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j.gene.2017.10.066

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Accepted Manuscript
SFRP5 is a target gene transcriptionally regulated by PPARγ in
3T3-L1 adipocytes
Jun Zeng, Jiongyu Hu, Yu Lian, Youzhao Jiang, Bing Chen
PII:
DOI:
Reference:
S0378-1119(17)30912-5
doi:10.1016/j.gene.2017.10.066
GENE 42287
To appear in:
Gene
Received date:
Revised date:
Accepted date:
5 June 2017
16 October 2017
20 October 2017
Please cite this article as: Jun Zeng, Jiongyu Hu, Yu Lian, Youzhao Jiang, Bing Chen ,
SFRP5 is a target gene transcriptionally regulated by PPARγ in 3T3-L1 adipocytes. The
address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.066
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TITLE PAGE
Title: SFRP5 is a target gene transcriptionally regulated by PPARγ
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in 3T3-L1 adipocytes
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Authors:
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Jun Zeng, Jiongyu Hu, Yu Lian, Youzhao Jiang*, Bing Chen*
Department of Endocrinology, Southwest Hospital, Third Military Medical University,
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Chongqing, 400038, China.
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* Corresponding authors:
Address correspondence and reprint requests to Prof. Bing Chen (Department of
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Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing,
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400038, China. Tel/Fax: +86 23 68754138, Email: chenbing1960@163.com.) and
Prof. YouZhao Jiang (Department of Endocrinology, Southwest Hospital, Third
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Military Medical University, Chongqing, 400038, China. Tel: +86 23 68765721,
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Email: jyzh1980@163.com.)
Declaration of interest. We declared that we have no relevant conflicts of interest
Funding
This work was supported by the National Natural Science Foundation of China (No.
81400845)
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SFRP5 is a target gene transcriptionally regulated by PPARγ in 3T3L1 adipocytes
adipokine.
SFRP5
expression
increases
during
the
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identified
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[Abstract] Secreted frizzled-related protein 5 (SFRP5) is a newly
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differentiation and maturation of adipocytes, but the factors regulating
SFRP5 expression during this process remain unclear. This study showed
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that peroxisome proliferator-activated receptor γ (PPARγ) adenovirus
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transfection could enhance the SFRP5 expression of 3T3-L1 adipocytes.
Three potential binding sites of PPARγ in the SFRP5 promoter domain
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were found by bioinformatics analysis. Luciferase reporter gene assay
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demonstrated that PPARγ regulated the activity of the SFRP5 promoter
through cis-acting elements at −2,284–−1,500 bp. Further experiments
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verified that PPARγ could specifically bind to the SFRP5 promoter at
bp
using
chromatin
immunoprecipitation
and
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−2,284–−2,263
electrophoretic mobility shift assay. These results suggest that SFRP5 be
a target gene of PPARγ, and its expression may be under the
transcriptional regulation of PPARγ.
Abbreviations
SFRP5, secreted frizzled-related protein 5; PPARγ, peroxisome
proliferator-activated receptor γ; ChIP, chromatin immunoprecipitation;
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EMSA, electrophoretic mobility shift assay; CRD, cysteine-rich domain;
RSG, rosiglitazone; JNK, c-Jun N-terminal kinase.
Keywords: Secreted frizzled-related protein 5; Peroxisome proliferatoractivated receptor γ; Promoter; Chromatin immunoprecipitation;
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Electrophoretic mobility shift assay
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1. Introduction
Secreted frizzled-related protein 5 (SFRP5), a newly identified anti-
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inflammatory adipokine, can inhibit chronic inflammation, increase
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insulin sensitivity and improve glucolipid metabolism of obese mice
(Ouchi et al., 2010). The insulin sensitization effect of SFRP5 is mainly
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realized by binding with Wnt5a to inhibit the activation of c-Jun N-
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terminal kinase (JNK) in non-canonical Wnt pathway to reduce secretion
of inflammatory factors. In addition, SFRP5 plays an important role in
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adipocyte differentiation (Mori et al., 2012). Our previous research
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demonstrated that SFRP5 was not expressed in preadipocytes, but its
mRNA and protein expression levels gradually increased during the
differentiation and maturation of adipocytes (Lv et al., 2012). Other
researchers also demonstrated that SFRP5 was a marker of mature
adipocytes that was highly increased in mature adipocytes rather than
preadipocytes (Wang et al., 2014), which was consistent with our results.
