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Induction of apoptosis in bovine articular chondrocyte by prostaglandin E2through cAMP-dependent pathway
Osteoarthritis and Cartilage (2000) 8, 17–24 © 2000 OsteoArthritis Research Society International Article No. joca.1999.0266, available online at http://www.idealibrary.com on
1063–4584/00/010017+08 $35.00/0
Induction of apoptosis in bovine articular chondrocyte by prostaglandin E2 through cAMP-dependent pathway M. Miwa*, R. Saura†, S. Hirata†, Y. Hayashi*, K. Mizuno† and H. Itoh*
*First Department of Pathology, Kobe University School of Medicine, †Department of Orthopaedic Surgery, Kobe University School of Medicine, Japan Summary Objective: Regulation of important biological processes such as proliferation and differentiation of articular chondrocytes is known to be mediated by prostaglandin E2 (PGE2) in both normal and pathological states. Articular chondrocytes also undergo apoptosis, a biological phenomenon implicated in many physiological processes. Whether or not PGE2 induces apoptosis in articular chondrocytes, however, is not known. Design: Bovine articular chondrocytes were cultured with or without PGE2 for 24 h and amounts of fragmented DNA, which is a distinct characteristic of apoptosis, were measured by enzyme-linked immunosorbent assay. Also effect of cyclic AMP (cAMP), which is one of the intracellular downstream mediator of PGE2, on chondrocyte apoptosis was investigated. Results: Administration of exogenous PGE2 on bovine articular cartilage grown as a monolayer culture resulted in the induction of DNA fragmentation. This DNA fragmentation was accompanied with a marked dose-dependent increase in intracellular cAMP. Also cultured cells were treated with cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase, and the incidence of apoptosis in the chondrocytes was determined. As well as PGE2, dibutyryl-cAMP and forskolin stimulated chondrocyte DNA fragmentation. Conclusions: It is the first report that PGE2 can induce articular chondrocyte apoptosis in vitro. It is also suggest that apoptosis of chondrocytes by PGE2 is linked with cAMP-dependent pathway. © 2000 OsteoArthritis Research Society International Key words: Bovine articular chondrocytes, Prostaglandin E2, Apoptosis, Cyclic AMP.
of animal models, which may be correlated with the increased risk of degenerative joint disease seen in elder people.9 These observations suggest that apoptosis of chondrocytes is critical for endochondral ossification during skeletal growth and also in the development of the degenerative change in articular cartilage during inflammation. However, the mechanism that induces articular chondrocyte apoptosis has, to date, not been fully understood. Prostaglandins (PGs) are important regulators of cellular function in a variety of tissues, including bone and cartilage. Numerous reports have suggested that PGs play a significant role in both the normal cartilage metabolism and the pathogenesis of joint disorders. For instance, the physeal cartilage responds to mechanical forces through modulation of PG synthesis.10 PGE2 was also reported to be the most potent cytokine, causing significant effects in both DNA synthesis and sulfate incorporation into the growth plate chondrocytes.11 This observation indicates that PGs may have an important role in chondrocyte metabolism in the areas of endochondral ossification integral to growth plate development and fracture repair of long bones. PGE2 is detectable at a high level in the fluid of knee joints in OA12,13 and rheumatoid arthritis (RA).14,15 PGs including PGE2 are synthesized from eicosatetraenoic acids in the presence of cyclooxygenase (COX). The expression of the inducible COX isoform, COX-2, but not constitutive form, COX-1, was found to be elevated in a disease-related pattern in the synovial tissue from patients with RA in comparison with OA.16 It is also reported that
Introduction Cartilage is one of the tissues with the highest susceptibility to age-related pathology. Osteoarthritis (OA) represents not only the most frequent muscloskeletal disorder but also the most common disease in the aging population.1 Very remarkable changes that occur with aging in the human articular cartilage include both profound loss in tissue cellularity and deleterious change of the extracellular matrix.2,3 At advanced stages of OA, the number of chondrocytes per unit volume diminishes, leading to the failure in the maintenance of the cartilage matrix. This corresponds to an age-related decline in responsiveness to growth factors in human articular chondrocytes which could be due to a decreased rate of cell division or an increased rate of cell death.4,5 Apoptosis has been involved in a wide variety of physiological regulatory processes and the pathology of various diseases. In terms of the cartilaginous tissue, apoptosis plays an important role in the differentiation of hypertrophic chondrocytes followed by the ossification of growth plate.6,7 Also, the number of apoptotic chondrocytes in the articular cartilage observed in OA tissues is much higher than that in the healthy ones.8 In addition, aging is reported to increase the ratio of apoptotic cells in articular cartilage Received 26 February 1999; accepted 9 June 1999. Address correspondence to: Ryuichi Saura, Department of Orthopaedic Surgery, Kobe University School of Medicine, 7-5-1 Kusunoki-cho Chuo-ku Kobe Hyogo Japan 650-0017.
17
18 cartilage specimens from OA-affected patients spontaneously released PGE2 at levels higher than in cytokinetreated normal cartilage due to upregulation of COX-2.17 These results suggested that PGE2 might exacerbate joint inflammation and be involved in the disease process of degenerative arthritis. PGE2 plays a key role in chondrocyte metabolism and increases concentration of cyclic adenosine monophosphate (cAMP) in these cells. Cyclic AMP generation is linked to the stimulation of proteoglycan synthesis in rabbit articular chondrocytes.18 There is considerable evidence that PGs mediate many of the effects of polypeptides, steroid hormones, growth factors, cytokines and mechanical forces on articular chondrocytes.19–22 For PGE2, however, the net effects on articular chondrocyte, which play a central role in the physiology and pathology of the articular cartilage, are less clear.23,24 Recently, the administration of nonsteroidal antiinflammatory drugs (NSAIDs) is reported to reduce the risk of fatal colon cancer.25,26 Though it is still elusive why NSAIDs can prevent the carcinogenesis of colon, the following observations could support these epidemiological reports. Enhanced expression of COX-2 polypeptides in human colon cancer tissues were detected using immunohistochemistry, whereas COX-1 expression was weak in both normal and cancerous specimens.27 It is also shown that overexpression of COX-2 leads to phenotypic changes involving the inhibition of apoptosis in intestinal epithelial cells that could enhance their tumorigenic potential.28 Thus, COX-2 may be strongly related to the cell proliferation including colon carcinogenesis. On the other hand, PGE2 is reported to be an antiproliferative molecule and one of the inducers of apoptotic change in various types of cells. For instance, PGE2 induces cAMP accumulation and inhibits the growth of the most differentiated breast cancer cells due to loss and probably dysfunction of PGE2 receptors.29 PGE2 also suppressed RA synovial cell proliferation through intracellular cAMP accumulation.30 In terms of the apoptosis by PGE2, it is involved in apoptotic alterations typical of ovarian surface epithelium associated with ovulatory ovine follicles31 and induction of thymocyte apoptosis for negative selection of T lymphocytes.32 During skeletal development, chondrocytes in the growth plate undergo a sequence of events that include proliferation, hypertrophy and cell death which have morphologically been demonstrated to be evidence of apoptosis.6,7 PGE2 has also been found to increase cartilaginous matrix surrounding the hypertrophic chondrocytes in the growth plate.33,34 These findings suggest that increased PGE2 could be closely related to the induction of apoptosis in growth plate chondrocytes. Thus, the effects of PGE2 and COX-2 in the regulation of apoptosis are cell types specific. However, there is no report as to whether or not PGE2 induces articular chondrocyte apoptosis. The present study was undertaken to address this issue.
