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Cardiovascular Research Advance Access first published online on September 24, 2008
This version [Corrected Proof] published online on October 16, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn264
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

PARP-1 suppresses adiponectin expression through poly(ADP-ribosyl)ation of PPAR{gamma} in cardiac fibroblasts

Dan Huang{dagger}, Chongzhe Yang{dagger}, Yan Wang, Yuhua Liao and Kai Huang*

Department of Cardiology, Institute of Cardiovascular Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China

* Corresponding author. Tel: +86 27 85726360; fax: +86 27 85756636. E-mail address: huangkai1{at}yahoo.com

Received 19 June 2008; revised 23 September 2008; accepted 23 September 2008

Time for primary review: 24 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Our aim was to explore the mechanism underlying the transcriptional regulation of adiponectin and its receptors (AdipoR) in cultured rat cardiac fibroblasts.

Methods and results: Using western blot and real-time RT–PCR assays, the expression of adiponectin and its receptors was determined. Using Southwestern blot and electrophoretic mobility shift assays, the DNA binding activity of peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) was determined. The results showed that adiponectin and AdipoR1 were highly expressed in cultured rat cardiac fibroblasts. Inhibition of poly(ADP-ribose) polymerase 1 (PARP-1) by 3-aminobenzamide, PJ34, or PARP-1 siRNA markedly increased the transcription of adiponectin and AdipoR1 in cultured fibroblasts, mature 3T3 L1 adipocytes, rat myocardium, and white adipose tissue. PPAR{gamma} was poly(ADP-ribosyl)ated by PARP-1 in cardiac fibroblasts under basal conditions. Poly(ADP-ribosyl)ation of PPAR{gamma} prevented its binding to DNA. Inhibition of PARP-1 enhanced the DNA binding and transactivation of PPAR{gamma} and increased the transcription of PPAR{gamma}-target genes including CD36, lipoprotein lipase, and leptin in cultured fibroblasts.

Conclusion: PARP-1 inhibits adiponectin and AdipoR1 expression as well as PPAR{gamma} transactivation through poly(ADP-ribosyl)ation of PPAR{gamma} in cultured rat cardiac fibroblasts.

KEYWORDS Adiponectin; Poly(ADP-ribose) polymerase; Peroxisome proliferator activated receptor-gamma; Cardiac fibroblast; Transcriptional regulation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Cardiac fibroblasts, which make up nearly two-thirds of the total myocardial cell number, are the major source of extracellular matrix (ECM) production in the heart and contribute importantly to structural, biochemical, mechanical, and electrical properties of the myocardium.1 Cardiac fibroblasts synthesize, release and appear to be functionally responsive to a variety of hormones and growth factors which play key roles in cardiac hypertrophy, fibrosis, and the deterioration of heart function.2 Recent studies have revealed that adiponectin protects the heart against adverse ventricular hypertrophy and interstitial fibrosis in cardiac diseases.3–5 Data suggest that adiponectin contributes critically to the maintenance of cardiac fibroblast function. Adiponectin is abundantly produced by differentiated adipocytes, liver cells, and cardiomyocytes,6–9 and plays an important role in fatty acid oxidation and glucose uptake.10 It has been reported that adiponectin is a potent anti-inflammatory adipocytokine.11,12 Adiponectin has two receptors, AdipoR1 and AdipoR2,13 which are abundantly expressed in cardiomyocytes,9 skeletal muscle (AdipoR1) and liver (AdipoR2).14 Among these, AdipoR1 is critical for the anti-inflammatory effects of adiponectin.15 Although some studies have shown that the transcription of adiponectin and its receptors were regulated by peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) and retinoid X receptor (RXR),16,17 the expressional regulation of adiponectin and its receptors in cardiac fibroblasts still remain to be defined.

Poly(ADP-ribose) polymerase 1 (PARP-1), a ubiquitous nuclear enzyme present in eukaryotes, accounts for about 90% of cellular poly(ADP-ribose) polymerase activity. PARP-1 normally functions in genomic stabilization through successive transfer of ADP-ribose moiety of nicotinamide dinucleotide (oxidized, NAD+) to a variety of nuclear proteins which participate in DNA repair. It has been demonstrated that activation of PARP-1 contributes importantly to the pathogenesis of cardiac dysfunction associated with cardiovascular diseases.18–21 Pharmacological inhibition of PARP provides significant benefits in animal models with cardiovascular disorders.18–21 Although PARP-1 is implicated in the transcriptional regulation of many genes through collaboration with transcription factors,21,22 it remains unclear whether PARP-1 is involved in the transcriptional regulation of adiponectin and its receptors.

