The authors have no conflict of interest.
PGE2 Signal Through EP2 Promotes the Growth of Articular Chondrocytes†
Article first published online: 29 NOV 2004
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 3, pages 377–389, March 2005
How to Cite
Aoyama, T., Liang, B., Okamoto, T., Matsusaki, T., Nishijo, K., Ishibe, T., Yasura, K., Nagayama, S., Nakayama, T., Nakamura, T. and Toguchida, J. (2005), PGE2 Signal Through EP2 Promotes the Growth of Articular Chondrocytes. J Bone Miner Res, 20: 377–389. doi: 10.1359/JBMR.041122
- Issue published online: 4 DEC 2009
- Article first published online: 29 NOV 2004
- Manuscript Accepted: 15 OCT 2004
- Manuscript Revised: 13 SEP 2004
- Manuscript Received: 6 APR 2004
- prostaglandin E2;
- articular cartilage;
EP2 was identified as the major PGE2 receptor expressed in articular cartilage. An EP2 agonist increased intracellular cAMP in articular chondrocytes, stimulating DNA synthesis in both monolayer and 3D cultures. Hence, the EP2 agonist may be a potent therapeutic agent for degenerative cartilage diseases.
Introduction: Prostaglandin E2 (PGE2) exhibits pleiotropic effects in various types of tissue through four types of receptors, EP1-4. We examined the expression of EPs and effects of agonists for each EP on articular chondrocytes.
Materials and Methods: The expression of each EP in articular chondrocytes was examined by immunohistochemistry and RT-PCR. A chondrocyte cell line, MMA2, was established from articular cartilage of p53−/− mice and used to analyze the effects of agonists for each EP. A search for molecules downstream of the PGE2 signal through the EP2 agonist was made by cDNA microarray analysis. The growth-promoting effect of the EP2 agonist on chondrocytes surrounded by cartilage matrix was examined in an organ culture of rat femora.
Results and Conclusion: EP2 was identified as the major EP expressed in articular cartilage. Treatment of MMA2 cells with specific agonists for each EP showed that only the EP2 agonist significantly increased intracellular cAMP levels in a dose-dependent manner. Gene expression profiling of MMA2 revealed a set of genes upregulated by the EP2 agonist, including several growth-promoting and apoptosis-protecting genes such as the cyclin D1, fibronectin, integrin α5, AP2α, and 14-3-3γ genes. The upregulation of these genes by the EP2 agonist was confirmed in human articular chondrocytes by quantitative mRNA analysis. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of 5-bromo-2-deoxyuracil (BrdU), and the organ culture of rat femora showed an increase of proliferating cell nuclear antigen (PCNA) staining in articular chondrocytes surrounded by cartilage matrix, suggesting growth-promoting effects of the PGE2 signal through EP2 in articular cartilage. These results suggested that the PGE2 signal through EP2 enhances the growth of articular chondrocytes, and the EP2 agonist is a candidate for a new therapeutic compound for the treatment of degenerative cartilage diseases.
PROSTAGLANDINS (PGs) ARE a group of lipid mediators produced from arachidonic acid in various tissues under both physiological and pathological conditions. The role of PGs in the metabolism of articular cartilage is still a matter of debate. Some reports indicated that PGs participated in the destruction of articular cartilage by degrading cartilage extracellular matrix,(1,2) whereas others showed that PGs promoted chondrogenesis and terminal differentiation.(3–5) The confusion may stem from the complexity of the family of PGs and also from the complexity of receptors. PGE2 is a major PG, the receptors of which are pharmacologically subdivided into four subtypes; EP1, EP2, EP3, and EP4.(6) Each receptor subtype has a distinct pharmacological signature based on its pharmacophore and PGE2-evoked signal transduction.(6) EP1 couples to Gq protein and increases the intracellular calcium concentration. EP2 and EP4 couple to Gs protein and increase the intracellular cAMP concentration. Several EP3 splicing variants have different signaling pathways, involving an increase or decrease in intracellular cAMP and an increase in the intracellular calcium concentration.(7,8) PGE2 is known to have a variety of actions in the growth plate cartilage,(5,9–11) and the expression of EPs in cartilage tissues has been shown in several reports. EP1 and EP2 were expressed in rat resting zone chondrocytes of the growth plate and a costochondral cell line.(5,9–11) An EP1 agonist increased alkaline phosphatase activity in resting rat chondrocytes,(5) decreased [3H]thymidine incorporation, and increased proteoglycan synthesis and alkaline phosphatase activity in a rat costochondral cartilage cell line.(10) These results suggested that the PGE2 signal through EP1 induces the maturation and differentiation of growth plate chondrocytes. As for EP2, an EP2 agonist enhanced the expression of the Col2a1 gene in growth plate-derived chondrocytes only when an EP4 agonist was simultaneously administered.(11) It should be noted that most of these experiments in vitro were performed using chondrocytes derived from the growth plate, not chondrocytes from articular cartilage. There is, however, plenty of evidence of biological differences between chondrocytes from the growth plate and from articular cartilage,(12,13) and it is rational to use chondrocytes from articular cartilage for dissecting the role of the PGE2 signal in articular cartilage.
