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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Feb 1, 2010.
Published in final edited form as:
PMCID: PMC2659545
NIHMSID: NIHMS87449

PGE2 And Its Cognate EP Receptors Control Human Adult Articular Cartilage Homeostasis and Are Linked to the Pathophysiology of Osteoarthritis

Abstract

Objective

To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes.

Methods

Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonist, antagonist of EP receptors and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and PI3k/Akt signaling were assessed by real-time PCR and western blotting, respectively, using a monolayer cell culture model.

Results

Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor (i) suppresses proteoglycan accumulation and synthesis, (ii) suppresses aggrecan gene expression, (iii) does not appreciably affect expression of matrix-degrading enzymes; and (iv) decreases the collagen II:I ratio. EP2 and EP4 receptors are expressed at higher levels in knee compared to ankle cartilage, and in a grade-dependent fashion. PGE2 titration combined with IL-1 synergistically accelerates expression of pain-associated molecules such as inducible nitric oxide synthase (iNOS) and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 agonist inhibits activation of Akt that is induced by insulin-like growth factor (IGF-1).

Conclusion

PGE2 exerts an anti-anabolic effect on human adult articular cartilage in vitro, and EP2/4 receptor antagonists may represent effective therapeutic agents for the treatment of osteoarthritis.

Introduction

Osteoarthritis (OA) is a disabling disease that is highly prevalent in elderly patients (1). It is a complex process involving a combination of cartilage degradation, reparation, and inflammation, and the pathogenesis of OA is not yet fully understood. Normal articular chondrocytes maintain a dynamic equilibrium between synthesis and degradation of extracellular matrix (ECM) components, which includes type II collagen fibrils surrounding and restraining large, hydrated aggregates of the proteoglycan aggrecan, allowing normal cartilage to function as a natural ‘shock absorber’ and withstand compressive loads (2). However, in OA there is a disruption of the matrix equilibrium leading to progressive loss of cartilage tissue. Chondrocyte metabolism is unbalanced due to excessive production of catabolic factors, including matrix metalloproteinases (MMPs), aggrecanases (ADAMTS), and other cytokines and growth factors released by chondrocytes that aid in the destruction of proteoglycans and the ECM (36). Recently, synovial inflammation has been found to contribute to the pathogenesis of OA via the release of catabolic and pro-inflammatory mediators that alter matrix homeostasis (7). Studies have shown increased expression of pro-inflammatory proteins in human OA joint cartilage compared to normal cartilage (8), and others have revealed a correlation between increased expression of inflammatory mediators and degradation of cartilage matrix macromolecules (9).

Prostaglandins are pro-inflammatory lipid mediators locally increased in the synovial membrane and synovial fluid of patients with OA (8). The role of prostaglandins in the metabolism of articular cartilage is still a matter of debate. Some reports indicate that prostaglandins participate in the destruction of articular cartilage by degrading cartilage ECM (10, 11), while others show that they promote chondrogenesis and terminal differentiation (12, 13). The opposing biological roles attributed to these compounds is a direct reflection of the molecular complexity of prostaglandins and their unique cognate receptors (14).

Prostaglandin E2 (PGE2) is one of the major catabolic mediators involved in cartilage degradation and the progression of OA (1517). PGE2 is a prostanoid derived from arachidonic acid that is released from membranes by phospholipase A2. In the initial step in prostaglandin biosynthesis, arachidonic acid is metabolized by cyclooxygenase (COX) activity to form prostaglandin H2 (PGH2), which is subsequently metabolized by PGE synthase to form PGE2 (18). Previous studies have shown that PGE2 is involved in inflammation, apoptosis, and angiogenesis (19, 20). However, the precise biological role of PGE2 in articular cartilage is still unclear. PGE2 has been associated with structural changes seen in OA tissues (21) and characterized as a catabolic mediator in cartilage homeostasis (10, 1517). In contrast, others have demonstrated an anabolic effect of PGE2 in articular cartilage (22, 23). The PGE2-mediated signal is transduced by four different EP receptor subtypes (EP1-EP4), which cause distinct and sometimes opposing effects on cell metabolism depending on the cell/tissue types (23), and, at this point, it is not clear which of these EP receptor subtypes contribute to the pathogenesis of OA.

