Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2dependent proliferation of growth plate chondrocytes
© Brochhausen et al.; licensee BioMed Central Ltd. 2006
Received: 26 August 2005
Accepted: 28 March 2006
Published: 28 April 2006
Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.
Prostaglandins, especially prostaglandin E2 (PGE2), play an important role in bone and cartilage metabolism. Although PGE2 was initially described as a potent bone-resorbing substance , several studies have demonstrated its activity in bone-forming processes [2, 3]. In osteoblast-like cells, endogenous PGE2 was shown to affect proliferation and differentiation by stimulation of DNA synthesis and alkaline phosphatase activity . An interesting aspect in the investigation of the function of prostaglandins in cartilage or bone tissue is their possible role in the growth plate. This special cartilage tissue is responsible for the endochondral ossification of long bones and represents all differentiation steps in distinguishable layers, from undifferentiated reserve zone cells to proliferative and hypertrophic chondrocytes, which initiate cartilage mineralisation. Due to this complex structure of the growth plate, cellular effects of prostaglandins on growth plate chondrocytes have been examined using various in vitro systems. PGE2 elicits differentiation of chondrocytes, as previously shown for the chondrocyte cell line RCJ3.1C5.18  and rat growth plate chondrocytes . In the latter, the effect of PGE2 was mediated by cAMP and protein kinase C. Furthermore, PGE2 also makes an important contribution to cartilage formation and promotes DNA and matrix synthesis in growth plate chondrocytes . In addition to various findings in vitro, the physiological role of prostaglandins was clarified by its stimulating effect on bone formation and by the increase in bone mass after systemic administration of PGE2 to infants  and animals . Furthermore, local administration of PGE2 resulted in osteogenesis in situ [10, 11].
The rate-limiting step for the synthesis of PGE2 and other prostaglandins is the conversion of arachidonic acid to prostaglandin endoperoxide by cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2 . These enzymes are differentially regulated. Previous in vitro analysis demonstrated the functional importance of COX-1 for proliferation, differentiation and matrix production in cultured growth zone chondrocytes . In various chondrocyte cell models, as well as in fracture callus formation, COX-2 may also be important for prostaglandin synthesis . Moreover, the expression of COX-2 is regulated by different stimuli, such as tumour necrosis factor-α  or shear stress . The induction of COX-2 is regarded as an important step in inflammatory situations. COX-1 and COX-2 are expressed in inflamed bone tissue  and COX inhibitors are extensively used in the treatment of rheumatoid arthritis. However, inadequate information is available on in situ expression of both COX-1 and COX-2 within the growth plate to correlate in vitro findings with the in situ situation.
PGE2, the principal product of bone prostaglandin synthesis, acts locally on target cells by binding to prostaglandin E (EP)-type G protein-coupled receptors. Four different EP receptors are known, which are linked to different intracellular signal transduction pathways . The EP1 receptor is coupled to intracellular Ca2+ mobilization, while the EP2 and EP4 receptors increase intracellular cAMP accumulation. By contrast, EP3 inhibits intracellular cAMP accumulation. Regarding bone formation and bone resorption, the EP4 receptor has been shown to be essential in terms of PGE2 action in bone . Recently, the EP2 and EP4 receptors were shown to be required for PGE2-dependent chondrocyte differentiation . In previous studies, we demonstrated that stimulation of growth plate chondrocyte proliferation by both calciotropic hormones, 1,25 (OH)2D3 and parathyroid hormone, is dependent on an increase in intracellular calcium and activation of protein kinase C . On the other hand, an increase in intracellular cAMP concentration was without any effect on proliferation , but was able to stimulate matrix synthesis . In the present study, we were interested in whether PGE2 acts in a proliferative and stimulatory fashion on growth plate chondrocyte function. We therefore investigated the effects of PGE2 and prostaglandin receptor agonists and antagonists on cultured growth plate chondrocytes. Furthermore, we analysed the expression and spatial distribution of COX-1 and COX-2 and the PGE2 receptors EP1, EP2, EP3, and EP4 in the growth plate and compared this profile with their expression in cultured growth plate chondrocytes in order to give innovative insights into in situ -in vitro correlations.
