SEARCH

SEARCH BY CITATION

Keywords:

  • prostaglandin E2;
  • EP2;
  • articular cartilage;
  • chondrocyte;
  • microarray

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.

Immunohistochemistry

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).

Western blotting

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.

RT-PCR

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.

Table Table 1. Information on Primers Used in the RT-PCR Analyses
Thumbnail image of

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.

Statistics

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.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.

thumbnail image

Figure Fig. 1.. Expression of EPs in normal human cartilage. (A) Immunohistochemical staining of EP1, EP2, EP3, and EP4 in articular cartilage tissues of KS834 (39 years old; original magnification, ×400). EP2 was strongly expressed, and the expression of EP3 was faintly detected. Nuclei were counterstained with hematoxylin. (B) mRNA expression of EP1, EP2, EP3, and EP4 genes in articular cartilage tissues (left) and chondrocytes (right). RNA was extracted from articular cartilage tissue of four individuals (1, KS847; 2, KS730; 3, KS834; and 4, KS574), and also from primary cultured chondrocytes isolated from corresponding tissues. (C) Expression of EP protein in primary cultured chondrocytes. Whole cell lysates were prepared from primary cultured chondrocytes of patients analyzed in B, and 20 μg of total protein was loaded in each lane. The expression of each EP protein was analyzed by Western blotting using antibodies for each EP protein.

Download figure to PowerPoint

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.

thumbnail image

Figure Fig. 2.. In situ hybridization of EP genes in epiphyseal tissues. The mRNA expression of each EP gene in the epiphyseal portion of the distal femora and proximal tibias isolated from newborn mice was investigated by in situ hybridization. (Left) Hybridization with antisense probes. (Right) Hybridization with sense probes.

Download figure to PowerPoint

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).

thumbnail image

Figure Fig. 3.. Characteristics of MMA2. (A) In vitro morphology. MMA2 was seeded at a density of 5 × 104 cells/60-mm dish, and phase-contrast micrographs were taken at days 2 and 21. No calcified nodule was formed. (B) Nodule formation of MMA2 in 3D culture. 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, embedded in paraffin, and stained with H&E (left) or 0.1% alcian blue and counterstained with nuclear fast red (right).

Download figure to PowerPoint

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.

thumbnail image

Figure Fig. 4.. mRNA expression of cartilage-related genes in MMA cell lines. Each clone was seeded at a density of 3 × 105 cells/100-mm culture dish and cultured using standard medium supplemented with 10% FBS. Total RNA was extracted from each cultured cell line on day 2 and processed for reverse transcription. One microliter of the reverse transcription product was used for each PCR, and PCR products were separated in a 1.5% agarose gel and stained with ethidium bromide. (A) Extracellular matrix-related genes. (B) Signal transduction genes.

Download figure to PowerPoint

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).

thumbnail image

Figure Fig. 5.. mRNA expression of EP genes in cell lines and cartilage tissues. The mRNA expression of EP genes in MMA cell lines and (A) mouse articular cartilage tissues and (B) rat articular cartilage tissues was analyzed using primers for each species. RNA extracted from rat kidney tissues was used as positive controls for rat EP genes. Experiments were performed as described in the legend of Figure 4.

Download figure to PowerPoint

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).

thumbnail image

Figure Fig. 6.. Effects of EP agonists on the intracellular cAMP concentration of MMA2. (A) Specific induction of cAMP by the EP2 agonist. Cells were seeded at a density of 1 × 104 cells/well on 24-well culture dishes. After the cells had attached to the plate, each EP agonist (1 μM) was added and the plate was incubated for 24 h. The Intracellular cAMP concentration was increased by the treatment with the EP2 agonist (*p < 0.05). (B), Dose-dependent increase of cAMP induced by the EP2 agonist. MMA2 cells were incubated with the indicated concentration of the EP2 agonist for 24 h.

Download figure to PowerPoint

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.

Table Table 2. Genes Up- and Downregulated in Response to the EP2 Agonist
Thumbnail image of

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.

thumbnail image

Figure Fig. 7.. Quantitative mRNA expression analyses of genes identified by gene expression profiling. The mRNA expression of EP2-regulated genes identified by gene expression profiling in MMA2 and primary chondrocytes was analyzed before and after the treatment of with the EP2 agonist (1 μM). As for the upregulated genes, (A) cyclin D1, (B) integrin α5, (C) fibronectin, (D) AP2α, and (E) 14-3-3γ were analyzed, and (F) osteopontin was also analyzed as the downregulated gene. ▴, MMA2; ○, KS874; •, KS834; □, KS730; ▪, KS574.