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2 Biochemical Corp., Freehold. NJ) in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY) for 6 h at 37°C. Chondrocytes were harvested by filtration through gauze to remove tissue fragments, followed by centrifugation (450 g, 10 min). The isolated cells were washed three times and resuspended in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY) and antibiotics (20 units/ml penicillin and 100 ìg/ml streptomycin). They were then checked for viability by exclusion of trypan blue, and inoculated into culture plate at high density (1.2×105 cells/cm2) in a monolayer fashion. This high density primary cell culture can maintain the in vitro phenotype of bovine articular chondrocytes up to 14 days.36,37 Immediately after cells were attached to the bottom of culture plate, monolayer cultures were subjected to the experiments described below. All experiments were performed with chondrocytes in primary culture.
ASSAY FOR CELL PROLIFERATION
Chondrocytes plated into 96-well plates (1.2×105 cells/ cm2) were cultured in total volume of 200 ìl of DMEM containing 10% FBS and antibiotics with varying concentration of PGE2 (SIGMA, St. Louis, MO) for 72 h. Fifteen hours before terminating the proliferation assay of each incubation time, 37 KBq of [3H]thymidine ([3H]TdR; DuPont/NEN Research Products, Boston, MA) was added to each well. At the end of assay, cells were washed three times with distilled water. One hundred microliter of scintillation liquid was then added and total radioactivity of cell associated [3H]TdR was quantified by liquid scintillation counting (Packard Instrument Company, Meriden, CT).
QUANTIFICATION OF NITRITES
Chondrocytes were inoculated into 24-well culture plates at high density (1.2×105 cells/cm2) in DMEM containing 10% FBS and antibiotics. After 24 h in culture, the plating medium was removed and replaced with fresh serum-free medium containing varying concentrations of PGE2 and cultured for an additional 48 h. Generation of nitric oxide (NO) by cells was detected by its stable oxidative product, nitrite accumulation of which was measured in the culture supernatants by the Griess reaction as described previously.38 Briefly, at the end of incubation, sample aliquots were mixed with an equal volume of reaction regents composed of 0.1% N-1naphthylethylenediamine dihydrochloride and 1% sulfanilamide and in 5% phosphoric acid (Wako Pure Chemical Industries Ltd, Osaka, Japan). The absorbance at 550 nm was measured, and the nitrite concentration was determined using a curve calibrated on sodium nitrite as standards.
DNA FRAGMENTATION ASSAY
Materials and methods CHONDROCYTE CELL CULTURE
Articular cartilage was shaved from the bovine carpometatarsal joints and cut into thin sections. Cells were isolated from the tissue by a collagenase digestion procedure as described with some modifications.35 Briefly, the tissue was treated with 0.1% collagenase (Washington
Chondrocytes (1.2×105 cells/cm2) were inoculated into 24-well culture plates in DMEM containing 10% FBS and antibiotics. After 24 h culture, the plating medium was changed with serum free medium containing varying concentrations of either PGE2, dibutyryl-cAMP or forskolin (Sigma, St Louis, MO). For examining the effect of serum on DNA fragmentation induced by PGE2, the medium containing FBS was substituted for serum-free conditions.
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After an additional 24-h exposure to various stimuli, cells were washed with 0.05 M phosphate buffered saline (PBS; pH 7.4) and lysed in PBS containing 1% sodium dodecyl sulfate and 1% Tween 20 (Wako Pure Chemical Industries Ltd, Osaka, Japan) at 4°C for 30 min. The cell lysate were centrifuged at 200 g at 4°C for 10 min to remove cell debris and high molecular weight DNA. Fragmented DNA was measured by quantitation of cytosolic oligonucleosome-bound DNA with a Cell Death Detection enzyme-linked immunosorbent assay kit (Boehringer Mannheim GmbH, Germany) according to the manufacturer’s instructions. This assay is based on the quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. This allowed the specific determination of mono- and oligo-nucleosomes in the cytoplasmic fraction of cell lysate. INTRACELLULAR CAMP MEASUREMENT
Chondrocyte monolayer culture (1.2×105 cells/cm2) was also prepared in 24-well culture plates with DMEM containing 10% FBS and antibiotics. After 24 h, the medium in culture plates was changed with 1.0 ml of serum-free medium per well. The cultures were allowed to equilibrate for 1 h, then 10 ìl isobutylmethylxanthine (10 mM in 95% ethanol) was added and the cultures were incubated for 20 min at 37°C. PGE2 was then added at the indicated concentrations. After 20 min of incubation at 37°C, the reaction was stopped by aspirating the medium, adding 0.5 ml of 95% ethanol per well, and allowing cultures to air dry. The dried cell samples were reconstituted in 300 ìl sodium acetate buffer (pH 5.8) for enzyme immunoassay (EIA) using the commercial reagent for cAMP-EIA (Amersham, U.K.). STATISTICAL ANALYSIS
Statistical analysis was carried out on all data points with regard to control by an unpaired Student’s t-test. Each data point represented the mean of four or six separate samples with the corresponding standard error of the mean (SEM). P values under 0.05 were considered significant statistically.
Results EFFECT OF PGE2 ON PROLIFERATION AND DNA FRAGMENTATION OF BOVINE ARTICULAR CHONDROCYTES
To investigate whether PGE2 suppresses chondrocyte proliferation and induces DNA fragmentation in these cells, chondrocytes were treated with PGE2 at doses ranging from 10 −10–10 −6 M and for varying periods of time. Incubation with a varying concentration of PGE2 for 72 h demonstrated that [3H]TdR incorporation into the chondrocytes was decreased by PGE2 in dose dependent manner up to 10 −6 M. At 10 −8 M PGE2, [3H]TdR incorporation was reduced by 25% compared to the control and at 10 −7 M PGE2, incorporation of [3H]TdR was further reduced (60% of the control). However, further increases in PGE2 concentration did not produce any greater effect. Statistical significance of the results was observed up to 10 −8 M of PGE2 while the maximum effect was noted at 10 −7 M (Fig. 1). The concentration of PGE2 in this range is reported to
Fig. 1. Effect of PGE2 on [3H]TdR incorporation into bovine articular chondrocytes. Chondrocytes were plated into 96-well plates in total volume of 200 ìl of DMEM containing 10% FBS and antibiotics with various concentration of PGE2. Total radioactivity of cell associated [3H]TdR was quantified by liquid scintillation counting after 72 h incubation as described in Materials and methods. Data are mean±SEM of six replicates. *P< 0.05 (vs. control).
be detectable in the joint fluids of OA12,13 and RA patients.14,15 Intracellular DNA fragmentation, which is a hallmark of apoptosis, was induced in chondrocytes by PGE2 treatment and showed a statistically significant increase of apoptotic rate at doses over 10 −9 M (Fig. 2). A maximal 4-fold increase in DNA fragmentation was detected at a concentration of 10 −6 M of PGE2; this dose of PGE2 was also used over varying periods of time, and a timedependent rate of chondrocyte DNA fragmentation reached a maximum at 24 h (data not shown). In order to examine the effect of serum factors on PGE2 induced chondrocyte DNA fragmentation, articular chondrocytes were cultured with PGE2 in the presence of FBS. As shown Fig. 3, supplemented with 10% FBS, DNA fragmentation of articular chondrocyte was reduced by 50% compared to serum free conditions. However, further increase of FBS concentration did not reduce DNA fragmentation in chondrocytes. Though FBS markedly decreased DNA fragmentation, 10 −8 M of PGE2 induced small but statistically significant fragmentation of articular chondrocyte DNA. The assay for DNA fragmentation applied in the present study does not define the number of apoptotic cells. Therefore, the data on DNA fragmentation do not provide information on the percentage of cells that underwent apoptosis in a given culture. The percentage of cells which synthesize DNA and incorporate [3H]TdR is also not indicated. It is possible that PGE2 may affect distinct subpopulations of chondrocytes and suppress DNA synthesis in one subset and induce apoptosis in another. In order to detect which cells in culture as apoptotic and which is in S phase, further
20
Fig. 2. Effect of PGE2 on apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10% FBS and after 24 h cells were exposed to varying concentrations of PGE2 in identical fresh medium for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control).
experiments should be performed with an assay that allows localization and quantification of the number of cells that undergo apoptosis in response to PGE2.