Our current studies demonstrate the pathophysiologic links between PGE2 and OA. We also identify which specific EP receptors may be responsible for the biological effect of PGE2 in human articular cartilage, and we elucidate which of these receptors may contribute to the generation of OA symptoms via stimulation of nociceptive pathways in arthritic joints.

Materials and Methods

Synovial Fluid Analysis

Human synovial fluid was aspirated within 24 hours of death from the knee joints of asymptomatic human organ donors with no history of joint diseases (N=9, 45–60 years old, grade 0/1 degeneration) using approved institutional protocols (the Gift of Hope Organ & Tissue Donor Network). Synovial fluid was also obtained with appropriate consent from OA (N=8, 50–65 years old, advanced OA requiring surgery), and RA (N=18, 50–65 years old) patients from the Rush University Section of Rheumatology who were undergoing diagnostic or therapeutic arthrocentesis. The level of PGE2 was measured by ELISA (R&D System; standard curve units of pg/ml) following the instructions provided by the manufacturer.

Chondrocyte Isolation and Culture

Human articular cartilage from knee or ankle was obtained from tissue donors through the Gift of Hope Organ and Tissue Donor Network. Each donor specimen was graded for gross degenerative changes based on a modified version of the 5-point scale of Collins (24). The cells were released by enzymatic digestion as previously described (25, 26). Alginate beads and monolayers were made for long-term and short-term analysis, respectively. For alginate bead culture, grade 0 or 1 knee chondrocytes were isolated and re-suspended in 1.2% alginate, and beads were formed using a CaCl2 solution, as previously described (27). Cells were treated with PGE2 (Sigma, St. Louis, MO), EP2 agonist (Butaprost, Cayman Chemical, Ann Arbor, Michigan), EP3 agonist (Sulprostone, Sigma) and IL-1 (Amgen, Thousand Oaks, CA), a well-known catabolic cytokine used for control.

For monolayer culture, isolated chondrocytes were counted and plated onto 12-well plates at 8×105 cells/cm2 as previously described (5, 25). Cells were treated with PGE2, basic fibroblast growth factor (bFGF), IGF-1 (Austral Biologicals, San Ramon, CA), EP2 agonist, EP3 agonist, EP1/2 antagonist AH6809 (Sigma), EP1 antagonist SC19220 (Sigma), and IL-1. Cells were harvested at different time points and subjected to western blotting as described below. Cells were treated for 24 hours before total RNA harvesting.

Reverse Transcription and Real-Time Polymerase Chain Reaction

Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) following the instructions provided by the manufacturer. Reverse transcription (RT) was carried out with 1 µg total RNA using ThermoScript TM RT-PCR system (Invitrogen) for first strand cDNA synthesis. For real-time PCR, cDNA was amplified using MyiQ Real-Time PCR Detection System (Bio-Rad Hercules, CA). Relative mRNA expression was determined using the ΔΔCT method, as detailed by manufacturer (Bio-Rad). GAPDH was used as internal control. The deviations in samples represent three different donors in three separate experiments. The primer sequences and their conditions for use are summarized in Table 1.

Table 1
Primer sequence for RT-PCR

Dimethylethylene Blue (DMMB) assay for Proteoglycan Production and DNA Assay for Cell Numbers

At Day 21 of culture, the alginate beads were collected and processed for proteoglycan assays using the DMMB assay, as previously described (27). The proteoglycan levels measured in the cell-associated matrix (CM) were quantified per DNA to give the total amount of proteoglycans produced and retained in the alginate beads per cell (25). Using PicoGreen (Molecular Probes, Carlsbad, CA), cell numbers were determined by assay of total DNA in the cell pellets, as previously described (25).