In the present study, the expression and transcriptional regulation of adiponectin and its receptors in cultured cardiac fibroblasts were explored. Our results demonstrated that adiponectin and AdipoR1 were abundantly expressed in cultured fibroblasts under basal condition. Inhibition of PARP-1 enhanced the transcription of adiponectin and AdipoR1, and promoted PPAR{gamma} transactivation in cultured cardiac fibroblasts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Animal model
Wistar rats, male, 220–260 g, were randomly divided into three groups. The animals received an intraperitoneal injection once a day for 14 days with 3-aminobenzamide (3AB, 30 mg/kg/day, Sigma), or N-(6-oxo-5, 6-dihydro-phenanthridin-2-yl)-2-(N, N-dimethylamino) acetamide (PJ34, 10 mg/kg/day, Sigma), or an equal volume of physiological saline. After treatment, the hearts and white adipose tissues (WAT) were isolated and stored in liquid nitrogen. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and is approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

2.2 Cell culture
Rat cardiac fibroblasts were isolated from the ventricles of 1–2-day-old Wistar rats. The technical details were described in Supplementary material online. The 3T3-L1 preadipocytes (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, China) were cultured and differentiated as described previously.23

2.3 RNA interference and transfection
Small interfering RNA (siRNA) for PARP-1 (sense 5'-GGAUGAUCUUCGA CGUGGA-3', antisence 5'-UCCACGUCGAAGAUCAUCC-3') was designed and synthesized by Invitrogen (Carlsbad, CA). The cultured cells were transfected with 50 nM siRNA with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

2.4 Preparation of whole extracts and nuclear extracts
Whole extracts and nuclear extracts of fibroblasts were prepared as described.24 The details were described in Supplementary material online.

2.5 Poly(ADP-ribosyl)ation of nuclear extracts and recombinant peroxisome proliferator activated receptor {gamma}
Assays were performed as described previously.25 Twenty micrograms of nuclear extracts were incubated with 10 µM NAD+ in incubation buffer (containing 100 mM Tris–HCl (pH 8.0), 20 mM MgCl2, and 1 mM dithiothreitol). Recombinant PPAR{gamma} protein (400 ng, Cayman, Ann Arbor, MI) or p65 protein (400 ng, Cell Signaling, Danvers, MA) was incubated with recombinant PARP-1 protein (40 ng, Trevigen, Gaithersburg, MD), 10 µM NAD+ and 20 µg/mL activated DNA (Trevigen) in incubation buffer. In control assay, nuclear extracts or recombinant proteins were pre-incubated with 10 mM 3AB for 5 min at 37°C before the addition of NAD+. Reaction was carried out at 37°C for 20 min.

2.6 Transient transfection and dual luciferase reporter assays
The luciferase reporter plasmid PPRE3-TK-LUC [containing three copies of PPRE (PPAR response elements) consensus sequence] and control plasmid TK-LUC were kindly provided by Dr M.R. Evans (Salk Institute for Biological Studies). The full length PPAR{gamma} cDNA was subcloned into pcDNA3.1 mammalian expression plasmid (Invitrogen). pcDNA3.1-mPPAR{gamma} expressing plasmids (0.5 µg) were cotransfected with PPRE-TK-LUC (0.9 µg) and pRL-SV40 plasmid (6 ng, internal control for normalization of transfection efficiency, Promega, Madison, WI) into cultured fibroblasts using lipofectamine 2000 (Invitrogen). TK-LUC plasmid and empty pcDNA3.1 vector were used as control.

The human adiponectin promoter region spanning from –908 to +14 bp was amplified by PCR with the human genomic DNA of adiponectin26 and inserted into the pGL3 basic luciferase expression vector (Promega).27 Two micrograms of pGL3-basic plasmid containing human adiponectin promoter or control pGL3-basic plasmid was cotransfected with 6 ng of pRL-SV40 plasmid into cultured fibroblasts using lipofectamine 2000 (Invitrogen).

The luciferase activity was determined with Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instruction.

2.7 Poly(ADP-ribose) polymerase activity assay
PARP activity was assayed using the universal colorimetric PARP assay kit (Trevigen), based on the incorporation of biotinylated ADP-ribose onto histone proteins. Cell lysates from cardiac fibroblasts containing 50 µg of protein were loaded into a 96-well plate coated with histones and biotinylated poly ADP-ribose, allowed to incubate for 1 h, treated with strep-HRP, and read at 450 nm in a spectrophotometer.

2.8 Western blot
Fifty micrograms of denatured cell lysates were separated on a 9% SDS–PAGE gel and transferred to nitrocellulose membranes (Pierce, Rockford, IL). After blocking for 3 h with 5% non-fat milk in TBS, the membranes were incubated with 1:1000 primary antibodies against PARP-1, PAR (Trevigen), adiponectin, AdpoR1, and AdipoR2 (Chemicon, Temecula, CA) at 4°C overnight, respectively. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibody (1:5000) for 2 h at room temperature. Specific bands were detected using the ECL detection system (Pierce). Band intensities were quantified with NIH Image Version 1.61.

