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.
Subject(s)
Adiponectin/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Heme Oxygenase-1/metabolism , Hepatocytes/drug effects , Iron-Dextran Complex/toxicity , PPAR alpha/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Cell Proliferation/drug effects , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Enzyme Induction/drug effects , Fluorescent Antibody Technique , Hematinics/toxicity , Hepatocytes/cytology , Hepatocytes/metabolism , Immunoenzyme Techniques , Iron Overload/metabolism , Iron Overload/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , PPAR alpha/antagonists & inhibitors , PPAR alpha/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal TransductionABSTRACT
Adiponectin (APN)-mediated cyclooxygenase (COX)-2 induction is known to have various protective effects on cardiovascular diseases. However, the molecular mechanisms of APN-mediated COX-2 induction and its protection against iron-mediated injury in hepatocytes are still unclear. Herein, we show that AMP-mediated peroxisome proliferator-activated receptor (PPAR)alpha activation was attributable to COX-2 induction by APN, which was further confirmed by identifying novel functional PPAR responsive elements (PPREs) in the mouse COX-2 promoter region. Prostaglandin (PG)I2 and PGE2, metabolites of COX-2, time-dependently increased in hepatocytes treated with APN. Interestingly, beraprost and misoprostol, respective agonists for PGI2 and PGE2, mimicked the protective effects of APN in iron-mediated inflammation in hepatocytes. The iron dextran-activated nuclear factor (NF)-kappaB pathway was correlated with the increased production of inflammatory cytokines including tumor necrosis factor-alpha, intercellular adhesion molecule-1, and monocyte chemotactic protein-1. This was eliminated by administration of APN, whereas blockage of PPARalpha activation, an upstream regulator of COX-2 induction by APN, and COX-2 activation reversed the anti-inflammatory effect of APN, suggesting the crucial role of COX-2 in this event. Herein, we demonstrate that APN-mediated COX-2 induction through a PPARalpha-dependent mechanism, and COX-2 exerted an anti-inflammatory effect of APN in hepatocytes subjected to iron challenge.
Subject(s)
Adiponectin/metabolism , Cyclooxygenase 2/metabolism , Gene Expression Regulation, Enzymologic , Hepatocytes/drug effects , Hepatocytes/metabolism , Iron/toxicity , Adiponectin/genetics , Animals , Apoptosis/physiology , Cyclooxygenase 2/genetics , Dinoprostone/metabolism , Enzyme Activation , Enzyme Induction , Epoprostenol/metabolism , Hepatocytes/cytology , Humans , Mice , PPAR alpha/agonists , PPAR alpha/genetics , PPAR alpha/metabolism , Promoter Regions, Genetic , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction/physiologyABSTRACT
We previously demonstrated the antiproliferative and antiangiogenic effects of 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, in human umbilical vascular endothelial cells (HUVECs). Herein, we unraveled its molecular mechanisms in inhibiting HUVEC motility. 3MC down-regulated FAK, but up-regulated RhoA, which was rescued by AhR knockdown. It led us to identify novel AhR binding sites in the FAK/RhoA promoters. Additionally, 3MC increased RhoA activity via suppression of a negative feedback pathway of FAK/p190RhoGAP. With an increase in membrane-bound RhoA, subsequent stress fiber and focal adhesion complex formation was observed in 3MC-treated cells, and this was reversed by a RhoA inhibitor and AhR antagonists. Notably, these compounds significantly reversed 3MC-mediated anti-migration in a transwell assay. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR's genomic regulation of FAK/RhoA, together with RhoA activation, is ascribable to the anti-migration effect of 3MC in HUVECs.
Subject(s)
Cell Movement/drug effects , Endothelium, Vascular/drug effects , Focal Adhesion Kinase 1/physiology , Receptors, Aryl Hydrocarbon/agonists , rhoA GTP-Binding Protein/physiology , Animals , Binding Sites , Cell Movement/physiology , Cells, Cultured , Chromatin Immunoprecipitation , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Feedback, Physiological , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Gene Expression Regulation , Humans , Methylcholanthrene/pharmacology , Mice , Mice, Inbred BALB C , Promoter Regions, Genetic , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/physiology , Umbilical Cord/cytology , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
We previously showed that an increase in the peroxisome proliferator-activated receptor-delta (PPARdelta), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPARdelta activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPARdelta activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPARdelta interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPARdelta activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS.