35S-sulfate Incorporation into Newly-Synthesized Proteoglycans

At Day 7 of culture in alginate, the medium was removed and replaced by fresh medium. One hour later, this medium was replaced with fresh medium containing [35S]-sulfate at 20 µ;Ci/ml (Amersham Corp, Arlington Heights, IL). After 4 hours incubation, the labeling medium was removed and beads were rinsed and dissolved to separate out the CM and digested with papain (20 µg/ml in 0.1M sodium acetate, 0.05M EDTA, pH 5.53). Sulfate incorporation into proteoglycans was measured using the Alcian blue precipitation method (28). All samples were analyzed in duplicate and normalized for DNA content using Hoechst 33258 as previously described (28).

Western blotting

Cell and tissue lysates were prepared using modified RIPA buffer as previously described (6). Protein was resolved by 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane for western blot analyses as described previously (5).

Statistical Analysis

Analysis of variance was performed using StatView 5.0 software (SAS Institute, Cary,NC). P values less than 0.05 were considered significant.

Results

PGE2 is Upregulated in Arthritic Joints and Suppresses Proteoglycan in Human Articular Chondrocytes via Inhibition of Aggrecan Gene Expression

Synovial fluid collected from knee joints of asymptomatic individuals (normal), or RA and OA patients were subjected to ELISA to assess endogenous levels of PGE2 (Fig 1A). Compared to normal joints, the levels of PGE2 were at least two times higher in patients with OA and RA, with a greater effect seen in OA synovial fluid, suggesting a pathologic role of PGE2 in human knee joints.

Fig 1
Upregulated PGE2 in Human Arthritic Joints: Suppression of Proteoglycan Accumulation and Synthesis via Inhibition of Aggrecan Expression

To assess the biological impact of PGE2 on human articular cartilage, chondrocytes isolated from knee (grade 0–1) cartilage were encapsulated in 3-dimensional alginate beads in the presence or absence of PGE2 and analyzed for proteoglycan accumulation and synthesis (Fig 1B). In our initial dose-dependent studies (data not shown), >100 nM PGE2 significantly decreased (p<0.05) the amount of proteoglycan accumulation per cell without noticeable changes in cellular proliferation and survival (data not shown). Therefore, PGE2 was used at a 1 µM concentration in subsequent in vitro studies to maximize the biological effect (Fig 1B:[DMMB/DNA]). Further, to determine if the reduction in proteoglycan accumulation was mediated by PGE2-dependent inhibition of synthesis, the incorporation of 35S-sulfate by articular chondrocytes into proteoglycans was quantified. The results show that proteoglycan synthesis was indeed suppressed in the presence of PGE2 (Fig 1B:[Cmp/DNA]), suggesting that PGE2 contributes to imbalanced cartilage homeostasis.

Using real-time PCR, we investigated whether PGE2 suppresses aggregan gene expression (Fig 1C) and/or matrix-degrading enzyme expression (Fig 1D), thereby accelerating proteoglycan depletion. IL-1 and bFGF were used in parallel as catabolic experimental controls (5). Compared to control (untreated), the presence of PGE2 (1 µM) significantly decreased the aggrecan mRNA level by 60% (Fig 1C), and this suppression was similar to that seen after treatment with IL-1 and bFGF. Surprisingly, the presence of PGE2 failed to upregulate matrix-degrading enzymes, including MMP-13, MMP-1, MMP-14, MMP-3, ADAMTS4, and ADAMTS5 (29) (Fig 1D) while stimulation with both bFGF (100 ng/ml) and IL-1 (10 ng/ml), well-known stimulators of cartilage-degrading enzyme expression (5, 6), significantly increased expression of these genes. Taken together, these results demonstrate that PGE2 inhibits proteoglycan production mainly by downregulating the expression of matrix components (e.g., aggrecan) with minimal effects on the expression of cartilage-degrading enzymes.