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2
Fig. 3. Effect of serum on PGE2 induced apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10% FBS and after 24 h cells were exposed to 10 −8 M of PGE2 in fresh medium containing up to 50 % FBS for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control). Table I Effect of PGE2 on articular chondrocyte NO generation PGE2 (logM)
Concentration of nitrite (ìM)
P values
0.80±0.25 0.66±0.25 0.22±0.29 0.41±0.22
— 0.71 0.18 0.29
EFFECT OF PGE2 ON NO GENERATION IN BOVINE ARTICULAR CHONDROCYTES
It is reported that chondrocyte apoptosis induced by interleukin-1 (IL-1) is mediated by NO,39 thus the possibility of PGE2-induced chondrocyte DNA fragmentation mediated by NO synthesis was investigated. There was no increase of nitrite accumulation in the culture medium when articular chondrocytes were cultured in presence of PGE2 for 48 h. In articular chondrocytes, PGE2 failed to induce NO generation at a concentration of up to 10 −6 M (Table I). These results suggest that PGE2 induce DNA fragmentation of articular chondrocytes independently of NO generation.
No PGE2 −8 −7 −6
After 48-h incubation of articular chondrocytes with serum-free medium containing varying concentrations of PGE2, nitric oxide generation of the chondrocytes was detected in the culture supernatants by the Griess reaction stated in Materials and methods. Statistical analysis was carried out on all data points with regard to control by a Student’s t-test. Each data point represented the mean of four separate samples with the corresponding standard error of the mean (SEM). P values (vs. no PGE2) under 0.05 were considered to be statistically significant.
EFFECT OF ELEVATED INTRACELLULAR CAMP LEVEL ON DNA FRAGMENTATION IN BOVINE ARTICULAR CHONDROCYTE
Since PGE2 has been demonstrated to exert its biologic effects in various cells through the stimulation of cAMPmediated biochemical pathways, the ability of PGE2 to induce DNA fragmentation was investigated as a function of cAMP concentration in articular chondrocytes. As shown in Fig. 4, 20-min exposure of articular chondrocytes to PGE2 elevated intracellular cAMP concentration in a dosedependent fashion such that nearly 3.3-fold increase of cAMP level was observed at 10 −6 M of PGE2. A statistically
significant elevation of cAMP was observed at doses over 10 −8 M of PGE2 and a maximal increase of about 4-fold over control level was caused by 10 −6 M of PGE2. Therefore, in order to investigate the relationship between the elevation of intracellular cAMP and induction of the chondrocyte DNA fragmentation, articular chondrocytes were treated with the membrane-permeant cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase and examined for changes in their DNA fragmentation after 24 h using sandwich ELISA. Both dibutyryl-cAMP and forskolin induced chondrocyte DNA
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Fig. 4. Effects of PGE2 on intracellular cAMP accumulation in bovine chondrocytes. Chondrocytes plated in monolayer cultures were exposed to varying concentrations of PGE2 in serum free medium for an additional 20 minutes after 20 minutes incubations performed in the presence of 100 ìM 3-isobutyl-1-methylxanthine. Intracellular cAMP of chondrocyte was determined by EIA, as described in Materials and methods. Each value represents the mean±SEM of four replicates. **P< 0.01 (vs. control).
fragmentation in a dose-dependent fashion (Fig. 5a, b) such that dibutyryl-cAMP increased DNA fragmentation 8-fold over control, whereas forskolin increased it by 2.6fold. Significant effect of the dibutyryl-cAMP was detected over a concentration of 10 −8 M with a maximal effect at 10 −6 M. Also, over 10 −7 M of folskolin significantly induced DNA fragmentation of chondrocytes. In fact, we did not assay adenylate cyclase activity directly in cultured chondrocytes. However, forskolin that activates adenylate cyclase induced chondrocyte DNA fragmentation. Also, cAMP analogue, dibutyryl-cAMP, which is substitute for the intracellular cAMP, induced it. These results suggest that PGE2 could induce DNA fragmentation of articular chondrocyte through the elevation of intracellular cAMP level by activation of adenylate cyclase.
Discussion We show that increased DNA fragmentation in articular chondrocytes could be observed by adding exogenous PGE2 into the cells. Increased DNA fragmentation, which is a hallmark of apoptosis, could be also observed by increased cAMP level achieved through the use of dibutyryl-cAMP or forskolin. Thus, present study helps us to suggest that PGE2 induces articular chondrocyte apoptosis through the elevation of intracellular cAMP concentration followed by activation of protein kinase A (PKA) second messenger system. As to the aging alterations of articular cartilage, increasing number of the apoptotic chondrocytes may be correlated with the risk of degenerative joint disease seen in the elderly people.9 Recently, it was demonstrated that a high
21 number of apoptotic chondrocytes was histologically present at the pannus-cartilage junction in experimentally induced osteoarthritic cartilage and that the prevalence of apoptotic cells was significantly correlated with the levels of nitrite production in ex vivo explants and its OA grade.40 Also, the effectiveness of a selective inhibitor of inducible NO synthase (iNOS) was reported in attenuating the progression of experimental OA, which indicated the diminution of chondrocyte apoptosis due to inhibition of NO synthesis.41 Thus, articular chondrocyte apoptosis seems to be related to the pathology of degenerative joint diseases. In relation to the pathogenesis of OA, it is reported that NO generated from articular chondrocytes in the presence of inflammatory cytokines such as IL-1 induces articular chondrocyte apoptosis in vitro.39 IL-1 is one of the most potent cytokines, which can deteriorate the functional property of articular cartilage. Among its catabolic effects on cartilage are the stimulation of matrix metalloproteinase synthesis and inhibition of extracellular matrix production of articular chondrocytes. Inhibition of chondrocyte proliferation is another important effect of IL-1, which may prevent compensatory repair responses under conditions that are associated with inflammatory cartilage remodeling.42 Thus, chondrocyte replication, it is important not only during skeletal development but also cartilage repair to maintain cartilage cellularity in degenerative joint diseases. Previous studies have suggested that the antiproliferative effect of IL-1 on articular chondrocytes might be mediated by the induction of PGE2. IL-1 increases COX-2 mRNA expression in articular chondrocytes, which lead to generate much amount of PGE2.43 In order to address the potential interactions between PGE2 and NO, we examined whether PGE2 is involved with the induction of NO. In the present study, PGE2 alone failed to stimulate NO synthesis of articular chondrocytes as previously reported,42 which indicate that PGE2-induced DNA fragmentation of articular chondrocyte was not mediated by NO but PGE2 itself. Supplementation with FBS was reported to induce a dose-dependent increase in [3H]TdR incorporation into the chondrocytes. The present study showed that exogenously added PGE2 inhibited chondrocyte proliferation in the presence of 10% FBS and induced small but statistically significant DNA fragmentation of chondrocyte while supplementation with FBS markedly decreased it. FBS may include several chondrocyte growth factors such as transforming growth factor â (TGFâ). Some serum factors may protect cultured cells from apoptosis. Previous reports suggested that TGFâ increased PGE2 synthesis by articular chondrocytes and induced cell proliferation.44,45 This would be inconsistent with the present results. It is supposed that PGE2, of which levels as induced by TGFâ are relatively low, do not interfere with chondrocyte proliferation, whereas at high levels PGE2 as seen in response to IL-1 are growth inhibitory. Levels of PGE2 applied in this study seem to be higher than that induced by TGFâ. However, PGE2 does indeed chondrocyte DNA fragmentation in the presence of up to 50% FBS. The present studies were performed with primary chondrocytes in serum starved condition to avoid antagonistic effects of PGE2 and other serum factors including TGFâ in the regulation of chondrocyte proliferation. The actions of PGE2 are mediated by four distinct classes of PGE2 E-prostanoid (EP) receptors (EP1 through EP4). Increased cAMP generation by PGE2 may be due to EP2 or EP4 receptor activation. Though it is still unclear which receptors are expressed on the surface of articular
22
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2
Fig. 5. Effect of dibutyryl-cAMP and forskolin on apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10 % FBS, and after 24 h cells were exposed to varying concentrations of (a) dibutyryl-cAMP or (b) forskolin in identical fresh medium for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control).