2.9 Enzyme-linked immunosorbent assay for adiponectin
Adiponectin levels in rat sera or supernatants of cell cultures were analysed using a Rat Adiponectin ELISA kit (Chemicon) according to the manufacturer’s protocol. The results were compared with a standard curve. Each assay was carried out in triplicate for each sample. Absorbance was measured at 450 nm by means of a spectrophotometer.

2.10 Real-time RT–PCR
Total RNA from cultured cells or tissues was isolated using Trizol reagent (Invitrogen) according to manufacturer’s instruction, respectively. One microgram of total RNA was reverse transcripted using RNA PCR Kit (Takara Biotechnology, Dalian, China) and the resulting cDNA was used as a PCR template. The mRNA levels were determined by real-time PCR with ABI PRISM 7900 Sequence Detector system (Applied Biosystem, Foster City, CA) according to the manufacturer’s instructions. GAPDH was used as endogenous control. PCR reaction mixture contained the SYBR Green I (Takara Biotechnology), cDNA, and the primers. Relative gene expression level (the amount of target, normalized to endogenous control gene) was calculated using the comparative Ct method formula 2–{Delta}{Delta}Ct. The primer sequences are listed in Supplementary material online, Table S1.

2.11 Immunoprecipitation assay
Immunoprecipitation assays were performed as described.28 Briefly, 100 µg of nuclear extracts were incubated with 5 µg of unspecific IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A-Sepharose beads (Santa Cruz) for 30 min at 4°C with rocking. After centrifugation, supernatants were incubated with antibodies against PARP-1, PAR or unspecific IgG at 4°C for 1 h, respectively. Protein A-Sepharose beads (1:1 slurry) were added next, and the mixtures were incubated at 4°C for another 1 h. After centrifugation at 5000g for 1 min, the beads were washed and the bead-bound proteins were extracted with SDS gel loading buffer and subjected to western blot analysis.

2.12 In vitro protein–protein interaction assay (far-western blot)
Nuclear extracts were resolved on a 9% SDS–PAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked with 5% BLOTTO in Hyb-75 buffer followed by incubation with 1 µg/mL PARP-1 (Trevigen) at room temperature for 1 h. After washing with Hyb-75 buffer, membranes were incubated with anti-PARP-1 antibody and then secondary antibody, visualized by enhanced chemiluminescence and X-ray film, and quantified using NIH image version 1.61 as detailed in Supplementary material online.

2.13 Electrophoretic mobility shift assay and supershift assay
LightShiftTM Chemiluminescent EMSA kit (Pierce) was used to detect DNA-protein interaction. The sequences of PPRE consensus oligonucleotides were: forward 5'-GGTAAAGGTCAAAGGTCAATCGGC-3'; reverse 5'-GCCGATTGACC TTTGACCTTTACC-3'.29 Additional details are shown in Supplementary material online.

2.14 Southwestern blot
Southwestern blot was performed according to the procedure of Butler and Ordahl28 with slight modifications. Nuclear proteins (35 µg) were resolved on a 9% SDS–PAGE and then electrotransferred to a nitrocellulose membrane. Membranes were blocked with 5% BLOTTO–0.1% bovine serum albumin–1 mg/mL poly(dI–dC) in binding buffer [30 mM HEPES (pH 7.6), 1 mM dithiothreitol], followed by incubation with 1.0 pmol biotin-labelled PPRE oligonucleotides in Hyb-50 buffer [30 mM HEPES (pH 7.6), 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 5% BLOTTO, 0.1% bovine serum albumin, and 1 mg/mL poly(dI–dC)] at 4°C overnight. After washing three times [30 mM HEPES (pH 7.6), 50 mM NaCl, 1% BLOTTO], membranes were incubated with streptavidin-horseradish peroxidase conjugate in blocking buffer (Pierce) for 15 min. Specific binding was detected with ECL detection reagents (Pierce) and band intensities were quantified as described earlier.

2.15 Statistic analysis
Results are shown as mean±SEM of at least three independent experiments. The significance of differences was estimated by ANOVA followed by Student–Newmann–Keuls multiple comparison tests. P < 0.05 was considered significant. All statistical analyses were performed with SPSS software (version 11.0, SPSS Inc., Chicago, IL).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Expression of adiponectin and AdipoR1 in cardiac fibroblasts
We observed that adiponectin and AdipoR1, but not AdipoR2 were abundantly expressed at the mRNA and protein levels in the cultured fibroblasts (Figure 1). We further detected the released adiponectin in the supernatants (2.91 ± 0.23 ng/mL). The levels of adiponectin were 30-fold higher than that in the unused culture medium (0.097 ± 0.0070 ng/mL) (P < 0.05).


