were replaced with phenol-free DMEM that contained 0.5 µg/ml rhodamine 123 (Molecular Probes, Eugene, OR). After 20 min, the dye was removed, and the cells were washed twice with phenol-free DMEM. Mitochondrial fluorescence was then evaluated using an Olympus Fluoview inverted confocal microscope (Olympus, Melville, NY). The wells were analyzed using a ×40 long-working- distance lens with a specialized cap for plastic dishes.
Measurement of glutathione. Cell glutathione levels were measured by high-performance liquid chromatography. Samples were injected onto a reverse-phase column (Alltech Adsorbosphere HS C18 7U; 250 × 4.6 mm) at a flow rate of 0.9 ml/min. The solvent comprised 0.1 M phosphoric acid, 3 mM heptane sulfonic acid, and 5% methanol, pH 2.0; the system was helium sparged. Thiol peaks were measured using an electrochemical detection system (35).
Statistical analysis. All experiments were repeated 3-5 times. MTT absorbance values were normalized to 100%; experimental values (treated) were expressed as percentage of the control. Significant differences between sets of values for control and test groups were assessed by ANOVA. A P value refers to a comparison of a measured parameter in the experimental group with that of the appropriate control; significance was set at P < 0.05. The means were tested for normalcy by Scheffé's test.
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RESULTS |
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Embryonic hypertrophic chondrocytes treated with
Pi exhibit a profound decrease in cell viability. Figure
1 shows that when the medium
Pi concentration is raised to 5 mM, there is a fivefold fall in MTT values; at 7 mM Pi, almost all the cells are
dead. In other studies, we (22) have shown that these
cells die through apoptosis. Pi-induced
apoptosis was associated with an elevation in
NO
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PFA, an NaPi transport inhibitor, inhibited NO metabolite
generation (NO
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Inhibition of NO generation by NMMA or L-NAME prevented
Pi-dependent apoptosis. Figure
3A indicates that the NOS
inhibitor L-NAME blocks cell death. At 5 mM
L-NAME, 100% of cells are viable. The effects of
L-NMMA are similar to L-NAME, except that at
the highest concentration used (5 mM), there is ~80% protection.
Exogenous NO also caused an increase in chondrocyte apoptosis.
Figure 3B shows that as the SNOG concentration is raised to
5 mM, there is a dose-dependent increase in cell death. At 5 mM SNOG,
almost all of the chondrocytes are dead.
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The mechanism of NO-mediated chondrocyte death involves caspases 1 and
3. Inhibition of these enzymes protects cells from Pi-induced cell death. Figure
4 indicates that in the absence of the
inhibitors, almost all of the cells are apoptotic. However, the
presence of 50-300 µM caspase 1 or caspase 3 inhibitor results in a dose-dependent increase in cell viability. At the highest dose,
the caspase 3 inhibitor increases chondrocyte viability above control
values, possibly by inhibiting spontaneous apoptosis.
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We confirmed that caspases 1 and 3 were involved in the death process
by measuring caspase activity in chondrocytes treated with modulators
of apoptosis. Table 1 shows the
change in caspase activity of cells treated with the apoptogens
Pi, SNOG, and staurosporine. Expressed as a percentage of
control value, the presence of 5 mM Pi increases the
activity of caspase 3 2.7-fold, whereas caspase 1 is increased almost
2-fold. Staurosporine (1 µM) also elevates caspase 3 and 1 activities. The NO donor SNOG caused an almost twofold rise in caspase
3 activity, but appeared to exert little effect on caspase 1. When
Pi apoptosis is blocked by treating cells with
either PFA or L-NAME, caspase 1 and 3 activity is not increased above control values.
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Treatment of chondrocytes with Pi disrupts mitochondrial
function and may predispose cells to apoptosis
(29). Figure 5 shows that
when chondrocytes are treated with 5 mM Pi for 24 h,
there is a significant decrease in rhodamine 123 fluorescence (compare Fig. 5, A and B). However, in the presence of the
NOS inhibitor NMMA, mitochondrial fluorescence is maintained (Fig.
5D). Similar results are seen when chondrocytes are treated
with L-NAME. In the same study, cells were treated with the
apoptogen in the presence of PFA. Figure 5C shows that this
transport inhibitor prevents Pi-dependent loss of
mitochondrial fluorescence.
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We reported earlier that treatment of maturing chondrocytes with
Pi caused a decrease in cellular glutathione levels
(35). Glutathione levels in response to Pi
were next related to NO generation. Figure
6 shows that 5 mM Pi reduced
the glutathione concentration by almost 90%. However, when cells were
treated with Pi in the presence of PFA and
L-NAME, these agents completely inhibited Pi-induced thiol depletion of tibial chondrocytes.
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DISCUSSION |
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In a previous investigation, we reported that terminally differentiated cells of the epiphyseal growth plate undergo apoptosis (13). In a search for factors that could cause chondrocyte death in vivo, we found that hypertrophic cells were sensitive to Pi and that a rise in the anion concentration mediated chondrocyte apoptosis (22). Because Pi as well as Ca2+ is accumulated at the cartilage calcification front and around cells at the epiphyseal-metaphyseal junction, we reasoned that these ions could also serve as apoptogens in vivo. Results of the study reported here focus on one of these ions, Pi. Analysis of the data revealed that Pi can induce apoptosis by activating NO generation in chondrocytes. The study also showed that an elevation in NO levels occurred in concert with other downstream events, including activation of caspases and inhibition of mitochondrial function. It is noteworthy that similar changes are seen in other cell types in which NO upregulates apoptosis (19, 24, 31, 44). Thus our findings not only provide an insight into the mechanism of chondrocyte death but indirectly lend some strength to the notion that NO regulates cell number in the growth plate, and as such, controls bone development and growth.
