Dan Huang

PARP-1 suppresses adiponectin expression through poly(ADP-ribosyl)ation of PPARγ in cardiac fibroblasts

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.

Angiotensin II promotes poly(ADP-ribosyl)ation of c-Jun/c-Fos in cardiac fibroblasts.

Although c-Jun/c-Fos (activator protein 1, AP1) contributes importantly to Ang II-induced cardiac fibrosis through induction of extracellular matrix protein over-expression in cardiac fibroblasts, the mechanism by which Ang II promotes c-Jun/c-Fos transactivation remains unclear. In this study, we demonstrated that c-Fos and c-Jun were poly(ADP-ribosyl)ated in cultured cardiac fibroblasts. Southwestern blot and EMSA assays showed that incubation of nuclear extracts with NAD(+) and active DNA increased the basal DNA binding activities of c-Jun (31.0+/-1.0%, P<0.01) and AP1 (14.2+/-3.1%, P<0.01); incubation of recombinant c-Fos or/and c-Jun with PARP-1, NAD(+) and active DNA increased the basal DNA binding activities of c-Jun (48.3+/-4.2%, P<0.01) and AP1 (21.2+/-1.5%, P<0.01). Treatment with Ang II promoted PARP-1 activation and enhanced poly(ADP-ribosyl)ation of c-Fos (14.1+1.1%, P<0.01) and c-Jun (15.5+/-5.6%, P<0.01). Ang II also increased the basal DNA binding activities of c-Jun (13.5+/-2.4%, P<0.01) and AP1 (18.7+/-3.5%, P<0.01) in cultured cells. Inhibition of PARP-1 by PJ34 or siRNA effectively prevented Ang II-induced increases in the DNA binding of c-Jun and AP1, and decreased AP1-driven transcription (including collagen Ialpha1 and IIIalpha1, MMP-9 and TIMP-1). This study illustrated that c-Jun and c-Fos were poly(ADP-ribosyl)ated by PARP-1, and poly(ADP-ribosyl)ation enhanced the DNA binding of AP1. Ang II promoted poly(ADP-ribosyl)ation of c-Jun and c-Fos through activation of PARP-1 and, subsequently, enhanced AP1-driven transcription in cardiac fibroblasts.

Poly(ADP-ribose) Polymerase 1 Is Indispensable for Transforming Growth Factor-β Induced Smad3 Activation in Vascular Smooth Muscle Cell

Background Transforming growth factor type-β (TGF-β)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs). Methods and Results TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs. Conclusions PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.

Activation and Overexpression of PARP-1 in Circulating Mononuclear Cells Promote TNF-α and IL-6 Expression in Patients with Unstable Angina

BACKGROUND Proinflammatory cytokines are involved in the development of unstable angina (UA). Poly(ADP-ribose) polymerase-1 (PARP-1) contributes importantly to regulating the transcription of inflammatory cytokines. This study aims to investigate the relationship of PARP-1 in circulating mononuclear cells (MNCs) and plasma TNF-alpha and IL-6 in UA patients and to elucidate the mechanism that PARP-1 promotes TNF-alpha and IL-6 expression via NF-kappaB pathway. METHODS Twenty six Braunwald class IIIB UA patients, 25 stable angina patients and 25 healthy volunteers were enrolled in this study. Plasma TNF-alpha and IL-6 were determined with ELISA. Circulating MNCs were analyzed for PARP activity, PARP-1 expression and NF-kappaB DNA binding activity. MNCs from healthy subjects were cultured to investigate the direct effects of PARP-1 on NF-kappaB DNA binding activity and the expression of TNF-alpha and IL-6. RESULTS PARP activity and PARP-1 expression in circulating MNCs were increased and positively correlated with plasma TNF-alpha and IL-6, respectively, in UA patients. Spontaneous NF-kappaB activation in MNCs was demonstrated in UA patients. In cultured MNCs from healthy subjects, inhibition of PARP-1 prevented lipopolysaccharide-induced increase in DNA binding activity of NF-kappaB and the expression of TNF-alpha and IL-6. Supershift assay demonstrated that PARP-1 was a component of NF-kappaB/DNA complex. Addition of recombinant human PARP-1 protein to nuclear extracts of MNCs significantly increased the DNA binding activity of NF-kappaB. CONCLUSIONS Activation and overexpression of PARP-1 are demonstrated in circulating MNCs of UA patients. Overexpressed PARP-1 promotes PARP-1/NF-kappaB/DNA complex formation, thereby enhancing the expression of TNF-alpha and IL-6 in circulating MNCs of UA patients.