However, the factors regulating SFRP5 expression during the
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differentiation and maturation of adipocytes remain unclear.
Peroxisome proliferator-activated receptor γ (PPARγ) is a critical
regulating factor in adipocyte differentiation (Wang et al., 2016). As a
transcription factor, PPARγ exerts its biological activity by binding with
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the promoter of target genes (Kim et al., 2015). Rosiglitazone (RSG), a
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selective agonist of PPARγ, could significantly enhance the mRNA and
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protein expression of SFRP5 in 3T3-L1 cells in our previous study (Lv et
al., 2012), and the variation of PPARγ expression was consistent with that
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of SFRP5 during adipocyte differentiation (Lv et al., 2012). These results
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suggest that SFRP5 expression might be regulated by PPARγ. However,
there is no data about the correlation of SFRP5 with PPARγ activation in
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vivo research. Furthermore, many research studies have shown that RSG
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exerts PPARγ-independent activity (Lin et al., 2014; Wang et al., 2016).
It is necessary to find out whether SFRP5 is directly transcriptionally
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regulated by PPARγ. In the present study, through luciferase reporter
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gene assay, chromatin immunoprecipitation (ChIP), and electrophoretic
mobility shift assay (EMSA), we found PPARγ could directly bind with
the SFRP5 promoter domain and regulate the SFRP5 promoter activity.
These results suggest that SFRP5 be a target gene of PPARγ.
2. Materials and methods
2.1. Reagents and antibodies
The mouse adipocyte 3T3-L1, human hepatoma cell line HepG2, and
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human embryonic kidney HEK293 cells were preserved in our laboratory
(central lab at the first affiliated hospital of the Third Military Medical
University, Chongqing, China). GW9662 (a PPARγ antagonist) and RSG
were obtained from Sigma Corporation (St. Louis, MO). The dual
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luciferase detection kit was obtained from Promega Corporation
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(Wisconsin, US). The ChIP kit, chromatin extraction kit, PPARγ antibody,
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and SFRP5 antibody were obtained from Abcam Corporation (Cambridge,
England). Biotin-labeled probe, non-labeled competitive probe, and non-
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labeled mutant probe were obtained from Viagene Corporation (Shanghai,
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China).
2.2. Cell culture
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The 3T3-L1, HepG2 and HEK293 cells were cultured in Dulbecco’s
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modified Eagle’s medium containing 10% fetal calf serum at 37 °C. The
cells in the logarithmic growth phase were used in all experiments.
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2.3. Establishment of luciferase reporter gene plasmid
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The primer of the deletion fragment at the mouse SFRP5 (Gene ID:
54612) 5’ end was designed through Primer 5.0 software. The genomewide DNA was used as a template, and the PCR amplification of
sequence in the 5’ flanking domain of the SFRP5 promoter was PCR
amplified. The PCR reaction condition was as follows: hot start for 3 min
at 94 °C, denaturation for 30 s at 94 °C, annealing for 1 min at 58 °C, and
extension for 1 min at 72 °C for 32 cycles. The obtained PCR
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amplification products and pGL3-basic plasmids were subjected to
double enzyme digestion by restriction enzymes KpnI and XhoI,
respectively. After purification, recycling, and double enzyme digestion,
the PCR amplification products and pGL3-basic plasmids were connected
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through the T4 DNA ligase and then transformed into competent cells.
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LB solid medium containing ampicillin was used to select positive clones,
Finally,
double
enzyme
digestion
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which were used to culture and extract plasmids, namely, pSFRP5-1.
