Cellular Signalling 53 (2019) 190–200 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig Protective effect of lodoxamide on hepatic steatosis through GPR35 T ⁎ So-Yeon Nam, Soo-Jin Park, Dong-Soon Im College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea ARTICLE INFO ABSTRACT Keywords: Although GPR35 is an orphan G protein-coupled receptor, synthetic agonists and antagonists have been de- GPR35 veloped. Recently, cromolyn, a mast cell stabilizer, was reported as an agonist of GPR35 and was shown to Steatosis exhibit antifibrotic effects through its actions on hepatocytes and stellate cells. In this study, the roleofGPR35in Lodoxamide hepatic steatosis was investigated using an in vitro model of liver X receptor (LXR)-mediated hepatocellular Hepatocyte steatosis and an in vivo model of high fat diet-induced liver steatosis. GPR35 was expressed in Hep3B human Fatty liver hepatoma cells and mouse primary hepatocytes. A specific LXR activator, T0901317, induced lipid accumulation GPCR in Hep3B cells. Lodoxamide, the most potent agonist of GPR35, inhibited lipid accumulation in a concentration- dependent manner. The protective effect of lodoxamide was inhibited by a specific GPR35 antagonist, CID2745687, and by siRNA-mediated knockdown of GPR35. The expression of SREBP-1c, a key transcription factor for lipid synthesis, was induced by T0901317 and the induction was inhibited by lodoxamide. Through the use of specific inhibitors of cellular signaling components, the lodoxamide-induced inhibition of lipid accu- mulation was found to be mediated through p38 MAPKs and JNK, but not through Gi/o proteins and ERKs. Furthermore, the protective effect of lodoxamide was confirmed in mouse primary hepatocytes. Lodoxamide suppressed high fat diet-induced fatty liver development, which suggested the protective function of GPR35 in liver steatosis. Therefore, the present data suggest that GPR35 may function to protect against fatty liver de- velopment. 1. Introduction quently found to be in the micromolar range, which was not supportive of their role as GPR35 ligands [17]. Non-alcoholic hepatic steatosis is characterized by the presence of Recently, the chemokine CXCL17 was reported to be an endogenous steatosis in hepatocytes [1], which leads to the development of fatty ligand of GPR35 [18], although this was refuted by a later study [19]. liver [2]. It is strongly associated with obesity, insulin resistance, and However, many synthetic surrogate agonists and antagonists have been type II diabetes [3]. They are major risk factors of non-alcoholic hepatic identified or developed [20–29]. Lodoxamide and CID-2745687 have steatosis [4], which may progress to steatohepatitis, cirrhosis, and he- been identified as a potent agonist and a selective antagonist forGPR35 patocellular carcinoma [1]. [19,26]. Cromolyn, a mast cell stabilizer was identified as an agonist of De novo lipogenesis contributes to hepatic steatosis [5,6]. A nuclear GPR35 and was also shown to have antifibrotic effects through its ac- receptor, liver X receptor α (LXRα), plays an important role in the tions on both hepatocytes and hepatic stellate cells [11,30]. Therefore, regulation of fatty acid synthesis in hepatocytes through activating the function of GPR35 in hepatocytes especially in hepatic steatosis was sterol regulatory element binding protein 1c (SREBP-1c), a key tran- investigated. scription factor regulating hepatic lipogenesis [7]. The hepatic expres- sion of LXRα and SREBP-1c is increased in patients with steatosis [8]. 2. Materials and methods Human GPR35, an orphan G protein-coupled receptor, is expressed in lung, stomach, small intestine, colon, spleen, and immune cells in 2.1. Materials humans [9–12]. Although several endogenous molecules, such as, cGMP, kynurenic acid, lysophosphatidic acid, and reverse T3, were Lodoxamide was purchased from Toronto Research Chemicals Inc. reported as ligands of GPR35 [10,13–16], their potencies were subse- (North York, ON, Canada), and CID-2745687 from Tocris (Ellisville, ⁎ Corresponding author at: Laboratory of Signal Transduction, College of Pharmacy, Pusan National University, 63 Beon-gil 2, Busandaehag-ro, Geumjeong-gu, Busan 609-735, Republic of Korea. E-mail address: [email protected] (D.-S. Im). https://doi.org/10.1016/j.cellsig.2018.10.001 Received 26 June 2018; Received in revised form 1 October 2018; Accepted 1 October 2018 Available online 07 October 2018 0898-6568/ © 2018 Elsevier Inc. All rights reserved. S.-Y. Nam et al. Cellular Signalling 53 (2019) 190–200 Missouri, USA). The antibody used for the detection of GPR35 (NBP2- CA, USA), or β-actin (Cell Signaling Technology, Danvers, MA, USA) at 24640) was purchased from Novus Biologicals (Littleton, CO, USA). 