Renalase is a novel target gene of hypoxia-inducible factor-1 in protection against cardiac ischaemia-reperfusion injury

AIMS Renalase, an enzyme that can metabolize catecholamine, was recently reported to attenuate the ischaemia/reperfusion (I/R)-induced cardiac injury. This work was undertaken to investigate the functions and regulation mechanisms of renalase in protection against cardiac I/R injury. METHODS AND RESULTS An elevated level of renalase was found in C57BL/6 mice challenged with I/R injury. Then, we generated a mouse model with cardiac administration of cholesterol-conjugated renalase siRNA followed by I/R operation. The mice treated with renalase siRNA exhibited increased infarction size and decreased cardiac function compared with the scramble siRNA group. Subsequently, we identified four potential hypoxia-inducible factor-1 alpha (HIF-1α)-binding motifs in the promoter of renalase through bioinformatics approaches. Dual-luciferase reporter assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and western blot were conducted and demonstrated that renalase was a novel target gene of HIF-1α. Furthermore, administration of renalase reduced the infarct area and rescued the deterioration of cardiac function in myocardial HIF-1α knockdown mice subjected to I/R injury. In addition, the levels of norepinephrine in serum as well as nicotinamide adenine dinucleotide (NAD(+)) and ATP in myocardium were determined, which implied that cardiac protection of renalase against I/R may be related, at least in part, to its metabolism of catecholamine and regulation of energy. CONCLUSION These findings have revealed renalase as a novel target gene of HIF-1α in protection against myocardial I/R injury, which provided a basis for therapeutic strategies for enhancing cardiomyocyte survival in patients associated with ischaemic heart diseases.

Poly(ADP-ribose) polymerase 1 promotes oxidative-stress-induced liver cell death via suppressing farnesoid X receptor α.

ABSTRACT Farnesoid X receptor α (FXR) is highly expressed in the liver and regulates the expression of various genes involved in liver repair. In this study, we demonstrated that activated poly(ADP-ribose) polymerase 1 (PARP1) promoted hepatic cell death by inhibiting the expression of FXR-dependent hepatoprotective genes. PARP1 could bind to and poly(ADP-ribosyl)ate FXR. Poly(ADP-ribosyl)ation dissociated FXR from the FXR response element (FXRE), present in the promoters of target genes, and suppressed FXR-mediated gene transcription. Moreover, treatment with a FXR agonist attenuated poly(ADP-ribosyl)ation of FXR and promoted FXR-dependent gene expression. We further established the CCl4-induced acute liver injury model in wild-type and FXR-knockout mice and identified an essential role of FXR poly(ADP-ribosyl)ation in CCl4-induced liver injury. Thus, our results identified poly(ADP-ribosyl)ation of FXR by PARP1 as a key step in oxidative-stress-induced hepatic cell death. The molecular association between PARP1 and FXR provides new insight into the mechanism, suggesting that inhibition of PARP1 could prevent liver injury.

PARP1-mediated PPARα poly(ADP-ribosyl)ation suppresses fatty acid oxidation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS PARP1 is a key mediator of cellular stress responses and critical in multiple physiological and pathophysiological processes of cells. However, whether it is involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains elusive. METHODS We analysed PARP1 activity in the liver of mice on a high fat diet (HFD), and samples from NAFLD patients. Gain- or loss-of-function approaches were used to investigate the roles and mechanisms of hepatic PARP1 in the pathogenesis of NAFLD. RESULTS PARP1 is activated in fatty liver of HFD-fed mice. Pharmacological or genetic manipulations of PARP1 are sufficient to alter the HFD-induced hepatic steatosis and inflammation. Mechanistically we identified peroxisome proliferator-activated receptor α (PPARα) as a substrate of PARP1-mediated poly(ADP-ribosyl)ation. This poly(ADP-ribosyl)ation of PPARα inhibits its recruitment to target gene promoters and its interaction with SIRT1, a key regulator of PPARα signaling, resulting in suppression of fatty acid oxidation upregulation induced by fatty acids. Moreover, we show that PARP1 is a transcriptional repressor of PPARα gene in human hepatocytes, and its activation suppresses the ligand (fenofibrate)-induced PPARα transactivation and target gene expression. Importantly we demonstrate that liver biopsies of NAFLD patients display robust increases in PARP activity and PPARα poly(ADP-ribosyl)ation levels. CONCLUSIONS Our data indicate that PARP1 is activated in fatty liver, which prevents maximal activation of fatty acid oxidation by suppressing PPARα signaling. Pharmacological inhibition of PARP1 may alleviate PPARα suppression and therefore have therapeutic potential for NAFLD. LAY SUMMARY PARP1 is activated in the non-alcoholic fatty liver of mice and patients. Inhibition of PARP1 activation alleviates lipid accumulation and inflammation in fatty liver of mice.