Figure 1
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  		Figure 1 Expression of adiponectin and AdipoR1 in cultured rat cardiac fibroblasts. (A) Western blot analysis of adiponectin, AdipoR1, and AdipoR2 protein in cytoplastic extracts and nuclear extracts from cultured rat cardiac fibroblasts or cardiomyocytes. β-actin and Hist1 served as loading control for whole extracts and nuclear extracts, respectively. (B) RT–PCR analysis of adiponectin, AdipoR1, and AdipoR2 mRNA in cultured rat cardiac fibroblasts. GAPDH served as endogenous control. (C) Quantitative real-time RT–PCR analysis of AdipoR1 mRNA and AdipoR2 mRNA relative to GAPDH with equal loading of samples from cultured rat cardiac fibroblasts. Data are expressed as mean±SEM, n = 6, *P < 0.01.

 
3.2 Inhibition of poly(ADP-ribose) polymerase-1 promotes transcription of adiponectin and AdipoR1 genes
We next showed that treatment with PARP inhibitor (3AB or PJ34) or PARP-1 siRNA significantly enhanced the transcription of adiponectin (Figure 2A) and AdipoR1 (Figure 2B) in cardiac fibroblasts and mature 3T3 L1 adipocytes (see Supplementary material online, Figure S1B). Inhibition of PARP-1 also significantly enhanced the transcription of the above-mentioned genes in the cultured rat cardiomyocytes (data not shown). Treatment with Ang II did not influence the transcription of adiponectin and AdipoR1 in fibroblasts. Neither 3AB nor PARP-1 siRNA influenced the transcription of TNF-{alpha}, IL-6, and IL-1β in the cultured fibroblasts under basal condition (Figure 2F).


Figure 2
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  		Figure 2 Effects of 3AB, PJ34, and poly(ADP-ribose) polymerase-1 siRNA on transcription of adiponectin and AdipoR1 genes in cultured rat cardiac fibroblasts. (A and B) Quantitative real-time RT–PCR analysis of adiponectin and AdipoR1 in cultured rat cardiac fibroblasts treated with 3-aminobenzamide (3AB(5): 5 mmol/L, 3AB(10): 10 mmol/L, 3AB(20): 20 mmol/L), or PJ34 (PJ34(1): 1 µmol/L, PJ34(5): 5 µmol/L, PJ34(10): 10 µmol/L), or pioglitazone (PTZ(5): 5 µmol/L, PTZ(10): 10 µmol/L, PTZ(15): 15 µmol/L), or AngII (0.1 µmol/L) for 24 h, or transfected with poly(ADP-ribose) polymerase-1 siRNA (50 nmol/L) or unrelated siRNA (50 nmol/L) for 48 h. (C and D) Quantitative real-time RT–PCR analysis of adiponectin and AdipoR1 in hearts of rat administered with 3-aminobenzamide (30 mg/kg/day) or PJ34 (10 mg/kg/day) for 0 day, 7 days, 14 days, respectively. (E) ELISA analysis of adiponectin secreted into sera of rat treated with 3-aminobenzamide (30 mg/kg/day) or PJ34 (10 mg/kg/day) for 0 day, 7 days, 14 days, respectively. (F) ELISA analysis of TNF-{alpha}, IL-6, and IL-1β secreted into culture media in cultured rat cardiac fibroblasts. Data are expressed as mean±SEM, n = 6. **, ## P < 0.01; *, # P < 0.05 compared with control group.

 
In vivo administration of rats with 3AB or PJ34 significantly increased adiponectin expression in myocardium (Figure 2C), serum (Figure 2E), and WAT (see Supplementary material online, Figure S1A) on day 7 and day 14, AdipoR1 expression in myocardium on day 14 (Figure 2D), and AdipoR1 and AdipoR2 expression in WAT on day 7 and day 14 (see Supplementary material online, Figure S1A).

The data indicated that PARP-1 siRNA inhibited PARP-1 expression and biological activity, whereas 3AB and PJ34 decreased PARP activity, but did not influence PAPR-1 expression in fibroblasts under basal conditions (see Supplementary material online, Figure S2).

3.3 Inhibition of PARP-1 increased PPRE directed reporter expression and adiponectin promoter activity
Activation of PPAR{gamma} promotes the transcription of adiponectin and AdipoR1.16,17 In this study, treatment of cultured fibroblasts with 3AB, PJ34, PARP-1 siRNA, or PPAR{gamma} activator pioglitazone (PTZ) significantly enhanced PPRE-directed luciferase gene expression, respectively (Figure 3A). Thereafter, forced expression of PPAR{gamma} was used to explore the influence of PARP inhibition on PPAR{gamma} transactivation in cultured fibroblasts. Our results showed that treatment with 3AB, PJ34, or PARP-1 siRNA significantly enhanced PPRE-directed reporter expression in pcDNA3.1-mPPAR{gamma}-transfected fibroblasts (Figure 3B).