Download figure to PowerPoint

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.

thumbnail image

Figure Fig. 8.. EP2 agonist stimulates DNA synthesis in primary chondrocytes. The uptake of BrdU was measured after the cells were treated with 10−6 M of the EP2 agonist for 24 h. The data are presented relative to control values (*p < 0.05). (○, KS874; •, KS574; □, KS574; ▪, KS834).

Download figure to PowerPoint

thumbnail image

Figure Fig. 9.. Effects of EP agonists on cell growth. (A) MMA2 cells were seeded at a density of 5 × 104 cells/60-mm dish, and indomethacin (5 μM) and each EP agonist (1 μM) were added every other day. Cells were enumerated on day 7, and the fold increase was calculated using the cell number for the control (untreated) as a standard. *p < 0.05. (B) Primary short-term cultured chondrocytes were seeded at a density of 5 × 104 cells/60-mm dish, and indomethacin (5 μM) and each EP agonist (1 μM) were added every other day. Cell counting was performed in the same way. *p < 0.05 (○, KS847; •, KS730; ▪, KS834).

Download figure to PowerPoint

thumbnail image

Figure Fig. 10.. Effects of EP2 agonist on chondrocytes in organ culture. One femur taken from each rat was incubated in medium containing EP2 agonist (10−5, 10−6, or 10−7 M, n = 3 for each concentration), and another from the same rat was incubated without EP2 agonist. The medium was changed every 48 h until day 14. (A) Immunohistochemical staining of PCNA. (Top) Control sample. (Bottom) Experimental sample treated with EP2 agonist (10−5 M). (B) Relative increase of PCNA+ cells. Cells positive for PCNA staining were counted under the microscope, and a labeling index was calculated for each sample. The relative increase in PCNA+ cells was shown as described in the Materials and Methods section.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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)