The p53 tumor suppressor gene is one of most important genes in human oncogenesis, and approximately one-half of all human tumors carry some type of mutation in this gene.(14) In normal cells, the p53 protein has roles in regulating the cell cycle, assembling the machinery to repair DNA damage, and inducing apoptosis.(15) Cells isolated from p53−/− mice are prone to be spontaneously immortalized without losing their differentiated phenotype.(16–19) We previously established a cell line from calvariae of p53−/− mice (MMC2), which showed the phenotypes of matured osteoblasts,(20) and three cell lines from the growth plate of ribs (MMR32, MMR14, and MMR17), which showed the phenotypes of matured growth plate chondrocytes.(21) Based on these previous successes, we attempted to establish cell lines from articular cartilage of p53−/− mice and analyze the effect of the PGE2 signal on articular chondrocytes.
MATERIALS AND METHODS
Tissue samples, cell culture, and reagents
Human articular cartilage tissues were obtained from knee joints of four individuals who underwent a prosthetic replacement of the knee joint caused by malignant bone tumors (KS574, 69 years old; KS730, 10 years old; KS834, 39 years old; KS847, 6 years old). Informed consent was obtained from the patients or their parents, and tissue materials were approved for analysis by the Ethics Committee of the Faculty of Medicine, Kyoto University. Cartilage tissues were subsequently cut with scissors into small pieces, dispersed by 0.1% collagenase (Nitta Gelatin, Osaka, Japan), and cultured in DMEM/Ham's F12 (1:1; Life Technologies, Rockville, MD, USA) supplemented with 10% FBS (Hyclone, Road Logan, UT, USA) and antibiotics at 37°C in a humidified atmosphere of 5% CO2/95% air. Subsequent experiments were done in the same medium under the same conditions. Chondrocytes were isolated from epiphyses of femora and tibias of 4-week-old p53−/− mice using the same method. When the cells were 80–90% confluent, they were seeded at a density of 103/100-mm culture dish to isolate single cell clones. Specific agonists for each EP were provided by Ono Pharmaceutical Co., Osaka, Japan: ONO-DI-004 for EP1, ONO-AE-259-01 for EP2, ONO-AE-248 for EP3, and ONO-AE1-329 for EP4. In the culture experiments using these agonists, indomethacin (5 μM; Sigma Chemical, St Louis, MO, USA) was simultaneously added to the culture medium to inhibit the endogenous PGE2 production.
Cartilage samples were fixed overnight at 4°C in a periodate-lysine-paraformaldehyde solution and embedded in paraffin. Longitudinal serial sections were cut at 6 μm, and the immunostaining was performed using the DAKO Envision plus System (Dako, Glostrup, Denmark). Deparaffinized sections were treated with hydrogen peroxide for 10 minutes to reduce endogenous peroxidase activity and washed in Tris-buffered saline. Sections were treated with anti-EP1, EP2, EP3, or EP4 antibody (Cayman Chemical Co., Ann Arbor, MI, USA) at a dilution of 1:500 and further incubated overnight at 4°C. Preimmune rabbit IgG (Dako) was used as a negative control. Stained slides were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted in Entellan (Merck, Darmstadt, Germany).