Figure 3
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  		Figure 3 Inhibition of poly(ADP-ribose) polymerase-1 promoted transactivation of peroxisome proliferator activated receptor {gamma}. (A) Cardiac fibroblasts were co-transfected with PPRE-TK-LUC or TK-LUC plasmid (0.9 µg), and pRL-SV40 (6 ng). After transfection, fibroblasts were treated with 3AB (3AB(10): 10 mmol/L, 3AB(20): 20 mmol/L), or PJ34 (PJ34(5): 5 µmol/L, PJ34(10): 10 µmol/L), or/and piglitazone (15 µmol/L), or poly(ADP-ribose) polymerase-1 siRNA (50 nmol/L) or unrelated siRNA for 24 h. (B) pcDNA3.1-mPPAR{gamma} expression plasmids (0.5 µg) were co-transfected with PPRE-TK-LUC or TK-LUC plasmid (0.9 µg) and pRL-SV40 (6 ng) into cultured fibroblasts. After transfection, fibroblasts were treated with 3AB (20 mmol/L), PJ34 (10 µmol/L), poly(ADP-ribose) polymerase-1 siRNA (50 nmol/L), or unrelated siRNA for 24 h. (C) pGL3 plasmid containing human adiponectin promoter (Adn-pGL3) or control pGL3-basic plasmid (pGL3 basic) was co-transfected with pRL-SV40 plasmid into cultured fibroblasts. After transfection, fibroblasts were treated with 3AB (10, 20 mmol/L), PJ34 (5, 10 µmol/L), poly(ADP-ribose) polymerase-1 siRNA (50 nmol/L), or unrelated siRNA (50 nmol/L), or piglitazone (15 µmol/L) for 24 h. Data are expressed as mean±SEM, n = 6, * P < 0.05, ** P < 0.01 compared with the control group.

 
The influence of PARP-1 on adiponectin promoter directed luciferase expression was also investigated. Results showed that 3AB, PJ34, PARP-1 siRNA, and PTZ significantly enhanced adiponectin promoter directed luciferase gene expression, respectively (Figure 3C).

3.4 Interaction between poly(ADP-ribose) polymerase-1 and peroxisome proliferator activated receptor {gamma}
To explore the potential interaction between PARP-1 and PPAR{gamma}, nuclear extracts were subjected to co-immunoprecipitation with antibody specific for PARP-1. Immunoblot analysis revealed that PPAR{gamma} protein was co-immunoprecipitated with PARP-1 (Figure 4A). Far-western blot assay showed that recombinant PARP-1 protein can directly bind to PPAR{gamma}. However, auto-poly(ADP-ribosyl)ated PARP-1 failed to bind to PPAR{gamma} (Figure 4B).


Figure 4
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  		Figure 4 Specific binding of peroxisome proliferator activated receptor {gamma} to PARP-1 in cultured rat cardiac fibroblasts. (A) After co-immunoprecipitation (IP) of nuclear extracts with anti-PAR antibody, or anti-poly(ADP-ribose) polymerase-1 antibody or unspecific IgG, respectively, the eluted proteins were submitted to western blot (WB) with anti-peroxisome proliferator activated receptor {gamma} antibody. (B) Western blot (upper): anti-peroxisome proliferator activated receptor {gamma} antibody was used to detect peroxisome proliferator activated receptor {gamma} protein in nuclear extracts (left) or recombinant peroxisome proliferator activated receptor {gamma} protein (right). Far western blots (lower): after incubation with auto-modified (AM)-poly(ADP-ribose) polymerase-1 or non-modified (NM)-poly(ADP-ribose) polymerase-1, anti-poly(ADP-ribose) polymerase-1 antibody was used to detect the band of peroxisome proliferator activated receptor {gamma}•poly(ADP-ribose) polymerase-1 complex. (C). Poly(ADP-ribosyl)ation levels of peroxisome proliferator activated receptor {gamma}. Left lanes, western blot (WB): anti-peroxisome proliferator activated receptor {gamma} antibody was used to detect peroxisome proliferator activated receptor {gamma} (MW 67 kd). Right lanes, western blots (WB): anti-PAR antibody was used to detect poly(ADP-ribosyl)ation of peroxisome proliferator activated receptor {gamma} (MW 67 kDa). Control, cells were treated with vehicle (PBS); 3AB, cells were treated with 20 mmol/L of 3-aminobenzamide; poly(ADP-ribose) polymerase-1 siRNA, cells were treated with 50 nmol/L of poly(ADP-ribose) polymerase-1 siRNA.

 
To further explore whether PPAR{gamma} was poly(ADP-ribosyl)ated in cultured cells, nuclear extracts were subjected to immunoprecipitation with antibody specific for PAR. Immunoblot analysis showed that PPAR{gamma} was immunoprecipitated with anti-PAR antibody (Figure 4A). Additionally, western blot analysis with anti-PAR antibody also demonstrated that PPAR{gamma} was poly(ADP-ribosyl)ated in cultured fibroblasts under basal conditions (Figure 4C). In addition, 3AB or PARP-1 siRNA significantly inhibited poly(ADP-ribosyl)ation of PPAR{gamma} (Figure 4C).

