Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E2 (PGE2) by rheumatoid arthritis synovial fibroblasts (RASFs).
Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E2 (PGE2) by rheumatoid arthritis synovial fibroblasts (RASFs).
Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. Fibroblast-like cells from the third or fourth passage were used as RASFs. Expression of adiponectin receptor messenger RNA (mRNA) and protein was detected. PGE2 (converted from arachidonic acid) was measured by enzyme-linked immunosorbent assay (ELISA). Expression of mRNA and protein for cyclooxygenase 2 (COX-2) and membrane-associated PGE synthase 1 (mPGES-1), key enzymes involved in PGE2 synthesis, was detected in RASFs. The effects of RNA interference (RNAi) targeting the adiponectin receptor genes and the receptor signal inhibitors were examined. The influence of adiponectin on NF-κB activation in RASFs was measured with an ELISA kit.
Adiponectin receptors were detected in RASFs. Adiponectin increased both COX-2 and mPGES-1 mRNA and protein expression by RASFs in a time- and concentration-dependent manner. PGE2 production by RASFs was also increased by the addition of adiponectin, and this increase was inhibited by RNAi for the adiponectin receptor gene, or coincubation with the receptor signal inhibitors. Enhancement of NF-κB activation by adiponectin as well as by interleukin-1β was observed in RASFs.
Our findings indicate that adiponectin induces COX-2 and mPGES-1 expression, resulting in the enhancement of PGE2 production by RASFs. Thus, adiponectin may play a role in the pathogenesis of synovitis in RA patients.
Adipose tissue has long been considered to be a structural component of many organs and a site for energy storage. Recently, however, some studies have demonstrated that the major cellular component of adipose tissue, the adipocyte, has the ability to synthesize and release physiologically active molecules such as adiponectin, leptin, and resistin, as well as cytokines such as interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) (1). These molecules are called adipokines or adipocytokines. Several adipokines, such as adiponectin, may play a central role in the regulation of insulin resistance (2), as well as being involved in many aspects of inflammation and immunity (3, 4).
Rheumatoid arthritis (RA) is characterized by extensive inflammation and proliferation of the synovium in various joints. Since proinflammatory cytokines, such as TNFα, IL-1β, and IL-6, play a central role in the pathophysiologic mechanisms of RA, novel strategies that neutralize these cytokines by using monoclonal antibodies or soluble receptors have recently been developed as new treatments for RA (5). Although blockade of these cytokines is beneficial, it is not curative and the effect is only partial, with failure to respond being common (5). Therefore, it seems possible that other proinflammatory cytokines may contribute to the pathophysiology of inflammation in RA patients.
Some previous studies provoked our interest in the role of adiponectin in the pathogenesis of arthritis. For instance, the concentration of adiponectin in RA synovial fluid was shown to be significantly higher than in that of patients with osteoarthritis (OA) (6–8). Moreover, serum and plasma concentrations of adiponectin are higher in RA patients than in healthy controls (7, 9) and are significantly correlated with the C-reactive protein level (9). Ehling et al (10) showed that adiponectin exists in cells from the synovial lining layer and in articular adipose tissue. Furthermore, adiponectin induces proinflammatory molecules, such as IL-6 and matrix metalloproteinase 1, in RA synovial fibroblasts (RASFs). Moreover, adiponectin enhances the expression of monocyte chemoattractant protein 1 and IL-6 by RASFs (11). Recently, Giles et al (12) reported that adiponectin may represent a mechanistic link between low adiposity and increased radiographic damage in RA. The results of these studies suggest that adiponectin might play a role in the pathogenesis of RA.
In the synovial tissue of RA patients, we previously found that proinflammatory cytokines, such as IL-1β, increased the expression of cyclooxygenase 2 (COX-2) and membrane-associated PGE synthase 1 (mPGES-1), resulting in increased production of prostaglandin E2 (PGE2) (13). We also found that PGE2 was a strong enhancer of IL-1β–induced mPGES-1 expression in RASFs (14). In the present study, we examined the effects of adiponectin on these key enzymes that contribute to the inflammatory response of RASFs.