Materials and methods
Polyclonal rabbit antibodies against the EP1, EP2, EP3 and EP4 receptors and COX-1 and COX-2 were described previously [23, 24]. Polyclonal rabbit antibodies against collagen (Col) type I and type II were purchased from Biotrend Chemicals GmbH (Cologne, Germany). Monoclonal anti-collagen type X antibody (mouse) was from Quartett (Berlin, Germany). All other antibodies used were obtained from DAKO (Glostrup, Denmark). DNase (10 U/μl) for cartilage digestion was from Amersham Pharmacia Biotech (Piscataway, NY, USA) and CaCl2 was from Serva (Heidelberg, Germany). FCS and culture dishes were from Greiner (Frickenhausen, Germany), and culture media were obtained from PAA GmbH (Linz, Austria). Butaprost, misoprostole, sulprostone and PGE2 were purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). Ligands for the PGE2 receptors (ONO D1-004, ONO AE1-259-001, ONO AE-248, ONO AE1-329, and ONO-8711) have been described previously [25–27] and were kindly provided by Dr Maruyama (ONO Pharmaceuticals, Osaka, Japan). PicoGreen for double-stranded (ds)DNA quantification was obtained from Mobitec (Göttingen, Germany). Gene Amp RNA-PCR kit, DNA Polymerase (Ampli taq Gold), reverse transcriptase (MuLV RT) and oligo d(T)16 were purchased from Perkin Elmer, Roche Molecular Systems Inc. (Branchburg, NJ, USA). Other chemicals were of p.a. grade and purchased from Merck (Darmstadt, Germany), Gibco BRL Life Technologies (Karlsruhe, Germany) or Sigma Aldrich Chemistry (Steinheim, Germany).
Isolation of chondrocytes
Chondrocytes were isolated and cultured as described earlier by Benya and Shaffer  and modified according to Klaus and colleagues . Briefly, femurs of up to four week old Sprague Dawley rats (60 to 80 g each) were dissected. The epiphyseal growth plate of the tibiae was separated by cleaning the cartilage plate of muscular tissue, periosteum and perichondrium. The proximal epiphysis was divided by a transverse cut with a sharp scalpel, and the cartilage plate was separated distally from the calcification zone of the tibial metaphysis. Isolated growth plates were digested for 3 hours at 37°C by collagenase (0.12% w/v) and DNase (0.02% w/v) in 5 ml of serum free F12/DMEM medium. After thorough washing, cells were counted using a Neubauer chamber. Viability, examined by trypan blue exclusion, was > 95%.
Chondrocytes were cultured in flasks, 96-well-plates or 2-well cell-tissue-chambers containing F12/DMEM 1/1 medium supplemented with 10% FCS, 10 mM HEPES, 2 mM pyruvate, 2 mM L-glutamine, 0.7 μM CaCl2, 10 mg/ml penicillin/streptomycin and L-cysteine. Ionized calcium measured by a calcium-sensitive electrode was 1.2 mmol/l. During the first four days of cell culturing the serum substitute Ultroser-G (1%) was added to the medium. From day 5 on, β-glycerophosphate (10 mM) and L-thyroxine (100 μg/μl), as well as ascorbic acid (5 to 60 μg/ml) from day 11 on, were added to the culture medium. Medium was changed every 48 hours and cells became confluent within 6 to 12 days.
Assay of cell proliferation: semiquantitative dsDNA determination
Primary cultures of chondrocytes were transferred to 96-well-plates in serum-free medium without L-thyroxine, which is reported to exert antiproliferative effects . Cell cycles were synchronised for 24 hours as described earlier .
PGE2, EP receptor agonists, or vehicle were added with fresh medium, supplemented with 10% FCS and cells were stimulated for 24 or 48 hours. Incubation was stopped by aspiration of the supernatants and the culture plates were frozen at -80°C for 1 hour. Thereafter, cells were thawed and incubated with 200 μl staining solution (containing 2.5 μl/ml PicoGreen) for 10 minutes under light protection. Optical density was determined using a plate reader (excitation/emission, 485 nm/530 nm). Experiments were run with four to six parallel aliquots.
Assay of cell proliferation: [3H]-thymidine incorporation
Incorporation of [3H]-thymidine was determined in serum-free cultures as described previously . Cells were synchronised in serum-free medium for 24 hours. Thereafter, medium was changed to F-12/DMEM with 0.2% (w/v) bovine serum albumin and the substances or vehicles were added. Cells were incubated for 48 hours and 2 μCi [3H]-thymidine were added to each well 3 hours before stopping the incubation.
