General and Comparative Endocrinology 163 (2009) 251–258

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General and Comparative Endocrinology

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Molecular cloning and characterization of olive flounder (Paralichthys olivaceus)peroxisome proliferator-activated receptor c

Hyun Kook Cho a,1, Hee Jeong Kong b,1, Bo-Hye Nam b, Woo-Jin Kim b, Jae-Koo Noh b, Jeong-Ho Lee b,Young-Ok Kim b, JaeHun Cheong a,*a Dept. of Molecular Biology, Pusan National University, Busan 609-735, Republic of Koreab Biotechnology Research Center, National Fisheries Research and Development Institute, Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, Republic of Korea

a r t i c l e i n f o

Article history:Received 1 July 2008Revised 10 April 2009Accepted 17 April 2009Available online 23 April 2009

Keywords:PPARcNuclear hormone receptorOlive flounderCloningTransactivation

0016-6480/$ - see front matter � 2009 Published bydoi:10.1016/j.ygcen.2009.04.018

* Corresponding author. Address: Dept. of MolecuUniversity, Jang-Jeon Dong, Busan 609-735, Republic

E-mail address: molecule85@pusan.ac.kr (J. Cheon1 Both authors contributed equally to this work.

a b s t r a c t

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles inlipid and energy homeostasis. Olive flounder (Paralichthys olivaceus) PPARc cDNA (olPPARc) was isolatedby reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE). The full-length cDNA is 1667-bp long and encodes a polypeptide with 532 amino acids containinga C4-type zinc finger and a ligand-binding domain. Quantitative RT-PCR revealed that olPPARc transcrip-tion was detected from 7 days post-hatching, and its expression was increased under a starved condition.Overexpression of olPPARc stimulated PPAR response element (PPRE) activity, and treatment with rosig-litazone, a PPARc agonist, augmented olPPARc-stimulated PPRE activity in HINAE olive flounder cells.Cotransfection of olPPARc and olRXRb, in the absence or presence of rosiglitazone and ciglitazone, pro-duced a synergistic effect on PPRE transactivation in 3T3L1 adipocytes. Moreover, olPPARc, in the pres-ence or absence of rosiglitazone, regulated the expression of lipid synthesis- and adipogenesis-relatedproteins in NIH3T3 and 3T3L1 cells. Taken together, these results suggest that olPPARc is functionallyand evolutionarily conserved in olive flounder and mammals.

� 2009 Published by Elsevier Inc.

1. Introduction

Peroxisome proliferator-activated receptor c (PPARc), as its iso-types a and b, are members of the nuclear hormone receptor super-family. PPARc is activated by natural ligands such as arachidonicacid metabolites and fatty acid-derived components, and by rosiglit-azone (Ro), a thiazolidinedione (TZD; Spiegelman, 1998). PPARc is acritical transcription factor in adipogenesis, and its expression isgreatly increased during adipocyte differentiation (Rosen et al.,2002; Gregoire et al., 1998). By activating PPARc, Ro promotes adipo-cyte differentiation in vitro (Hutley et al., 2003; Shao and Lazar,1997). The overexpression of PPARc in fibroblasts induces adipogen-esis, whereas PPARc-null embryonic stem cells and fibroblastic cellsfrom PPARc-deficient mouse embryos cannot differentiate into adi-pocytes in vitro (Rosen et al., 2000; Kadowaki, 2000; Lee et al., 2003).

Transcriptional activation by PPARs requires the presence ofPPAR response elements (PPREs) in the promoter of the targetgene. PPREs are DR1-type direct repeat elements (direct repeatspaced by 1 bp). PPARs bind PPREs as heterodimers with any oneof three retinoid X receptor (RXR) isotypes (a, b, or c), which func-

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lar Biology, Pusan Nationalof Korea.g).

tion as receptors for the vitamin A metabolite 9-cis-retinoic acid(Mangelsdorf et al., 1995; Chambon, 1996; Desvergne and Wahli,1999). PPAR target genes for which a functional PPRE has beenidentified include acyl-CoA synthase (ACS), adipocyte lipid bindingprotein (ALBP/aP2), fatty acid transport protein (FATP), and liver fattyacid binding protein (L-FABP) (Schoonjans et al., 1995; Tontonozet al., 1994; Frohnert et al., 1999; Issemann et al., 1992).