Identification of poly(ADP-ribose) polymerase-1 as a cell cycle regulator through modulating Sp1 mediated transcription in human hepatoma cells.

The transcription factor Sp1 is implicated in the activation of G0/G1 phase genes. Modulation of Sp1 transcription activities may affect G1-S checkpoint, resulting in changes in cell proliferation. In this study, our results demonstrated that activated poly(ADP-ribose) polymerase 1 (PARP-1) promoted cell proliferation by inhibiting Sp1 signaling pathway. Cell proliferation and cell cycle assays demonstrated that PARP inhibitors or PARP-1 siRNA treatment significantly inhibited proliferation of hepatoma cells and induced G0/G1 cell cycle arrest in hepatoma cells, while overexpression of PARP-1 or PARP-1 activator treatment promoted cell cycle progression. Simultaneously, inhibition of PARP-1 enhanced the expression of Sp1-mediated checkpoint proteins, such as p21 and p27. In this study, we also showed that Sp1 was poly(ADP-ribosyl)ated by PARP-1 in hepatoma cells. Poly(ADP-ribosyl)ation suppressed Sp1 mediated transcription through preventing Sp1 binding to the Sp1 response element present in the promoters of target genes. Taken together, these data indicated that PARP-1 inhibition attenuated the poly(ADP-ribosyl)ation of Sp1 and significantly increased the expression of Sp1 target genes, resulting in G0/G1 cell cycle arrest and the decreased proliferative ability of the hepatoma cells.

Nardosinone protects H9c2 cardiac cells from angiotensin II-induced hypertrophy

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.SummaryPathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.

Inhibition of PARP prevents angiotensin II-induced aortic fibrosis in rats☆

BACKGROUND Fibrosis is one of the major pathological features of hypertensive vascular disease. In this study, we aim to explore the possible protective effects of poly(ADP-ribose) polymerase (PARP) inhibitor on angiotensin II (AngII)-induced aortic fibrosis. METHODS Sprague-Dawley rats were infused subcutaneously with AngII. PARP inhibitor was intraperitoneally injected once a day. Collagen deposition in thoracic aorta was assayed by Masson tricrome staining. The mRNA and protein expression of TGF-β target genes involved in extracellular matrix (ECM) remodeling in aorta was measured. Plasma level and aortic expression of TGF-β1 was assayed. Correlation of systolic blood pressure (SBP) with plasma level of TGF-β1 was analyzed. In cultured rat vascular smooth muscle cells (VSMCs), effects of PARP inhibition on TGF-β1 expression, Smad3 transactivity, and TGF-β/Smad3 target gene expression were investigated. RESULTS Infusion of AngII promoted aortic PARP activation. Treatment with PARP inhibitor alleviated AngII-induced collagen deposition and expression of TGF-β target genes involved in ECM remodeling in aorta of rat. AngII increased plasma level and aortic expression of TGF-β1. A positive correlation between SBP and plasma level of TGF-β1 was revealed. Treatment with PARP inhibitor prevented AngII-induced elevation of SBP. Further experiments uncovered that AngII treatment increased TGF-β dependent gene expression through Smad3 pathway in cultured VSMCs. Inhibition of PARP prevented AngII-induced increases in TGF-β1 expression, Smad3 transactivity and its target gene expression. CONCLUSIONS These data indicate that inhibition of PARP prevents aortic fibrosis in AngII-induced hypertension in rats. This beneficial effect is mediated by inhibiting TGF-β/Smad3 pathway.