International Immunopharmacology 22 (2014) 204–208

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International Immunopharmacology

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Involvement of glucocorticoid receptor activation on anti-inflammatoryeffect induced by peroxisome proliferator-activated receptor γ agonistin mice

Atsuki Yamamoto, Hiroki Kakuta, Yukio Sugimoto ⁎Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan

⁎ Corresponding author. Tel.: +81 86 251 7940; fax: +E-mail address: sugimoto@pharm.okayama-u.ac.jp (Y

http://dx.doi.org/10.1016/j.intimp.2014.06.0281567-5769/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 February 2014Received in revised form 4 June 2014Accepted 13 June 2014Available online 27 June 2014

Keywords:Glucocorticoid receptorPeroxisome proliferator activated receptor γRetinoid X receptorInflammationCarrageenan

Glucocorticoids are effective anti-inflammatory agents widely used for the treatment of acute and chronicinflammatory diseases. Recent in vitro studies have proposed that glucocorticoid receptor (GR) activation isinvolved in peroxisome proliferator-activated receptor γ (PPARγ) agonist-induced effects. In this study, toexamine the involvement of the GR in PPARγ agonist- and retinoid X receptor (RXR) agonist-mediated anti-inflammatory effects in vivo, we tested the anti-inflammatory effects of dexamethasone (a GR agonist) withpioglitazone (a PPARγ agonist) or 6-[N-ethyl-N-(3-isopropoxy-4-isopropylphenyl)-amino] nicotinic acid (NEt-3IP; an RXR agonist) by using an experimental model of carrageenan-induced inflammation. We also evaluatedthe effects of a GR antagonist on PPARγ agonist- or RXR agonist-induced anti-inflammatory effects. Resultsshowed that the GR antagonist RU486 reduced the anti-inflammatory effects of GR or PPARγ agonists but notthose of the RXR agonist. In addition, combinations of GR and PPARγ agonists or GR and RXR agonists had noeffect on carrageenan-induced paw edema. Moreover, the PPARγ antagonist GW9662 and RXR antagonist 6-[N-4-(trifluoromethyl)-benzenesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)-amino] nico-tinic acid (NS-4TF) had no effect on the anti-inflammatory effect of the GR agonist dexamethasone. Therefore,it is suggested that GR activation in vivo does not play a direct role in PPARγ/RXR heterodimer signaling. Incontrast, pioglitazone showed a partial anti-inflammatory effect via GR activation. These data provide evidencefor the pro-inflammatory activity of pioglitazone.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Glucocorticoids are effective anti-inflammatory agents widely usedfor the treatment of inflammatory and autoimmune diseases [1,2].Release of endogenous steroids, produced under the control of thehypothalamus-pituitary-adrenal-axis, also represents a physiologicalmechanism involved in the homeostatic control of inflammatoryprocess development [3,4]. Activated glucocorticoid receptor (GR) caninteract with other transcription factors such as nuclear factor kappa B(NF-κB), an important player in the inflammatory process, byprotein-to-protein interaction. Multiple mechanisms are involvedin glucocorticoid-mediated anti-inflammatory activity in additionto direct GR/NF-κB interaction. These mechanisms include upregula-tion of inhibitory kappa B (IκB) and leucine zipper, which are the 2 pro-teins capable of binding to NF-κB and inhibiting its activation [5–7].Because of the inhibition of NF-κB, a number of inflammatory para-meters are blocked, including cytokine production, cell migration,cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS)activation, and tissue damage [8,9].

81 86 251 7926.. Sugimoto).

The peroxisome proliferator-activated receptor γ (PPARγ) forms aheterodimeric complex with the retinoid X receptor (RXR) and acts asa transcriptional regulator of genes via cross-talk with the DNA bindingcomplex [10–12]. Studies have reported that PPARγ is expressed inmonocytes andmacrophages; therefore, there is a great deal of interestin the involvement of PPARγ in inflammatory processes [13–15].Previous studies have shown that PPARγ agonists effectively inhibitinflammatory responses. Moreover, Diab et al. reported that an endo-genous PPARγ agonist (15-deoxy-Δ12,14-PGJ2) and an RXR agonist (9-cisretinoic acid)weakly inhibited nitric oxide (NO) production bymicroglialcells when used alone but strongly when used in combination [16].