Recombinant human adiponectin, which was composed of 3 isoforms (low, middle, and high molecular weight), was purchased from Biovendor Laboratory Medicine. It was dissolved in deionized water to prepare a stock solution. Recombinant human IL-1β was purchased from R&D Systems and was dissolved in sterile phosphate buffered saline (PBS) containing 0.1% (volume/volume) bovine serum albumin to prepare a stock solution. Mouse anti-human COX-1 monoclonal antibody was purchased from Wako Pure Chemical Industries. Rabbit anti-human COX-2 polyclonal antibody, rabbit anti-human mPGES-1 polyclonal antibody, and rabbit anti-human cytosolic PGES (cPGES) polyclonal antibody were obtained from Cayman Chemical. Rabbit anti-human GAPDH polyclonal antibody, goat anti-human adiponectin receptor 1 (AdipoR1) polyclonal antibody, and goat anti-human AdipoR2 polyclonal antibody were obtained from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, and HRP-conjugated donkey anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories. ECL Western blotting detection reagent was purchased from GE Healthcare UK, and polyvinylidene difluoride membrane (Immobilon-P) was obtained from Millipore. Compound C (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine) was purchased from Merck. MK886 (1-[(4-chlorophenyl)methyl]-3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid, sodium salt) was purchased from Sigma-Aldrich. RPMI 1640 medium, penicillin/streptomycin solution, fetal bovine serum (FBS), and 0.25% trypsin/EDTA were obtained from Invitrogen. PBS was purchased from Takara Shuzo. All other chemicals were purchased from Wako Pure Chemical Industries.
RASFs were prepared from synovial tissue as previously described (15). RA and OA tissue specimens were obtained from patients undergoing total knee replacement surgery who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for RA or OA (16, 17). The protocol for this study was approved by the Toho University Ethics Committee, and all patients gave written consent for the use of their tissue in the present research. Synovial tissue was digested for 2 hours with 0.25% (weight/volume) bacterial collagenase (ImmunoBiological Laboratories) and then was suspended in RPMI 1640 medium with 10% (v/v) FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. The cells were incubated at 37°C in 5% CO2 for several days, after which nonadherent cells were removed. Fibroblast-like adherent cells from the third or fourth passages were used as RASFs. The concentration of RASFs was 2.5 × 106 cells/75-cm2 flask.
Cells were seeded in culture medium containing 10% (v/v) FBS, and total RNA was extracted with an RNeasy Mini kit according to the recommendations of the manufacturer (Qiagen). Reverse transcription was performed with a SuperScript first-strand synthesis system for RT-PCR according to the recommendations of the manufacturer (Invitrogen), with 1 μg of total RNA from the cells as a template. Equal amounts of each reverse-transcribed product were amplified by PCR with HotStar Taq polymerase (Qiagen). The primer sequences and numbers of cycles were as follows: for AdipoR1 (35 cycles), sense 5′-CCCTGACTGGCTAAAGGACA and antisense 5′-CAGTACAGCCGCCTTCTAGG; for AdipoR2 (35 cycles), sense 5′-TTTGGAGCCCATTTTAGAGG and antisense 5′-TCAACCAGCCTATCTGCCCTA; and for β-actin (28 cycles), sense 5′-CCTCGCCTTTGCCGATCC and antisense 5′-GGATCTTCATGAGGTAGTCAGTC. After initial denaturation at 95°C for 15 minutes, PCR involved amplification for a variable number of cycles of 30 seconds at 95°C, 30 seconds at 56°C, and 45 seconds at 72°C, followed by elongation for 5 minutes at 72°C. The amplified complementary DNA (cDNA) fragments were resolved by electrophoresis on a 2% (w/v) agarose gel, and were detected under ultraviolet light using LAS-3000 (Fujifilm) after staining the gel with ethidium bromide.
To evaluate the expression of messenger RNA (mRNA) for AdipoR1, AdipoR2, COX-2, and mPGES-1, real-time PCR was performed using real-time TaqMan technology with a Sequence Detection System model 7000 according to the recommendations of the manufacturer (Applied Biosystems). Cells were cultured under various conditions in medium containing 1% (v/v) FBS, and extraction of total RNA and synthesis of cDNA were performed as described above. The specific probes for AdipoR1, AdipoR2, COX-2, and mPGES-1 were obtained from TaqMan Gene Expression Assay (Applied Biosystems). The ID numbers of the products were Hs00360422_m1 for AdipoR1, Hs00226105_m1 for AdipoR2, Hs00153133_m1 for COX-2, and Hs00610420_m1 for mPGES-1. The threshold cycle was calculated from a standard curve. Expression of the target mRNA was normalized to the expression of β-actin mRNA.