Reverse transcriptase-polymerase chain reaction
Primers used for RT-PCR
Sequence of primer
5' -GCT GTA CGC CTC GCA TCG TGG-3'
5' -GTG TTT CGA GCA TCC CAT GTA TCT-3'
5' -GAA CGC TAC CTC TCC ATC GG-3'
5' -TGA TGG TCA TAA TGG-3'
5' -GTTTGGTCTG GCGTCTTAGA AC-3'
5' -CTTGGAACAG GACCTTCTGA GT-3'
5' -AATGAGTACC GCAAA-3'
5' -ATCTAGTCTG GAGCGGGAGG-3'
5' -TGGTGACAAG GGTGAGACAG-3'
5' -TGAGGCAGGA AGCTGAAGTC-3'
5' -GTTTGGTCTG GCGTCTTAGA AC-3'
5' -CTTGGAACAG GACCTTCTGA GT-3'
5' -CTCCAGGTGT GAAGGGTGAG-3'
5' -GAACCTTGAG CACCTTCAGG-3'
5' -AATGAGTACC GCAAA-3'
5' -ATCTAGTCTG GAGCGGGAGG-3'
5' -TGCCTCTTGT CAGTGCTAAC C-3'
5' -GCGTGCCGTT CTTATACAGG-3'
5' -TGGTGACAAG GGTGAGACAG-3'
5' -TGAGGCAGGA AGCTGAAGTC-3'
5' -CATCACCATT GGCAATGAGC G-3'
5' -CTAGAAGCAT TTGCGGTCGG AC-3'
5' -CTCCAGGTGT GAAGGGTGAG-3'
5' -GAACCTTGAG CACCTTCAGG-3'
5' -TGCCTCTTGT CAGTGCTAAC C-3'
5' -GCGTGCCGTT CTTATACAGG-3'
5' -CATCACCATT GGCAATGAGC G-3'
5' -CTAGAAGCAT TTGCGGTCGG AC-3'
For immunohistochemistry, the epiphyseal plate with neighbouring bony metaphysis and epiphysis including the knee joint were dissected. The isolated tissue was immediately frozen in isopentane at -80°C. For detection of EP1, EP2, EP3, EP4, COX-1, COX-2, Col II and Col X, the alkaline-phosphatase-anti-alkaline-phosphatase method was used according to Cordell and colleagues  as modified by Bittinger and colleagues . Frozen sections (4 μm) were fixed in paraformaldehyde (4%). Polyclonal rabbit antibodies against EP1 (1:300), EP2 (1:200), EP3 (1:300), EP4 (1:300), COX-1 (1:100), COX-2 (1:100) and Col II (1:800) as well as a monoclonal mouse antibody against Col X (1:200) were incubated for 16 hours at 4°C. After staining, these sections were counter-stained with hemalaun. For the antibodies directed against the EP receptors, the following controls were performed. Firstly, the primary antibody was omitted; under this condition no staining was visible. Secondly, the antibodies were preabsorbed with the corresponding peptide against which they are directed as described previously ; under this condition staining was completely blocked.
Determination of PGE2
PGE2 was determined in cell supernatants as described previously .
Statistical analysis was carried out by t test or ANOVA as appropriate. P values are < 0.05 or < 0.001.
Collagen expression in cultured chondrocytes
PGE2production and COX-1 and COX-2 expression
Release of PGE2 into the supernatant of cultured rat chondrocytes
120 ± 20
530 ± 270a
150 ± 30
Effect of PGE2and analogues on proliferation of growth plate chondrocytes
Expression of EP1 and EP2 receptors
Expression of EP3 and EP4 receptors
The present study clearly demonstrates that growth plate chondrocytes are capable of secreting PGE2. The effects of PGE2 are mediated by G-protein-coupled receptors with different pathways of signal transduction. The present data show for the first time expression of COX-1 and COX-2, as well as EP1, EP2, EP3 and EP4, in the intact growth plate in situ in comparison with the expression in cultured growth plate chondrocytes. COX enzymes are expressed in situ in a characteristic spatial distribution: whereas COX-1 is homogenously expressed in all zones of the growth plate, COX-2 showed moderate expression in the reserve zone and strong expression in the other zones. Regarding EP receptor expression, EP1 expression in situ was mainly restricted to the proliferative and hypertrophic zone. Contrasting with this, EP2, EP3 and EP4 receptors in situ were homogeneously expressed by all chondrocytes, but in vitro by a subpopulation of cells only.