Johnson et al. also showed that dexamethasone augmented osteo-blastic differentiation induced by the PPARγ agonist thiazolidinedione[17]. In addition, Iarenti et al. reported that the PPARγ agonistrosiglitazone enhanced GR nuclear translocation [18]. Moreover, theGR antagonist RU486 reduced the inhibitory effect of rosiglitazone onbacterial endotoxins, lipopolysaccharide (LPS)-mediated interleukin-6(IL-6), and iNOS expression. In contrast, Johnson et al. describedthat thiazolidinedione-induced alkaline phosphatase activity wassuppressed by dexamethasone, while thiazolidinedione potentiatedglucocorticoid-mediated transcription [17]. Thus, reports on the effectsof coadministration of PPARγ agonists and GR agonists are inconsistent.

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Fig. 1. Anti-inflammatory effect of GR agonist, PPARγ agonist, and RXR agonist. (A) Dose-dependence of GR agonist, dexamethasone on carrageenan-induced paw edema in mice.Dexamethasone was orally administrated 3 h before carrageenan-injection at doses of0.1, 0.3, and 1 mg/kg. (B) Dose-dependence of PPARγ agonist, pioglitazone oncarrageenan-induced paw edema in mice. Pioglitazone was orally administrated 3 hbefore carrageenan-injection at doses of 1, 3, and 10 mg/kg. (C) Dose-dependence ofRXR agonist, NEt-3IP on carrageenan-induced paw edema in mice. NEt-3IP was orallyadministrated 3 h before carrageenan-injection at doses of 1, 3, and 10 mg/kg. The controlgroup received 0.5% carboxymethylcellulose. Each column and vertical bar represents themeans± S.E.M. (n= 7). *,**: Significantly different from the control group at P b 0.05 andP b 0.01, respectively (Dunnett's test).

205A. Yamamoto et al. / International Immunopharmacology 22 (2014) 204–208

We previously reported that both 6-[N-ethyl-N-(3-isopropoxy-4-isopropylphenyl)-amino] nicotinic acid (NEt-3IP; an RXR agonist) andpioglitazone (a PPARγ agonist) showed anti-inflammatory effects viathe PPARγ/RXRpathway [19]. Injection of carrageenan induces a varietyof chemical mediators resulting in vascular extension and leakage ofplasma. The carrageenan-induced paw edema test is very useful toevaluate anti-inflammatory action as well as to study the mechanismsinvolved in inflammation [19]. In the present study, in order to exam-ine the involvement of GR in PPARγ agonist- and RXR agonist-mediated anti-inflammatory effects in vivo, we tested the anti-inflammatory activities of dexamethasone (a GR agonist) withpioglitazone (a PPARγ agonist) or NEt-3IP (an RXR agonist) usingthe carrageenan-induced experimental inflammation model. In ad-dition, we showed the impact of PPARγ and RXR activation onglucocorticoid-induced anti-inflammatory effects.

2. Materials and methods

2.1. Animals

Five-week-old male ICR mice (body weight, 23–28 g) were pur-chased from Japan SLC, Shizuoka, Japan. The animals were kept in anair-conditioned room at a controlled temperature (24 °C ± 2 °C) andhumidity (55% ± 15%). They were housed in plastic cages lined withsawdust and kept under a light-dark cycle (lights on from 0700 to1900). Food andwater were freely available, except during test periods.All procedures involving animals were conducted in accordancewith the Guidelines for Animal Experiments at Okayama UniversityAdvanced Science Research Center, and all procedures were licensedby the Animal Research Control Committee of Okayama University(permission No. OKU-2009134).

2.2. Reagents and drugs

λ-Carrageenan (Wako, Osaka, Japan) was dissolved in physiologicalsaline. NEt-3IP and 6-[N-4-(trifluoromethyl)-benzenesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)-amino] nicotinicacid (NS-4TF) were synthesized at Okayama University [20,21]. Dexa-methasone (Wako), pioglitazone (Actos; Takeda Pharmaceutics, Osaka,Japan), andNEt-3IPwere suspended in 0.5% carboxymethylcellulose solu-tion. NS-4TF, GW9662 (Sigma, St. Louis, MO, USA), and RU486 (Sigma)were dissolved in physiological saline containing 10% dimethyl sulfoxide.