Cells (at a density of 5 × 104/cm2) were cultured under various conditions in medium containing 1% (v/v) FBS. Subsequently, the cells were lysed in Tris buffered saline (TBS) containing 0.1% (w/v) sodium dodecyl sulfate (SDS) for COX and PGES as reported previously (14). For AdipoR1 and AdipoR2, the cells were lysed in Triton lysis buffer containing 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, and a protease inhibitor cocktail (Pierce Biotechnology) as reported previously (18). The protein content of the lysates was determined with the bicinchoninic acid protein assay reagent (Pierce Biotechnology), using bovine serum albumin as the standard. Then cell lysates were adjusted to 10 μg of protein and were applied to SDS polyacrylamide gel (10–15% [w/v]) for electrophoresis. Next, the proteins were electroblotted onto Immobilon-P polyvinylidene difluoride membranes with a semidry blotter (Atto). After the membranes had been blocked in 10 mM TBS containing 0.1% (v/v) Tween 20 (TBST) and 5% (w/v) skim milk, the primary antibody (goat anti-human AdipoR1 antibody, goat anti-human AdipoR2 antibody, rabbit anti-human GAPDH antibody, mouse anti-human COX-1 antibody, rabbit anti-human COX-2 antibody, rabbit anti-human mPGES-1 antibody, or rabbit anti-human cPGES antibody) was added at a dilution of 1:200 (AdipoR1, AdipoR2, GAPDH, COX-1, and COX-2) or 1:500 (mPGES-1 and cPGES) in TBST, and incubated for 1.5 hours. After the membranes had been washed with TBST, the secondary antibody (HRP-conjugated donkey anti-goat antibody, HRP-conjugated goat anti-rabbit antibody, or HRP-conjugated goat anti-mouse antibody) was added (at a dilution of 1:10,000 or 1:5,000 in TBST) and incubation was performed for 1 hour. After further washing with TBST, protein bands were detected with enhanced chemiluminescence Western blotting detection reagents (GE Healthcare UK) using LAS-3000 (Fujifilm).
Cells were plated in 24-well plastic plates (1 × 105/well) and cultured for 18 hours under various conditions in medium containing 1% (v/v) FBS in an atmosphere of 5% CO2. After washing with PBS, 3 μM arachidonic acid (Cayman Chemical) was added to each well. After incubation for 30 minutes, the culture medium was harvested using a syringe and filtered through a 0.22-μm filter (Millipore). Then PGE2 concentrations in the medium were measured by an enzyme-linked immunosorbent assay (ELISA) kit according to the recommendations of the manufacturer (Cayman Chemical). Experiments using RASFs and OASFs were conducted in triplicate wells, and PGE2 concentration was measured in triplicate.
Antiadiponectin antibody was used to neutralize adiponectin as described previously (18). Adiponectin was incubated with mouse antiadiponectin monoclonal antibody (Millipore), mouse monoclonal IgG1 negative control (Millipore), or PBS and Protein G–Sepharose beads (GE Healthcare UK) at 4°C overnight. The supernatant was collected and added to RASFs cultured in 96-well plates (2 × 104/well) for measurement of PGE2 levels in culture medium. After 18 hours of incubation, PGE2 production from arachidonic acid was analyzed as described above.
An RNAi assay was performed to down-regulate the expression of AdipoR1 or AdipoR2 by RASFs. Small interfering RNA (siRNA) for AdipoR1 and AdipoR2 (Stealth RNAi) were purchased from Invitrogen. For gene knockdown experiments, RASFs were plated in 10-cm plastic dishes (3 × 105/dish) in RPMI 1640 medium with 10% (v/v) FBS and cultured for 18 hours. Then the medium was changed to serum-free RPMI 1640 medium, and the cells were transfected with siRNA (10 pmoles/ml) for adiponectin receptors or with control siRNA (10 pmoles/ml; Invitrogen) using Lipofectamine RNAiMAX according to the recommendations of the manufacturer (Invitrogen). After 72 hours, the cells were replated into 35-mm plastic dishes for PCR or into 96-well plastic plates for PGE2 ELISA and receptor protein analyses.
RASFs, which were transfected with siRNA for AdipoR1, AdipoR2, or negative control were plated into 96-well plates (2 × 104/well) for cell-based ELISA (R&D Systems) and cultured for 18 hours. The cells were fixed with 4% formaldehyde for 20 minutes at room temperature. After washing, cells were blocked for 1 hour at room temperature. Cells were incubated overnight at 4°C with primary antibody (anti-AdipoR1 antibody, anti-AdipoR2 antibody, or anti-GAPDH antibody). Alkaline phosphatase–conjugated secondary antibody and HRP-conjugated secondary antibody were added to the wells, and incubation at room temperature for 2 hours was carried out. After incubation, fluorogenic substrates for each secondary antibody were added to the wells. Fluorescence was measured according to the recommendations of the manufacturer. Experiments were performed using triplicate samples from each of 3 patients.