chondrocyte, PGE2 induced chondrocyte apoptosis is supposed to be mediated by EP2 or EP4 receptor activation46 because the elevation of intracellular cAMP concentration was observed in articular chondrocytes as shown in Fig. 4. The further investigation for detecting the type of EP receptor on articular chondrocyte should be required in order to elucidate the mechanism of PGE2-induced chondrocyte apoptosis in detail. It has been suggested that the DNA cleavage induced by cAMP in thymocytes and peripheral lymphocytes may depend on either the induction of the new endonuclease gene products or the activation of endonuclease activities24,47 such a mechanism can be suggested for PGE2induced apoptosis of chondrocytes. Another possibility is that cAMP may inhibit a physiologically functioning mechanism by which chromatin is protected from DNA fragmentation when cell death signals are turned on.48 Phosphorylation of cellular substrates by cAMP dependent protein kinase A (PKA) is also reported to induce apoptosis in other cells. Apoptosis induced in thymocytes by gliotoxin, which is a fungal toxin, is calcium-independent and unaffected by protein synthesis inhibitors. It has shown that gliotoxin results in rapid phosphorylation of histone H3 in thymocytes.49 Cyclic AMP levels and activity of PKA were raised in cells treated with gliotoxin. Cells treated with gliotoxin showed an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation. Forskolin is reported to show some increase in phosphorylation of H3 after 8-hour treatment with this agent. Forskolin also induces apoptosis in thymocytes but over a
longer time course than gliotoxin. In both cases levels of apoptosis correlated with degree of H3 phosphorylation. It is presumed that PGE2 induced chondrocyte apoptosis may be mediated by phosphorylation of H3 because forskolin showed some increase in DNA fragmentation of chondrocytes. The proto-oncogene bcl-2 is considered important in determining whether cells enter an apoptotic pathway or survive. Increased expression of Bcl-2 in immature B-cell lymphomas is reported to completely inhibit PGE2- and cAMP-mediated DNA fragmentation and nuclear condensation.50 Also, cell cycle analysis demonstrated that PGE2 increased the percentage of cells in G1, and analysis of synchronized populations revealed that PGE2 acts at all phases of the cell cycle to delay normal progression in immature B cell lymphomas. The microtubule-damaging drugs, paclitaxel and vincristine induced activation of PKA, which induced Bcl-2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells.51 These microtubule-damaging drugs caused growth arrest in G2-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation by PKA mediated inactivation of Bcl2 and apoptosis without the involvement of p53. In relation to regulation of chondrocyte apoptosis in articular cartilage, the importance of Bcl-2 was also reported. Serum withdrawal and retinoic acid treatment induced apoptosis of primary human articular chondrocytes, when the expression of Bcl-2 was downregulated by both agents.52 The results of FBS effect on DNA fragmentation in this study are consistent with this report. Bcl-2 may
Osteoarthritis and Cartilage Vol. 8 No. 1 play an important role in the maintenance of articular chondrocyte survival. It has not been fully investigated in this study that the detailed intracellular mechanisms of the induction of the DNA fragmentation by PGE2 and whether PGE2 may inactivate Bcl-2 in articular chondrocytes through cAMPPKA second messenger system. The results of the present study, however, provide the first report that PGE2 induces apoptosis in articular chondrocyte through intracellular accumulation of cAMP and suggest that the cAMP-PKA second messenger system plays a major role in regulating chondrocyte cell metabolism including apoptosis. Efforts are underway to determine if the mechanism of PGE2induced chondrocyte apoptosis is also operational in vivo.
Acknowledgments We are grateful to thank Professor S. Nakamura for critical reading of the manuscript and helpful discussions.
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-2 in human colorectal cancer. Cancer Res 1995;55:3785–9. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493–501. Planchon P, Veber N, Magnien V, Israel L, Starzec AB. Alteration of prostaglandin E receptors in advanced breast tumour cell lines. Mol Cell Endocrinol 1995;111:219–23. Yamamoto M, Yasuda M, Shiokawa S, Nobunaga M. Intracellular signal transduction in proliferation of synovial cells. Clin Rheumatol 1992;11:92–6. Ackerman RC, Murdoch WJ. Prostaglandin-induced apoptosis of ovarian surface epithelial cells. Prostaglandins 1993;45:475–85. Mastino A, Piacentini M, Grelli S, Favalli C, Autuori F, Tentori L, et al. Induction of apoptosis in thymocytes by prostaglandin E2 in vivo. Dev Immunol 1992;2:263–71. Northington FK, Oglesby TD, Ishikawa Y, Wuthier RE, et al. Localization of prostaglandin synthetase in chicken epiphyseal cartilage. Calcif Tissue int 1978;26:227–36. Kemick ML, Chin JE, Wuthier RE. Role of prostaglandins in differentiation of growth plate chondrocytes. Adv Prostaglandin Thromboxane Leukot Res 1989;19:423–6. Saura R, Uno K, Satsuma S, Kurz EU, Scudamore RA, Cooke TD. Mechanisms of cartilage degradation in inflammatory arthritis: interaction between chondrocytes and immunoglobulin G. J Rheumatol 1993;20:336–43. Kuettner KE, Pauli BU, Gall G, Memoli VA, Schenk RK. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology. J Cell Biol 1982;93:743–50. Kuettner KE, Memoli VA, Pauli BU, Wrobel NC, Thonar EJ, Daniel JC. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype. J Cell Biol 1982;93:751–7. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131–8. Blanco FJ, Ochs RL, Schwarz H, Lotz M. Chondrocyte apoptosis induced by nitric oxide. Am J Pathol 1995;146:75–85. Hashimoto S, Takahashi K, Amiel D, Coutts RD, Lotz M. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis. Arthritis Rheum 1998;41:1266–74. Pelletier JP, Jovanovic D, Fernandes JC, Manning P, Connor JR, Currie MG, et al. Reduced progression of
42. 43.