Expression of EP Receptors in Human Adult Articular Cartilage

To identify which of the four EP receptors are expressed in normal human articular cartilage, we performed real-time PCR experiments using total RNA extracted from grade-matched (0 or 1) and age-matched (25–40 years old) knee and ankle articular cartilage. The EP2 and EP4 receptors were most prominently expressed in both knee and ankle cartilage (Fig 2A), and knee cartilage expresses strikingly higher mRNA levels of the EP2 and EP4 receptors compared to ankle cartilage. Based on these findings, we investigated the pathogenic links of EP2 and EP4 receptors in the progression of OA by monitoring their expression patterns in progressively degenerated (grades 0/1, 2 and 3) or OA cartilage (Fig 2B). Age-matched (25–40 years old) ankle cartilage (grades 0/1, 2 and 3) and knee cartilage (grade 1 and OA) were analyzed by real-time PCR analysis. In ankle cartilage, grade 2 tissue exhibits a >2-fold increase in the basal expression of EP2 and EP4 receptors compared to grades 0/1 cartilage, and this induction is grade-dependent as grade 3 cartilage exhibits a >10-fold increase of both EP2 and EP4 expression, compared to control (Fig 2B:[ankle]). Similar grade-dependent results were obtained using knee cartilage (Fig 2B:[knee]). Western blot analysis (Fig 2C) corroborates these observations, revealing increased EP2 and EP4 protein expression in OA compared to grade 1 knee cartilage.

Fig 2
The Expression of EP Receptors in Human Adult Articular Cartilage

The Biological Effects of PGE2 are Mediated by Activation of the EP2 Receptor

In arthritic joints (both OA and RA), expression of catabolic factors such as PGE2 (see Fig 1A), bFGF (4), and IL-1 (30) are significantly increased. Therefore, we determined which of the four EP receptors are most responsive to these exogenous stimuli in human articular chondrocytes. Cells (knee G:1) in monolayer were challenged with PGE2 (1 µM), bFGF (100 ng/ml) and IL-1 (10 ng/ml) for 24 hrs and the stimulatory effects on expression of EP receptors were analyzed by real-time PCR (Fig 3A). The EP receptors most responsive to all exogenous catabolic stimuli were the EP2 and EP4 receptors. More specifically, the presence of PGE2, bFGF and IL-1 stimulated EP2 receptor expression, respectively, by 2-, 3.5-, and 4.8-fold, while EP4 receptor expression was increased, respectively, by 3-, 5-, and 3-fold, compared to untreated controls. In contrast, changes in the expression of the EP1 or EP3 receptor were negligible.

Fig 3
The Biological Effects of PGE2 are Mediated by EP2 Receptor Activation

Based on these observations, we further analyzed the contribution of the EP2 receptor on proteoglycan accumulation (Fig 3B), aggrecan gene expression (Fig 3C), and collagen production (Fig 3D). Human adult articular chondrocytes encapsulated in 3-dimensional alginate beads were cultured in the presence or absence of butaprost (an EP2 receptor-specific agonist), sulprostone (an EP3 receptor-specific agonist), or IL-1 as positive control for 21 days. The treatment of cells with butaprost (1 µM) significantly decreased (p<0.05) the amount of proteoglycan accumulation per cell without a noticeable change in cellular proliferation and survival (data not shown), and this suppressive effect was similar to that seen after stimulation with PGE2 and IL-1 (Fig 3B). In contrast, activation of EP3 receptor by sulprostone had no significant effect on proteoglycan accumulation. Further, real-time PCR results demonstrated that treatment of chondrocytes with butaprost led to a dose-dependent decrease in aggrecan gene expression (Fig 3C). Together, these results suggest that the PGE2-mediated suppression of proteoglycan production and aggrecan gene expression occurs via the EP2 receptor, and this receptor may be an important initiator of the PGE2 signaling pathway in human articular cartilage.

Ratio of collagen type II relative to type I is an important factor for the proper function of mature articular cartilage. In this study, we investigated whether PGE2 and activation of EP receptors have a biological influence on modulating this ratio. Our real-time PCR results show that both PGE2 and butaprost decrease levels of collagen I and collagen II (data not shown) compared to control (untreated). Interestingly, the relative expression ratio of collagen type II compared to collagen type I was decreased by butaprost, and to a lesser extent by PGE2 (Fig 3D), suggesting a dual role for PGE2-activation of EP2 receptor: suppression of proteoglycan synthesis/production coupled with a decrease in the collagen II:collagen I ratio.

