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General and Comparative Endocrinology 163 (2009) 251–258
Contents lists available at ScienceDirect
General and Comparative Endocrinology
journal homepage: www.elsevier .com/locate /ygcen
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: [email protected] (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
mailto:[email protected]://www.sciencedirect.com/science/journal/00166480http://www.elsevier.com/locate/ygcen
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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
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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
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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.
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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