Collagen expression was analysed as a parameter of the phenotypic integrity of the chondrocytes and Col II and Col X are expressed in specific maturation states. In our system, the differentiation state of the majority of cells corresponded to cells in the proliferative layer, as shown previously . This is confirmed not only by the proliferative activity but also by the production of Col II, and the lack of Col X, which is a specific marker of late hypertrophic chondrocytes . Col I is not believed to be characteristically expressed in the growth plate and costochondral cartilage, but rather in the superficial layer of mandibular and articular cartilage . Col I was also detectable in our cultured cells, which indicates the presence of 'de-differentiated' chondrocytes  in the absence of Col X expression.
PGE2 is produced by COX, of which two isoforms – COX-1 and COX-2 – exist. However, its protein expression has not been demonstrated previously in the growth plate, despite the fact that secreted prostanoids, which were generated by COX-1 and/or COX-2, were shown to modulate chondrocyte proliferation and function in in vitro systems. These results can only be extrapolated to the in situ situation if COX is expressed in the intact growth plate. Using polyclonal antibodies to COX-1 and COX-2, we were able to demonstrate COX-1 and COX-2 immunoreactivity in growth plate chondrocytes. Paralleling the in situ situation, both COX-1 and COX-2 mRNA as well as COX-1 and COX-2 protein were expressed in cultured chondrocytes. Concluding from the observed inhibitory effect of the COX-2 inhibitor SC-236, but not of the COX-1 inhibitor SC-560, on chondrocyte proliferation, we suggest that, at least for the cultured chondrocytes, COX-2 is the responsible enzyme driving PGE2 formation.
In our primary culture system, PGE2 stimulated DNA synthesis in a bell-shaped manner, the strongest effect being observed at concentrations that are higher than those physiologically found in the circulation . These results are in accordance with studies by O'Keefe and colleagues  and Schwartz and colleagues , describing a growth-stimulatory effect of PGE2 at similar concentrations. We speculate, therefore, that secreted PGE2 could function as an autocrine/paracrine mediator of chondrocyte proliferation. From in vitro studies it is well known that PGE2may have different concentration-dependent effects on cell proliferation and matrix synthesis. This implies that local PGE2 concentrations in the various zones of the growth plate may differ. In fact, bovine chondrocytes isolated from the 'superficial zone' of the growth plate, that is, mainly reserve zone cells, were shown to produce less PGE2 than proliferating and early hypertrophic cells isolated from the 'deep zone' .
The proliferative action of PGE2 was mimicked by sulprostone, which was shown to selectively bind to EP1 and EP3 receptors  and only a minor stimulatory effect was provoked by misoprostole. Furthermore, a selective EP1 agonist provoked a similar proliferative effect in rat cultured chondrocytes compared to PGE2 and the growth-promoting effect of PGE2 could be completely blocked by a specific EP1 antagonist. We conclude that PGE2 mediates its proliferative effect primarily via the EP1 receptor. It has to be noted that a minor growth-promoting effect was also seen by the addition of EP2, EP3 and EP4 specific ligands. The minor growth-promoting effect observed with the EP3 agonist might be due to the presence of endogenously produced PGE2. EP3 receptor activation causes a decrease in intracellular cAMP levels. We speculate that in cultured chondrocytes, EP3 activation might promote an EP1 signalling pathway, triggered by endogenously formed PGE2, by ablation of cAMP, the opponent of the Ca2+ signalling pathway. Alternatively, it has been shown that different splice variants do exist for the EP3 receptor, which in part may evoke a phosphatidyl-inositol response . However, we can not exclude that different subpopulations within our cell culture system are regulated in a different way by PGE2, as we did not observe a homogenous expression of the different EP receptors in the cultured chondrocytes. Differences in responsiveness to PGE2 has, for example, also been reported for mouse chondroprogenitors and chondrocytes .
The second messenger of the EP1 receptor is free ionised intracellular calcium . An increase of intracellular calcium was shown to be necessary for chondrocyte proliferation in response to the calciotropic hormones parathormone and 1,25(OH)2D3 [21, 41]. The latter is thought to stimulate cell growth via generation of PGE2 . To our knowledge, an increase of intracellular calcium in response to PGE2 has not been measured in growth plate chondrocytes. Contrasting with this hypothesis, PGE2 was found to have no effect on intracellular calcium in cultured articular bovine cartilage cells .