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experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum 1988;41:1275–86. Blanco FJ, Lotz M. IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. Exp Cell Res 1995;218:319–25. Morisset S, Patry C, Lora M, de Brum Fernandes AJ. Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17 beta-estradiol. J Rheumatol 1998;25:1146–53. Galera P, Vivien D, Pronost S, Bonaventure J, Redini F, Loyau G, et al. Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing. J Cell Physiol 1992;153:596–606. Bandara G, Lin CW, Georgescu HI, Evans CH. The synovial activation of chondrocytes: evidence for complex cytokine interactions involving a possible novel factor. Biochim Biophys Acta 1992;1134:309– 18. de Brum Fernandes AJ, Morisset S, Bkaily G, Patry C. Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture. Br J Pharmacol 1996;118:1597–1604. Ushikubi F, Aiba Y, Nakamura K, Namba T, Hirata M, Mazda O, et al. Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis. J Exp Med 1993;178:1825–30. Kizaki H, Suzuki K, Tadakuma T, Ishimura Y. Adenosine receptor-mediated accumulation of cyclic AMP-induced T-lymphocyte death through internucleosomal DNA cleavage. J Biol Chem 1990;265:5280–4. Waring P, Khan T, Sjaarda A. Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J Biol Chem 1997;272:17929–36. Brown DM, Phipps RP. Bcl-2 expression inhibits prostaglandin E2-mediated apoptosis in B cell lymphomas. J Immunol 1996;157:1359–70. Srivastava RK, Srivastava AR, Korsmeyer SJ, Nesterova M, Cho Chung YS, Longo DL. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998;18:3509–17. Feng L, Precht P, Balakir R, Horton WE Jr. Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment. J Cell Biochem 1998;71:302–9.
1063–4584/00/010017+08 $35.00/0
Induction of apoptosis in bovine articular chondrocyte by prostaglandin E2 through cAMP-dependent pathway M. Miwa*, R. Saura†, S. Hirata†, Y. Hayashi*, K. Mizuno† and H. Itoh*
*First Department of Pathology, Kobe University School of Medicine, †Department of Orthopaedic Surgery, Kobe University School of Medicine, Japan Summary Objective: Regulation of important biological processes such as proliferation and differentiation of articular chondrocytes is known to be mediated by prostaglandin E2 (PGE2) in both normal and pathological states. Articular chondrocytes also undergo apoptosis, a biological phenomenon implicated in many physiological processes. Whether or not PGE2 induces apoptosis in articular chondrocytes, however, is not known. Design: Bovine articular chondrocytes were cultured with or without PGE2 for 24 h and amounts of fragmented DNA, which is a distinct characteristic of apoptosis, were measured by enzyme-linked immunosorbent assay. Also effect of cyclic AMP (cAMP), which is one of the intracellular downstream mediator of PGE2, on chondrocyte apoptosis was investigated. Results: Administration of exogenous PGE2 on bovine articular cartilage grown as a monolayer culture resulted in the induction of DNA fragmentation. This DNA fragmentation was accompanied with a marked dose-dependent increase in intracellular cAMP. Also cultured cells were treated with cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase, and the incidence of apoptosis in the chondrocytes was determined. As well as PGE2, dibutyryl-cAMP and forskolin stimulated chondrocyte DNA fragmentation. Conclusions: It is the first report that PGE2 can induce articular chondrocyte apoptosis in vitro. It is also suggest that apoptosis of chondrocytes by PGE2 is linked with cAMP-dependent pathway. © 2000 OsteoArthritis Research Society International Key words: Bovine articular chondrocytes, Prostaglandin E2, Apoptosis, Cyclic AMP.
of animal models, which may be correlated with the increased risk of degenerative joint disease seen in elder people.9 These observations suggest that apoptosis of chondrocytes is critical for endochondral ossification during skeletal growth and also in the development of the degenerative change in articular cartilage during inflammation. However, the mechanism that induces articular chondrocyte apoptosis has, to date, not been fully understood. Prostaglandins (PGs) are important regulators of cellular function in a variety of tissues, including bone and cartilage. Numerous reports have suggested that PGs play a significant role in both the normal cartilage metabolism and the pathogenesis of joint disorders. For instance, the physeal cartilage responds to mechanical forces through modulation of PG synthesis.10 PGE2 was also reported to be the most potent cytokine, causing significant effects in both DNA synthesis and sulfate incorporation into the growth plate chondrocytes.11 This observation indicates that PGs may have an important role in chondrocyte metabolism in the areas of endochondral ossification integral to growth plate development and fracture repair of long bones. PGE2 is detectable at a high level in the fluid of knee joints in OA12,13 and rheumatoid arthritis (RA).14,15 PGs including PGE2 are synthesized from eicosatetraenoic acids in the presence of cyclooxygenase (COX). The expression of the inducible COX isoform, COX-2, but not constitutive form, COX-1, was found to be elevated in a disease-related pattern in the synovial tissue from patients with RA in comparison with OA.16 It is also reported that
Introduction Cartilage is one of the tissues with the highest susceptibility to age-related pathology. Osteoarthritis (OA) represents not only the most frequent muscloskeletal disorder but also the most common disease in the aging population.1 Very remarkable changes that occur with aging in the human articular cartilage include both profound loss in tissue cellularity and deleterious change of the extracellular matrix.2,3 At advanced stages of OA, the number of chondrocytes per unit volume diminishes, leading to the failure in the maintenance of the cartilage matrix. This corresponds to an age-related decline in responsiveness to growth factors in human articular chondrocytes which could be due to a decreased rate of cell division or an increased rate of cell death.4,5 Apoptosis has been involved in a wide variety of physiological regulatory processes and the pathology of various diseases. In terms of the cartilaginous tissue, apoptosis plays an important role in the differentiation of hypertrophic chondrocytes followed by the ossification of growth plate.6,7 Also, the number of apoptotic chondrocytes in the articular cartilage observed in OA tissues is much higher than that in the healthy ones.8 In addition, aging is reported to increase the ratio of apoptotic cells in articular cartilage Received 26 February 1999; accepted 9 June 1999. Address correspondence to: Ryuichi Saura, Department of Orthopaedic Surgery, Kobe University School of Medicine, 7-5-1 Kusunoki-cho Chuo-ku Kobe Hyogo Japan 650-0017.
