Poly (ADP-ribose) Polymerase 1 As A Transcription Factor Regulates Arginase ? Expression And Vascular Endothelial Function

BackgroundImpairment of vascular endothelial function is undoubtedly the primary factor of atherosclerosis (AS). NO plays an important role in the maintenance of vasodilatation, inhibition of platelet aggregation, inflammatory response and regulation of intravascular homeostasis.Arginase ? modulates eNOS activity by regulating intracellular L-arginine bioavailability, which results in atherosclerosis. Recent studies have reported that oxLDL decreases endothelial NO in HAECs by increasing arginase ? activity reciprocally and the supply of L-arginine to eNOS is impaired in the presence of oxLDL. As arginase ? is an important regulator of NO production in endothelial cells of humans and other species, suppression of arginase ? expression has been effective in preventing endothelial dysfunction and the progression of atherosclerosis. However, the regulatory mechanisms of arginase ?transcription are still poorly understood. PARP-1 is a kind of DNA repair enzyme that can participate in the inflammatory reaction and affect the survival of the cell. It affects the development of atherosclerosis and can play a wide range of gene transcriptional regulation. Our previous mass spectrometric analysis revealed that poly (ADP-ribose) polymerase 1 (PARP-1) specifically binds to the arginase ? promoter region as a transcription factor. We aimed to determine whether PARP-1 is a key factor responsible for the basal transcription of arginase?and for oxLDL induced upregulation of arginase ?. We investigated the core promoter region of arginase?and identified PARP-1 as the direct binding transcriptional factor responsible for arginase ? basal transcription, as well as oxLDL mediated increase in arginase ?production. We validated these primary findings in both in vitro and in vivo.In present study, we propose the following assumptions:1) inhibition of PARP-1 can reduce the endothelial-derived arginase ? expression, and then affect the activity of eNOS and NO production.2) PARP-1 plays an important role in the regulation of arginase ? expression and vascular endothelial function. Based on the above ideas, the aim of this study is to investigate the role of PARP-1 in the regulation of arginase ? expression and endothelial function by using luciferase reporter assay, chromatin immunoprecipitation and protein immunoprecipitation. We expect our study could verify that Poly (ADP-ribose) Polymerase 1 as a transcription factor regulating arginase ? expression and vascular endothelial function.Materials and Methods1 plasmid and Luciferase AssayWe constructed pGL3 vector containing different sites of the upstream promoter region of arginase ?, transfection HEACs, luciferase reporter system was used to screen of arginase ? core promoter region. Luciferase activity was detected with use of a luminometer with luciferase assay reagent (Promega) as previously described. The proteins binding to the -774 to -738 bp DNA element were purified by use of a DNA binding protein purification kit (Roche, Indianapolis, IN) following the manufacturer's instructions.2 Cell Culture, Transfection and TreatmentAll plasmids and the siRNA duplex were transfected with use of Lipofectamine 2000. To examine the dose dependent effects, HAECs were treated with 0,5,10,15, 30, and 50 ug/ml oxLDL 24 h before cells were harvested for measurement of target gene mRNA and protein levels. To examine the time dependent effects, cells were treated for 0,0.5,1,6,12, and 24 h with 50?g/ml oxLDL. Protein and mRNA were harvested for Western blot and RT-PCR.3 ChIP AssayChromatin immunoprecipitation (ChIP) assay was performed using a kit (Millipore). To verify whether PARP-1 binds to the active portion of the arginase II promoter. Immunoprecipitated DNA and input DNA were quantified with use of the quantitative RT-PCR detection system (Bio-Rad). The relative levels of DNA were normalized to the input DNA and expressed as percentage of the nontreatment control. The PCR products were separated on a 1.5%agarose gel.4 Real-time quantitative RT-PCRTotal RNA was extracted by Trizol method according to the manufacturer's protocol. The mRNA expression of PARP-1 and arginase ? were detected. qRT-PCR involved the iCycler iQ RT-PCR detection system (Bio-Rad). The mRNA levels were acquired by normalizing the threshold cycle (Ct) of PARP-1 to that of P-actin.5. Western blottingProtein were extracted from the cultured cells or aortas tissues. The expressions of PARP-1, arginase ?, eNOS and iNOS were detected by SDS-PAGE.6. ImmunoprecipitationTo investigate the interaction between PARP-1 and phosphorylated ERK1/2 and Myosin, HAECs were harvested in lysis buffer for immunoprecipitation with a mouse anti-PARP-1 antibody. The immune complexes were further analyzed by immunoblotting with phosphorylated ERK1/2 and myosin antibodies.7. Animal experimentsApoE-/-PARP-1-/-mice were generated by crossing the offspring of ApoE+/-PARP-1+/-mice that resulted from mating their respective homozygous knockouts and were genotyped by PCR. Serum levels of total cholesterol (TC), triglycerides (TGs), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured. Vasoconstriction was induced by administration of a large dose of norepinephrine. Endothelium dependent vasorelaxant responses to increased doses of acetylcholine were measured. NO production by mouse aortas and cultured cells was determined with use of the Total Nitric Oxide Assay Kit. Aortas and cells were homogenized in lysis buffer and detected.8. Immunocytochemistry and immunohistochemistryThe localization and expression of CD31, arginase ? and eNOS in HAECs and aorta were detected. The results were observed by Laser scanning confocal microscopy.Result1. PARP-1 directly binds to arginase ? promoter at the region of -774?