Adiponectin-mediated heme oxygenase-1 induction protects against iron-induced liver injury via a PPARα dependent mechanism

Abstract

Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge.

Figure 1

Therapeutic effects and mechanisms of adiponectin (APN) associated with HO-1 induction in murine hepatic iron overload. A: Mice were intravenously injected with an AAV-APN gene for two weeks before SnPP and iron dextran challenge for another two weeks. The method for determining serum levels of GOT and GPT followed the manufacturer's instructions. GOT and GPT levels are expressed as ΔOD460/min/mg protein. B: Representative photomicrographs of iron-mediated apoptosis in hepatocytes stained with TUNEL, as indicated by arrows, and the nuclei marker, DAPI. C: Western blot analysis of AMPK phosphorylation and HO-1 induction by APN in mice with iron overload for one week. Liver tissues (50 μg) from each group were assessed for the induction of pAMPK and heme oxygenase (HO)-1. A representative result of three separate experiments is shown. D: Assessment of hepatic HO-1 activity after mice were exposed to iron dextran challenge for two weeks. E: Elimination of iron accumulation with hepatic iron overload by APN. Liver sections from various treatments were stained to determine the extent of iron deposition for one week. The green staining of deposited iron is indicated by arrows, and a representative result of three separate experiments is shown. Results are expressed as the mean ± SD (n = 15). *P < 0.005, vs. the given empty AAV alone and **P < 0.01 vs. AAV-APN with additional iron dextran challenge.

Figure 2

Regulation of heme oxygenase (HO)-1 induction by adiponectin (APN) in concentration- and time-dependent fashions. A: Concentration-dependent induction of HO-1 by APN. Left: The effect of increasing concentrations (0–50 μg/ml) of APN for six hours on the induction of the HO-1 protein level was analyzed by Western blotting. Right: Mouse hepatocytes were transiently transfected with pGL3/HO-1 and pRL-TK for 24 hours, followed by 30 μg/ml of APN treatment for six hours. The methods are described in Materials and Methods. Results are presented as the mean ± SEM of four independent experiments. *P < 0.05 vs. pGL3 alone and **P < 0.05 vs. pGL3 with additional APN treatment. Time-course induction of (B) HO-1 mRNA/protein and (C) HO-1 activity in hepatocytes by APN treatment. Cells were treated with adiponectin (30 μg) for the indicated time points and analyzed by RT-PCR and Western blotting. Equal loading in each lane or transfer was confirmed using GAPDH mRNA or by incubation with an anti-GAPDH antibody. Representative results of three separate experiments are shown. Additionally, the bilirubin concentration in 0.5 ml of cell medium from hepatocytes at various time points of 30 μg/ml of APN treatment was assessed, as described in Materials and Methods. Data were obtained from six independent experiments. Results are expressed as the mean ± SEM. Significantly different (*P < 0.05, **P < 0.01, and ***P < 0.005 vs. the control group). D: Verification of APN-induced HO-1 overexpression by an adenovirus carrying the APN gene in hepatocytes. After cells were infected with 30 MOI of Adv variants for one, three, and five days, cell lysates were prepared, and the expression of HO-1 protein was determined by Western blot analysis.

Figure 3

Determination of signal transduction pathways involved in adiponectin (APN)-mediated heme oxygenase (HO)-1 induction. Hepatocytes were pretreated with a pAMPK inhibitor, compound C (A), or a p38MAPK inhibitor, SB202190 (B), for 30 minutes before treatment with APN for 20 minutes or one or six hours to determine the respective active forms or protein levels by Western blot analysis. Phosphorylated and total forms of AMPK and p38MAPK were harvested at 20 minutes of APN treatment, while the activation of PPARα and consequent HO-1 protein level were determined at one and six hours of treatment, respectively. Three samples were analyzed in each group, and values are presented as the mean ± SEM. Representative results of three separate experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.005 vs. the control group; ^†P < 0.01 vs. the APN-treated group.

Figure 4

Involvement of PPARα activation in adiponectin (APN)-mediated heme oxygenase (HO)-1 induction. APN increased PPRE enhancer-driven luciferase activity (A) and nuclear translocation of PPARα activation (B) and subsequent HO-1 induction (C). A: HEK 293 cells were transiently simultaneously transfected with pBV-Luc-PPARα enhancer and Renilla control vectors or with additional transfection of pcDNA-PPARα, and pcDNA-RXR (0.5 μg/well) for five hours, after which fresh medium was added for overnight inoculation. Cell lysates were harvested after cells were treated with APN for six hours. B: Cells were respectively pretreated with either AMPK or PPARα antagonist, or a PPARα agonist for one hour before the administration of APN for another one hour for nuclear translocation of PPARα by Western blot analysis and (C) subsequent HO-1 induction at six hours of APN treatment. D: Elimination by PPARα knockdown of APN-mediated HO-1 induction. The method of cells with PPARα knockdown was described in Materials and Methods. Equal loading or transfer was confirmed by incubation with an anti-GAPDH or anti-lamin A/C antibody. Representative results of three separate experiments are shown, and data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control; ^†P < 0.05 vs. APN alone).

