View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE A GPR40 Agonist GW9508 Suppresses CCL5, CCL17, and CXCL10 Induction in Keratinocytes and Attenuates Cutaneous Immune Inflammation Tomoko Fujita1,2, Toshiyuki Matsuoka1,2, Tetsuya Honda3, Kenji Kabashima1,3, Takako Hirata1,2 and Shuh Narumiya1,2 G-protein coupled receptors (GPCR) exert diverse physiological functions, many of which are exploited therapeutically. The roles of GPCR in keratinocytes in immune response in the skin, however, remain poorly defined. In this study, we focused on Gi-coupled GPCR in keratinocytes and defined their actions in immunoactivation of cultured keratinocytes in vitro and immune reaction in the skin in vivo. We first activated HaCaT cells by tumor necrosis factor (TNF)-a and IFN-g and examined effects of various ligands for GPCR on production of CCL17 and CCL5. Agonists for Gi-coupled receptors, particularly GW9508 for GPR40, inhibited CCL17 and CCL5 expression in a pertussis toxin-sensitive manner. The inhibitory effect by GW9508 was abrogated by depletion of GPR40 with RNA interference. GW9508 further suppressed expression of IL-11, IL-24, and IL-33 induced in HaCaT cells by TNF-a and IFN-g. GW9508 also inhibited CCL5 and CXCL10 production by normal human epidermal keratinocytes. Administration of GW9508 topically to the skin in the challenging phase suppressed ear swelling in a repeated hapten application model and contact hypersensitivity with downregulation of CCL5 and CXCL10, respectively. Thus, in the skin, stimulation of Gi-coupled receptors attenuates induction of critical cytokines and chemokines by proinflammatory cytokines in keratinocytes and suppresses allergic inflammation in the skin. Journal of Investigative Dermatology (2011) 131, 1660–1667; doi:10.1038/jid.2011.123; published online 19 May 2011 INTRODUCTION such as taste or odor perception. Notably, about half of all G-proteins consisting of a, b, and g subunits are signal drugs currently used clinically target GPCR in various transducers and mediate actions of G-protein coupled systems from the central nervous system to the cardiovascular receptors (GPCR) with seven transmembrane domains in system. However, the role of GPCR in the immune system the plasma membrane (Neves et al., 2002). Each GPCR is remains obscure and their therapeutic application in this area coupled to either a different class of G-proteins, Gs, Gi, Gq, has been limited to those for chemokines and other or G12/13, or a combination thereof. Gsa activates adenylate chemotactic substances such as sphingosine-1-phosphate cyclase, and raises the intracellular concentration of cAMP. (Rosen et al., 2007). GPCR have been generally viewed as On the other hand, Gia inhibits adenylate cyclase and mediating immunosuppression through elevation of intra- decreases intracellular cAMP. Among more than 1,000 GPCR cellular cAMP in cells such as macrophages and T cells encoded by mammalian genomes, up to 400–500 GPCR (Nance and Sanders, 2007). However, contrary to this recognize neurotransmitters, hormones, and paracrine oversimplified view, recent studies have indicated that GPCR factors, whereas others are involved in sensory functions have diverse roles in immunity depending on the context of immune response. For example, we have recently shown that stimulation of the cAMP pathway by prostaglandin 1Center for Innovation in Immunoregulative Technology and Therapeutics, E receptors EP2 and EP4 exerts significant facilitation of Th17 2 Kyoto University, Kyoto, Japan; Department of Pharmacology, Faculty of expansion by regulating IL-23 expression by dendritic cells Medicine, Kyoto University, Kyoto, Japan and 3Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan and enhancing the IL-23-induced Th17 amplification Correspondence: Shuh Narumiya, Department of Pharmacology, Faculty of (Yao et al., 2009). These findings have questioned the simple Medicine, Kyoto University, Yoshida Konoecho, Kyoto, 606-8501 Japan. paradigm of the immunosuppression by cAMP and suggest E-mail: [email protected] that the action of a GPCR in the immune system has to be Abbreviations: AD, atopic dermatitis; CHS, contact hypersensitivity; GPCR, defined in the contexts of target cells and tissues, timing of G-protein coupled receptors; NHEK, normal human epidermal keratinocyte; stimulation, types of other combined stimuli, and output of PTX, pertussis toxin; QRT-PCR, quantitative RT-PCR; TNF, tumor necrosis factor; TNF-a þ IFN-g, TNF-a and IFN-g in combination the response at various levels. Received 17 November 2010; revised 24 February 2011; accepted 21 March The incidence and severity of allergic skin diseases such as 2011; published online 19 May 2011 contact hypersensitivity (CHS) and atopic dermatitis (AD) 1660 Journal