17
18 cartilage specimens from OA-affected patients spontaneously released PGE2 at levels higher than in cytokinetreated normal cartilage due to upregulation of COX-2.17 These results suggested that PGE2 might exacerbate joint inflammation and be involved in the disease process of degenerative arthritis. PGE2 plays a key role in chondrocyte metabolism and increases concentration of cyclic adenosine monophosphate (cAMP) in these cells. Cyclic AMP generation is linked to the stimulation of proteoglycan synthesis in rabbit articular chondrocytes.18 There is considerable evidence that PGs mediate many of the effects of polypeptides, steroid hormones, growth factors, cytokines and mechanical forces on articular chondrocytes.19–22 For PGE2, however, the net effects on articular chondrocyte, which play a central role in the physiology and pathology of the articular cartilage, are less clear.23,24 Recently, the administration of nonsteroidal antiinflammatory drugs (NSAIDs) is reported to reduce the risk of fatal colon cancer.25,26 Though it is still elusive why NSAIDs can prevent the carcinogenesis of colon, the following observations could support these epidemiological reports. Enhanced expression of COX-2 polypeptides in human colon cancer tissues were detected using immunohistochemistry, whereas COX-1 expression was weak in both normal and cancerous specimens.27 It is also shown that overexpression of COX-2 leads to phenotypic changes involving the inhibition of apoptosis in intestinal epithelial cells that could enhance their tumorigenic potential.28 Thus, COX-2 may be strongly related to the cell proliferation including colon carcinogenesis. On the other hand, PGE2 is reported to be an antiproliferative molecule and one of the inducers of apoptotic change in various types of cells. For instance, PGE2 induces cAMP accumulation and inhibits the growth of the most differentiated breast cancer cells due to loss and probably dysfunction of PGE2 receptors.29 PGE2 also suppressed RA synovial cell proliferation through intracellular cAMP accumulation.30 In terms of the apoptosis by PGE2, it is involved in apoptotic alterations typical of ovarian surface epithelium associated with ovulatory ovine follicles31 and induction of thymocyte apoptosis for negative selection of T lymphocytes.32 During skeletal development, chondrocytes in the growth plate undergo a sequence of events that include proliferation, hypertrophy and cell death which have morphologically been demonstrated to be evidence of apoptosis.6,7 PGE2 has also been found to increase cartilaginous matrix surrounding the hypertrophic chondrocytes in the growth plate.33,34 These findings suggest that increased PGE2 could be closely related to the induction of apoptosis in growth plate chondrocytes. Thus, the effects of PGE2 and COX-2 in the regulation of apoptosis are cell types specific. However, there is no report as to whether or not PGE2 induces articular chondrocyte apoptosis. The present study was undertaken to address this issue.
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2 Biochemical Corp., Freehold. NJ) in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY) for 6 h at 37°C. Chondrocytes were harvested by filtration through gauze to remove tissue fragments, followed by centrifugation (450 g, 10 min). The isolated cells were washed three times and resuspended in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY) and antibiotics (20 units/ml penicillin and 100 ìg/ml streptomycin). They were then checked for viability by exclusion of trypan blue, and inoculated into culture plate at high density (1.2×105 cells/cm2) in a monolayer fashion. This high density primary cell culture can maintain the in vitro phenotype of bovine articular chondrocytes up to 14 days.36,37 Immediately after cells were attached to the bottom of culture plate, monolayer cultures were subjected to the experiments described below. All experiments were performed with chondrocytes in primary culture.
ASSAY FOR CELL PROLIFERATION
Chondrocytes plated into 96-well plates (1.2×105 cells/ cm2) were cultured in total volume of 200 ìl of DMEM containing 10% FBS and antibiotics with varying concentration of PGE2 (SIGMA, St. Louis, MO) for 72 h. Fifteen hours before terminating the proliferation assay of each incubation time, 37 KBq of [3H]thymidine ([3H]TdR; DuPont/NEN Research Products, Boston, MA) was added to each well. At the end of assay, cells were washed three times with distilled water. One hundred microliter of scintillation liquid was then added and total radioactivity of cell associated [3H]TdR was quantified by liquid scintillation counting (Packard Instrument Company, Meriden, CT).
QUANTIFICATION OF NITRITES
Chondrocytes were inoculated into 24-well culture plates at high density (1.2×105 cells/cm2) in DMEM containing 10% FBS and antibiotics. After 24 h in culture, the plating medium was removed and replaced with fresh serum-free medium containing varying concentrations of PGE2 and cultured for an additional 48 h. Generation of nitric oxide (NO) by cells was detected by its stable oxidative product, nitrite accumulation of which was measured in the culture supernatants by the Griess reaction as described previously.38 Briefly, at the end of incubation, sample aliquots were mixed with an equal volume of reaction regents composed of 0.1% N-1naphthylethylenediamine dihydrochloride and 1% sulfanilamide and in 5% phosphoric acid (Wako Pure Chemical Industries Ltd, Osaka, Japan). The absorbance at 550 nm was measured, and the nitrite concentration was determined using a curve calibrated on sodium nitrite as standards.
DNA FRAGMENTATION ASSAY
Materials and methods CHONDROCYTE CELL CULTURE
Articular cartilage was shaved from the bovine carpometatarsal joints and cut into thin sections. Cells were isolated from the tissue by a collagenase digestion procedure as described with some modifications.35 Briefly, the tissue was treated with 0.1% collagenase (Washington
Chondrocytes (1.2×105 cells/cm2) were inoculated into 24-well culture plates in DMEM containing 10% FBS and antibiotics. After 24 h culture, the plating medium was changed with serum free medium containing varying concentrations of either PGE2, dibutyryl-cAMP or forskolin (Sigma, St Louis, MO). For examining the effect of serum on DNA fragmentation induced by PGE2, the medium containing FBS was substituted for serum-free conditions.
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After an additional 24-h exposure to various stimuli, cells were washed with 0.05 M phosphate buffered saline (PBS; pH 7.4) and lysed in PBS containing 1% sodium dodecyl sulfate and 1% Tween 20 (Wako Pure Chemical Industries Ltd, Osaka, Japan) at 4°C for 30 min. The cell lysate were centrifuged at 200 g at 4°C for 10 min to remove cell debris and high molecular weight DNA. Fragmented DNA was measured by quantitation of cytosolic oligonucleosome-bound DNA with a Cell Death Detection enzyme-linked immunosorbent assay kit (Boehringer Mannheim GmbH, Germany) according to the manufacturer’s instructions. This assay is based on the quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. This allowed the specific determination of mono- and oligo-nucleosomes in the cytoplasmic fraction of cell lysate. INTRACELLULAR CAMP MEASUREMENT
Chondrocyte monolayer culture (1.2×105 cells/cm2) was also prepared in 24-well culture plates with DMEM containing 10% FBS and antibiotics. After 24 h, the medium in culture plates was changed with 1.0 ml of serum-free medium per well. The cultures were allowed to equilibrate for 1 h, then 10 ìl isobutylmethylxanthine (10 mM in 95% ethanol) was added and the cultures were incubated for 20 min at 37°C. PGE2 was then added at the indicated concentrations. After 20 min of incubation at 37°C, the reaction was stopped by aspirating the medium, adding 0.5 ml of 95% ethanol per well, and allowing cultures to air dry. The dried cell samples were reconstituted in 300 ìl sodium acetate buffer (pH 5.8) for enzyme immunoassay (EIA) using the commercial reagent for cAMP-EIA (Amersham, U.K.). STATISTICAL ANALYSIS
Statistical analysis was carried out on all data points with regard to control by an unpaired Student’s t-test. Each data point represented the mean of four or six separate samples with the corresponding standard error of the mean (SEM). P values under 0.05 were considered significant statistically.