4 °C. The blots were incubated with HRP-conjugated secondary anti- T0901317 was obtained from Cayman Chemical (Ann Arbor, MI, USA). body (Cell Signaling Technology, Danvers, MA, USA) and subsequently Pertussis toxin (PTX) was purchased from Sigma-Aldrich (St. Louis, MO, developed with ECL detection reagents [33]. All antibodies were used USA). U0126, SB202190, and SP600125 were purchased from Tocris at a dilution of 1:1000. Luminescence was detected using a ChemiDoc Bioscience (Bristol, UK). Touch Imaging System (BioRad) and analyzed by using ImageLab software (BioRad). 2.2. Cell culture and treatment 2.6. Transfection for GPR35 silencing Hep3B and HepG2 human hepatocellular carcinoma were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) The following oligonucleotides were purchased from Bioneer and maintained in Dulbecco's modified Eagle's medium with high glu- (Korea); GPR35 siRNA sense 5′-CCA CAA AAG CCA GGA CUC U(dTdT)- cose with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 50 μg/ 3′, antisense 5′-AGA GUC CUG GCU UUU GUG G(dTdT)-3′. Briefly, mL streptomycin at 37 °C in a humidified atmosphere containing 5% Hep3B cells were seeded at 1.0 × 105 cells/well in 12-well plates; after CO2. Cells were seeded into 6-well culture plates and allowed to adhere 24 h, GPR35 siRNA (200 nM) was introduced into cells by using overnight (18 h). The medium was then changed to serum-free medium Lipofectamine LTX reagent (Life Technologies, Carlsbad, CA) in ac- with 0.1% bovine serum albumin (BSA). Lodoxamide was pretreated at cordance with the manufacturer's instructions. Non-silencing siRNA and indicated concentrations for 1 h prior to incubation in the presence or siRNAs specific for GPR35 were transfected. The knockdown ofthe absence of 1 μM T0901317. Each experiment was performed on three target gene and protein was confirmed by RT-PCR and Western blotting independent occasions. analyses at 24 h and 48 h after transfection, respectively. 2.3. Reverse transcription-PCR 2.7. Isolation of mouse primary hepatocytes Total RNA was isolated from the cells using Trizol reagent Mouse primary hepatocytes were isolated from 8 weeks old Balb/c (Invitrogen, USA). RNA concentrations were determined by a Nanodrop female mice by using a two-step collagenase perfusion method, as de- ND-1000 spectrophotometer. One microgram of RNA was transcribed scribed previously [34]. The livers were perfused for 3 min with pre- by using the Promega ImProm-II Reverse Transcription System warmed Hanks' Balanced Salt Solution (HBSS) without calcium and (Madision, WI, USA) in accordance with the manufacturer's protocol. magnesium, and then with HBSS containing 0.1% collagenase at a flow Synthesized cDNA products and primers for each gene were used for rate of 8 mL/min for 5 min. The excised liver was gently minced in PBS, PCR with Promega Go-Taq DNA polymerase (Madision, WI, USA). after which the suspension was filtered with a 70 μM filter and cen- Specific primers for mGAPDH (sense 5′-GCG CTA CCG GTC TTCTAT trifuged at 50g for 1 min. After three washes with PBS and centrifuging CA-3′, antisense 5′-TGC TGC CAA AAG ACA AGG G-3′), hGAPDH (sense steps to pellet the cells, the cell pellet was resuspended with DMEM 5′-GAG TCA ACG GAT TTG GTC GT-3′, antisense 5′-TTG ATT TTG GAG containing 10% FBS, 100 units/mL penicillin, and 50 μg/mL strepto- GGA TCT CG-3′), mGPR35 (sense 5′-CCA AGA TTC CCA GAT CCT GA-3′ mycin. Isolated mouse hepatocytes were seeded in collagen-coated 8- antisense 5′-GGG GAG GGG TGT ATC CTA AA -3′), hGPR35a sense 5′- well chambers (SPL, Pocheon, Korea) at a density of 3.6 × 105 cells/ GTG TTC GTG GTC TGC TTC CT-3′, hGPR35b sense 5′-GTC CTT GCG mL. TCT CTC TGA CC-3′, hGPR35 antisense 5′-GAG AGT CCT GGC TTT TGT GG-3′ were used to amplify gene fragments. For each sample, 7 μL of 2.8. Measurement of cellular triglyceride content in liver the PCR products were electrophoresed in 1.2% agarose gels and stained with nucleic acid gel stain (Real Biotech, Taiwan) [31]. Triglycerides in livers were extracted with methanol/chloroform (2:1; v/v). Solvent was evaporated in 60 °C and lipids were resuspended 2.4. Oil red O staining in deionized water. The levels of triglycerides were determined using a commercial kit from Asan Pharm (Chungcheong, South Korea). Oil red O staining was performed in accordance with a previously described method [32]. Briefly, cells were fixed with 10% formalin for 2.9. Animals and diets 15 min at room temperature and then rinsed with PBS. The slides were immersed in Oil red O working solution for 1 h, rinsed in tap water, Male C57BL/6 mice were purchased from Daehan Biolink (DBL, counterstained with hematoxylin for 5 s, rinsed again with tap water, Seoul, Korea) and housed under standard laboratory conditions and mounted in an aqueous mounting medium. (22 °C ± 2 °C under a 12-h light/dark cycle) with free access to food and water in the laboratory animal facility at Pusan National University 2.5. Western blotting (PNU).