Whole cell lysates were prepared from each primary chondrocyte, separated by SDS-PAGE using 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was treated with specific antibodies for EP1, EP2, EP3, or EP4 at a dilution of 1:1000, then with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) as a secondary antibody (Dako), and finally visualized using an ECL plus kit (Amersham Biosciences, Buckinghamshire, UK).
In situ hybridization
The catalyzed signal amplification system (GenPoint; Dako) was used for in situ hybridization. Knee joints of newborn mice were dissected, embedded in optimum cutting temperature (OCT) compound (Sakura Finetechnical Co., Tokyo, Japan), and cryosectioned 4 μm thick. The specimens were fixed with 4% paraformaldehyde/PBS for 15 minutes at room temperature, air dried, and digested with 20 μg/ml of proteinase K (Dako) for 10 minutes at room temperature. Each slide was covered with 100 μl of hybridization buffer (Dako) containing 5′-FITC labeled oligonucleotide probes (1 μg/ml) and incubated in a moist chamber at 37°C for 6 h. The probes used are EP1-antisense (5′-ACAGTACCCTGGCACCTGGTGTTTTATTAGCCTTGG-3′), EP1-sense (5′-CCAAGGCTAATAAAACACCAGGTGCCAGGGTACTGT-3′), EP2-antisense (5′-AAAGATTGTGAAAGGCAAGGAGCATATGGCGAAGGT-3′), EP2-sense (5′-ACCTTCGCCATATGCTCCTTGCCTTTCACAATCTTT-3′), EP3-antisense (5′-CAGCAGATAAACCCAGGGATCCAAGATCTGGTTCAG-3′), EP3-sense (5′-CTGAACCAGATCTTGGATCCCTGGGTTTATCTGCTG-3′), EP4-antisense (5′-GGAGGAGTCTGAGGTCTCGGAAATTCGCAAAGTTCT-3′), and EP4-sense (5′-AGAACTTTGCGAATTTCCGAGACCTCAGACTCCTCC-3′). After hybridization, the slides were washed twice with stringent wash solution (Dako) at 45°C for 20 minutes and incubated with rabbit anti-FITC/HRP antibody (Dako) for 60 minutes. For the catalyzed signal amplification, the slides were further incubated with biotinyl tyramide solution (Dako) and then with streptavidin/HRP (Dako). The signals were visualized by adding DAB chromogen solution (Dako).
High density suspension culture
The cell suspension was transferred into a 15-ml polypropylene centrifuge tube (2325-015; IWAKI, Tokyo, Japan) at a density of 1 × 105/ml/tube and centrifuged at 1500 rpm for 5 minutes. The resulting cell pellet was incubated with the same medium containing 50 μg/ml of ascorbate under the same conditions as for the monolayer culture. The culture medium was not changed for the first 6 days and thereafter was changed every other day. The cell pellet was centrifuged at 1500 rpm for 5 minutes after each change of medium. The 21-day cultured cell pellet was fixed with 20% formaldehyde and embedded on paraffin. Sections 6 μm thick were cut, stained with H&E or 0.1% alcian blue (Sigma Chemical) in 0.1N HCl, counterstained with nuclear fast red, and examined by light microscope.
Total RNA was extracted from cartilage tissues or cultured cells using Trizol reagent (Life Technologies) following the manufacturer's protocol. All RT reactions were performed using 1 μg of total RNA with a Super Script First Strand Synthesis System for RT-PCR kit (Life Technologies) according to the manufacturer's directions. PCR amplification was carried out using 1 μl of RT product in a final volume of 25 μl containing 20 pmol each of the sense and antisense primers, 25 mM MgCl2, 0.2 mM of each dNTP, and one unit of rTaq polymerase (Toyobo, Osaka, Japan). All reactions were performed using GeneAmp 9700 (PE Applied Biosystem, Foster City, CA, USA). Sequences of primers were described in Table 1. PCR products were separated in a 1.5% agarose gel and stained with ethidium bromide. PCR experiments for each gene were performed at least three times to confirm the consistency of results. Culture experiments were done at least twice in all cell lines, and the RNA extracted from cells in each experiment was analyzed separately.