PPARs have recently been discovered in several fish species,including tarafugu (Kondo et al., 2007), zebrafish (Ibabe et al.,2005), salmon (Leaver et al., 2007), goldfish (Mimeault et al.,2006), grey mullet (Raingeard et al., 2006), rainbow trout (Liuet al., 2005), sea bass (Boukouvala et al., 2004), plaice, and seabream (Leaver et al., 2005). Although these reports studied tissue-and/or developmental stage-specific gene expression, the regula-tion and function of each PPAR remain unknown. The aim of thepresent study was to clone and characterize PPARc from oliveflounder (Paralichthys olivaceus) in order to address its functionin the regulation of lipid homeostasis in fish.

2. Materials and methods

2.1. Reagents

Rosiglitazone and ciglitazone (PPARc ligands) was purchasedfrom Cayman Chemical (Michigan, USA). The transfection reagents

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252 H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258

SuperFect and PolyFect were purchased from Qiagen and JetPEIwas purchased from PolyPlus Transfection. All other reagents werepurchased from Sigma.

2.2. cDNA sequences of olive flounder PPARc (olPPARc)

Initial PCR was performed with specific primers to obtain thefragment sequences of olive flounder PPARc (P1: 50-GCC ATC CTCTCT GGG AAG ACC G -30, P2: 50-CAG CGC CAT GTC ACT GTC GTCC-30). 50-, 30-Rapid Amplification cDNA Ends (RACE) were per-formed using SMARTTM RACE cDNA amplification kit (Clontech),following the manufacturer’s instruction to obtain olive flounderPPARc cDNA full sequences. Based on the partial PPARc sequences,internal primers were designed (P3: 50-GCA ATT AAT GAA CTG CTCTCC TTC C-30, P4: 50-AGC TGT CGT CCA GCT CCG AGA G-30, P5: 50-CAT GAC GCG GGA GTT CCT CAA G-30, P6: 50-GTC AGA TGA TGGAAC CAA AGT TTG AG-30) and were used in combination with theuniversal primer supplied with the kit to amplify the 50- and 30-end of olPPARc transcript. DNA sequencing was performed withthe universal and the internal primers using an ABI 3100 autosequ-encer. The full-length of the olPPARc cDNA sequence was obtainedby combining the DNA sequences of the partial sequences and 50-,30-RACE PCR products.

2.3. Bioinformatic analysis

Analyses of potential open reading frames (ORFs) and compari-son of amino acid sequences (or nucleotide sequences) were per-formed with the ORF finder and BLAST programs on the NationalCenter for Biotechnology Information website. The multiple se-quence alignments and the construction of phylogenetic trees(using the neighbor-joining method) were performed with theMega 3.1 (http://www.megasoftware.net).

2.4. Fish rearing condition

Artificially fertilized flounder eggs were stocked in a tank with aflow through system of filtered seawater. A total of 98% of the eggshatched 3 days later. Feeding program was modified from Sakak-ura (2006). Enriched L-type rotifers (Brachionus plicatilis complex)were fed from day 3 to day 14; enriched Artemia franciscana naupliiwere supplied from day 13 to day 28; commercial fish diets (Mar-uwa Co., Ltd.; crude protein: 48–54%, crude fat: 9–12%) were of-fered from day 21. Feeding was given six times per day forensuring sufficient food supply. Temperature on rearing tankswas maintained at 18 �C.

2.5. Starvation protocol

Fish (approximately 16 cm in size) were randomly divided intotwo experimental groups (10 fish each). Starvation protocol wasmodified from Salem et al. (2005). Control group was fed a com-mercial fish diets (Suhyup Feed; crude protein: 52%, crude fat:11%) twice per day. Experimental group was subjected to a starva-tion regimen for 30 days. At the end of the experimental period,several tissues were collected from each group.