Results EFFECT OF PGE2 ON PROLIFERATION AND DNA FRAGMENTATION OF BOVINE ARTICULAR CHONDROCYTES
To investigate whether PGE2 suppresses chondrocyte proliferation and induces DNA fragmentation in these cells, chondrocytes were treated with PGE2 at doses ranging from 10 −10–10 −6 M and for varying periods of time. Incubation with a varying concentration of PGE2 for 72 h demonstrated that [3H]TdR incorporation into the chondrocytes was decreased by PGE2 in dose dependent manner up to 10 −6 M. At 10 −8 M PGE2, [3H]TdR incorporation was reduced by 25% compared to the control and at 10 −7 M PGE2, incorporation of [3H]TdR was further reduced (60% of the control). However, further increases in PGE2 concentration did not produce any greater effect. Statistical significance of the results was observed up to 10 −8 M of PGE2 while the maximum effect was noted at 10 −7 M (Fig. 1). The concentration of PGE2 in this range is reported to
Fig. 1. Effect of PGE2 on [3H]TdR incorporation into bovine articular chondrocytes. Chondrocytes were plated into 96-well plates in total volume of 200 ìl of DMEM containing 10% FBS and antibiotics with various concentration of PGE2. Total radioactivity of cell associated [3H]TdR was quantified by liquid scintillation counting after 72 h incubation as described in Materials and methods. Data are mean±SEM of six replicates. *P< 0.05 (vs. control).
be detectable in the joint fluids of OA12,13 and RA patients.14,15 Intracellular DNA fragmentation, which is a hallmark of apoptosis, was induced in chondrocytes by PGE2 treatment and showed a statistically significant increase of apoptotic rate at doses over 10 −9 M (Fig. 2). A maximal 4-fold increase in DNA fragmentation was detected at a concentration of 10 −6 M of PGE2; this dose of PGE2 was also used over varying periods of time, and a timedependent rate of chondrocyte DNA fragmentation reached a maximum at 24 h (data not shown). In order to examine the effect of serum factors on PGE2 induced chondrocyte DNA fragmentation, articular chondrocytes were cultured with PGE2 in the presence of FBS. As shown Fig. 3, supplemented with 10% FBS, DNA fragmentation of articular chondrocyte was reduced by 50% compared to serum free conditions. However, further increase of FBS concentration did not reduce DNA fragmentation in chondrocytes. Though FBS markedly decreased DNA fragmentation, 10 −8 M of PGE2 induced small but statistically significant fragmentation of articular chondrocyte DNA. The assay for DNA fragmentation applied in the present study does not define the number of apoptotic cells. Therefore, the data on DNA fragmentation do not provide information on the percentage of cells that underwent apoptosis in a given culture. The percentage of cells which synthesize DNA and incorporate [3H]TdR is also not indicated. It is possible that PGE2 may affect distinct subpopulations of chondrocytes and suppress DNA synthesis in one subset and induce apoptosis in another. In order to detect which cells in culture as apoptotic and which is in S phase, further
20
Fig. 2. Effect of PGE2 on apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10% FBS and after 24 h cells were exposed to varying concentrations of PGE2 in identical fresh medium for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control).
experiments should be performed with an assay that allows localization and quantification of the number of cells that undergo apoptosis in response to PGE2.
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2
Fig. 3. Effect of serum on PGE2 induced apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10% FBS and after 24 h cells were exposed to 10 −8 M of PGE2 in fresh medium containing up to 50 % FBS for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control). Table I Effect of PGE2 on articular chondrocyte NO generation PGE2 (logM)
Concentration of nitrite (ìM)
P values
0.80±0.25 0.66±0.25 0.22±0.29 0.41±0.22
— 0.71 0.18 0.29
EFFECT OF PGE2 ON NO GENERATION IN BOVINE ARTICULAR CHONDROCYTES
It is reported that chondrocyte apoptosis induced by interleukin-1 (IL-1) is mediated by NO,39 thus the possibility of PGE2-induced chondrocyte DNA fragmentation mediated by NO synthesis was investigated. There was no increase of nitrite accumulation in the culture medium when articular chondrocytes were cultured in presence of PGE2 for 48 h. In articular chondrocytes, PGE2 failed to induce NO generation at a concentration of up to 10 −6 M (Table I). These results suggest that PGE2 induce DNA fragmentation of articular chondrocytes independently of NO generation.
No PGE2 −8 −7 −6
After 48-h incubation of articular chondrocytes with serum-free medium containing varying concentrations of PGE2, nitric oxide generation of the chondrocytes was detected in the culture supernatants by the Griess reaction stated in Materials and methods. Statistical analysis was carried out on all data points with regard to control by a Student’s t-test. Each data point represented the mean of four separate samples with the corresponding standard error of the mean (SEM). P values (vs. no PGE2) under 0.05 were considered to be statistically significant.
EFFECT OF ELEVATED INTRACELLULAR CAMP LEVEL ON DNA FRAGMENTATION IN BOVINE ARTICULAR CHONDROCYTE
Since PGE2 has been demonstrated to exert its biologic effects in various cells through the stimulation of cAMPmediated biochemical pathways, the ability of PGE2 to induce DNA fragmentation was investigated as a function of cAMP concentration in articular chondrocytes. As shown in Fig. 4, 20-min exposure of articular chondrocytes to PGE2 elevated intracellular cAMP concentration in a dosedependent fashion such that nearly 3.3-fold increase of cAMP level was observed at 10 −6 M of PGE2. A statistically
significant elevation of cAMP was observed at doses over 10 −8 M of PGE2 and a maximal increase of about 4-fold over control level was caused by 10 −6 M of PGE2. Therefore, in order to investigate the relationship between the elevation of intracellular cAMP and induction of the chondrocyte DNA fragmentation, articular chondrocytes were treated with the membrane-permeant cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase and examined for changes in their DNA fragmentation after 24 h using sandwich ELISA. Both dibutyryl-cAMP and forskolin induced chondrocyte DNA
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Fig. 4. Effects of PGE2 on intracellular cAMP accumulation in bovine chondrocytes. Chondrocytes plated in monolayer cultures were exposed to varying concentrations of PGE2 in serum free medium for an additional 20 minutes after 20 minutes incubations performed in the presence of 100 ìM 3-isobutyl-1-methylxanthine. Intracellular cAMP of chondrocyte was determined by EIA, as described in Materials and methods. Each value represents the mean±SEM of four replicates. **P< 0.01 (vs. control).
fragmentation in a dose-dependent fashion (Fig. 5a, b) such that dibutyryl-cAMP increased DNA fragmentation 8-fold over control, whereas forskolin increased it by 2.6fold. Significant effect of the dibutyryl-cAMP was detected over a concentration of 10 −8 M with a maximal effect at 10 −6 M. Also, over 10 −7 M of folskolin significantly induced DNA fragmentation of chondrocytes. In fact, we did not assay adenylate cyclase activity directly in cultured chondrocytes. However, forskolin that activates adenylate cyclase induced chondrocyte DNA fragmentation. Also, cAMP analogue, dibutyryl-cAMP, which is substitute for the intracellular cAMP, induced it. These results suggest that PGE2 could induce DNA fragmentation of articular chondrocyte through the elevation of intracellular cAMP level by activation of adenylate cyclase.