Measurement of intracellular cAMP concentration
Cells were seeded at a density of 1 × 104 cells/well on 24-well culture dishes (Corning, Corning, NY, USA). After the cells had attached to the plate, each of the EP agonists was added, and the plate was incubated for 24 h. The cells were homogenized with 0.1 M Tris/HCl buffer, pH 7.2, and centrifuged at 2000g for 15 minutes. The concentration of intracellular cAMP was measured with a cAMP assay kit as recommended by the manufacturer (Cayman Chemical Co.).
Gene expression profiling
Gene expression profiling before and after the treatment with the EP2 agonist was compared using a custom made cDNA microarray, which consisted of 964 mouse genes of a standard chip (IntelliGene Mouse CHIP Ver. 1; TaKaRa Bio, Shiga, Japan) and an additional 78 bone- and cartilage-related mouse genes (the list of genes is available on request). MMA2 cells were cultured with or without the EP2 agonist (1 μM) for 72 h. Total RNA (20 μg) extracted from cells treated with or without the agonist were labeled by Molony-murine leukemia virus (M-MLV) RT (400U; TaKaRa Bio) with fluorescence-conjugated Cy3- or Cy5-dUTP (Amersham Biosciences), respectively. Equal amounts of Cy3- and Cy5-labeled cDNA was mixed in the reaction buffer (6× SSC with 0.2% SDS, 5× Denhardt's solution, and 0.1 mg/ml of denatured salmon sperm DNA) and hybridized to the cDNA chip at 65°C overnight. The chip was washed with 2× SSC with 0.2% SDS twice at 55°C for 5 minutes and once at 65°C for 5 minutes. Finally, the chip was washed with 0.05× SSC at room temperature for 1 minute. The hybridized signal was visualized and quantified using an Affymetrix 418 Array Scanner (Affymetrix, Santa Clara, CA, USA) and ImaGene software (BioDiscovery, Marina Del Rey, CA, USA).
Quantitative RT-PCR analysis
The levels of mRNA expression of genes identified by the gene expression profiling (osteopontin, cyclin D1, integrinα5, fibronectin, AP2α, and 14-3-3γ) were evaluated by SYBR Green real time PCR with the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). cDNA was synthesized from total RNA (1 μg) extracted from MMA2 or primary cultured chondrocytes, which were treated with or without the EP2 agonist (1 μM) for 72 h. One microgram of cDNA was added to a reaction mixture (25 μl) containing 12.5 μl of 2× SYBR Green mastermix and primers. After a preincubation at 95°C for 10 minutes, the cDNA was amplified for 40 cycles of 15 s at 95°C and 1 minute at an annealing-extension temperature, which was optimized for each gene. Information on primer sequences and PCR conditions for each gene is available on request. The intensity of amplified fragments of human β-actin was used as an internal control. All reactions were run in triplicate, and the mean value was used to calculate the ratio of target gene/β-actin expression in each sample. Using the ratio in untreated sample as a standard (1.0), the relative ratio of treated sample was presented as the relative expression level of the target gene.
Cell proliferation assay
Activity for DNA synthesis was analyzed by 5-bromo-2-deoxyuracil (BrdU) assay. Primary chondrocytes were seeded at a density of 2 × 103 cells/well on 96-well culture dishes (Corning) and incubated with each EP agonist (1 μM) for 24 h. Cells were labeled with BrdU for 8 h. Labeled nuclei were detected using a BrdU Detection Kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. Experiments were performed in triplicate in each of four primary samples.
Effects of each agonist on the cell growth were also analyzed by directly counting numbers of cells (MMA2 or primary chondrocytes). Cells were seeded at a density of 5 × 104 cells/well on 6-well culture dishes, and the number of cells was counted after a 7-day culture with each EP agonist.