verification
and
sequencing
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identification were conducted. Subsequently, various lengths of SFRP5
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promoter fragments were obtained by walking deletion assay with
pSFRP5-1 as a template. Then, luciferase reporter gene plasmids
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containing the above fragments were constructed, respectively. Namely,
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pSFRP5-2, pSFRP5-3, pSFRP5-4, pSFRP5-5, and pSFRP5-6. The
upstream primers for pSFRP5-1 to pSFRP5-6 are as follows: sfrp5-1F
5’-TTTCT CGAGGGCTGGATGTTGCGGCCT-3’, sfrp5-2F
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(3000)
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(2543) 5’-GGGGG TACCATCCAGGGCAAGGGCTGA-3’, sfrp5-3F
(2284)
5’-TTTGG TACCATGCCCAGCTCTGTCCAGGT-3’, sfrp5-4F
(1500)
5’-CCTGGTACCGGAGTGGATACTATAGTGTG-3’, sfrp5-
5F (1000) 5’-AAAGGTACCAGTTAACCCCACGTCTCCAA-3’, sfrp56F (500)
5’- TTTGGTACCGGGATTCATTAAATAGAAGC-3’. The
downstream
primer
for
pSFRP5-1
to
TTTCTCGAGGGCTG GATGTTGCGGCCT-3’.
pSFRP5-6
is:
5’-
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2.4 Establishment of the PPARγ adenovirus
Mouse RNA was extracted, and cDNA was generated by reverse
transcription. Then, the primers (upstream primer: 5’-ATAGTGC
GGCCGCGTTTATGGGTGAAACTCTGGAGAT-3’ and downstream
5’-GCATGCTCGAGGGACTTTCCTGCTAATACAAG-3’)
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primer:
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were used to amplify PPARγ. PCR reaction condition was as follows: hot
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start for 2 min at 94 °C, denaturation for 30 s at 94 °C, annealing for 40 s
at 58 °C, and extension for 2 min at 72 °C for 35 cycles. BamHI and SalI
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were used for double enzyme digestion of the PPARγ adenovirus shuttle
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vector pAd-TO4. PCR cloning identification of the bacterial solution was
conducted after recycled fragments were connected, and sequencing
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identification was implemented after plasmid extraction through positive
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strains and double enzyme digestion identification. Furthermore, pAdTO4-PPARγ was transformed into the BJ5183 bacteria through PmeI
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enzyme digestion. Then, bacteria were selected for identification. The
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pAd-PPARγ plasmid was used to transfect HEK293 cells after PacI
enzyme digestion, and adenovirus Ad-PPARγ was packed. After the
amplification and purification of adenovirus, the 3T3-L1 cells were
transfected, and then Western blot was conducted to detect the influence
of the PPARγ adenovirus on PPARγ protein expression.
2.5 Luciferase reporter gene assay
HepG2 cells, which are widely used for luciferase reporter gene
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assay (Zhou et al., 2013; Thonpho et al., 2010), were cultured in a 96hole cell plate. Transfection was jointly implemented by luciferase
reporter gene recombinant plasmids of the SFRP5 promoter, pGL3-basic
control plasmids, and beta-actin pRL-TK plasmids. After 48 h of culture,
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the cells were collected, washed three times with PBS, and then oscillated
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for 10 min after addition of cell lysis buffer. Luciferase activity was then
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detected. The relative luciferase activities of recombined reporter
pSFRP5 plasmids, pGL3-basic control plasmids, and beta-actin pRL-TR
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plasmids were calculated. To observe the effect of PPARγ on the
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activities of the SFRP5 promoter, the PPARγ adenovirus (3×1010 PFU per
hole), GW9662 (5 µmol/L), and RSG (10 µmol/L) were added.
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2.6 Real-time PCR
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Total RNA was extracted, and the concentration was detected. Up to
1 μg of RNA was used for reverse transcription using the PrimeScriptTM
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RT reagent kit (TaKaRa). Real-time PCR was performed using the
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SYBR® Green Real-time PCR Master Mix kit (Toyobo). The PCR
reaction condition was as follows: hot start for 3 min at 95 °C,
denaturation for 10 s at 95 °C, annealing for 30 s at 55 °C, and extension
for 15 s at 72 °C for 39 cycles. Fluorescence signal and Ct value were
detected using iCycler by Bio-Rad Laboratories, Inc. The experiment was
repeated thrice for each group.
2.7 Western blot analysis
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The 3T3-L1 cell proteins in different groups were extracted, and
their concentrations were determined. The sample loading quantity was
decided by the BCA method. The protein sample was mixed with 5×SDS
buffer. Up to 35 µg of the protein sample was separated by 10% SDS-
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PAGE gel electrophoresis and then transferred onto a PVDF membrane.