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Lippiello L, Yamamoto K, Robinson D, Mankin HJ 1978 Involvement of prostaglandins from rheumatoid synovium in inhibition of articular cartilage metabolism. Arthritis Rheum 21: 909917.
  • 2
    Fulkerson JP, Damiano P 1983 Effect of prostaglandin E2 on adult pig articular cartilage slices in culture. Clin Orthop 179: 266269.
  • 3
    Kosher RA, Walker KH 1983 The effect of prostaglandins on in vitro limb cartilage differentiation. Exp Cell Res 145: 145153.
  • 4
    Biddulph DM, Sawyer LM, Dozier MM 1988 Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: Changes in cyclic AMP and in prostaglandin responsiveness. J Cell Physiol 136: 8187.
  • 5
    Del Toro F Jr, Sylvia VL, Schubkegel SR, Campos R, Dean DD, Boyan BD, Schwartz Z 2000 Characterization of prostaglandin E(2) receptors and their role in 24,25-(OH)(2)D(3)-mediated effects on resting zone chondrocytes. J Cell Physiol 182: 196208.
  • 6
    Negishi M, Sugimoto Y, Ichikawa A 1995 Prostaglandin E receptors. J Lipid Mediat Cell Signal 12: 379391.
  • 7
    Negishi M, Hasegawa H, Ichikawa A 1996 Prostaglandin E receptor EP3gamma isoform, with mostly full constitutive Gi activity and agonist-dependent Gs activity. FEBS Lett 386: 165168.
  • 8
    Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, Narumiya S 1993 Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature 365: 166170.
  • 9
    Sylvia VL, Del Toro F, Dean DD, Hardin RR, Schwartz Z, Boyan BD 2001 Effects of 1alpha,25-(OH)(2)D(3) on rat growth zone chondrocytes are mediated via cyclooxygenase-1 and phospholipase A(2). J Cell Biochem 81: 3245.
  • 10
    Sylvia VL, Del Toro F Jr, Hardin RR, Dean DD, Boyan BD, Schwartz Z 2001 Characterization of PGE(2) receptors (EP) and their role as mediators of 1alpha,25-(OH)(2)D(3) effects on growth zone chondrocytes. J Steroid Biochem Mol Biol 78: 261274.
  • 11
    Miyamoto M, Ito H, Mukai S, Kobayashi T, Yamamoto H, Kobayashi M, Maruyama T, Akiyama H, Nakamura T 2003 Simultaneous stimulation of EP2 and EP4 is essential to the effect of prostaglandin E2 in chondrocyte differentiation. Osteoarthritis Cartilage 11: 644652.
  • 12
    Iwamoto M, Higuchi Y, Koyama E, Enomoto-Iwamoto M, Kurisu K, Yeh H, Abrams WR, Rosenbloom J, Pacifici M 2000 Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development. J Cell Biol 150: 2740.
  • 13
    Iwamoto M, Higuchi Y, Enomoto-Iwamoto M, Kurisu K, Koyama E, Yeh H, Rosenbloom J, Pacifici M 2001 The role of ERG (ets related gene) in cartilage development. Osteoarthritis Cartilage 9 (Suppl A): S41S47.
  • 14
    Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, Hovig E, Smith-Sorensen B, Montesano R, Harris CC 1994 Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res 22: 35513555.
  • 15
    Levine AJ 1997 p53, the cellular gatekeeper for growth and division. Cell 88: 323331.
  • 16
    Shimoji M, Hattori K, Itoh S, Nakayama K, Katsuki M, Aizawa S, Yokoi T, Kamataki T 1995 Establishment of immortal hepatocytes from a CYP3A7-transgenic/p53-knockout mouse. Biochem Biophys Res Commun 217: 10011005.
  • 17
    Yahanda AM, Bruner JM, Donehower LA, Morrison RS 1995 Astocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas. Mol Cell Biol 15: 42494259.
  • 18
    Ghosh-Choudhury N, Harris MA, Wozney J, Mundy GR, Harris SE 1997 Clonal osteoblastic cell lines from p53 null mouse calvariae are immortalized and dependent on bone morphogenetic protein 2 for mature osteoblastic phenotype. Biochem Biophys Res Commun 231: 196202.
  • 19
    Thompson DL, Lum KD, Nygaard SC, Kuestner RE, Kelly KA, Gimble JM, Moore EE 1998 The derivation and characterization of stromal cell lines from the bone marrow of p53−/− mice: New insights into osteoblast and adipocyte differentiation. J Bone Miner Res 13: 195204.
  • 20
    Nakayama T, Kanoe H, Sasaki MS, Aizawa S, Nakamura T, Toguchida J 1997 Establishment of an osteoblast-like cell line, MMC2, from p53-deficient mouse. Bone 21: 313319.
  • 21
    Nakamata T, Aoyama T, Okamoto T, Hosaka T, Nishijo K, Nakayama T, Nakamura T, Toguchida J 2003 In vitro demonstration of cell-to-cell interaction in growth plate cartilage using chondrocytes established from p53−/− mice. J Bone Miner Res 18: 97107.
  • 22
    Hiraki Y, Inoue H, Iyama K, Kamizono A, Ochiai M, Shukunami C, Iijima S, Suzuki F, Kondo J 1997 Identification of chondromodulin I as a novel endothelial cell growth inhibitor. Purification and its localization in the avascular zone of epiphyseal cartilage. J Biol Chem 272: 3241932426.
  • 23
    Breyer RM 2001 Prostaglandin EP(1) receptor subtype selectivity takes shape. Mol Pharmacol 59: 13571359.
  • 24
    Gomi K, Zhu FG, Marshall JS 2000 Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism. J Immunol 165: 65456552.
  • 25