of Investigative Dermatology (2011), Volume 131 & 2011 The Society for Investigative Dermatology T Fujita et al. A GPR40 Agonist Suppresses Skin Inflammation have increased extensively over the last 20 years in keratinocytes (NHEKs), and mouse models of AD and CHS, developed countries, and drugs targeted to these diseases and addressed these issues. are urgently demanded. In these allergic diseases, keratino- cytes have a crucial role in disease progression (Girolomoni RESULTS et al., 2004; Pastore et al., 2006). We previously reported that GPR40 agonist GW9508 suppresses CCL17 production through administration of an agonist of prostaglandin E receptor EP3, pertussis toxin-sensitive Gi protein a Gi-coupled receptor, attenuates allergic asthma (Kunikata We first used HaCaT human epidermal keratinocyte cell line. et al., 2005), CHS (Honda et al., 2009), and allergic Previous reports showed that HaCaT cells produce CCL17 in conjunctivitis (Ueta et al., 2009). Stimulation of cannabinoid response to stimulation with tumor necrosis factor (TNF)-a CB1 and CB2 receptors, both coupled to Gi, has also been or IFN-g alone, or TNF-a and IFN-g in combination shown to induce attenuation of DNFB-induced CHS (Karsak (TNF-a þ IFN-g; Saeki and Tamaki, 2006). We exploited this et al., 2007). Although EP3 is expressed on epithelial cells in finding and reproduced synergistic production of CCL17 by the airway and conjunctiva and keratinocytes in the skin TNF-a þ IFN-g in HaCaT cells, both at mRNA and protein and EP3 in these cells is suggested to mediate the above levels (Figure 1a). We also found that TNF-a and IFN-g therapeutic action, a direct proof of the involvement of synergistically enhance CCL5 production by HaCaT cells Gi-coupled receptors in keratinocytes in such action is (Figure 1b). CCL17 and CCL5 are produced by keratinocytes lacking. In this study, we first sought to define the actions in inflammatory states and have crucial roles in Th2-type of Gi-coupled receptors in cultured keratinocytes and then to immune responses such as AD (Pastore et al., 2006; Saeki analyze the effects of such pathway in elicitation of immune and Tamaki, 2006). To screen GPCR ligands that inhibit the inflammation in the skin. Here, we used HaCaT, a human TNF-a þ IFN-g-induced chemokine expression, we examined epidermal keratinocyte cell line, normal human epidermal the activity of compounds in the Tocriscreen Mini library in *** ** * * * ) 70 * 35 * ) 1,250 ** –1 125 –1 60 30 1,000 50 100 25 (a.u.) 40 75 (a.u.) 20 750 15 30 50 500 20 10 250 25 5 10 Ccl5/Gapdh Ccl17/Gapdh 0 CCL5 protein (pg ml 0 0 CCL17 protein (pg ml 0 None TNF-α IFN-γ TI None TNF-α IFN-γ TI None TNF-α IFN-γ TI None TNF-α IFN-γ TI 90 125 80 ) 70 ) –1 1,250 70 100 60 –1 1,000 (a.u.) 60 50 75 (a.u.) 50 *** *** 40 * 750 40 ** ** * ** 30 30 50 ** 500 20 20 25 Ccl17/Gapdh 250 10 Ccl5/Gapdh 10 CCL17 protein (pg ml 0 0 0 CCL5 protein (pg ml 0 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI TI+veh –8 –7 –6 TI + GW9508 (log[M]) TI + GW9508 (log [M]) TI + GW9508 (log [M]) TI + GW9508 (log [M]) 250 ** 70 *** 150 *** ) 1,250 200 60 * –1 * (a.u.) 50 1,000 150 100 ** 40 (a.u.) 750 100 ** 30 500 20 50 50 Ccl17/Gapdh 10 250 Ccl5/Gapdh 0 CCL17 protein (pg/ml) 0 0 CCL5 protein (pg ml TI+veh 0 1 10 TI+veh 0 1 10 0 TI+veh 0 1 10 TI+veh 0 1 10 μ –1 TI + 1 M GW9508 + PTX (ng ml ) TI + 1 μM GW9508 + PTX (ng ml–1) –1 TI + 1 μM GW9508 + PTX (ng ml ) TI + 1 μM GW9508 + PTX (ng ml–1) 150 *** 125 *** 100 (a.u.) 100 (a.u.) 75 50 50 25 Ccl5/Gapdh Ccl17/Gapdh 0 0 TI+veh –8 –7 –6 TI+veh –8 –7 –6 TI + forskolin (log[M]) TI + forskolin (log [M]) Figure 1. Inhibition by GW9508 of tumor necrosis factor (TNF)-a þ IFN-c-induced production of CCL17 and CCL5 in HaCaT cells. CCL17 (a) and CCL5 (b) production by TNF-a and/or IFN-g. Inhibitory effect of GW9508 on TNF-a þ IFN-g-induced CCL17 (c) and CCL5 (d) production. Effect of pertussis toxin (PTX) (e and f) and forskolin (g) on TNF-a þ IFN-g-induced CCL17 and CCL5 production. TI, TNF-a þ IFN-g; veh, vehicle. Quantitative real-time RT-PCR data are shown as arbitrary units (a.u.) where the value for unstimulated sample is set at 1. Results show a representative of three independent experiments. Data show mean±SEM (n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 versus TI þ veh in (c–f). www.jidonline.org 1661 T Fujita et al. A GPR40 Agonist Suppresses Skin Inflammation this system with CCL17 as a parameter. This screening GPR40 (–) GPR120 (–) GAPDH (–) revealed that several agonists for putative Gi-coupled receptors, such as ST91 for a2 adrenergic receptor, GP1a and GP2a for CB2, 30-fluorobenzyl-spiperone for dopamine D2, and GW9508 for GPR40 potently suppressed the 1.0 TNF-a þ IFN-g-induced CCL17 production (Supplementary Table S1 online).