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The sample was then placed for 2 h at room temperature with 10%
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skimmed milk powder solution. The primary antibodies (1:1000) were
added and incubated at 4 °C overnight. After the membrane was washed
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with TBST (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.05% Tween
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20), the secondary antibodies (1:5000) were added and then incubated at
room temperature for 1 h. This procedure was repeated thrice. Finally, the
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PVDF film and chemiluminescent reagent were incubated at room
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temperature for 5 min. The film was exposed to X-ray in a dark room.
After X-ray film scanning, the optical density of the target band was
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determined using Quantity One image analysis software.
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2.8 ChIP assay
After the PPARγ adenovirus was infected for 24 h in 3T3-L1 cells,
the cells were fixed with 1% formalin and chromatin was extracted. The
optimal ultrasonic conditions of ChIP were obtained by selecting
different total volumes, ultrasonic power intensities, durations, interval
times, and cycle numbers. The optimal ultrasonic conditions were 400 µL
volume, 40% power, 10 s on/60 s off, and 20 circulations. DNA
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precipitation was conducted, and two pairs of primers were designed
(Primers 1 and 2). Upstream primer of primer 1 was 5’CCCTGGGTTTACCTCAT-3’ and downstream primer of primer 1 was
5’- GGAGAGCCCTCTATAAGG-3’. Upstream primer of primer 2 was
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5’-GGAGAGCCCTCTATAAGG-3’ and downstream primer of primer 2
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was 5’-GGTGGCTGTGTAAGGTGT-3’. Afterward, ordinary PCR and
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real-time PCR experiments were performed, and the operation was
repeated thrice for each group. The ordinary PCR reaction condition was
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as follows: hot start for 3 min at 95 °C, denaturation for 30 s at 95 °C,
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annealing for 30 s at 50 °C, and extension for 30 s at 72 °C for 30 cycles.
2.9 EMSA experiment
experiment.
Biotin-labeled
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early-stage
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PPARγ binding site was determined as aagggc on the basis of an
ACCACAGAAGGGCA-3’),
(5’- CAGGGCAAGGGCTG
non-labeled
competitive
(5’-
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CAGGGCAAGGGCTGACCACAGAAGGGCA-3’), and non-labeled
AC
mutant (5’-CAGG GCAAGGGCTAAAGACAGAAGGGCA-3’) probes
for binding the SFRP5 promoter domain and PPARγ were prepared and
verified. Cell nuclear protein was extracted after the PPARγ agonist
rosiglitazone was used to treat the 3T3-L1 cells. Concentration
determination (BCA method) of the extracts was completed. The reaction
systems were incubated for 20 min with the antibodies at room
temperature. For competition, a
200-fold
excess
of unlabeled
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oligonucleotides
was
incubated
with
nuclear
extract
before
oligonucleotides were added. Shift reactions were loaded onto a 4%
polyacrylamide gel and run at 200 V for 3 h in 0.25× TBE buffer. Gels
were dried and exposed for 12 h at −80 °C.
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2.10 Statistical analysis
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Data were expressed as mean ± standard deviation. SPSS 17.0
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software was used to for statistical analysis of data. One-way ANOVA
was used for comparison among the groups, and the SNK method was
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used for comparison between each two groups. P<0.05 indicated
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statistical difference.
3. Results
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3. 1 Construction of the PPARγ adenovirus
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PPARγ gene fragments were obtained by PCR (Fig. 1A). Enzyme
digestion was conducted on pAd-TO4-PPARγ for the confirmation of
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successful connection (Fig. 1B). The enzyme digestion of the extracted
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plasmids proved the successful recombination of the adenovirus
expression vector (pAd-PPARγ) (Fig. 1C). The fluorescence of the
transfected 293 cells verified the successful packing of the adenovirus
(Fig. 1D). PPARγ protein level increased significantly (P<0.05) in 3T3L1 after PPARγ adenovirus transfection compared with that in the control
group (Fig. 1E and 1F).