2.3. Drug administration

Dexamethasone (0.1, 0.3, and 1 mg/kg), pioglitazone (1, 3, and10 mg/kg), and NEt-3IP (1, 3, and 10 mg/kg) were administered orally3 h before carrageenan injection. RU486 (3 and 10 μg/paw), GW9662(3 and 10 μg/paw), and NS-4TF (10 and 30 μg/paw) were injectedinto the subplantar of the hind paw 15 min before the carrageenaninjection.

2.4. Mouse paw edema

After the carrageenan injection, the volume of the hind paw wasmeasured at 3 h. The paw volume was determined volumetrically byusing plethysmometry (TK-101; UNICOM, Chiba, Japan). The basalvolume of the hind paw was determined before the administration ofany of the drugs. After determination of the basal volume, the animalswere divided into experimental groups in a way that themean volumesof the different groups were similar. A 1% λ-carrageenan solutiondissolved in saline (0.05 mL/animal) was injected subcutaneously intothe right hind paw of themice. The difference in the paw volume beforeand after the carrageenan injection was taken as “paw edema” andexpressed as Δ paw volume.

2.5. Statistical analysis

All data are presented as the mean ± standard error of the mean(S.E.M.). Statistical analysis was performed using one-way analysis ofvariance (ANOVA) with the Dunnett's test or Student's unpaired t test.When the probability (P) value was less than 0.05, the difference wasconsidered significant.

3. Results

3.1. Anti-inflammatory effects of the GR agonist, PPARγ agonist, andRXR agonist

As illustrated in Fig 1A, the GR agonist dexamethasone showed adose-dependent anti-inflammatory effect. Oral administration of dexa-methasone at doses of 0.3 and 1 mg/kg resulted in a significant in-hibition of carrageenan-induced paw edema as compared to thatobserved in the control group,which received 0.5% carboxymethylcellu-lose solution. As illustrated in Fig 1B, the PPARγ agonist pioglitazone

206 A. Yamamoto et al. / International Immunopharmacology 22 (2014) 204–208

also showed a dose-dependent anti-inflammatory effect. Oral adminis-tration of pioglitazone at doses of 3 and 10 mg/kg resulted in a signifi-cant inhibition of carrageenan-induced paw edema as compared tothat observed in the control group. Similarly, as illustrated in Fig 1C,the RXR agonist NEt-3IP showed a dose-dependent anti-inflammatoryeffect. Oral administration of NEt-3IP at doses 3 and 10 mg/kg resultedin a significant inhibition of carrageenan-induced paw edema as com-pared to that observed in the control group.

3.2. Effect of the GR antagonist on the anti-inflammatory effects of GR,PPARγ, and RXR agonists

Fig 2 shows the effects of the GR antagonist RU486 on the anti-inflammatory effects of dexamethasone, pioglitazone, and NEt-3IP.The intraplantar injection of RU486 at doses of 3 and 10 μg/paw

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Fig. 2. Effect of GR antagonist on anti-inflammatory effect induced by GR agonist, PPARγagonist, and RXR agonist. (A) Effect of GR antagonist, RU486 on GR agonist induced anti-inflammatory effect. Dexamethasone was orally administrated 3 h before carrageenan-injection at a dose of 10 mg/kg. RU486 was injected 15 min before carrageenan-injection at doses of 10 and 30 μg/paw. (B) Effect of GR antagonist on PPARγ agonistinduced anti-inflammatory effect. Pioglitazone was orally administrated 3 h beforecarrageenan-injection at a dose of 10 mg/kg. RU486 was injected 15 min beforecarrageenan-injection at doses of 10 and 30 μg/paw. (C) Effect of GR antagonist on RXRagonist induced anti-inflammatory effect. NEt-3IP was orally administrated 3 h beforecarrageenan-injection at a dose of 10 mg/kg. RU486 was injected 15 min beforecarrageenan-injection at doses of 10 and 30 μg/paw. The vehicle group received physio-logical saline including 10% dimethylsulphoxide. Each column and vertical bar representsthe means ± S.E.M. (n = 7). **: Significantly different from the control group at P b 0.01(Student's unpaired t test). †,††: Significantly different from the vehicle group at P b 0.05and P b 0.01, respectively (Dunnett's test).

significantly reduced the anti-inflammatory effects of dexamethasoneandpioglitazone, respectively, as compared to that in the vehicle controlgroup. In contrast, administration of RU486 at a dose of 10 μg/paw didnot modify the anti-inflammatory effect of NEt-3IP.