2.6. Quantitative real-time RT-PCR analysis

Total RNA was prepared from cell lines or tissues using TRIzol�

reagent (Invitrogen) following the manufacturer’s instructions. Thesizes of flounders which were used for tissue sampling during earlydevelopment are approximately 3.7 mm at D7, 8 mm at D18, and12 mm at D33. One microgram of total RNA was DNase treated,and cDNA was synthesized using the Advantage� RT-for-PCR kit(BD Biosciences). The dilution factor of the cDNA used quantitative

RT-PCR is 1. Quantitative real-time PCR was performed using Light-Cycler� FastStart DNA Master SYBR Green I (Roche) and the follow-ing forward and reverse primers: olPPARc F, 50-GCC ATC CTC TCTGGG AAG ACC G-30, olPPARc R, 50-CAG CGC CAT GTC ACT GTCGTC C-30, ol18S RNA F, 50-ATG GCC GTT CTT AGT TGG TG-30,ol18S RNA R, 50-CAC ACG CTG ATC CAG TCA GT-30, mFASN F, 50-GCT GTG CTT GCA GCT TAC TG-30, mFASN R, 50-CGG ATC ACCTTC TTG AGA GC-30. mActin F, 50-GAC TAC CTC ATG AAG ATC-30,mActin R, 50-GAT CCA CAT TTG CTG GAA-30. Following an initial10-min Taq activation step at 95 �C, LightCycler PCR was conductedvia 40 cycles under the following cycling conditions: 95 �C for 15 s,60 �C for 5 s, 72 �C for 10 s, and fluorescent reading. Immediatelyafter the PCR, the machine performed a melting curve analysis bygradually (0.1 �C/s) increasing the temperature from 65 to 95 �C,with a continuous registration of changes in fluorescent emissionintensity.

2.7. Construction of the expression plasmid

Amplification of the open reading frame (ORF) of the oliveflounder PPARc was carried out using the Ex Taq DNA polymerase(TaKaRa) and primers specific to the 50 (starting at the ATG initiatorcodon) and 30 ends of the olPPARc cDNA based on nucleotide se-quence. The primers used were designed so that the amplifiedDNA would contain EcoRI and XbaI restriction endonuclease sitesat its 50 and 30 ends, respectively. The primer sequences were asfollows: forward, 50-AAG AAT TCA TGG TGG ACA CCC AGC AG-30;reverse, 50-CCT CTA GAC TAA TAC AAG TCC TTC ATG ATC TC-30.The amplified cDNA fragment was cloned into pcDNA3-HA vector.The construct was confirmed by DNA sequencing.

2.8. Cell culture

HINAE flounder embryonic cells, a gift from Takashi Aoki, weremaintained in Leibovitz L-15 medium (L-15; GIBCO BRL) with 10%heat-inactived fetal bovine serum (FBS; GIBCO BRL) and 1% (v/v)penicillin–streptomycin (PS; GIBCO BRL) at 20 �C. 3T3L1 andNIH3T3 cells were propagated in growth medium consistingDMEM, 10% FBS and 1% PS at 37 �C in humid atmosphere 5% CO2.Medium was changed every second day in all experiments.

2.9. Transient transfection and luciferase assay

The PPRE-driven luciferase reporter vector J3-TK-Luc containingthe three copies of J-site (�737 to �715 site) of the human apoA-IIgene promoter upstream of the thymidine kinase (TK) promoterand expression vector pSV SPORT1-mPPARc1 were kindly giftedfrom Bruce M. Spiegelman. And expression vector pcDNA3-RXRawas kindly gifted from Hueng-Sik Choi. Cells were seeded in 24-well culture plate and transfected with reporter vector andb-galactosidase expression plasmid, along with each indicatedexpression plasmids using SuperFect or Polyfect (Qiagen). Totalamounts of expression vectors were kept constant by pcDNA3.1(Invitorgen). Twenty-four hours after transfection, cells were incu-bated in the presence or absence of rosiglitazone (Ro) and ciglitaz-one (Ci) for 24 h. After 48 h of transfection, the cells were lysed inthe cell culture lysis buffer (Promega). Luciferase activity was deter-mined using an analytical luminescence luminometer according tothe manufacturer’s instructions. Luciferase activity was normalizedfor transfection efficiency using the corresponding b-galactosidaseactivity. All assays were performed at least in triplicate.

2.10. SDS–PAGE and Western blot analysis

The cells were prepared by washing with cold-PBS and lysed.The protein concentration was determined using Bradford reagent

http://www.megasoftware.net

H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258 253

(Bio-Rad). Equal amount of proteins was loaded and separated bySDS–PAGE and the gels were transferred to polyvinylidene fluoride(PVDF) membrane (Millipore). For western blotting, the mem-branes were incubated with anti-HA (Roche), anti-C/EBPa (Santa

Fig. 1. Cloning of the olive flounder PPARc cDNA. (A) Alignment of salmon PPARc (AJ2929are shaded in grey. Boxes indicate the primers used for PCR. (B) Locations of the primer

Fig. 2. Nucleotide and deduced amino acid sequences of PPARc. Start and stop codons arthe box, while the ligand-binding domain is underlined.