    Kurihara Y, Endo H, Akahoshi T, Kondo H 2001 Up-regulation of prostaglandin E receptor EP2 and EP4 subtypes in rat synovial tissues with adjuvant arthritis. Clin Exp Immunol 123: 323330.
  • 26
    Paralkar VM, Borovecki F, Ke HZ, Cameron KO, Lefker B, Grasser WA, Owen TA, Li M, DaSilva-Jardine P, Zhou M, Dunn RL, Dumont F, Korsmeyer R, Krasney P, Brown TA, Plowchalk D, Vukicevic S, Thompson DD 2003 An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci USA 100: 67366740.
  • 27
    Li M, Ke HZ, Qi H, Healy DR, Li Y, Crawford DT, Paralkar VM, Owen TA, Cameron KO, Lefker BA, Brown TA, Thompson DD 2003 A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing. J Bone Miner Res 18: 20332042.
  • 28
    Yoshida K, Oida H, Kobayashi T, Maruyama T, Tanaka M, Katayama T, Yamaguchi K, Segi E, Tsuboyama T, Matsushita M, Ito K, Ito Y, Sugimoto Y, Ushikubi F, Ohuchida S, Kondo K, Nakamura T, Narumiya S 2002 Stimulation of bone formation and prevention of bone loss by prostaglandin EP4 receptor activation. Proc Natl Acad Sci USA 99: 45804585.
  • 29
    Pullig O, Weseloh G, Gauer S, Swoboda B 2000 Osteopontin is expressed by adult human osteoarthritic chondrocytes: Protein and mRNA analysis of normal and osteoarthritic cartilage. Matrix Biol 19: 245255.
  • 30
    Attur MG, Dave MN, Stuchin S, Kowalski AJ, Steiner G, Abramson SB, Denhardt DT, Amin AR 2001 Osteopontin: An intrinsic inhibitor of inflammation in cartilage. Arthritis Rheum 44: 578584.
  • 31
    Yumoto K, Ishijima M, Rittling SR, Tsuji K, Tsuchiya Y, Kon S, Nifuji A, Uede T, Denhardt DT, Noda M 2002 Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice. Proc Natl Acad Sci USA 99: 45564561.
  • 32
    Beier F, Ali Z, Mok D, Taylor AC, Leask T, Albanese C, Pestell RG, Lu Valle P 2001 TGFbeta and PTHrP control chondrocyte proliferation by activating cyclin D1 expression. Mol Biol Cell 12: 38523863.
  • 33
    Beier F, Lee RJ, Taylor AC, Pestell RG, Lu Valle P 1999 Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. Proc Natl Acad Sci USA 96: 14331438.
  • 34
    Davies SR, Sakano S, Zhu Y, Sandell LJ 2002 Distribution of the transcription factors Sox9, AP-2, and [delta]EF1 in adult murine articular and meniscal cartilage and growth plate. J Histochem Cytochem 50: 10591065.
  • 35
    Pfisterer P, Ehlermann J, Hegen M, Schorle H 2002 A subtractive gene expression screen suggests a role of transcription factor AP-2 alpha in control of proliferation and differentiation. J Biol Chem 277: 66376644.
  • 36
    Yasuda T, Poole AR 2002 A fibronectin fragment induces type II collagen degradation by collagenase through an interleukin-1-mediated pathway. Arthritis Rheum 46: 138148.
  • 37
    Homandberg GA, Wen C, Hui F 1998 Cartilage damaging activities of fibronectin fragments derived from cartilage and synovial fluid. Osteoarthritis Cartilage 6: 231244.
  • 38
    Homandberg GA 1999 Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments. Front Biosci 4: D713D730.
  • 39
    Homandberg GA, Costa V, Ummadi V, Pichika R 2002 Antisense oligonucleotides to the integrin receptor subunit alpha(5) decrease fibronectin fragment mediated cartilage chondrolysis. Osteoarthritis Cartilage 10: 381393.
  • 40
    Enomoto-Iwamoto M, Iwamoto M, Nakashima K, Mukudai Y, Boettiger D, Pacifici M, Kurisu K, Suzuki F 1997 Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes. J Bone Miner Res 12: 11241132.
  • 41
    Forsyth CB, Pulai J, Loeser RF 2002 Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes. Arthritis Rheum 46: 23682376.
  • 42
    Pulai JI, Del Carlo M Jr, Loeser RF 2002 The alpha5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro. Arthritis Rheum 46: 15281535.
  • 43
    Masters SC, Yang H, Datta SR, Greenberg ME, Fu H 2001 14–3-3 inhibits Bad-induced cell death through interaction with serine-136. Mol Pharmacol 60: 13251331.
  • 44
    Masters SC, Fu H 2001 14–3-3 proteins mediate an essential anti-apoptotic signal. J Biol Chem 276: 4519345200.
  • 45
    Masters SC, Subramanian RR, Truong A, Yang H, Fujii K, Zhang H, Fu H 2002 Survival-promoting functions of 14–3-3 proteins. Biochem Soc Trans 30: 360365.
  • 46
    Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN 1998 Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 58: 362366.
  • 47
    Sanchez T, Moreno JJ 2002 Role of EP(1) and EP(4) PGE(2) subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation. Am J Physiol Cell Physiol 282: C280C288.
  • 48
    Kawamori T, Uchiya N, Sugimura T, Wakabayashi K 2003 Enhancement of colon carcinogenesis by prostaglandin E2 administration. Carcinogenesis 24: 985990.
  • 49
    Jabbour HN, Boddy SC 2003 Prostaglandin E2 induces proliferation of glandular epithelial cells of the human endometrium via extracellular regulated kinase 1/2-mediated pathway. J Clin Endocrinol Metab 88: 44814487.
  • 50
    Yau L, Zahradka P PGE(2) stimulates vascular smooth muscle cell proliferation via the EP2 receptor. Mol Cell Endocrinol 203: 7790.