RASFs were incubated without serum for 18 hours, and then were incubated with or without adiponectin (2 μg/ml) or IL-1β (1 ng/ml) for 3 hours. Next the cells were lysed, and nuclear extracts were obtained with a Nuclear Extract Kit according to the recommendations of the manufacturer (Active Motif). These nuclear extracts were diluted and applied to an NF-κB Family Transcription Factor Assay Kit (Active Motif). Nuclear translocation of NF-κB subunits was measured by ELISA using antibodies for each subtype of NF-κB.
Data are expressed as the mean ± SEM. Groups were compared using the Kruskal-Wallis test or Tukey's multiple comparison test. P values less than 0.05 were considered significant.
To determine whether the 2 adiponectin receptors were expressed by RASFs, we performed RT-PCR and Western blotting. Messenger RNA for both adiponectin receptors, AdipoR1 and AdipoR2, was expressed in RASFs from each of 3 RA patients (data not shown), as previously demonstrated (10, 19). RASFs also expressed adiponectin receptor proteins (Figure 1A).
To determine whether adiponectin increased the production of PGE2 from arachidonic acid by RASFs, we measured PGE2 concentrations in the culture medium of RASFs incubated with adiponectin (Figure 1B). We found that adiponectin significantly increased PGE2 production by RASFs in a concentration-dependent manner. The effect of 5 μg/ml of adiponectin was equal to the effect of 1 ng/ml of IL-1β. In OASFs, adiponectin also stimulated PGE2 production, but its effect was weaker. Production of 2,3-dinor-6-keto-PGF1α (a metabolic product of PGI2), PGD2, PGF2α, and thromboxane B2 (a metabolic product of thromboxane A2) by RASFs was not enhanced after adiponectin treatment (data not shown). Adiponectin-induced PGE2 production was inhibited by the presence of antiadiponectin antibody (Figure 1C).
To determine whether adiponectin increased the expression of enzymes related to PGE2 synthesis, we performed Western blotting with selective antibodies for COX-1, COX-2, mPGES-1, and cPGES. As shown in Figure 2, adiponectin increased the expression of COX-2 protein in a concentration-dependent manner. The expression of mPGES-1 protein was also increased by adiponectin, whereas COX-1 and cPGES protein levels were unchanged, as measured by densitometry analyses of the enzyme:GAPDH expression ratio (data not shown).
Figures 3A and B show that adiponectin caused a concentration-dependent increase in the expression of COX-2 and mPGES-1 mRNA, as detected by real-time PCR. As shown in Figures 3C and D, COX-2 and mPGES-1 mRNA expression were both increased by adiponectin treatment in a time-dependent manner. COX-2 mRNA expression was detected after 3 hours of incubation with adiponectin and was maximal at 18 hours; mPGES-1 mRNA expression also peaked after 18 hours of treatment.
To determine whether the induction of PGE2 production by adiponectin occurred via adiponectin receptors, we examined the effect of RNAi with the 2 adiponectin receptors (AdipoR1 and AdipoR2). RASFs were transfected with siRNA for AdipoR1 or AdipoR2, or with negative control siRNA, and then expression of AdipoR1 or AdipoR2 mRNA was detected by RT-PCR (Figure 4A) and real-time PCR (Figure 4B). When cells were seeded in 96-well plates and incubated with adiponectin for 18 hours, PGE2 production by RASFs transfected with the siRNA for AdipoR1 or AdipoR2 was significantly reduced compared with that by RASFs transfected with control siRNA (Figure 4C). The mRNA knockdown of both of the adiponectin receptor genes also decreased adiponectin-induced PGE2 production (Figure 4C).
AdipoR1 and AdipoR2 protein expression in RASFs transfected with siRNA for each receptor were measured using cell-based ELISA. The siRNA for AdipoR1 down-regulated mean ± SEM AdipoR1 protein expression by 34.6 ± 18.8% (n = 3 patients) compared with the negative control, whereas the siRNA for AdipoR2 down-regulated mean ± SEM AdipoR2 protein expression by 8.3 ± 11.3% (n = 3 patients) compared with the negative control. However, these differences were not statistically significant.