3.5 Inhibition of poly(ADP-ribose) polymerase-1 activity increases the DNA binding activity of peroxisome proliferator activated receptor {gamma}
It has been demonstrated that PPAR{gamma} binds to PPRE motif in the promoter and initiates gene transcription.27 We observed that treatment of fibroblasts with 3AB or PARP-1 siRNA significantly increased the DNA binding activity of PPAR{gamma} (Figure 5A and B). We investigated the influences of PARP-1 on the expression and nuclear translocation of PPAR{gamma}. Neither 3AB nor PARP-1 siRNA influenced PPAR{gamma} expression and nuclear translocation in cells under basal conditions (Figure 5C).


Figure 5
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  		Figure 5 Binding of peroxisome proliferator activated receptor {gamma} to PPRE in nuclear extracts from cultured rat cardiac fibroblasts was detected by electrophoretic mobility shift assay (EMSA). (A) Lane 1, cardiac fibroblasts treated with PBS as control. Lane 2, cardiac fibroblasts treated with 3AB (20 mmol/L) for 24 h. Lane 3, cardiac fibroblasts treated with pioglitazone (PTZ, 15 µmol/L) for 24 h. (B) Lane 1, cardiac fibroblasts treated with PBS as control. Lane 2–3, cardiac fibroblasts transfected with unrelated siRNA (50 nmol/L) or poly(ADP-ribose) polymerase-1 siRNA (50 nmol/L) for 48 h, respectively. (A–B) Data are expressed as mean±SEM, n = 6, * P < 0.05’** P < 0.01 compared with control group. (C) Western blot analysis with equal loading of proteins from cultured rat cardiac fibroblasts, was used to detect expression of peroxisome proliferator activated receptor {gamma} in whole extracts (left lanes) and nuclear extracts (right lanes) (MW 67 kDa). Control, cells were treated with vehicle (PBS); 3AB, cells were treated with 20 mmol/L of 3AB; poly(ADP-ribose) polymerase-1 siRNA, cells were treated with 50 nmol/L of poly(ADP-ribose) polymerase-1 siRNA. (D) Binding of peroxisome proliferator activated receptor {gamma} to PPRE and supershift of peroxisome proliferator activated receptor {gamma} in nuclear extracts from cultured cardiac fibroblasts were detected by EMSA. Lanes 1–4, supershift assay of nuclear extracts with anti-poly(ADP-ribose) polymerase-1 antibody (Lane 1), anti-PAR antibody (Lane 2), anti-peroxisome proliferator activated receptor {gamma} antibody (Lane 3), and unspecific IgG (Lane 4). Lane 5, competition of biotin-labelled probe with a 200-fold excess of unlabeled probes. Lane 6, further incubation of nuclear extracts with 10 µM NAD+ and 10 mM 3AB together. Lane 7, further incubation of nuclear extracts with 10 µM NAD+. Lane 8, cardiac fibroblasts treated with PBS as control. Data are expressed as mean±SEM. n = 6.

 
To investigate whether PARP-1 was involved in the binding of PPAR{gamma} to PPRE, supershift assay was performed. Results showed that anti-PARP-1 antibody failed to shift or abrogate the band of PPAR{gamma}/PPRE complex (Figure 5D, lane 1). Although PPAR{gamma} could be poly(ADP-ribosyl)ated in nucleus, anti-PAR antibody failed to influence the band of PPAR{gamma}/PPRE complex yet (Figure 5D, lane 2), suggesting that poly(ADP-ribosyl)ated PPAR{gamma} could not bind to PPRE. In accordance with this conclusion, incubation of NAD+ with nuclear extracts significantly inhibited binding of PPAR{gamma} to PPRE (Figure 5D, lane 7); and the NAD+-induced inhibition was effectively reversed by co-incubation with 3AB (Figure 5D, lane 6).

The direct influence of poly(ADP-ribosyl)ation on the DNA binding of PPAR{gamma} was studied. Treatments of fibroblasts with 3AB or PARP-1 siRNA significantly inhibited poly(ADP-ribosyl)ation of PPAR{gamma} and enhanced direct binding of PPAR{gamma} to PPRE (Figure 6A). Incubation of recombinant PPAR{gamma} protein with PARP-1, NAD+, and activated DNA promoted poly(ADP-ribosy)-lation of PPAR{gamma} and prevented binding of PPAR{gamma} to PPRE; and the NAD+-induced decrease in the DNA binding of PPAR{gamma} was reversed by co-incubation with 3AB (Figure 6B). Further, recombinant NF-{kappa}B p65 subunit could not be poly(ADP-ribosyl)ated by PARP-1, and the DNA binding activity of p65 subunit was not altered (Figure 6C).