Cruz Biotechnology), anti-FASN (BD Biosciences), anti-Actin (Sig-ma) in TBST supplemented with 3% non-fat dry skim milk for over-night at 4 �C. After washing three times with cold TBST, the blottedmembranes were incubated with peroxidase-conjugated second-

63), zebrafish PPARc (ABI30002), and plaice PPARc (CAD62449). Conserved residuess used for 50- and 30-RACE of the olive flounder PPARc.

e indicated by bold letters. The conserved zinc finger domain (C4-type) is shown in

254 H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258

ary antibody (Santa Cruz Biotechnology) for 30 min at room tem-perature. After washing three times with cold TBST, the proteinswere visualized by the enhanced chemiluminescent developmentreagent (Amersham Pharmacia Biotech). Visualized bands werequantified and normalized relative to the actin bands with ImageJversion 1.35d (NIH Image).

3. Results

3.1. Cloning of the cDNA encoding olive flounder peroxisomeproliferator-activated receptor c (olPPARc)

The cDNA or genomic sequence of olPPARc has not yet beenreported. To obtain a partial sequence for olPPARc, PCR was per-formed using primers specific for a conserved region identifiedthrough multiple alignments of the PPARc mRNA sequences fromother species (Fig. 1A). From this fragment, the full-length CDSalong with the 50 and 30 flanking untranslated sequences of theolPPARc gene was established through 50- and 30-Rapid Amplifi-cation cDNA Ends (RACE; Fig. 1B). The cDNA is 1667 bp longand consists of a 1596-bp open reading frame that encodes a532 amino acid protein, preceded by a 20-bp 50-UTR and fol-lowed by a 51-bp 30-UTR (GenBank Accession No. FJ262993). Acomputer search using BLASTP (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) revealed that the deduced primary sequence ofolPPARc contains well-conserved C4-type zinc finger and li-gand-binding domains (Fig. 2).

Fig. 3. Multiple sequence alignment between PPARc from olive flounder and other spzebrafish, mouse, and human using ClustalW. Identical residues are indicated by asteriskPoPPARc, Paralichthys olivaceus PPARc; PpPPARc, Pleuronectes platessa PPARc (CAD62(EDK99525); HsPPARc, Homo sapiens PPARc (CAA62153).

3.2. Characterization of the olPPARc cDNA

Using BioEdit software, we assessed the percent identity of theolive flounder PPARc to that from the other species (Fig. 3). Pairwisealignments revealed identities of 93.8%, 60%, 60%, and 58% betweenthe PPARc of olive flounder and that of plaice (GenBank AccessionNo. CAD62449), zebrafish (ABI30002), mouse (EDK99525), and hu-man (CAA62153), respectively. In particular, the C4-type zinc fingerdomain of PPARc showed high levels of identity (100–95%), whilethe ligand-binding domain showed identities of 97–74% and simi-larities of 98–91%. Thus, the C4-type zinc finger and ligand-bindingdomains of olPPARc have been highly conserved throughout theevolutionary process. The full-length primary sequence of PPARcfrom olive flounder was used along with those from various fish,amphibians, reptiles, birds, and mammals to generate a phyloge-netic tree. As shown in Fig. 4, the tree shows clear and robust clus-tering of the PPARc sequences into two groups: those from fish andthose from the other species. Among the fishes, olPPARc was mostclosely related to flatfish PPARc, and was divergent from salmonPPARc.

3.3. mRNA expression profile of olPPARc

To determine the stage of development in which transcriptionof olPPARc occurs, we analyzed its expression by real-time RT-PCR at 7, 14, 21, 27, and 34 days post-hatching (dph). As shownin Fig. 5A, olPPARc mRNA was detected from 7 dph, and increasedto 4.1-fold by 34 dph. To examine the tissue distribution of olP-

ecies. Alignment of the primary sequences of PPARc from olive flounder, plaice,s (*); conservative substitutions are shown by dots (.:). Abbreviations are as follows:449); DrPPARc, Danio rerio PPARc (ABI30002); Mm PPARc, Mus musculus PPARc

http://www.ncbi.nlm.nih.gov/blast/Blast.cgihttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi

Fig. 4. Phylogenetic tree depicting the evolutionary relationships between various PPARcs. An unrooted phylogenetic tree was constructed by the neighbor-joining methodafter alignment. The sequences were extracted from GenBank: Pleuronectes platessa (CAD62449), Danio rerio (ABI30002), Mus musculus (EDK99525), Homo sapiens(CAA62153), Platichthys flesus (CAB51396), Lateolabrax japonicus (ABC70398), Pagrus major (BAF80459), Sparus aurata (AAT85618), Dicentrarchus labrax (AAT85617), Dentexdentex (ABO69005), Chelon labrosus (ABM66074), Bos taurus (NP_851367), Sus scrofa (BAD20642), Ovis aries (NP_001094391), Rattus norvegicus (BAA32540), Anasplatyrhynchos (ABQ23994), Xenopus laevis (AAH60474), Eublepharis macularius (BAF79869), Cavia porcellus (AAG60685), Salmo salar (CAC95230), and Oncorhynchus keta(BAD94509).

Fig. 5. mRNA expression of olPPARc. (A) Quantitative RT-PCR was performed on equal amounts of total whole-body RNA isolated at different developmental stages. The timepoints are expressed as days post-hatching (dph). 18S rRNA was used as an internal control. Expression level of olPPARc mRNA in D7 was arbitrarily defined as 1. The valuesrepresent the means ± SD (n = 3). **P < 0.01, *P < 0.05 compared with the corresponding value for D7. (B) Quantitative RT-PCR was performed on equal amounts of total RNAisolated from the internal organs of normally fed or starved fish. 18S rRNA was used as an internal control. K, kidney; Sp, spleen; St, stomach; L, liver; I, intestine; Sk, skin; G,gill. Expression level of olPPARc mRNA in the kidney of normally fed flounder was arbitrarily defined as 1. The values represent the means ± SD (n = 3). **P < 0.01, *P < 0.05compared with the corresponding value for fed fish.

H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258 255

PARc, real-time RT-PCR was performed using various olive floundertissues from normally fed and starved fish. olPPARc mRNA washighly expressed in the stomach, intestine, and gills in both groups,but increases of olPPARc mRNA were detected in the tested tissuesin the starved fish group: a 1.56-, 3.4-, 1.92-, 1.75-, 1.52-, 6.71-, and1.27-fold increase in kidney, spleen, stomach, liver, intestine, skin,and gill, respectively (Fig. 5B). These results suggest that olPPARcmay be necessary for early olive flounder development and understarved conditions.

3.4. Functional analysis of olPPARc

The significant identity of the C4-type zinc finger and ligand-binding domains of olPPARc with those in other species led us tospeculate that olPPARc, like other PPARcs, regulates lipid metabo-

lism in olive flounder. To investigate whether olPPARc can activatetranscription through a PPAR response element (PPRE)-driven pro-moter, we used a luciferase assay. Mammalian expression vectorscarrying olPPARc were transfected into HINAE cells, concomitantwith a PPRE-driven luciferase reporter plasmid. olPPARc or olP-PARc plus Ro, but not Ro alone, drove the expression of the repor-ter gene (Fig. 6A). Since PPARs bind PPREs as heterodimers withRXRs (Mangelsdorf et al., 1995; Chambon, 1996; Desvergne andWahli, 1999), we then examined whether olPPARc interacts witholive flounder RXRb (olRXRb) for DNA binding and transactivation.olPPARc alone or olPPARc and olRXRb isolated in our laboratory(unpublished result; GenBank Accession No. FJ262992) were trans-fected into 3T3L1 adipocytes, concomitant with a PPRE-drivenluciferase reporter plasmid. Cotransfection of olPPARc and olRXRb,in the absence or presence of Ro or Ci, produced a synergistic effect