Discussion We show that increased DNA fragmentation in articular chondrocytes could be observed by adding exogenous PGE2 into the cells. Increased DNA fragmentation, which is a hallmark of apoptosis, could be also observed by increased cAMP level achieved through the use of dibutyryl-cAMP or forskolin. Thus, present study helps us to suggest that PGE2 induces articular chondrocyte apoptosis through the elevation of intracellular cAMP concentration followed by activation of protein kinase A (PKA) second messenger system. As to the aging alterations of articular cartilage, increasing number of the apoptotic chondrocytes may be correlated with the risk of degenerative joint disease seen in the elderly people.9 Recently, it was demonstrated that a high
21 number of apoptotic chondrocytes was histologically present at the pannus-cartilage junction in experimentally induced osteoarthritic cartilage and that the prevalence of apoptotic cells was significantly correlated with the levels of nitrite production in ex vivo explants and its OA grade.40 Also, the effectiveness of a selective inhibitor of inducible NO synthase (iNOS) was reported in attenuating the progression of experimental OA, which indicated the diminution of chondrocyte apoptosis due to inhibition of NO synthesis.41 Thus, articular chondrocyte apoptosis seems to be related to the pathology of degenerative joint diseases. In relation to the pathogenesis of OA, it is reported that NO generated from articular chondrocytes in the presence of inflammatory cytokines such as IL-1 induces articular chondrocyte apoptosis in vitro.39 IL-1 is one of the most potent cytokines, which can deteriorate the functional property of articular cartilage. Among its catabolic effects on cartilage are the stimulation of matrix metalloproteinase synthesis and inhibition of extracellular matrix production of articular chondrocytes. Inhibition of chondrocyte proliferation is another important effect of IL-1, which may prevent compensatory repair responses under conditions that are associated with inflammatory cartilage remodeling.42 Thus, chondrocyte replication, it is important not only during skeletal development but also cartilage repair to maintain cartilage cellularity in degenerative joint diseases. Previous studies have suggested that the antiproliferative effect of IL-1 on articular chondrocytes might be mediated by the induction of PGE2. IL-1 increases COX-2 mRNA expression in articular chondrocytes, which lead to generate much amount of PGE2.43 In order to address the potential interactions between PGE2 and NO, we examined whether PGE2 is involved with the induction of NO. In the present study, PGE2 alone failed to stimulate NO synthesis of articular chondrocytes as previously reported,42 which indicate that PGE2-induced DNA fragmentation of articular chondrocyte was not mediated by NO but PGE2 itself. Supplementation with FBS was reported to induce a dose-dependent increase in [3H]TdR incorporation into the chondrocytes. The present study showed that exogenously added PGE2 inhibited chondrocyte proliferation in the presence of 10% FBS and induced small but statistically significant DNA fragmentation of chondrocyte while supplementation with FBS markedly decreased it. FBS may include several chondrocyte growth factors such as transforming growth factor â (TGFâ). Some serum factors may protect cultured cells from apoptosis. Previous reports suggested that TGFâ increased PGE2 synthesis by articular chondrocytes and induced cell proliferation.44,45 This would be inconsistent with the present results. It is supposed that PGE2, of which levels as induced by TGFâ are relatively low, do not interfere with chondrocyte proliferation, whereas at high levels PGE2 as seen in response to IL-1 are growth inhibitory. Levels of PGE2 applied in this study seem to be higher than that induced by TGFâ. However, PGE2 does indeed chondrocyte DNA fragmentation in the presence of up to 50% FBS. The present studies were performed with primary chondrocytes in serum starved condition to avoid antagonistic effects of PGE2 and other serum factors including TGFâ in the regulation of chondrocyte proliferation. The actions of PGE2 are mediated by four distinct classes of PGE2 E-prostanoid (EP) receptors (EP1 through EP4). Increased cAMP generation by PGE2 may be due to EP2 or EP4 receptor activation. Though it is still unclear which receptors are expressed on the surface of articular
22
M. Miwa et al.: Articular chondrocyte apoptosis by PGE2
Fig. 5. Effect of dibutyryl-cAMP and forskolin on apoptosis in bovine articular chondrocytes. Chondrocytes were plated in monolayer cultures in DMEM plus 10 % FBS, and after 24 h cells were exposed to varying concentrations of (a) dibutyryl-cAMP or (b) forskolin in identical fresh medium for an additional 24 h. Apoptosis was determined by sandwich ELISA, as described in Materials and methods. Data are mean±SEM of four replicates. *P< 0.05; **P< 0.01 (vs. control).
chondrocyte, PGE2 induced chondrocyte apoptosis is supposed to be mediated by EP2 or EP4 receptor activation46 because the elevation of intracellular cAMP concentration was observed in articular chondrocytes as shown in Fig. 4. The further investigation for detecting the type of EP receptor on articular chondrocyte should be required in order to elucidate the mechanism of PGE2-induced chondrocyte apoptosis in detail. It has been suggested that the DNA cleavage induced by cAMP in thymocytes and peripheral lymphocytes may depend on either the induction of the new endonuclease gene products or the activation of endonuclease activities24,47 such a mechanism can be suggested for PGE2induced apoptosis of chondrocytes. Another possibility is that cAMP may inhibit a physiologically functioning mechanism by which chromatin is protected from DNA fragmentation when cell death signals are turned on.48 Phosphorylation of cellular substrates by cAMP dependent protein kinase A (PKA) is also reported to induce apoptosis in other cells. Apoptosis induced in thymocytes by gliotoxin, which is a fungal toxin, is calcium-independent and unaffected by protein synthesis inhibitors. It has shown that gliotoxin results in rapid phosphorylation of histone H3 in thymocytes.49 Cyclic AMP levels and activity of PKA were raised in cells treated with gliotoxin. Cells treated with gliotoxin showed an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation. Forskolin is reported to show some increase in phosphorylation of H3 after 8-hour treatment with this agent. Forskolin also induces apoptosis in thymocytes but over a
longer time course than gliotoxin. In both cases levels of apoptosis correlated with degree of H3 phosphorylation. It is presumed that PGE2 induced chondrocyte apoptosis may be mediated by phosphorylation of H3 because forskolin showed some increase in DNA fragmentation of chondrocytes. The proto-oncogene bcl-2 is considered important in determining whether cells enter an apoptotic pathway or survive. Increased expression of Bcl-2 in immature B-cell lymphomas is reported to completely inhibit PGE2- and cAMP-mediated DNA fragmentation and nuclear condensation.50 Also, cell cycle analysis demonstrated that PGE2 increased the percentage of cells in G1, and analysis of synchronized populations revealed that PGE2 acts at all phases of the cell cycle to delay normal progression in immature B cell lymphomas. The microtubule-damaging drugs, paclitaxel and vincristine induced activation of PKA, which induced Bcl-2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells.51 These microtubule-damaging drugs caused growth arrest in G2-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation by PKA mediated inactivation of Bcl2 and apoptosis without the involvement of p53. In relation to regulation of chondrocyte apoptosis in articular cartilage, the importance of Bcl-2 was also reported. Serum withdrawal and retinoic acid treatment induced apoptosis of primary human articular chondrocytes, when the expression of Bcl-2 was downregulated by both agents.52 The results of FBS effect on DNA fragmentation in this study are consistent with this report. Bcl-2 may
Osteoarthritis and Cartilage Vol. 8 No. 1 play an important role in the maintenance of articular chondrocyte survival. It has not been fully investigated in this study that the detailed intracellular mechanisms of the induction of the DNA fragmentation by PGE2 and whether PGE2 may inactivate Bcl-2 in articular chondrocytes through cAMPPKA second messenger system. The results of the present study, however, provide the first report that PGE2 induces apoptosis in articular chondrocyte through intracellular accumulation of cAMP and suggest that the cAMP-PKA second messenger system plays a major role in regulating chondrocyte cell metabolism including apoptosis. Efforts are underway to determine if the mechanism of PGE2induced chondrocyte apoptosis is also operational in vivo.
Acknowledgments We are grateful to thank Professor S. Nakamura for critical reading of the manuscript and helpful discussions.
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