Figure 6
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  		Figure 6 (A) Left lanes, western blot: anti-peroxisome proliferator activated receptor {gamma} antibody was used to detect peroxisome proliferator activated receptor {gamma} (MW 67 kDa) in nuclear extracts. Right lanes, Southwestern blot: biotin-labelled PPRE oligonucleotide was used to detect binding of peroxisome proliferator activated receptor {gamma} to PPRE in nuclear extracts. 3AB, fibroblasts were treated with 20 mmol/L of 3AB; Control, fibroblasts were treated with vehicle (PBS); poly(ADP-ribose) polymerase-1 siRNA, fibroblasts were treated with 50 nmol/L of poly(ADP-ribose) polymerase-1 siRNA. (B) Left lanes, western blot: anti-peroxisome proliferator activated receptor {gamma} Ab was used to detect peroxisome proliferator activated receptor {gamma} (MW 67 kDa). Middle lanes, western blot: anti-PAR Ab was used to detect poly(ADP-ribosyl)ation level of peroxisome proliferator activated receptor {gamma}. Right lanes, Southwestern blot: biotin-labelled PPRE oligonucleotide was used to detect binding of peroxisome proliferator activated receptor {gamma} to PPRE. Data are expressed as mean±SEM, n = 6, *P < 0.05 compared with control group; # P < 0.05 compared with 3AB group. (C) Left lanes, western blot: anti-p65 Ab was used to detect NF-{kappa}B p65 (MW 65 kDa). Middle lanes, western blot: anti-PAR Ab was used to detect poly(ADP-ribosyl)ation level of p65. Right lanes, Southwestern blot: biotin-labelled NF-{kappa}B oligonucleotide was used to detect binding of p65 to NF-{kappa}B oligonucleotide. Data are expressed as mean±SEM, n = 6.

 
3.6 Inhibition of poly(ADP-ribose) polymerase-1 activity promotes peroxisome proliferator activated receptor {gamma}-target gene transcription
The influence of PARP-1 on PPAR{gamma}-target gene transcription30 in cultured fibroblasts was investigated. Results showed that inhibition of PARP-1 by 3AB, PJ34, or siRNA markedly increased the mRNA expression of leptin, CD36, and lipoprotein lipase in fibroblasts (see Supplementary material online, Figure S3). Interestingly, the mRNA expression of aP2, another PPAR{gamma}-target gene, was barely detected in cultured cardiac fibroblasts (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
In this study, we have demonstrated that adiponectin and AdipoR1 are abundantly expressed in cultured cardiac fibroblasts. Inhibition of PARP-1 enhances adiponectin, AdipoR1 expression, and adiponectin promoter activity. The data suggest that PARP-1 is a transcriptional repressor of adiponectin and AdipoR1 under basal condition. We also provide the first evidence that PARP-1 inhibits PPAR{gamma} transactivation through poly(ADP-ribosyl)ation of PPAR{gamma} in cardiac fibroblasts.

Adiponectin, AdipoR1, and AdipoR2 are expressed abundantly in cardiomyocytes.8,9 It has been reported that adiponectin has many protective actions in heart diseases.31,32 In this study, we showed that adiponectin and AdipoR1 were also abundantly expressed in cultured cardiac fibroblasts, suggesting that adiponectin may exert protective effects on the heart through autocrine and/or paracrine manner. We also showed that AdipoR2 was barely expressed in cardiac fibroblast. The result is consistent with Hattori’s study showing that the influence of AdipoR2 antibody on NF-kcyB activation in cardiac fibroblast was much weaker than that of AdipoR1 antibody.33

We also observed that inhibition of PARP-1 enhances adiponectin and AdipoR1 expression. It has been demonstrated that treatment of adipocytes with TNF-{alpha} effectively inhibited adiponectin production through NF-{kappa}B signalling pathway.34 However, as inhibition of PARP-1 did not influence the expression of TNF-{alpha}, IL-1β, and IL-6 in cultured fibroblasts under basal conditions, the data suggest that the influence of PARP-1 on adiponectin expression may not be mediated by the increases of pro-inflammatory cytokines.

Multiple transcription factors participate in the transcriptional regulation of adiponectin and AdipoR1.8,9,16,17,35,36 Among them, PPAR{gamma} is the most important because point mutation of PPRE motif markedly reduced the basal transcriptional activity and completely blocked thiazolidinedione-induced transactivation of adiponectin promoter.27 We also demonstrated that treatment with PPAR{gamma} agonist PTZ significantly enhanced adiponectin and AdipoR1 expression, and inhibition of PARP-1 markedly increased the DNA binding and the transactivity of PPAR{gamma}. Our data indicate that PARP-1 might influence adiponectin and AdipoR1 expression through PPAR{gamma} pathway. We have observed that PPAR{gamma} could directly bind to non-modified PARP-1, but not auto-modified PARP-1. Thus, it is postulated that PARP-1 may affect the DNA binding and the transactivation of PPAR{gamma} through physical interaction with PPAR{gamma}. Based on this hypothesis, PARP inhibitors, which inhibited auto-poly(ADP-ribosyl)ation of PARP-1, would enhance the DNA binding of PPAR{gamma} by increasing binding of non-modified PARP-1 to PPAR{gamma}. However, we observed that PARP-1 siRNA, which dramatically inhibited PARP-1 expression, also increased the DNA binding of PPAR{gamma}. Moreover, our supershift assay showed that PARP-1 was not a component of PPAR{gamma}/PPRE complex. The data indicate that physical interaction between PARP-1 and PPAR{gamma} did not enhance the DNA-binding of PPAR{gamma}.