3.2 SFRP5 expression was enhanced by PPARγ transfection
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The 3T3-L1 cells were treated with DMSO, GW9662, PPARγ
adenovirus transfection with or without RSG, and RSG alone. Then the
protein expression of SFRP5 was detected by Western blot. SFRP5
expression was significantly elevated (P<0.05) (Fig. 2A and 2B) after
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treatment of the PPARγ adenovirus, RSG, and RSG plus PPARγ
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adenovirus. By contrast, SFRP5 expression was down-regulated by
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GW9662. These results indicated that PPARγ could enhance SFRP5
expression.
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3.3 Bioinformatics analysis identified potential binding sites of
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PPARγ in the SFRP5 promoter domain
The NUBIScan software was adopted for the online prediction of
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binding sites in the SFRP5 promoter domain. A similarity grade score
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greater than 0.8 was considered a selection criterion (Ramani et al., 2012).
Promoter sequence analysis revealed three potential binding sites of
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PPARγ in the SFRP5 promoter domain (Table 1).
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3.4 Identification of the SFRP5 promoter and influence of PPARγ on
the promoter activity
In HepG2 cells, the relative fluorescence values of the above pGLSFRP5 of different truncation lengths were obviously higher than those of
the pGL-3-basic empty vectors (P<0.05) (Fig. 3A). This result indicated
that the above fragments promoted the transcription of pGL-3-basic
reporter genes. No statistical differences were observed among pGL-3-
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3000, pGL-3-2543, and pGL-3-2284, and neither between pGL-3-1500
and pGL-3-1000. However, the comparison between pGL-3-3000, pGL3-2543, and pGL-3-2284 and pGL-3-1500 and pGL-3-1000 showed
statistical difference (P<0.05) (Fig. 3A). This finding suggests that an
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important transcriptional activation of cis-acting elements exist within
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−2284–−1500 bp. No statistical difference in the relative fluorescence
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values was found among the non-treatment, RSG, and PPARγ adenovirus
groups (P>0.05) (Fig. 3B); however, the relative fluorescence values of
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RSG plus PPARγ adenovirus group was higher than those of the two
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other groups (P<0.05) (Fig. 3B). Conversely, GW9662 decreased the
relative fluorescence values (P<0.05) (Fig. 3B). These results indicated
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that PPARγ could enhance the SFRP5 promoter activity.
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3.5 PPARγ can bind with the SFRP5 promoter at the −2284–−2263
bp domain
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DNA binding with PPARγ was used as template, and the expected
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bands were obtained in the electrophoresis channel of primer 1 (designed
according to −2284–−2263 bp region) rather than primer 2 (designed
according to −2543–−2515 bp region) (Fig. 4A). This result indicated that
PPARγ could bind with the SFRP5 promoter domain of −2284–−2263 bp.
Fold enrichment by real-time PCR experiment indicated and SFRP5 was
enriched by 23.1 times after using PPARγ antibody (Fig. 4B).
After the treatment of PPARγ agonist rosiglitazone, cell nuclear
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extracts of 3T3-L1 cells were obtained, and the EMSA probe was
synthesized through biotin labeling. After the co-incubation of the biotinlabeled probe and the 3T3-L1 nuclear protein, compared with the
unbounded probe, a hysteretic band appeared (Fig. 4C. lane 2). This
bound with each other to form the
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of −2284–−2263bp domain
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result indicated that the 3T3-L1 nuclear protein and the SFRP5 promoter
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DNA/protein compounds. Specific/non-specific competitive binding tests
were conducted. The addition of 200-fold cold probes (non-labeled
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probes) could enable the disappearance of the blocked bands (Fig. 4C
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lane 3), demonstrating that the DNA/protein compounds were formed by
binding between the 3T3-L1 nuclear protein and the SFRP5 promoter
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−2284–−2263 bp domain. The bands disappeared when labeled mutant
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probes were added, suggesting that these DNA/protein compounds were
specific and were formed by binding PPARγ with the SFRP5 promoter
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−2284–−2263 bp domain (Fig. 4C lane 4). A more hysteretic band
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appeared after the PPARγ antibody was added in the reactant, verifying
the specificity of binding between the PPARγ protein and the probes (Fig.
4C lane 5). These results confirmed that PPARγ could specifically bind to
the SFRP5 promoter at −2284–−2263 bp domain in the extracellular
aspect.