Fig. 6. Overexpression of PPARc increases PPRE-luc reporter activity in olive flounder embryonic (HINAE) and mouse adipocytes (3T3L1) cells. (A) HINAE cells weretransfected with expression vectors encoding olive flounder PPARc and a reporter plasmid containing a PPAR response element (PPRE). At 24 h post-transfection, the cellswere incubated in the presence or absence of rosiglitazone (Ro) for 24 h. Relative luciferase activity in the cells is presented as the fold-induction with respect to that seen inmock transfectants in the absence of Ro. 3T3L1 cells were transfected with the expression vector of the indicated genes and a reporter plasmid carrying a PPRE. At 24 h post-transfection, the cells were incubated in the presence or absence of Ro (B) or ciglitazone (Ci) (C) for 24 h. The data are representative of three independent experiments. Thevalues represent the means ± SD (n = 3). *P, **P < 0.05, ***P < 0.01 compared with the corresponding value for olPPARc-, olPPARc/olRXRb-, or mPPARc1-transfected and DMSO-treated cells, respectively. ****P, *****P < 0.01 compared with the corresponding value for mock or olPPARc-transfected and DMSO- or Ro-treated cells. #P, ##P, ###P < 0.01compared with the corresponding value for olPPARc-, olPPARc/olRXRb-, or mPPARc1-transfected and DMSO-treated cells, respectively. ####P, #####P < 0.01 compared withthe corresponding value for mock or olPPARc-transfected and DMSO- or Ro-treated cells.

256 H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258

on PPRE transactivation (Fig. 6B and C). These results indicate thatolPPARc recognizes and binds PPREs in conjunction with olRXRb,and that Ro and Ci may act as an olPPARc ligand.

3.5. Regulation of the expression of lipid synthesis and adipogenesis-related proteins by olPPARc

PPARc regulates lipogenesis and lipid accumulation in the liver(Gavrilova et al., 2003; Schadinger et al., 2005), and plays a crucialrole in adipocyte differentiation (Ren et al., 2002; Lowell, 1999;Valyasevi et al., 2002). The fatty acid synthase (FASN), a major lipo-genic enzyme catalyzing the synthesis of long-chain saturated fattyacids from the 2-carbon donors malonyl-CoA and acetyl-CoA(Bressler and Wakil, 1961; Wakil, 1989), is distributed mainly incells with high lipid metabolism (Kusakabe et al., 2000). The mRNAand protein expression of FASN was up-regulated in olPPARc-over-expressing and/or Ro-treated NIH3T3 fibroblasts and 3T3L1 adipo-cytes (Fig. 7). Expression of the CCAAT/enhancer binding proteinalpha (C/EBPa) transcription factor, a key regulator of adipogenesis(Rosen et al., 2002; Shao and Lazar, 1997), was also increased inNIH3T3 and 3T3L1 cells (Fig. 7C and D). These results suggest thatolPPARc plays a critical role in lipid metabolism and adipogenesis,similar to the role played by mammalian PPARc.

4. Discussion

Here, we describe the isolation of the PPARc cDNA from oliveflounder (P. olivaceus). The open reading frame consists of1596 bp encoding 532 amino acids. The theoretical mol weight ofolPPARc is 60.3 kDa, and the mol weight as determined by Western

blot is approximately 60 kDa (data not shown). Although the over-all primary sequence of olPPARc shows 58% identity with humanPPARc, the zinc finger (C4-type) and ligand-binding domains show95% and 74% identities, respectively. In addition, two LXXLL motifsin the ligand-binding domain are perfectively matched to humanPPARc-1 and -2. The deduced amino acid sequence of olPPARcshows 60% and 93.8% identity with the PPARcs of zebrafish andplaice, respectively. Multiple sequence alignment demonstratedthat the fish PPARc protein is longer than its mammalian counter-part due to the presence of approximately 30 amino acid residues(in the N-terminal region and between the C4-type zinc finger do-main and ligand-binding domain). olPPARc was more closely re-lated to the PPARcs of fish than to those of other species.

Since olPPARc was detected from the early larval stage (Fig. 5A)and PPARc-knockout mice showed a lethal phenotype (Barak et al.,1999), we speculate a possible role of olPPARc in flounder develop-ment. olPPARc was highly expressed in the intestine and gills,moderately in the stomach and liver, and weakly in the kidney,spleen, and skin of normally fed flounder, but its expression wasincreased in all tested tissues, especially the spleen and skin, undera starved condition. In humans, PPARc is strongly expressed in adi-pocytes and weakly expressed in the bone marrow, spleen, testis,brain, skeletal muscle, and liver (Elbrecht et al., 1996). A recentstudy demonstrated that PPARc protects cells from serum starva-tion-induced apoptosis in human T lymphoma cell lines, andshowed that the survival effect of PPARc is mediated through itsactions on cellular metabolic activities (Jo et al., 2006). Our obser-vation of increased expression of olPPARc under the starved condi-tion suggests that olPPARc may be necessary for the protection offlounder from starvation.