Organ culture, sample preparation, and proliferating cell nuclear antigen staining
Animal studies were approved by the institutional animal research committee and performed according to the Guidelines for Animal Experiments of Kyoto University. Entire femora were aseptically isolated from 12-week-old Wister rats under deep anesthesia, and connective tissues attached to bones were completely removed. One of the femora taken from each rat was incubated in a 100-mm dish with DMEM/Ham's F12 (1:1) supplemented with 20% FBS, and the other was treated with the EP2 agonist at concentrations of 10−5, 10−6, and 10−7 M (n = 3 for each concentration) in the same medium. The medium was changed every 48 h for 14 days, when the femora were fixed in 10% formalin solution at 4°C for 24 h, decalcified with 10% EDTA for 21 days, and embedded in paraffin. The femoral head was divided into two parts through the long axis of femoral head and neck, and 5-μm-thick sections were prepared along the central coronal surface. A monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) antibody (M0879; Dako) was used at a dilution of 1:200, and the sections were counterstained with hematoxylin. Under the microscope (×400), total and PCNA+ cells were counted by two individuals who were unaware of the contents of specimens. Three visual fields were randomly selected by each observer, and therefore each specimen was evaluated six times, and the labeling index was calculated as a mean of six values. The relative increase in PCNA+ cells was expressed as the ratio of the labeling index of EP2-treated sample versus that of control sample.
Results are expressed as the mean ± SE. Statistical analysis was performed with Student's t-test. A significant difference was accepted at the p < 0.05 level. Each experimental group was compared with its own control and prepared and analyzed simultaneously.
Expression of EPs in human articular cartilage
Immunohistochemical analysis revealed that chondrocytes in human articular cartilage expressed EP2 and EP3 with less intensity, whereas EP1 and EP4 were not detected in any cells (Fig. 1A). RT-PCR analyses of RNA extracted from human articular cartilage tissue and primary short-term cultured chondrocytes showed the expression of the EP2 and EP3 genes, although the latter was expressed at much lower levels (Fig. 1B). No signal for EP1 or EP4 was detected in either cartilage tissue or primary cultured cells (Fig. 1B). Western blot analyses using proteins extracted from primary short-term cultured chondrocytes showed signal for EP2 and EP3, but not EP1 or EP4 (Fig. 1C), which was consistent with the results of the RT-PCR analysis. These data indicated that EP2 was the main EP expressed in human articular chondrocytes, whereas EP3 was also expressed but with much less intensity.
Expression of EPs in epiphyseal cartilages of newborn mice
The expression of each EP in epiphyseal cartilage tissues was examined by in situ hybridization using distal femora and proximal tibias of newborn mice (Fig. 2). The EP2 gene was strongly and exclusively expressed in epiphyseal cartilage tissue, and the expression of EP3 was also observed in epiphyseal cartilage. mRNA signals for the EP1 and EP4 genes were observed mainly in bone tissues and faintly in epiphyseal cartilage.
Isolation of clonal cell lines from articular cartilage of p53−/− mice
Chondrocytes were isolated from articular cartilage tissues of distal femora of p53−/− mice, and a total of 11 clonal cell lines was established, among which three cell lines, designated MMA2, MMA4, and MMA5, showed vigorous growth and therefore were investigated further. All three cell lines showed stable growth in medium supplemented with 10% FBS, with a doubling time of 19.9, 19.5, and 24.9 h, respectively. No significant change in growth potential was shown after >60 population doublings during periods of >12 months (data not shown). During the growing phase, all of these cell lines showed a polygonal shape and adopted a cobblestone appearance after becoming confluent (Fig. 3A, represented by MMA2). None of these cell lines showed calcified nodules, even after prolonged culture. The ability of the three cell lines to produce a matrix was analyzed in a 3D culture. After 21 days of high density suspension culture without any growth factors, MMA2 formed a solid mass, which contained extracellular matrix positive for alcian blue staining (Fig. 3B). Neither MMA4 nor MMA5 produced a similar solid mass (data not shown).