4. Discussion
In this research, western blot revealed that the PPARγ adenovirus
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could enhance SFRP5 expression. PPARγ could regulate the activity of
the SFRP5 promoter through the luciferase reporter gene technology.
Binding sites of PPARγ were predicted through bioinformatics analysis in
the SFRP5 promoter. Meanwhile, ChIP and EMSA experiments further
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indicated that PPARγ could bind to the SFRP5 promoter at the −2284–
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−2263 bp domain. These results confirmed that SFRP5 was a target gene
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under the direct transcriptional regulation of PPARγ.
SFRP5, a member of the SFRP superfamily, is expressed in the liver,
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gastric mucosa, intestinal epithelial cells, and adipose tissues (Wang et al.,
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2014; Gutierrez-Vidal et al., 2015; Anunciado-Koza et al., 2016; Bie et
al., 2016). SFRP5 contains a cysteine-rich domain (CRD), which is of
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high homology with the CRD of the Wnt receptor frizzled protein. Hence,
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SFRP5 can compete with the frizzled protein receptor for the Wnt ligand
to exert a regulating effect on the Wnt pathway. SFRP5 can also inhibit
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non-canonical Wnt pathway to regulate glucolipid metabolism. However,
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there were few studies referring the upstream pathways regulating the
SFRP5 expression. It has been reported SFRP5 is a gene regulated by
DNA methylation, and high methylation in the promoter domain can
inhibit its expression (Xie et al., 2014). It had been reported that the
methylation in the SFRP5 promoter domain was related to the
development of gastric carcinoma, colorectal carcinoma, and leukemia
(Silva et al., 2013; Zhao et al., 2013; Wang et al., 2014; Liang et al.,
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2015). However, studies on adipocytes have found that demethylase
could not enhance SFRP5 expression in 3T3-L1, and the SFRP5
expression induced by high-fat diet without the alteration of methylation
level in its promoter domain (Okada et al., 2009). These data suggest that
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SFRP5 expression be influenced by other factors other than methylation
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regulation.
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The PPARγ selective agonist RSG could enhance SFRP5 expression,
indicating that SFRP5 expression might be regulated by PPARγ (Lv et al.,
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2012). However, in addition to exertion of biological effect as a ligand of
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PPARγ, RSG also possesses PPARγ-independent pathway (Lin et al.,
2014; Wang et al., 2016). In the present research, the PPARγ adenovirus
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itself could directly enhance SFRP5 expression, and the administration of
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RSG plus PPARγ adenovirus obviously increased SFRP5 expression
compared with single treatment. The above results indicated that SFRP5
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expression was regulated by PPARγ, but further research is required to
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determine whether this regulation was direct.
Through bioinformatics analysis, multiple binding sites of PPARγ
were found on the upstream of the transcriptional initiation point of the
SFRP5 gene, where the −2284–−2263 site belonged to a highly conserved
sequence. The luciferase reporter gene experiment verified the presence
of cis-acting elements regulating the transcriptional activity of SFRP5 in
the −2284–−1500 bp domain, which includes the −2284 –−2263 domain.
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Notably, this experiment was conducted in HepG2 cells instead of mouse
cell lines. Although HepG2 is one of the most widely used cell types for
this assay, not only for genes of homo sapiens, but also for mouse genes
(Zhou et al., 2013; Thonpho et al., 2010), it is still a limitation of this
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study. The results of ChIP and EMSA showed that PPARγ could directly
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regulate the transcription of the SFRP5 gene.
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bind to the −2284–−2263 bp site in the SFRP5 promoter domain to
First, SFRP5 was reported as an anti-inflammatory adipokine in
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animal experiments (Ouchi et al., 2010). Subsequently, several studies
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focused on the correlation of SFRP5 with metabolic syndrome in humans.
A previous study (Hu et al., 2013) revealed that plasma SFRP5 was
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decreased in Chinese obese and type 2 diabetes mellitus (T2DM) subjects
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and was an independent factor affecting glucolipid metabolism,
inflammation, and insulin resistance (IR). Circulating SFRP5 was
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significantly lower in both impaired glucose intolerance and newly
AC
diagnosed type 2 diabetes mellitus than in individuals with normal
glucose
tolerance.
Furthermore,
overweight/obese
subjects
had
significantly lower Sfrp5 levels than lean individuals (Hu et al., 2013).
