| Accumulating evidence suggests that an excess of reactive oxygen and nitrogen species are generated in cardiomyocytes cardiac fibroblasts, smooth muscle cells, endothelial cells and infiltrating inflammatory cells during myocardial ischemia/reperfusion injury, various forms of heart failure or cardiomyopathies, circulatory shock, cardiovascular aging, myocardial hypertrophy, atherosclerosis and vascular remodeling following injury. An appropriate amount of reactive oxygen/nitrogen species can act as signal molecules in cell physiological processes. However, excessive reactive oxygen/nitrogen species can directly cause cell dysfunction and affect the transcriptional regulation of genes through the activation of its downstream signaling pathway. The abnormal expression of the protein is the basis of the development of many cardiovascular diseases. Looking for the oxidative stress associated downstream signaling pathway molecules will provide a new approach as an intervention target for the treatment of cardiovascular disease.The nuclear enzyme poly(ADPribose)polymerase1(PARP-1), as a DNA damage sensor and signaling molecule, plays an important role in maintaining chromosome stability, DNA replication, apoptosis, cell death, and activation of gene transcription. PARP-1over activation can catalyzes the formation of Poly (ADP-ribose)(PAR) chain using intracellular NAD as a substrate. Meanwhile, PARP-1can also poly(ADP-ribosyl)ate target protein and lead to functional changes. And this chemical modification reaction can be inhibited by PARP inhibitors. In addition, PARP-1can also serve as a cofactor involved in regulating the functions of transcription factors.In recent years, the study found that PARP-1play an important role in a variety of cardiovascular diseases by regulating the transcription of multiple genes.In related animal models of cardiovascular diseases, the PARP-1catalytic activity was significantly enhanced and the PARP inhibitors treatment showed a significant therapeutic effect. But, the mechanism of PARP-1regulating transcription of genes in cardiovascular system remain unclear. This study will discuss the mechanisms of PARP-1in regulating the cardiovascular-related genes and the therapeutic effect of PARP-1inhibitors. These will provide theoretical and experimental basis for clinical studies of PARP inhibitors. Part I The mechanism of PARP-1induce the expression of HSP70in human peripheral blood mononuclear cellsAims:We aimed to investigate the role of poly (ADP-ribose) polymerase-1(PARP-1) in LPS-induced expression of heat shock protein70(HSP70) in cultured human peripheral blood mononuclear cells (PBMC).Methods and results:Primary cultured human PBMC were divided into3groups. Group1(control group) received vehical (PBS) treatment. Group2received treatment with LPS alone (1μg/ml). Group3were pretreated with10mmol/L PARP-1inhibitor3-aminobenzamide (3AB) for1hour, followed by additional incubation with LPS (1μg/ml). After LPS treatment, culture supernatants were collected at6h,18h,24h,48h respectively. The concentrations of HSP70were measured with enzyme linked immunosorbent assay (ELISA). Cellular PARP activity was determined using universal colorimetric PARP assay kit. The DNA binding activity of heat shock factor (HSF) was determined by Electrophoretic Mobility Shift Assay (EMS A) and Southwestern blot. The interaction of PARP-land HSF-1was determined by Far-western blot. Compared with control group, the expression of HSP70(at6h,18h,24h,48h) in LPS-stimulated group was significantly increased to115.44±1.92(P<0.05),195.96±5.33(P<0.05),233.83±15.90(P<0.05),239.43±3.07(P<0.05), respectively. Pretreatment of cells with3AB significantly inhibited LPS-induced HSP70expression at24h,48h, respectively. At the same time point, the DNA binding activity of HSF was significantly increased by LPS treatment, and pretreatment of cells with3AB obviously decreased the DNA binding activity of HSF. The result of Far-western blot showed that PARP-1could bind to HSF-1.The Southwestern-blot confirmed that the DNA binding activity of HSF-1recombinant protein could be increased when interacting with activated PARP-1, and could be abolished by pretreating with3AB.Conclusions:PARP-1play an important role in LPS-induced expression of HSP-70through regulating the DNA binding activity of HSF-1in primary cultured human PBMC. Part Ⅱ The role of PARP1in regulating TGF(3/Smad3pathway in rat VMSCsAims:This study aims to investigate the influence of poly(ADP-ribose) polymerase1(PARP-1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3pathway in rat vascular smooth muscle cells (VSMCs).Methods and results:TGF-β1treatment promoted PARP1activation through induction of ROS generation in rat VSMCs. TGF-1β-induced phosphorylation and nuclear accumulation of Smad3was prevented by treatment of cells with PARP inhibitor,3-aminobenzamide (3AB) orN-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1siRNA. TGF-β1treatment promoted poly(ADP-ribosy)lation of Smad3via activation of PARP1in 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 with3AB, PJ34, or PARP1siRNA prevented TGF-β1-induced Smad3transactivation and expression of Smad3target genes, including collagen Ial, collagen Ⅲal and tissue inhibitor of metalloproteinase1, in rat VSMCs.Conclusions:PARP1is indispensable for TGF-β1induced Smad3activation in rat VSMCs. Targeting PARP1may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3pathway.
|
PDF Full Text Request