mRNA expression of cartilage-related molecules
To analyze the properties of articular cartilage-derived cells, the expression of cartilage-related genes of MMA cell lines was analyzed by RT-PCR and compared with that of articular cartilage (Fig. 4). As for the extracellular matrix molecules (Fig. 4A), the expression of the Col2a1, Col11a1, and aggrecan genes was detected in all three cell lines, whereas only MMA2 expressed the Col9a1 gene. Expression of the alkaline phosphatase gene was detected only in MMA5, which was the only cell line negative for the chondromodulin-I gene, which encodes a cartilage-specific matrix protein with inhibitory activity for angiogenesis.(22) The expression of signal molecules, which have been shown to be involved in the metabolism of cartilage, was also examined (Fig. 4B). Expression of Sox9, Tgfb3, and Erg with its splicing variant form C1-1(12,13) was detected in all cell lines as well as articular cartilage. MMA2 shared the expression pattern of the Ihh and Pthrp genes with articular cartilage, being negative for the former and positive for the latter. From these data, the expression profiles of MMA2 seemed to be compatible with those of articular cartilage.
Expression of EP genes in chondrocyte cell lines and cartilage tissues
The expression of each EP was analyzed by RT-PCR in three cell lines along with mouse and rat articular cartilage (Fig. 5). Expression of the EP2 and EP3 genes, especially the γ form of EP3, was clearly detected in mouse articular cartilage, whereas the signals for the EP1 and EP4 genes were only faintly observed (Fig. 5A). Expression of EP2 was detected in all cell lines (Fig. 5A). MMA2 showed a similar expression pattern of EP genes as articular cartilage except for the expression of EP4, whereas MMA4 and MMA5 showed a different pattern, being positive for the EP1 gene and negative or faintly positive for the EP3 gene (Fig. 5A). As in human and mouse articular cartilage, the EP2 gene was the major EP gene expressed in rat articular cartilage, whereas the EP3 gene was also expressed, but with much less intensity (Fig. 5B).
Effect of EP agonists on intracellular cAMP concentration in MMA2
To investigate the functional significance of these EPs, the amount of intracellular cAMP was measured when MMA2 was treated with specific agonists for each EP (Fig. 6A). Signals through EP2 and EP4 are known to increase the intracellular cAMP concentration, whereas those through EP1 and EP3 are known to increase the intracellular calcium concentration.(23) Treatment with the EP2 agonist (1 μM) dramatically increased the intracellular cAMP concentration in MMA2. The EP4 agonist also evoked an increase, although to a lesser extent, the level being compatible with the expression of this receptor in MMA2. Neither the EP1 nor EP3 agonist had an effect on the concentration of intracellular cAMP. The effect of the EP2 agonist on intracellular cAMP was observed even at a lower concentration (10−7 M), and increased in a dose-dependent manner, suggesting functional activity of EP2 in MMA2 (Fig. 6B). None of the four types of EP agonists had an effect on the intracellular Ca2+ concentration (data not shown).
Identification of upregulated and downregulated genes on treatment with the EP2-specific agonist by cDNA microarray assay
The above data suggested that EP2 was expressed in articular cartilage, and MMA2 cells can be used as a representative for articular chondrocytes expressing the EP2 gene. Therefore, molecules downstream of the PGE2 signal through EP2 in MMA2 were investigated by gene expression profiling using a cDNA microarray. Gene expression profiles of MMA2 were compared before and after the treatment with EP2 agonist (1 μM), and genes whose expression increased >2-fold or decreased by less than one-half were identified as upregulated or downregulated, respectively. As a result, 22 upregulated genes and one downregulated gene were detected after the treatment with the EP2 agonist (Table 2). Among the 22 upregulated genes, 5 were ESTs and 17 were known genes, of which some have been shown to promote cell growth and/or protect cells from apoptosis, such as the cyclin D1, fibronectin, integrin α5, AP2α, and 14-3-3γ genes. The osteopontin gene was isolated as the sole downregulated gene.