These results were supported by some other studies (Yin et al., 2017; Xu
et al., 2017). Catalán V et al. demonstrated that down-regulation
of SFRP5 might promote a proinflammatory state in visceral adipose
tissue in humans contributing to the development of obesity-associated
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comorbidities (Catalán et al., 2014). However, there were still some other
researchers obtained inconsistent results. Carstensen et al. revealed
that serum SFRP5 was directly related to HOMA-IR in humans
(Carstensen et al., 2013). In another study, serum SFRP5 levels were
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elevated in T2DM patients as compared with prediabetic individuals and
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controls (Canivell et al., 2015). Most of the above studies were based on
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small sample sizes (Carstensen-Kirberg et al., 2017). A recent study
involving a large population-based cohort revealed that high serum
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SFRP5 was inversely associated with multiple risk factors for type 2
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diabetes and cardiovascular diseases (Carstensen-Kirberg et al., 2017).
Overall, SFRP5 was considered to be a target for the treatment of obesity
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and related diseases, such as T2DM and nonalcoholic steatohepatitis.
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In the current study, we revealed that SFRP5 was the target gene
under the direct transcriptional regulation of PPARγ, whose agonists are
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widely used for the treatment of type 2 diabetes. Despite the lack of
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evidence that RSG treatment in humans affects SFRP5 level, our research
provided the possibility that SFRP5 upregulation is an important pathway
through which RSG exerts its anti-diabetic effect. Recent studies have
found that RSG and piglitazone increased cardiovascular events, heart
failure, and osteoporosis (Standl et al., 2016; Adil et al., 2017), and their
use is limited in many countries. Whether SFRP5 can be an alternative
drug of glitazones for the treatment of type 2 diabetes and related diseases
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with alike therapeutic effect and less side effect is intriguing.
5. Conclusion
SFRP5 is the target gene under the direct transcriptional regulation of
PPARγ, providing a new clue for the treatment of obesity and related
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diseases.
Declaration of interest. We declared that we have no relevant conflicts of interest
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
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NU
81400845)
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Pharmacother 94 (2017), pp. 1010-1019.
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Anunciado-Koza, R.P., Higgins, D.C. and Koza, R.A. Adipose tissue Mest and Sfrp5 are
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Figures/Tables
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Fig. 1. Construction of PPARγ adenovirus. (A) Amplification of PPARγ fragment. (B)
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Identification of pAd-TO4-PPARγ. (C) Enzyme digestion analysis of pAd-PPARγ by Pac I.
(D) Adenovirus packing in HEK293 cells. (E) and (F) PPARγ expression was enhanced by
PPARγ adenovirus transfection in 3T3-L1 cells. (n=3 for each). *P<0.05 compared with
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Fig.2. SFRP5 expression was enhanced by PPARγ in 3T3-L1 cells. (n=3 for each). *P <0.05
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compared with DMSO group.
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Fig.3. Identification of sfrp5 promoter and the effect of PPARγ on sfrp5 promoter activity in
HepG2 cells. (A) Luciferase activity of different lengths of promoter. (B) PPARγ enhanced
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the sfrp5 promoter activity. (n=3 for each). #P<0.05 compared with PGL-3-1500; *P<0.05
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compared with PGL-3-Basic (A) or DMSO (B).
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Fig.4. PPARγ binds with the promoter region containing PPRE element between −2,284 –
−2,263. (A) Target band was obtained using PPARγ-bing DNA fragment as templet by PCR.
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(B) Fold enrichment detection by real-time PCR. (C) Binding of PPARγ with sfrp5 promoter
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was verified by ectrophoretic mobility shift assay.
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Table 1 Potential binding sites of PPARγ with SFRP5 promoter
Score
P value
Site sequence
-2543
0.811344
0.016892
AGGGCAagggcTGACCA
-2529
0.811344
0.016892
TGACCAcagaAGGGCA
-2284
0.803993
0.0139931
TGCCAgctctgtccAGGTCA
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Position
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Highlights
 SFRP5 expression was enhanced by PPARγ transfection
 PPARγ could regulate the activity of the SFRP5 promoter
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 PPARγ could bind with the SFRP5 promoter at −2,284 – −2,263bp
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domain
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