Treatment of cells with 3AB or PARP-1 siRNA inhibited poly(ADP-ribosyl)ation of PPAR{gamma} and promoted direct binding of PPAR{gamma} protein alone (without RXR protein) to PPRE oligonucleotide. EMSA assay also demonstrated that inhibition of PARP-1 significantly increased the DNA binding activity of PPAR{gamma}. In addition, incubation of nuclear extracts with NAD+ significantly decreased the DNA binding of PPAR{gamma}; and co-incubation with 3AB effectively reversed the NAD+-induced decrease. In cell-free experiment, poly(ADP-ribosyl)ation of PPAR{gamma} by PARP-1 inhibited binding of PPAR{gamma} to PPRE oligonucleotide. Thus, poly(ADP-ribosyl)ation of PPAR{gamma} decreases its DNA binding activity. Consistent with this possibility, supershift assay showed that poly(ADP-ribosyl)ated PPAR{gamma} was not a component of PPAR{gamma}/PPRE complex, implicating that poly(ADP-ribosyl)ated PPAR{gamma} could not bind to PPRE. Therefore, our results have illustrated that PARP-1 prevented PPAR{gamma} transactivation by poly(ADP-ribosyl)ation of PPAR{gamma}. Since PPAR{gamma} plays an important role in the transcriptional regulation of adiponectin and AdipoR1,16,17 it suggests that PARP-1 might suppress adiponectin and AdipoR1 transcription through poly(ADP-ribosyl)ation of PPAR{gamma}.

PPAR{gamma} belongs to the nuclear receptor superfamily and is a member of the NR1C subgroup that includes PPAR{alpha} and PPARβ/{delta}. These receptors form heterodimers with RXR and modulate the transcription of target genes.37,38 Miyamoto reported that PARP-1 can directly bind to RXR to form a PARP-1/RXR/thyroid hormone receptor heterotrimer to bind to promoter. PARP activity can be targeted to specific DNA sequences and thereby represses gene expression.39 These results suggested that physical interaction between PARP-1 and RXR may mediate the influence of PARP-1 on the transactivation of nuclear receptors. However, in the current study, PARP-1 was not a component of PPAR{gamma}/PPRE complex. Further, poly(ADP-ribosyl)ation inhibited the DNA binding of PPAR{gamma}; and inhibition of PARP-1 activity significantly increased the DNA binding and the transactivity of PPAR{gamma}. Thus, our results revealed a new mechanism underlying the regulation of PPAR{gamma} transactivation. In agreement with this conclusion, our results have demonstrated that inhibition of PARP-1 significantly enhanced the transcription of other PPAR{gamma}-target genes including leptin, CD36, and lipoprotein lipase. As RXR also contributes importantly to the transactivation of PPAR{gamma}, it will be worthy of further study of the effects of PARP-1 on the interaction between RXR and PPAR{gamma}.

In summary, we show for the first time that PARP-1 inhibits the expression of adiponectin and AdipoR1 in cardiac fibroblasts. As adiponectin exerts protective effects on the heart, the inhibition of adiponectin synthesis might be involved as a part of deleterious effects of PARP-1 activation on the pathogenesis of cardiac fibrosis and other heart diseases. Moreover, we have provided the first evidence that PPAR{gamma} is poly(ADP-ribosyl)ated by PARP-1 and poly(ADP-ribosyl)ation prevents PPAR{gamma} transactivation in cardiac fibroblasts under basal condition. As PPAR{gamma} plays a crucial role in the transcriptional regulation of adiponectin and AdipoR1, inhibition of PPAR{gamma} by PARP-1 should contribute to its inhibitory effects on adiponectin and AdipoR1 expression in cardiac fibroblasts.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary Material is available at Cardiovascular Research Online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by National Natural Science Foundation of China (30400175, 30770881 to K.H.) and Open Foundation of Hubei Key Laboratory of Biological Targeted Therapy of China (2007B03 to D.H.).


   Acknowledgements
 
We thank R.M. Evans for kindly providing PPRE-TK-Luc and TK-Luc plasmids and help from Charles P. Ordahl and Weiping Zou.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors have contributed equally. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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