Figure 5

Adiponectin (APN) increased nuclear translocation of exogenous PPARα and subsequent heme oxygenase (HO)-1 induction in hepatocytes with PPARα overexpression by (A) Western blot analysis of cytosol and nuclear fractions of PPARα and total cell lysates of HO-1 protein level and (B) fluorescent confocal microscopy. A: Cells were treated with APN for one or six hours and harvested for cytosolic-nuclear partitioning of PPARα. Weights of the cell lysates were 50 μg for the cytosolic and nuclear fractions analyzed, and GAPDH and lamin A/C were used as internal controls for these fractions. Data are presented as the mean ± SEM (*P < 0.01, and **P < 0.005 vs. the control; ***P < 0.01 vs. cells transfected with pcDNA3 with additional APN treatment). B: Cells were treated with APN (30 μg/ml) for one hour, then immunostained with an anti-Flag antibody followed by incubation with a second antibody conjugated with Texas red. Red color represents Flag-positive staining in the cytosol or nuclei. Identical fields stained with Flag were also stained using DAPI to reveal the positions of cell nuclei. Micrographs of representative fields were recorded. Representative results of three separate experiments are shown.

Figure 6

Adiponectin (APN)-mediated increase in the binding activity of PPARα to the PPRE of the heme oxygenase (HO)-1 promoter region by an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). A: Cells were cultured and pretreated with a PPARα or AMPK antagonist for 1 hour before the addition of 30 μg/ml APN for one hour, with Wy14643 used as a positive control. The putative PPRE-binding activity derived from the HO-1 promoter region of nuclear proteins was assayed by EMSA in cells with the indicated treatments. 100× cold denotes a 100-fold molar excess of unlabeled oligonucleotides relative to the biotin-labeled probe; this was added to the binding assay for competition with the unlabeled oligonucleotide. The mobility of specific PPARα complexes is indicated. B: Cell lysate was subjected to a ChIP assay. The DNA associated with the PPRE was immunoprecipitated with an anti-PPARα antibody, and PCR amplification was used to determine the extent of PPARα association with the functional PPRE in an HO-1 promoter fragment of 162 bp. Distilled water (ddH2O) and anti-GAPDH were used, respectively, as negative controls for the PCR and ChIP assays. Representative results of three separate experiments are shown, and data are presented as the mean ± SEM (*P < 0.01 vs. the control; **P < 0.01 vs. APN alone).

Figure 7

Adiponectin (APN) attenuates iron dextran-induced caspase 3 activation and reactive oxygen species (ROS) production. A: Therapeutic effect of APN in caspase 3 activation induced by varying concentrations of iron dextran. Cells were pretreated with 30 μg/ml of APN for one hour, followed by challenge with various concentrations of iron (0–40 μmol/L) B: Representative CM-H2DCFDA fluorescent photomicrographs of hepatocytes challenged with 20 μmol/L of iron dextran. Results are representative data from three separate experiments.

Figure 8

Elimination of iron dextran-mediated caspase 3 activation and apoptosis by adiponectin (APN) through AMPK-mediated PPARα activation and subsequent heme oxygenase (HO)−1 induction. A: Cells were pretreated with compound C, GW6471, or DMSO as the control for one hour before another one hour of APN or phosphate-buffered saline administration, followed by iron dextran challenge for 18 hours. Additionally, cells were treated with WY-14643 as a positive control for HO-1 induction. B: Effect of HO-1 on APN-mediated protection against iron-mediated hepatic injury. Cells were pretreated with SnPP for one hour to block HO-1 activity before APN administration. Fifty micrograms of total cell lysate was analyzed for the protein level of apoptotic-related molecules by Western blotting. Membranes were probed with an anti-GAPDH antibody to verify equivalent loading. Bar charts in the lower panel show the band intensities of indicated molecules by densitometry. Data were derived from three independent experiments and are presented as the mean ± SEM. Significantly different (*P < 0.01, and **P < 0.005 vs. the control; ^†P < 0.05 vs. iron dextran alone; ^‡P < 0.01 vs. APN and iron dextran-treated group). C: Cells grown on coverslips with the above-mentioned various pretreatments, then challenged with 20 μmol/L of iron dextran for two days. Nuclei with positive stains are indicated as having undergone cell apoptosis by the TUNEL assay. Representative results of three separate experiments are shown.

Figure 9

Effects of adiponectin (APN)-mediated heme oxygenase (HO)-1 induction in apoptosis and iron accumulation in hepatocytes by iron dextran challenge. A: Hepatocytes permanently transformed with HO-1 or small hairpin (sh)HO-1 were challenged with 20 μmol/L iron dextran for two days. Data are presented as the mean ± SEM (*P < 0.05, and **P < 0.01 vs. the control; ^†P < 0.01 vs. cells overexpressing HO-1; ^‡P < 0.005 vs. iron alone; ^§P < 0.001 vs. cells overexpressing HO-1 with the additional iron insult). B: The mechanism of HO-1 in iron dextran-mediated apoptosis by the administration of CO or bilirubin, end-products of HO-1 reaction. Cells were treated with CO (CORMII) or bilirubin to mimic the antiapoptotic effect of HO-1, followed by iron challenge for 18 hours for the Western blot analysis of Bcl-XL or cleaved caspase 3, respectively. Equal loading or transfer was confirmed by incubation with an anti-GAPDH antibody. C: Effect of APN (upper) and hepatocytes overexpressing or knocking down HO-1 (lower) on 20 μmol/L of iron dextran insult in vitro. Intracellular iron deposition was examined by Perls' iron staining in iron challenge for two days. Original magnification: ×200. Representative results of three separate experiments are shown.