That epiphyseal chondrocytes were able to generate NO was not unexpected. A plethora of studies show that articular chondrocytes respond to inflammatory stimuli by generating NO and that NO production is linked to apoptosis (20). Indeed, NO has been demonstrated to play a role in almost every aspect of the pathophysiology of osteoarthritis, from chondrocyte proliferation to extracellular matrix synthesis and degradation (20). What is surprising is that to date, there have been few reports of the generation of NO in the growth cartilage. While the work reported here relates to tibial chondrocytes, studies in progress indicate that a number of synthases are active in cells in the epiphysis, and, most importantly, products of NO metabolism such as S-nitrosocysteine and nitrotyrosine accumulate in the hypertrophic zone. Thus experiments on tibial chondrocytes in vitro probably mirror events that occur in maturing epiphyseal cells in situ. Against this background, it was interesting to note that the actual level of NO metabolites generated by the Pi-treated cells was similar to that reported for articular chondrocytes (14, 27, 34) and other cell types (5, 21). Because NO functions in these tissues to regulate a number of key physiological processes, it is possible that in the hypertrophic zone of the growth plate, NO serves to regulate not only apoptosis but other critical terminal activities.
To test the hypothesis that NO promoted apoptosis by activating caspases, we measured enzyme activity in cells that were undergoing apoptosis. We noted that staurosporine and Pi induced a similar pattern of changes in caspase activity, and in both cases, caspase 3 activity was considerably higher than caspase 1. In addition, both the NOS inhibitor and the NaPi transport blocker prevented a rise in caspase 3 activity. Relevant to this observation, the Pi-induced elevation in caspase 3 activity is similar to that observed in other tissues that are undergoing apoptosis (3). It is likely that activation of caspase 3 serves as a prerequisite step in the sequence of events that leads to endonuclease activation and subsequently cleavage of DNA. Moreover, the observation that we can block Pi-induced apoptosis by inhibiting caspase activity mirrors studies of other tissues in which apoptosis induced by NO donors have been obviated (3, 32, 33, 36-38, 41, 42).
It is important to note that while NO may serve to activate downstream effectors and upregulate apoptosis, NO can also serve to modulate cell survival signals. In signaling terms, depending on the cell type, NO activation has been described as either cGMP dependent or independent, and the activation of the cGMP pathway can either protect cells or induce death. We have explored the role of cGMP in Pi-induced death of tibial chondrocytes and found that NO-mediated apoptosis in our system is cGMP independent. Thus when cells were treated with inhibitors of the soluble guanylate cyclase or the cGMP-dependent kinase before induction of apoptosis with Pi, there were absolutely no alterations in the rate or extent of cell death. The same result was obtained when cells were treated with the cGMP analog 8-bromo-cGMP (data not shown). Thus while NO triggers chondrocyte apoptosis, details of the pathway have yet to be delineated.
In terms of the apoptotic cascade, NO could activate downstream
effectors directly or catalyze the formation of other highly reactive
intermediates, like peroxynitrite (ONOO). This radical
could then serve to promote oxidation processes that lead to cell death
(30). Despite the fact that NO can inhibit caspase 3 activity in vitro (18), ONOO
has been shown
to induce caspase 3 and other caspases (26, 41). To
investigate the possibility that Pi-induced
apoptosis is mediated through changes in oxidative metabolism,
mitochondrial activity was examined using a fluorescent
voltage-sensitive dye (rhodamine 123). We showed that Pi
treatment caused a decrease in the mitochondrial transmembrane
potential (
m). That a NO intermediary was formed was
supported by the observation that when cells were pretreated with NOS
inhibitors, there was preservation of the
m. Although we did not
assess how the loss of mitochondrial function could induce
apoptosis, it is known that ONOO
and NO donors
can potently and irreversibly deenergize mitochondria. In addition, NO
can inhibit a number of mitochondrial enzymes, including aconitase and
cytochrome c oxidase (6). There is also strong
evidence to indicate that NO and NO-derived reactive species can cause
oxidative stress by mediating nitrosative reactions and oxidizing
glutathione (11, 40, 43). Undoubtedly, nitrosative reactions, inhibition of tricarboxylic acid cycle and respiratory enzymes, and defective oxidative activity would serve to sensitize chondrocytes to apoptotic stimuli. In fact, we have previously reported that thiol levels are low when chondrocytes are treated with
Pi (35). On this basis, we argue that the loss
of reductive reserve, the decrease in protection against oxidative
stress, and compromised mitochondrial function result in activation of caspase 3. At this point in time, chondrocytes are irreversibly committed to the apoptotic pathway.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DE-13319, DE-10875, HL-05496, and HL-59664 and the FCT of Portugal.
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FOOTNOTES |
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H. Ischiropoulos is an American Heart Association Established Investigator.
Address for reprint requests and other correspondence: I. M. Shapiro, Dept. of Biochemistry, School of Dental Medicine, Univ. of Pennsylvania, 4001 Spruce St., Philadelphia, PA 19104-6003 (E-mail: ishap{at}biochem.dental.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 January 2001; accepted in final form 23 April 2001.
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