3.3. Combined effects of the GR and PPARγ agonists or the GR and RXRagonists on carrageenan-induced paw edema

Fig 3A shows the combined effect of dexamethasone and pio-glitazone on carrageenan-induced paw edema. Coadministration ofdexamethasone (0.1 mg/kg) and pioglitazone (1 mg/kg), whichshowed no inhibition when administered alone, had no effect oncarrageenan-induced paw edema. Fig 3B shows the combined effect ofdexamethasone and NEt-3IP on carrageenan-induced paw edema. Co-administration of dexamethasone (0.1 mg/kg) and NEt-3IP (1 mg/kg),which showed no inhibition when administered alone, also had noeffect on carrageenan-induced paw edema.

3.4. Effect of the PPARγ antagonist or RXR antagonist on theanti-inflammatory effect of the GR agonist

Fig 4 shows the effects of GW9662 (a PPARγ antagonist) and NS-4TF(an RXR antagonist) on the anti-inflammatory effect of dexamethasone.Intraplantar injection of GW9662 or NS-4TF at a dose of 30 μg/paw hadno effect on the anti-inflammatory effect of dexamethasone.

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Fig. 3.Combination effect ofGR agonist and PPARγ agonist or RXRagonist on carrageenan-induced paw edema. (A) The combination effect of GR agonist and PPARγ agonist oncarrageenan-induced paw edema. Coadministration of dexamethasone (0.1 mg/kg) andpioglitazone (1 mg/kg), which showed no inhibition each alone, was not affected oncarrageenan induced paw edema. (B) The combination effect of GR agonist and RXRagonist on carrageenan-induced paw edema. Coadministration of dexamethasone(0.1 mg/kg) and NEt-3IP (1 mg/kg), which showed no inhibition each alone, was notaffected on carrageenan induced paw edema. The control group received 0.5%carboxymethylcellulose solution. Each column and vertical bar represents the means ±S.E.M. (n = 7).

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Fig. 4. Effect of RXR antagonist or PPARγ antagonist on anti-inflammatory effect inducedby GR agonist. (A) Effect of RXR antagonist, NS-4TF on anti-inflammatory effect ofdexamethasone. Intraplantar injection of NS-4TF at doses of 10 and 30 μg/paw show noeffect on anti-inflammatory effect of dexamethasone. (B) Effect of PPARγ antagonist,GW9662 on anti-inflammatory effect of dexamethasone. Intraplantar injection ofGW9662 at doses of 10 and 30 μg/paw show no effect on anti-inflammatory effect ofdexamethasone. The vehicle group received physiological saline including 10% dimethylsulfoxide. Each column and vertical bar represents the means± S.E.M. (n= 7). **: Signif-icantly different from the control group at P b 0.01 (Student's unpaired t test).

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4. Discussion

In this study, we investigated the effect of a GR agonist, a PPARγagonist, and an RXR agonist on carrageenan-induced paw edema. Eachdrug had an inhibitory effect on carrageenan paw edema. In addition,the GR antagonist RU486 reduced GR agonist- or PPARγ agonist-mediated anti-inflammatory activity but did not reduce RXR agonist-mediated anti-inflammatory activity. In previous studies, we reportedthat pioglitazone exerted an anti-inflammatory effect via PPARγ/RXRactivation [19]. However, in this study, the pioglitazone-induced anti-inflammatory effect was inhibited by the GR antagonist RU486.Furthermore, RU486 did not inhibit the RXR agonist-induced anti-inflammatory effect. If RU486 had an influence on the PPARγ/RXR path-way, then it should prevent the anti-inflammatory effects of bothagents. Therefore, we hypothesized that pioglitazone was acting viaanother pathway to induce its anti-inflammatory effect. However,Yamaguchi et al. reported that dexamethasone dose-dependentlyinduced the expression of RXRα mRNA, which was not inhibited bythe protein synthesis inhibitor cyclohexamide in rat hepatocytes [22].Furthermore, studies have shown that a combination of dexamethasoneand fatty acids upregulated RXRα protein levels [23,24]. Therefore, itappeared that some kind of interaction occurred between RXR and GR.