Synergism by the Combination of PGE2 and IL-1 on IL-6 and iNOS Expression

Both nitric oxide (NO) and IL-6 are known to play an important role in cartilage metabolism and may mediate pain signaling (3133). We therefore examined the relationship between PGE2, IL-6 and iNOS, a gene responsible for the production of NO, in human articular chondrocytes (knee G:1). After co-stimulation with PGE2 and IL-1, the induction of IL-6 mRNA levels was dramatic, showing synergistic upregulation by a factor of 33 compared to control, significantly higher than stimulation with either factor alone (Fig 4A). Synergistic augmentation of iNOS was also observed when cells were stimulated with a combination of PGE2 and IL-1, despite a modest reduction in iNOS mRNA expression after treatment with PGE2 alone (Fig 4B). These striking results demonstrate a robust biological correlation between PGE2 and the pain mediators IL-6 and NO that may reflect regulatory interplay between PGE2 and pain pathways in articular cartilage.

Fig 4
Synergistic with IL-1, PGE2 Increases IL-6 and iNOS Gene Expression

PGE2 suppresses the PI3k/Akt pathway via the EP2 receptor

To characterize the intracellular signaling pathways that modulate proteoglycan expression in response to PGE2, we determined the effects of PGE2 on Akt phosphorylation, as stimulation of the PI3k/Akt pathway is required for production of proteoglycan by chondrocytes in response to insulin-like growth factor-1 (IGF-1) (38). When given exogenous PGE2, various cell types have different effects on the PI3k/Akt pathway via either an upregulation (34, 35) or downregulation (36, 37) of Akt phosphorylation. Thus, we elucidated whether the PGE2-mediated proteoglycan loss is associated with inhibition of PI3k/Akt pathway, and if so, whether the EP2 receptor is responsible for the biological consequences. Our western blot results showed that PGE2 inhibits Akt phosphorylation in <5 minutes without affecting total Akt levels (Fig 5A). Stimulation of the EP2 receptor (butaprost), but not EP3 receptor (sulprostone), also decreases phosphorylation of the Akt pathway (Fig 5B). A combination of PGE2 with an EP2 antagonist, but not an EP1 antagonist or EP4 antagonist, rescued the PGE2-induced suppression of the Akt pathway (Fig 5B). Further, co-incubation of cells with PGE2 in the presence of IGF-1 revealed that PGE2 inhibits not only basal levels of Akt, but also the IGF-1-activated Akt pathway which is required for chondrocytic anabolism by IGF-1 (38). This PGE2-mediated attenuation of the IGF-1-induced Akt pathway is dose-dependent (Fig 5C). Collectively, these results suggest that PGE2-mediated suppression of the PI3k/Akt pathway occurs via the EP2 receptor and may play a role in the suppression of proteoglycan production.

Fig 5
PGE2 suppresses the PI3k/Akt pathway

Discussion

This study reveals a significant correlation between arthritic joint diseases and PGE2-responsive signaling pathways in human articular cartilage. We demonstrate that PGE2 functions anti-anabolically in articular cartilage by suppressing aggregan synthesis and total proteoglycan accumulation, while only minimally stimulating the expression of cartilage-degrading enzyme expression (MMPs, ADAMTS). Equally important, this study provides the first evidence that the EP2 receptor is a major mediator of PGE2-induced suppression of proteoglycan accumulation in human adult articular chondrocytes. Signaling through the EP2 receptor also decreases the collagen type II:I ratio to render the ECM less chondrocytic and more fibroblast-like. Furthermore, PGE2 suppresses PI3k/Akt signaling via the EP2 receptor, which may be associated with PGE2-dependent suppression of aggrecan expression. We also provide support for the novel mechanistic concept that PGE2 may modulate pain generation in arthritic joints by upregulating IL-6 and iNOS expression in human articular cartilage.