Corresponding to the proposed proliferative action of PGE2 via the EP1 receptor, this receptor could be demonstrated at the mRNA and protein levels not only in vitro but also in situ. In the intact growth plate we observed a strong EP1 receptor immunoreactivity in proliferative and hypertrophic chondrocytes, but not in reserve zone cells. This is in line with the proliferative effect of PGE2 mediated via the EP1 receptor. In vitro, EP1 was expressed in all cells, although the intensity varied. Because in our culture system proliferative cells represented the majority of chondrocytes, the ubiquitous expression of EP1 receptor in vitro was in contrast to the in situ situation. This discrepancy indicates that extrapolation of the in vitro data to the in situ situation should be done with caution.
In addition, the EP2 receptor also showed a different expression pattern in situ and in vitro. The EP2 receptor was not uniformly detectable in vitro, although in situ all cells were positive. The highest expression was observed in dividing cells. It can be concluded from our data that EP2 receptor signalling also contributes to cell growth. The inhomogenous expression of EP2 in cultured chondrocytes may explain the lower proliferative effect achieved by the specific EP2 agonist. EP2 receptor expression has also been described in cultured articular chondrocytes  and fourth passage reserve zone cells . In the latter, PGE2 stimulated intracellular cAMP, which resulted in increased matrix synthesis. In a chondrocyte cell line, established from articular cartilage of p53-/- mice, the EP2 receptor was identified as the major PGE2 receptor . In this cell line, EP2 agonists evoked cAMP generation and promoted cell growth. In articular chondrocytes, PGE2 probably mediates its proliferative effect primarily via the EP2 receptor whereas in growth plate chondrocytes the EP1 receptor is dominant for PGE2-dependent growth. EP2 and EP4 receptors may also be involved in chondrogenesis . In limb bud mesenchymal cells, all four types of EP receptor are expressed and EP2 and EP4 receptor activation of cAMP metabolism was suggested to drive mesenchymal stem cells to chondrogenesis. We observed a weak expression of the EP4 receptor in our cultured chondrocytes. Most likely, EP receptors, and especially the EP4 type, are expressed depending on the cell differentiation state in culture. By contrast, in the growth plate tissue of the rat we observed EP4 expression in all layers. In a recent study, Miyamoto and colleagues  showed that the EP2 receptor promotes differentiation and synthesis of Col II and proteoglycans in cultured bovine growth plate cells. This effect was dependent on co-stimulation of the EP4 receptor; however, in rat, the EP4 receptor was not detected, at least in fourth passage chondrocytes . In view of these results, a role for the EP2 receptor in chondrocyte differentiation can be hypothesised. The differentiation-dependent expression of EP receptors might explain the contradictory results obtained in studies investigating the effects of PGE2. This indicates the crucial role played by species and culture conditions used in the various in vitro systems. According to our in vivo data, all types of EP receptors appeared to be expressed. Taking into account that the different EP receptors are coupled to different intracellular signalling pathways, we expect that other mechanisms, such as receptor activation, modulation of ligand affinity or selective access of PGE2 to the necessary receptor type, are involved in ensuring a coordinated action of PGE2 in growth plate physiology.
Cultured growth plate chondrocytes synthesized PGE2. Exogenous PGE2 stimulation had a proliferating-inducing effect in a dose-dependent manner on cultured growth plate chondrocytes via the EP1 receptor, which could be mimicked by EP agonists such as sulprostone and ONO-D1-004. The proliferating effects could be blocked by the EP1 antagonist ONO-8713.
Further analyses of the physiological and pathophysiological roles of EP1 and EP2, especially in chronic inflammatory disorders, are needed. From a therapeutic point of view, the long term effects of COX inhibitors and EP antagonists with respect to the integrity of the growth plate in the paediatric population is of special interest. Growth plate chondrocytes express COX-1, COX-2 and EP1, EP2, EP3, and EP4 in situ and in vitro with markedly different expression patterns. Therefore, the extrapolation from in vitro data to the in situ situation and the interpretation regarding physiological processes must be done with caution.
With respect to the possibilities for cartilage regeneration in the context of tissue engineering of bone and cartilage, the present data open interesting new aspects for optimising the seeding of scaffolds via stimulation of cell proliferation by PGE2 or EP1 ligands; at present, this is under investigation. The analysis of arachidonic metabolites in the growth plate in vitro and in situ presents a wide scope for further investigations with pathophysiological, therapeutic and regenerative end points.
= Dulbecco's modified Eagle's medium
= prostaglandin E receptor
= fetal calf serum
= prostaglandin E2.
We kindly thank Ulrike Hügel for her excellent technical assistance and Bernhard Watzer and Horst Schweer for their valuable help in PGE2 determination.
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