-738 bpThe pGL3 vector containing different fragments of arginase ? promoter was transfected into HAECs cells and the expression of luciferase activity was detected. The core region of arginase ? promoter was screened from-774 to-738bp. Nuclear proteins binding with a biotin labeled probe, were separated by SDS-PAGE and identified by MALDI-TOF-TOF MS/MS. PARP-1 was chosen from several identified proteins on the basis of the spectra of 3 peptides. ChIP assay was used to confirm the binding of PARP-1 with the arginase ? promoter region.2. PARP-1 regulates the transcription of arginase ?in aortic endothelial cells (HAECs)The mRNA and protein expression of arginase ? were inhibited by PARP-1 siRNA by 80% and 75%respectively. Luciferase mRNA level was suppressed after pGL-909 and pGL-774 transfection by nearly 80% with PARP-1 siRNA treatment (p<0.01). PARP-1 overexpression induced by transfection of the PARP-1/pCDNA3.1(-) vector into HAECs increased PARP-1 and arginase ? expression by 3 fold as compared with the control group.3.OxLDL upregulates PARP-1 expression in vitro and in vivo.PARP-1 mRNA levels were increased dose dependently 24 h after oxLDL treatment. The increased expression of PARP-1 mRNA in HAECs began as early as 0.5 h and lasted for 24 h under at 50 ?g/ml oxLDL treatment. Confocal microscopy revealed more than 3 fold higher PARP-1 expression in the endothelium of aortas with a high cholesterol than normal diet. Similar results were observed in human normal and atherosclerotic arteries. Thus, oxLDL may stimulate PARP-1 production in vitro and in vivo.4.PARP-1 mediates oxLDL induced arginase II expression and modulates vascular endothelial functionThe expression of arginase II in aorta of wild-type mice fed with high-cholesterol diet was significantly higher than that of normal diet, but the expression of arginase II in PARP-1-/-mice was lower than that in wild type Mice and did not differ from wild-type mice in the normal diet. The expression of eNOS in aorta of wild-type mice fed with high-fat diet was significantly lower than that in normal diet, but the expression of eNOS in aorta of PARP-1-/-mice was significantly higher than that of wild-type mice. The production of nitric oxide in the aorta of wild-type mice was significantly higher than that of high-fat diets, but the production of NO in PARP-1-/-mice was significantly higher than that of wild-type mice. The vasorelaxation of PARP-1-/-mice aortic rings was significantly higher than that of wild-type mice in the same diet (normal or high cholesterol diet). PARP-1 mediates oxLDL inhibition of eNOS/NO expression by modulating the production of arginase II.5. OxLDL upregulates Arginase II expression by phosphorylated ERK2 and PARP-1 interactionThe expression of PARP-1 was significantly increased when oxLDL was stimulated, and PD98059, an inhibitor of MEK1/2 phosphorylation, and U0126, an inhibitor of ERK1/2 phosphorylation, inhibited PARP-1 expression. Phosphorylated ERK2 translocated from the cytoplasm to the nucleus after oxLDL stimulation. PD98059 and U0126 but not DPQ inhibited ERK2 translocation in HAECs. Phosphorylated ERK2 was detected in proteins immunoprecipitated by anti-PARP-1 antibodies, and a higher level of phosphorylated ERK2 was found with oxLDL than control treatment. Western blot analysis revealed arginase II expression lower with PD98059, U0126 and DPQ than control treatment.6. OxLDL enhances the interaction of PARP-1 with myosin and regulates the expression of arginase ?Mass spectrometry analysis of proteins interacting with PARP-1 and confirmed by immunoprecipitation that myosin interact with PARP-1. OxLDL stimulated myosin expression in HAECs increased significantly by 4.37 folds compared with the control group. Immunohistochemistry showed that PARP-1 and myosin were co-located in the vascular endothelium, especially in the arteries of human atherosclerosis more than the normal arteries. High cholesterol diet in mice could induce aortic myosin expression. PARP-1 knockout mice significantly decreased the myosin expression in the aortic endothelium than that of wild-type mice.7. OxLDL up-regulates arginase ?expression through PARP-1-myosin complex-mediated chromatin remodelingAcetylation of histone 2A was enhanced by oxidized low-density lipoprotein, while PARP-1 siRNA could induce deacetylation of H2A lysine 5. C9873, a histone acetyltransferase inhibitor, inhibits histone 2A acetylation and inhibits arginase ? expression, significantly reversing oxidized LDL-induced arginase ? hypermethylation. The myosin inhibitor ML-7 could reverse the high expression of acetylated histone H2A induced by oxidized low-density lipoprotein in aortic endothelial cells. Myosin inhibitors also reversed the high expression of arginase ? in endothelial cells induced by oxidized -LDL stimulation.In conclusion1.PARP-1 can specifically bind to arginase ? promoter region as a transcription factor.2.1nhibition of PARP-1 can reduce endothelial-derived arginase ? expression, increase the activity of eNOS and NO production.3. OxLDL upregulates Arginase ? expression by the phosphorylation of ERK2 to nucleus migration and PARP-1 interaction, and then activation of arginase? transcription.