The biological activities of PGE2 in articular cartilage have sparked controversy over its precise role in matrix metabolism. Depending on the experimental system tested and the receptors utilized, PGE2 has been found to exert both anabolic and catabolic effects on articular cartilage. For example, PGE2 exerts chondroprotective effects in resting zone chondrocytes (13), human synovial fibroblasts (22), and at low concentrations in human OA explants (39), and has been found to downregulate matrix-degrading enzymes (MMP-1) in human chondrocytes (40). In contrast, other studies suggest that PGE2 elicits a major catabolic response that perturbs cartilage homeostasis (10, 15, 16). During inflammatory states, elevated production of PGE2 causes cartilage resorption by decreasing cellular proliferation, inhibiting aggrecan synthesis, and potentiating the effects of other inflammatory factors such as IL-1 (9, 16, 41). PGE2 has also been correlated with increased MMP production in various tissues, including human articular chondrocytes (42) and OA cartilage explants (43). The results from our study suggest an anti-anabolic role of PGE2 in human adult articular chondrocytes in vitro via downregulation of aggrecan gene expression and proteoglycan accumulation and synthesis, supporting findings of Hardy and colleagues, who found that PGE2 production from human synovial tissue corresponds with decreased proteoglycan accumulation (15). In addition, we suggest that PGE2 does not modulate expression of representative cartilage-degrading enzymes (e.g., MMP-13, -1, -14, -3, as well as ADAMTS4 and -5), and instead exerts its effects primarily by inhibiting aggrecan biosynthesis.

The actions of PGE2 depend on the expression of several distinct EP receptor subtypes on the cell surface, eliciting either catabolic or anabolic responses on cartilage homeostasis (44). EP1 receptors have been found to increase differentiation in growth plate chondrocytes (45), while EP2 and 4 receptors have been associated with both anabolic and catabolic effects in cartilage (44, 46). Aoyoma and colleagues reported that EP2 and EP3 are highly expressed in human and mouse articular chondrocytes, and PGE2 signals through EP2 to promote cell growth (14). However, based on our results, we conclude that EP2 and EP4, rather than EP3, are most abundantly expressed in human articular cartilage. This difference may perhaps be attributed to age variations between cartilage samples as we have used adult cartilage samples from normal and degenerative articular cartilage, while Aoyoma and colleagues studied normal cartilage from four individuals of varying ages (6, 10, 39 and 69 years of age). Thus, while EP2 and EP4 were highly expressed in our cartilage samples, EP3 was only minimally expressed but may be more prevalent in cartilage of a younger age.

Another intriguing finding of our study is that the EP2 receptor plays a pivotal biological role in articular cartilage homeostasis. EP2, which functions in an auto- and/or paracrine manner, transduces the PGE2 signal to suppress aggrecan expression in human articular chondrocytes. In addition, this receptor is involved in the PGE2-mediated decrease in collagen type II versus type I ratio in human cartilage. During the progression of OA in arthritic tissues, a decrease in this ratio is linked to perturbations in cartilage homeostasis (47), indicating a pathogenic role for ECM replacement through collagen I. This idea is consistent with findings suggesting that bFGF stimulates cell proliferation in cartilage tissue and decreases the collagen type II:I expression ratio (unpublished data). Replacement of collagen type II by type I may lead to formation of fibrocartilage rather than the stronger, more durable hyaline cartilage of a healthy joint. Our current results indicate that inhibition of PGE2/EP2-receptor signaling may be beneficial for therapy by antagonizing the suppression of aggrecan production and avoiding collagen substitution, thereby preserving stronger hyaline cartilage and mitigating its degeneration.

Our data reveal striking differences in both basal and PGE2-induced expression of the EP2 and EP4 receptors between knee and ankle cartilage (age- and grade-matched). Because of the long-standing clinical observation that some joints (e.g., knee) are more susceptible to OA than others (e.g., ankle) (1), and that EP2 and EP4 are more abundant in the knee than the ankle, we suggest that the PGE2/EP2 and/or PGE2/EP4 signaling pathways may be clinically involved in the onset and progression of OA. Further, we suggest that the catabolic activity of PGE2 in articular cartilage may be biologically linked to pain symptoms associated with OA in human joints. Pain pathways have been linked to increased levels of IL-6 and nitric oxide (NO) in mammalian knee joints (32, 33). Indeed, our studies show that the expression of both IL-6 and iNOS is synergistically increased by PGE2 and IL-1. Thus, our results indicate that PGE2 may participate in the generation of pain symptoms in human OA via activation of its cognate EP2 and EP4 receptors, leading to upregulation of both IL-6 and iNOS.