The data shown in Figure 4 suggest that both AdipoR1 and AdipoR2 participate in PGE2 production by RASFs exposed to adiponectin. Previous studies have shown that phosphorylation and activation of AMP-activated protein kinase (AMPK) are stimulated by adiponectin via AdipoR1 (20, 21). In the present study, we found that compound C, an inhibitor of AMPK, decreased adiponectin-induced PGE2 production (Figure 5A). Adiponectin has also been shown to enhance peroxisome proliferator–activated receptor α (PPARα) signaling via AdipoR2 (22). We examined the effect of MK886, an inhibitor of PPARα, on adiponectin-induced PGE2 production in RASFs. As shown in Figure 5B, MK886 significantly inhibited adiponectin-induced PGE2 production.
NF-κB is an essential transcription factor involved in the up-regulation of COX-2 (23) and mPGES-1 (24). To determine the association of NF-κB with adiponectin-induced PGE2 production in RASFs, we examined whether adiponectin activated the nuclear translocation of NF-κB. As shown in Figures 6A and B, adiponectin induced nuclear translocation of the p50 and p65 subunits of NF-κB, similar to the effects of IL-1β. However, translocation of the c-Rel, p52, and RelB subunits was not altered by adiponectin treatment (data not shown).
In the present study, we demonstrated that exposure to adiponectin induced the expression of mRNA and protein for COX-2 and mPGES-1, resulting in PGE2 overproduction by RASFs. Addition of antiadiponectin antibody or siRNA for adiponectin receptor gene decreased adiponectin-induced PGE2 production. Recently, we demonstrated that adiponectin stimulates IL-8 production by RASFs and that the culture supernatant of RASFs treated with adiponectin induces chemotaxis (18). These results may help to explain the contribution of adiponectin to inflammation in patients with RA.
With regard to its role in inflammation, physiologic concentrations of adiponectin have been shown to inhibit TNFα–induced adhesion of human monocytic THP-1 cells in a dose-dependent manner. Adiponectin also decreases TNFα-induced expression of vascular cell adhesion molecule 1, endothelial leukocyte adhesion molecule 1 (E-selectin), and intracellular adhesion molecule 1 by human aortic endothelial cells (25). In contrast, adiponectin activates NF-κB, an essential transcription factor for the expression of inflammatory proteins, in a time- and dose-dependent manner in U937 cells (26). These findings suggest that adiponectin might have antiinflammatory and/or proinflammatory properties under different experimental conditions.
Turner et al (27) reported that commercial recombinant adiponectin (Biovendor Laboratory Medicine) contained endotoxin at concentrations of 30 pg/μg of adiponectin. The endotoxin contamination of the adiponectin concentrations used in our study (1–10 μg/ml) can be estimated as 30–300 pg/ml. Picogram levels of lipopolysaccharide did not induce PGE2 production in previous studies using monocytes (28) or RASFs (29). To confirm that the induction of PGE2 by adiponectin is due to adiponectin itself, we conducted an experiment neutralizing adiponectin using antiadiponectin antibody. As shown in Figure 1C, antiadiponectin antibody significantly reduced adiponectin-induced PGE2 production, whereas negative control IgG did not decrease PGE2 production. Therefore, we confirmed that the induction of PGE2 production by recombinant adiponectin was caused by adiponectin itself and not by endotoxin or other contaminants.
The plasma concentration of adiponectin in RA patients and healthy controls has been shown to be ∼10 μg/ml (9). In our experiments, adiponectin (0.5–10 μg/ml) increased PGE2 production from RASFs by enhancing the expression of COX-2 and mPGES-1. However, leptin and resistin (2 other adipokines) did not increase PGE2 production by RASFs at levels up to 100-fold higher (1 μg/ml) (data not shown) than their serum concentrations in RA patients (9, 30). The potency of adiponectin for inducing these enzymes in RASFs was almost equal to that of IL-1β (1 ng/ml). Therefore, adiponectin may have a proinflammatory influence on RASFs in RA patients through induction of PGE2 production.
In our study, adiponectin also induced PGE2 production from OASFs. However, the PGE2 production seemed to be weaker than that from RASFs. Tan et al (11) reported that expression of mRNA for AdipoR1, but not AdipoR2, in RASFs was significantly higher than that in OASFs. This might explain the difference between RASFs and OASFs with regard to the degree of the effect of adiponectin on PGE2 production.