Confirmation of the results of the cDNA microarray assay by quantitative RT-PCR in MMA2 cells and human primary chondrocytes
Regulation of the mRNA expression of these genes by the EP2 agonist was confirmed in MMA2 cells and human primary articular chondrocytes by real time quantitative PCR analysis (Fig. 7). As upregulated genes, we selected the genes for cyclin D1, fibronectin, integrin α5, AP2α, and 14-3-3γ because of their known functions. The expression of these genes in MMA2 was downregulated by the treatment with indomethacin (5 μM) and upregulated by the treatment with the EP2 agonist (1 μM; Figs. 7A-7E, ▴). On the other hand, the expression of the osteopontin gene in MMA2 was upregulated by the treatment with indomethacin and downregulated by the treatment with the EP2 agonist (Fig. 7F, ▴). These results were compatible with the data from the gene expression profiling of MMA2. The expression of the same set of genes was investigated in four primary chondrocytes (Figs. 7A-7F, •, ○, ▪, and □). The changes in expression with indomethacin and with the EP2 agonist in the human articular chondrocytes were similar, although not identical, to those MMA2.
Growth promoting effects of the EP2 agonist on articular chondrocytes in vitro
The function of several upregulated genes was related to cell growth, suggesting a growth-promoting effect of the EP2 agonist on articular chondrocytes. The cellular incorporation of BrdU in primary human articular chondrocytes decreased on treatment with indomethacin (5 μM) and increased on treatment with the EP2 agonist (1 μM; Fig. 8). Consistent with the data from the BrdU assay, the administration of indomethacin (5 μM) resulted in a decrease of cell number in MMA2 (Fig. 9A) and primary chondrocytes (Fig. 9B), whereas the EP2 agonist (1 μM), but not the other agonists, reduced the inhibitory effect of indomethacin and significantly increased the cell number in MMA2 (Fig. 9A) and primary chondrocytes (Fig. 9B). To investigate whether the EP2 agonist exerts growth-promoting effects on chondrocytes surrounded by cartilage matrix, in vitro organ culture experiments using rat femora were performed. As shown in Fig. 5, EP2 was the major EP expressed in rat articular cartilage. The effect of the EP2 agonist was examined based on the immunoreactivity of PCNA, which is a marker for proliferating cells. After a 14-day incubation with the EP2 agonist, no apparent morphological change was observed in articular cartilage. There seemed to be a slight increase in cellularity in the EP2 agonist-treated articular cartilage tissues, but no significant difference was found in cell numbers between treated and control groups (data not shown). The ratio of PCNA+ cells, however, was significantly increased by the treatment with the EP2 agonist in a dose-dependent manner (Fig. 10), suggesting that the EP2 agonist was able to exert growth-promoting effects on cells surrounded by cartilage matrix. We have no clear explanation for why there were no morphological changes on treatment with the EP2 agonist in articular cartilage, despite the increase in PCNA+ cells. The proliferation of chondrocytes in normal cartilage matrix may require a much longer period of time and not be well supported in organ culture systems.
The effect of PGE2 depends on the type of receptor, and sometimes completely opposite effects will be achieved by different EPs. For example, PGE2 signals through EP1 and EP3 induced the production of interleukin (IL)-6 in synovial tissues,(24,25) whereas those through EP2 and EP4 inhibited the IL-1-induced production of IL-6.(25) The relative expression of EP receptor subtypes determines the effects of PGE2, and therefore it is important to understand the expression pattern of EPs in each tissue. We found that human and mouse articular chondrocytes expressed EP2 and EP3, but not EP1 or EP4. Expression of EP2 has been shown in fetal bone tissues (long bone and calvaria) and growth plate cartilage,(5,11) and recent reports indicated that an EP2 agonist can stimulate bone formation, although the induction required a relatively high concentration of EP2.(26,27) As far as we know, this is the first report to show the expression of EPs in human articular cartilage tissues. The negative expression of EP1 and EP4 in articular chondrocytes is in contrast with the expression pattern of EPs in growth plate-derived chondrocytes, as shown in previous reports.(5,9–11) Considering that EP1 and EP4 are involved in the calcification process,(5,28) the loss of expression of these molecules in articular chondrocytes may be involved in the mechanism to avoid calcification under physiological conditions.