We have also shown that stimulation of human articular chondrocytes with PGE2 suppresses Akt phosphorylation, which may be associated with decreased proteoglycan accumulation (38). Our data provide the first experimental evidence that exogenous PGE2 inhibits the phosphorylation of Akt via the EP2 receptor in human articular chondrocytes. Taken together with the findings of Starkman and colleagues (38), we suggest that PGE2–mediated activation of the EP2 receptor blocks Akt phosphorylation and subsequently suppresses proteoglycan synthesis. This inhibition can be effectively rescued by an EP2 antagonist, revealing the potential use of EP2 antagonists in the prevention of proteoglycan depletion found in arthritic cartilage.

One important limitation of this study must be taken into account. The present study uses concentrations of PGE2 in the micromolar range, and these concentrations are considerably higher than those found in endogenous samples in vivo (nanomolar range). For example, the endogenous range of PGE2 concentrations in synovial fluid measured in previous studies by ELISA varies from 10 pg/ml to 700 pg/ml (~20 nM) (48, 49). However, for in vitro studies, we and others (35, 40) used PGE2 concentrations significantly higher than reported physiological concentrations. These differences suggest that the currently available in vitro cell culture experimental models for OA apparently can not yet faithfully recapitulate biological responses with physiological doses of agents detected in synovial fluid. Several parameters may contribute to the necessity to use agents in excess of their physiological concentration in vitro. For example, like cytokines and growth factors, PGE2 operates in concert with other factors to exert its biological effects, and absence of these factors in vitro may diminish its efficacy. Alternatively, PGE2 effects may be masked in vitro by secreted inhibitory components that accumulate in the culture media. It is also possible that the specific activity of the exogenously administered PGE2 is less active than the same compound present endogenously in synovial fluid. Regardless of these possibilities, however, the concentration used in the current studies yields informative results and permits meaningful interpretations that increase our understanding of the biological effect of PGE2 and its cognate EP receptors in both in vitro and ex vivo cell culture systems.

In summary, we have defined the role of PGE2 as a potent anti-anabolic factor at a dose of 1 µM in human adult articular cartilage via the suppression of proteoglycan synthesis and aggrecan gene expression. EP2 and EP4 are the predominantly expressed receptors in human articular chondrocytes and appear to be responsive to exogenous stimuli such as PGE2 and other catabolic cytokines, suggesting that these receptors may be important signaling initiators of the PGE2-signaling cascades and may serve as a potential target for therapeutic regimens aimed at preventing progression of arthritic disease in the future. Our findings also provide one plausible mechanism for why certain joints (knees) are more susceptible to OA than others (ankles), as EP2/4 receptors are highly upregulated in knee cartilage compared to ankle cartilage. We further propose that PGE2 may mediate pain pathways in articular cartilage via a synergistic stimulatory effect along with the pro-inflammatory cytokine IL-1 on both IL-6 and iNOS expression. Further investigation should be pursued to help gain a better understanding of the specific signaling cascades governing the complex interactions between PGE2 and EP receptors in human articular chondrocytes both in vitro and in vivo.

Acknowledgements

We would like to thank the tissue donors, Dr. Arkady Margulis, and the Gift of Hope Organ and Tissue Donor Network for tissue samples. We thank Dr. Joel Block (Section of Rheumatology, Rush) for human synovial fluids collected from OA and RA patients. We also thank the National Cancer Institute (NCI) for supporting this study by providing bFGF. This work was supported by NIH RO1AR053220 (HJ Im), Arthritis Foundation (HJ Im), National Arthritis Research Foundation (HJ Im), and Falk Foundation.

List of abbreviations

PGE2
Prostaglandin E2
COX
cyclooxygenase
IL-1
interleukin 1
IL-6
interleukin 6
bFGF
basic fibroblast growth factor
IGF-1
insulin-like growth factor-1
ECM
extracellular matrix
MMPs
matrix metalloproteinases
ADAMTS
a disintegrin and metalloproteinase with thrombospondin motifs
NO
nitric oxide
iNOS
inducible nitric oxide synthase
DMMB
Dimethylethylene Blue
CM
cell-associated matrix

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