Shibata et al (31) demonstrated that adiponectin induced COX-2–dependent synthesis of PGE2, resulting in the protection of cardiomyocytes against ischemia-reperfusion injury. Yokota et al (32) suggested that adiponectin prevents preadipocyte differentiation via induction of COX-2 expression and the release of PGE2 by stromal preadipocytes. In this study, we showed that treatment of RASFs with adiponectin induced 2 key enzymes related to PGE2 production, which were COX-2 and mPGES-1. Contributions of the PGE2 biosynthesis pathway, including cytosolic phospholipase A2 (33), COX-2 (34), mPGES-1 (35, 36), and EP4 receptor of PGE2 (37), to arthritis in mouse models have been reported, and mice with knockdown of each molecule show amelioration of arthritis compared with wild-type mice. Therefore, adiponectin-induced PGE2 production might be a factor that promotes aggravation of inflammation in RA patients.
Adiponectin has been shown to stimulate RANKL and to inhibit osteoprotegerin expression in human osteoblasts via the MAPK signaling pathway (38). Adiponectin also induces the expression of nitric oxide synthase and matrix metalloproteinases in chondrocytes (39). It has been suggested that adiponectin might play an important role not only as a proinflammatory molecule (such as in its effect on PGE2 production), but also in regulating bone metabolism.
Previous studies have demonstrated that the concentration of adiponectin in the synovial fluid of patients with RA is significantly higher than that in the synovial fluid of patients with OA (6–8) and that serum and plasma concentrations of adiponectin are higher in RA patients than in healthy controls (7, 9). These findings may indicate that adiponectin plays a role as a proinflammatory cytokine in RA. However, some studies have shown that the serum concentration of adiponectin in RA patients increases by ∼20% during anti-TNFα therapy (40–43). The mean adiponectin concentration detected before anti-TNFα therapy in these studies was higher than that in healthy controls in observational studies (7, 9). The reason the already high serum adiponectin concentration in RA patients increased further during anti-TNFα therapy cannot be explained at present. It is possible that adiponectin is not directly related to inflammation caused by TNFα.
In this study, we detected expression of protein and mRNA for 2 adiponectin receptors (AdipoR1 and AdipoR2) in RASFs, as has previously been shown in RASFs (10, 19) and in various other tissues (20). In addition, adiponectin-induced PGE2 synthesis was reduced by siRNA targeting of both adiponectin receptor genes. Reduction of PGE2 production by double knockdown of AdipoR1 and AdipoR2 genes showed almost the same results as knockdown of the individual receptor genes. These results demonstrate that adiponectin-induced PGE2 production was mediated, at least in part, by these adiponectin receptors in RASFs. Pathways other than AdipoR1 and AdipoR2 might exist in adiponectin-induced PGE2 production in RASFs. Although mRNA expression was reduced almost completely by transfection of siRNA for the target gene, the inhibitory effect of each receptor on protein expression was not significant in our experimental condition. Additional studies of receptor proteins are needed.
After adiponectin combines with AdipoR1, activation of AMPK occurs (20, 21). Therefore, we investigated the effect of compound C, an inhibitor of AMPK, on PGE2 production by RASFs stimulated with adiponectin. Adiponectin-induced PGE2 production was significantly decreased by compound C, suggesting that this PGE2 production at least involved signal transduction via AdipoR1. Yamauchi et al (22) demonstrated that the PPARα signaling pathway existed downstream of AdipoR2. In our study, MK886, an antagonist of the PPARα pathway, reduced the PGE2 production that was induced by adiponectin treatment in RASFs.
NF-κB is known to play a central role in the regulation of inflammatory reactions in various cells (44). With regard to PGE2 production by RASFs, NF-κB is an important factor in the transcriptional regulation of COX-2 (23). In the present study, adiponectin activated the translocation of NF-κB in RASFs. This suggests that adiponectin induced COX-2 expression in RASFs via activation of NF-κB translocation. Since the mPGES-1 promoter does not contain an NF-κB–responsive element, expression of mPGES-1 might be induced indirectly after activation of NF-κB (45), unlike COX-2. An increase in PGE2 production by COX-2 activation after adiponectin treatment could lead to autocrine enhancement (14) of mPGES-1 expression.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kawai had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Kusunoki, Kawai.
Acquisition of data. Kusunoki, Kitahara, Tanaka, Kaneko, Suguro.
Analysis and interpretation of data. Kusunoki, Kojima, Endo, Kawai.
We thank Sonoko Sakurai for secretarial assistance.