To investigate the effects of PGE2 signals through EP2 in articular chondrocytes more precisely using a stable cell line, we established MMA2 cells, and confirmed the results obtained from this cell line using primary cells. As for EP genes, expression of the EP4 gene was the only significant difference between MMA2 and articular cartilage. We have no explanation for this difference, which might be caused by the environment in vitro. Other phenotypes including the expression of the EP2 gene, however, were compatible with those of articular chondrocytes, and we used this cell line to isolate downstream molecules of the PGE2 signal through EP2. We have found that the EP2 agonist downregulated the mRNA expression of osteopontin. Osteopontin is a matrix protein with various functions. mRNA expression and protein production of osteopontin was increased in chondrocytes in osteoarthrotic joints,(29,30) and osteopontin-deficient mice did not develop the collagen-induced arthritis. These results suggested that the role of osteopontin in cartilage tissue is to destroy cartilage by activating the apoptotic pathway,(31) and therefore the downregulation of osteopontin expression by the EP2 signal may exert protective effects in articular cartilage. Although the functional role of most of the genes upregulated by the EP2 signal is unknown, the proposed function of several genes seems to be beneficial to articular chondrocytes by promoting cell growth and preventing apoptosis. Cyclin D1 regulates the progression of chondrocytes in G1 and promotes proliferation,(32) which may be relevant to the finding that the EP2 signal enhanced DNA synthesis and promoted cell cycle progression in chondrocytes. The expression of cyclin D1 is activated by transforming growth factor (TGF)β1 and parathyroid hormone-related peptide (PTHrP) mediated by ATF-2 and CREB.(33) Because we found that the EP2 signal increased intracellular cAMP concentration of chondrocytes, the upregulation of cyclin D1 may be a direct effect of EP signaling. AP2α is one of the transcription factors expressed in articular cartilage(34) and known to promote proliferation.(35) The function of fibronectin and its receptor integrin α5β1 in chondrocytes is controversial. Fibronectin fragments induce the degradation of matrix in cartilage through the activation of MMP and result in osteoarthritis.(36–39) The intact fibronectin, however, participates in the regulation of chondrocyte adhesion, spreading, and proliferation in association with integrin α5β1.(40) The binding to fibronectin through integrin α5 induces a survival signal in chondrocytes, protecting cells from apoptosis.(41,42) Therefore, the upregulation of fibronectin and integrin α5 by the EP2 signal may protect articular chondrocytes from apoptosis. The function of another upregulated gene, 14-3-3γ, is also to inhibit apoptosis.(43–45) The 14–3-3 protein is a family of phosphor-serine/phosphothreonine-binding proteins that promote survival, in part, by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase1.(43) The involvement of these molecules in the apoptosis of articular chondrocytes is an important issue to be investigated.
We have found that EP3 was also expressed in articular chondrocytes, although at a level much lower than that of EP2 in human articular cartilage. PGE2 signals through EP3 induce the production of IL-6 in synovial tissues,(23,24) but no data concerning the effects of EP3 on articular chondrocytes were published. The analysis of isoforms in MMA2 cells revealed the main subtype of EP3 to be the γ form, which has no effect on intracellular cAMP. The effect of the γ form of EP3 on the intracellular calcium concentration depends on cell type,(7,8) and no effects were observed in chondrocytes in this study. The roles of EP3 in articular chondrocytes are currently being studied using a similar experimental system.
Induction of cell proliferation and reduction of apoptosis by PGE2 have been reported in other types of tissues,(46–50) and the signal through EP2 was also reported to promote the growth of vascular smooth muscle cells.(50) Although whether the molecules isolated with the cDNA microarray are direct downstream targets remains to be investigated, it is clear that the PGE2 signal through EP2 promotes the growth of articular cartilage cells as a net result. Therefore, combined with suitable drug delivery systems, the EP2 agonist is a promising therapeutic compound for the degenerative cartilage diseases to be used instead of PGE2 itself, which can have undesirable additional effects such as the promotion of cytokine production in synovial cells through the EP3 receptor.(24)
We are grateful to Drs Takayuki Maruyama and Shin-ichi Yokota for generous support and helpful discussions. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences and from the Ministry of Health, Labor, and Welfare of Japan.
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