Fig. 7. olPPARc regulates lipid synthesis- and adipogenesis-related protein expression. (A and B) NIH3T3 and 3T3L1 cells were transfected with an expression vector encodingolPPARc or mock transfected, and were then maintained in the presence of 10 lM or the same amount of DMSO. At 48 h post-transfection, the cells were harvested forquantitative RT-PCR. Actin was used as an internal control. Expression level of FASN mRNA in mock transfected and DMSO-treated cells was arbitrarily defined as 1. (C and D)NIH3T3 and 3T3L1 cells were transfected with an expression vector encoding olPPARc or mock transfected, and were then maintained in the presence of 10 lM or the sameamount of DMSO. At 48 h post-transfection, the cells were harvested for Western blotting. Actin was used as a loading control. The expression level of C/EBPa or FASN proteinin mock transfected and DMSO-treated cells was arbitrarily defined as 1. The data are representative of two or three independent experiments. The values represent themeans ± SD (n = 3). *P < 0.05, **P < 0.01 compared with the corresponding value for mock transfected and DMSO-treated cells. #P < 0.05, ##P < 0.01 compared with thecorresponding value for olPPARc-transfected and DMSO-treated cells.

H.K. Cho et al. / General and Comparative Endocrinology 163 (2009) 251–258 257

The H323, H449, and Y473 residues of human PPARc are criticalfor hydrogen bonding with the acidic head group of PPARc ligands(Uppenberg et al., 1998; Nolte et al., 1998; Xu et al., 1999), andthey are conserved in all mammalian, avian, and amphibianPPARcs; in contrast, in fishes, H449 is conserved while H323 is re-placed by isoleucine and Y473 is replaced by methionine (Leaveret al., 2005). The replaced residues or the inserted residues closeto the N-terminus of the ligand-binding domain in olPPARc mightaffect ligand specificity due to decreased ligand binding affinity oraction as a structural barrier for ligand proximity, respectively. Inreporter assays using HINAE and 3T3L1 cells (Fig. 6), olPPARc(about 1.7-fold) had weak transactivity for Ro compared withmPPARc (about 4.5-fold). However, the transactivity of olPPARc(about 3-fold) for Ci was similar to that of mPPARc (about 3-fold).Although variation between the sequences of olPPARc and mam-malian PPARc exist, olPPARc had transactivity for mammalianPPARc ligands. PPARc alone does not form a complex with PPREs;instead, it requires the addition of an RXR, and, in fact, the additionof PPARc ligands with retinoids results in synergic activation(Mukherjee et al., 1997). In this study, cotransfection of PPARcand RXRb increased PPRE transactivity in the presence of Ro orCi, indicating that olPPARc and olRXRb may form a heterodimerthat promotes PPRE activity in the presence of Ro or Ci.

We confirmed that FASN and C/EBPa were induced by olPPARcin a mouse adipocyte cell line (Fig. 7). Overexpression of PPARcin PPARc�/� mice has been shown to induce hepatic steatosis(Yu et al., 2003), and cells lacking PPARc express greatly reduced

levels of C/EBPa (Barak et al., 1999; Rosen et al., 1999). The resultsof this study indicate that olPPARc may play an important role inlipid metabolism and adipogenesis, similar to that of mammalianPPARc, and that olPPARc is evolutionarily conserved compared toPPARc in mammals.

Acknowledgments

This work was supported by a fisheries and development fundsgranted the Korean Ministry of Maritime affairs and Fisheries andby a grant from the National Fisheries Research and DevelopmentInstitute (NFRDI), Republic of Korea.

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Molecular cloning and characterization of olive IntroductionMaterials and methodsReagentscDNA sequences of olive flounder PPARγ (olPPARγ)Bioinformatic analysisFish rearing conditionStarvation protocolQuantitative real-time RT-PCR analysisConstruction of the expression plasmidCell cultureTransient transfection and luciferase assaySDS–PAGE and Western blot analysis

ResultsCloning of the cDNA encoding olive flounder peroCharacterization of the olPPARγ cDNAmRNA expression profile of olPPARγFunctional analysis of olPPARγRegulation of the expression of lipid synthesis

DiscussionAcknowledgmentsReferences