Next, we evaluated the effect of RXR agonist and GR agonist inter-action. The combination of NEt-3IP (1 mg/kg) and dexamethasone(0.1 mg/kg) showed no amelioration of carrageenan-induced pawedema, although there was a slight difference when both agonistswere used at higher effective doses. We reported that combinationadministration of a PPARγ agonist and an RXR agonist at low doses

which showed no inhibition when administered alone, showed syner-gistic anti-inflammatory effects. If GR and PPARγ/RXR interacted,some difference would be evident with combination administration,even at low doses that showed no inhibition when administeredalone. Therefore, we concluded that GR had no influence on PPARγ/RXR activation. Chen et al. observed RXRα gene downregulation in ratlivers with sepsis induced by cecal ligation and puncture to simulateperitonitis [25]. Moreover, dexamethasone treatment restored theRXRα content in this model, whereas dexamethasone had no effect onthe RXRα content in normal rat liver [26,27]. They also reported thatdexamethasone reversed the sepsis-inhibited RXRα expression by in-creasing rBAT gene expression. In addition, Yamaguchi et al. foundthat dexamethasone enhanced T3-dependent RXRα-TR expression [22].Accordingly, it was thought that a combination of the GR agonist andthe RXR agonist would not show any synergistic effect in this study,because the GR agonist does not directly act on the RXR.

Furthermore, we hypothesized that GR activation plays some role inthe anti-inflammatory effect of pioglitazone in accordance with thereport that rosiglitazone as well as dexamethasone induced nucleartranslocation of GR [18]. Our results showed that RU486 reversed theanti-inflammatory effect of pioglitazone. Ialenti et al. also showed thatthe rosiglitazone-dependent suppressive effect on IL-6 mRNA expres-sionmediated by LPS was inhibited by RU486 [18]. Cheron et al. report-ed that the endogenous PPARγ ligand 15-deoxy-Δ12,14-PGJ2 inhibitedGR binding by dexamethasone [27]. In addition, this effect was reducedby the GR cysteine residues, 2-cyclopenten-1-one, and methylmethanethiosulfonate but not by the PPARγ antagonist BADGE. Thesefindings suggest that pioglitazone limited GR binding and signalingthrough a PPARγ-independent and cyclopentenone-dependentmecha-nism in this study.

Lahiri et al. showed that administration of pioglitazone reversed thedecreased levels of both PPARγ and GR in the gastric mucosa of ulcerat-ed rats [28]. In contrast, Johnson et al. showed that dexamethasonecounteracted the effect of the PPARγ agonist thiazolidinedione on alka-line phosphatase enzyme activity and osteoblastic gene expression,while thiazolidinedione enhanced glucocorticoid-mediated transcrip-tion [17]. Thus, there is no consensus regarding the interaction betweenPPARγ andGR. Next, we investigated the effect of PPARγ andGR agonistcoadministration on the results of the carrageenan test. The combina-tion of pioglitazone and dexamethasone showed no amelioration ofcarrageenan-induced paw edema. Accordingly, we hypothesized thatpioglitazone acted as a partial rather than a complete GR agonist. Mat-thews et al. discovered that rosiglitazone enhanced GR nuclear translo-cation and GR activation [29]. Moreover, dexamethasone-induced GRactivation was inhibited by both RU486 and rosiglitazone. The resultsof our study support their suggestion.

Furthermore, we established the involvement of RXR and PPARγ inthe anti-inflammatory effect of dexamethasone. The RXR antagonistNS-4TF and PPARγ antagonist GW9662 had no effect on the anti-inflammatory effect of dexamethasone. Therefore, it can be suggestedthat GR activation is not involved in PPARγ/RXR heterodimer signaling.These findings suggest that GR activation appears to play some rolein the anti-inflammatory effect of pioglitazone via a PPARγ/RXR-independent pathway.

In conclusion, GR had no influence on the anti-inflammatory effectof an RXR agonist. In contrast, pioglitazone showed a partial anti-inflammatory effect via GR activation. These data provide evidenceabout the anti-inflammatory activity of pioglitazone.

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