| Vasoactive peptides are a kind of low molecular weight peptides that regulate the vasodilative and vasoconstrictive function and maintain the vascular tone through the way of autocrine and/or paracrine. There are dynamic equilibrium in the excretion and activity between vasoconstrictive peptides, including angiotensin II (Ang II) and endothelin-1 (ET-1), and vasodilative peptides, including adrenomedullin (ADM) and atrial natriuretic factor (ANF). The complex positive feedback or negative feedback exists in different vasoactive peptides, which forms a network of regulation of vascular tone. In addition to promoting the vascular smooth muscle cell (VSMC) hypertrophy, proliferation and migration, the vasoconstrictive peptides usually resulte in many vascular remodeling diseases, whereas vasodilative peptides usually have reverse functions. To clarify the regulatiory mechanism of vasoactive peptides is helpful to comprehend the pathophysiological function of vasoactive peptides in processes of cardiovascular disease. To study the molecular mechanism of vasoactive peptide gene expression and the relationship between vasodilative and vasoconstrictive peptides, the cultured rat VSMCs were stimulated with Ang II. In the present study, we investigated the expression and release of Ang II, ET-1, ADM and ANF, the activity of activator protein-1 (AP-1) binding to its site, which is a cis-element characterized in the promoter of four vasoactive peptides, and the role of janus protein tyrosine kinase-signal transducers and activators of transcription pathway (JAK-STAT pathway) in transcriptional activation of vasodilative and vasoconstrictive peptide genes. Furthermore, we also studied the cross-talking between the AP-1 and STAT5 in the transcriptional activation of angiotensinogen gene. 1 Expression and regulation of vasodilative and vasoconstrictive peptides in vascular smooth muscle cell Gene sequence assays show that AP-1 binding site locates in the promoter of ET-1, angiotensinogen, ADM and ANF genes. The four genes expression is associated with activation of AP-1. To determine the relationship between the AP-1 binding activity and the transcriptional activation of four genes, and the role of VSMC in maintaining the balance of vasodilative peptides and vasoconstrictive peptides, expression and release of ET-1, Ang II, ADM and ANF were detected, and the activity of AP-1 binding to cis-acting element located in the four vasoactive peptide gene promoters was also detected. The results were as following: 1.1 Ang II induces the expression of four vasoactive peptides in VSMC Fluorescence quantitative RT-PCR showed that the expression of four vasoactive peptide genes kept a balance in quiescent VSMCs, while the expression of four genes was up-regulated after Ang II stimulation. After stimulated with Ang II, the mRNA level of vasoconstrictive peptides was quickly elevated, whereas the expression of vasodilative peptides declined first and then increased. But, Ang II did not alter the balance between vasodilative and vasoconstrictive peptides. VSMC was capable of regulation of the vasoactive peptides to balance at short time (12~24 h). 1.2 Effects of Ang II on synthesis and release of ET-1, Ang II, ANF and ADM in VSMC At different time after Ang II treatment, concentration of ET-1, Ang II, ANF and ADM in culture medium and VSMC was detected by radioimmunoassay (RIA). The data demonstrated that synthesis of the four vasoactive peptides in VSMC was increased after Ang II stimulation, but secretion of vasoactive peptides from VSMC was different from each other. The level of ET-1 and Ang II in medium of VSMC was elevated, but secretion of ADM and ANF from VSMC was gradually declined following Ang II stimulation. 1.3 Effects of Ang II on transcription factor AP-1 binding activity in VSMC AP-1 is involved in cellular proliferation, and the site of AP-1 binding islocated in the promoter region of four vasoactive peptide genes. To determine the role of AP-1 in regulation of vasoactive peptide genes, AP-1 binding activity was demonstrated by electrophoretic mobility shift assay (EMSA). The results showed that activity of the AP-1 binding to promoter in VSMC was increased at 3 h after Ang II treatment and then declined gradually to control level. The AP-1 binding activity in vitro was consistent with the expression level of the vasoactive peptides. 1.4 Effects of Ang II on AP-1 binding to cis-acting element in vivo To determine whether AP-1 is capable of binding to cis-element, the chromatin immunoprecipitation (ChIP) assay was performed, which is a method for analyzing the binding activity of transcription factors to DNA at chromatin level. The cross-linker complex of DNA-transcription factors that contained AP-1 binding site were immunoprecipitated and enriched by anti-c-Jun antibody. ChIP assay provided an analysis of the DNA binding activities of AP-1 in living cells. The results suggested that recruitment AP-1 at the regulatory regions of angiotensinogen, ET-1 and ADM genes was involved in the transcription activation of the vasoactive peptides. 2 JAK-STAT pathway is involved in transcription activation of vasodilative and vasoconstrictive peptide genes in vascular smooth muscle cells It has been known that the expression of ANF is inhibited strongly by JAK2 specific inhibitor AG490 in culture myocardial cell, suggesting that JAK-STAT pathway maybe involved in transcription activation of vasodilative and vasoconstrictive peptide genes. Seven STAT proteins have been identified in mammalian cells. STAT5 participates in transcriptional activation of angiotensinogen in liver and heart. However, the role of STAT5 in transcriptional activation of vasodilative and vasoconstrictive peptide genes in VSMC has not been reported. To determine the role of JAK2-STAT5 pathway in expression of vasoactive peptides, STAT5 activation and binding activity to cis-acting elements located in the vasoactive peptide gene promoter were detected.2.1 Ang II induces STAT5 tyrosine phosphorylation in VSMC To determine STAT5 tyrosine phosphorylation induced by Ang II in VSMC, the phosphorylated STAT5 levels were assessed on the basis of anti-phosphotyrosine (PY99) immunoblotting using anti-STAT5 immunoprecipitates of whole cell extracts. Ang II stimulation of VSMCs did not alter the total level of STAT5 protein over the examined time periods, but the phosphorylated STAT5 level was increased significantly after treated with Ang II for 5 min and reached to peak at 10 min, and then the phosphorylated STAT5 levels was decreased. If prolongated Ang II stimulus duration, we observed that the phosphorylated STAT5 level was reincreased at 2 h, and reached to another peak at 6 h. It indicated that phosphorylated STAT5 took part in the process of Ang II signal pathway. 2.2 Ang II induces nuclear translocation of phosphorylated STAT5 in VSMC For signal propagation through homodimers or heterodimers, nuclear translocation of STAT5 regulated the expression of target genes. To determine the role of phosphorylated STAT5 induced by Ang II in nuclear translocation, two methods had been used to detect the level of STAT5 in VSMC nuclear. Immunoprecipitation results showed that phosphorylated STAT5 was not detected in untreated VSMC nuclear, but the phosphorylated STAT5 level was increased quickly after treated with Ang II for 5 min and then decreased until 30 min after Ang II treatment. Similar results were verified by using laser scanning confocal microscopy, anti-STAT5 fluorescence stain was intensively increased in VSMC nuclear space at 30 min after Ang II stimulation. It indicated that STAT5 nuclear translocation correlated with phosphorylated STAT5 level in VSMC after Ang II treatment. 2.3 AG490 inhibites the Ang II-induced phosphorylation and nuclear translocation of STAT5 The activated JAK2 subsequently phosphorylated targets, including STAT5; nevertheless STAT5 phosphorylation was complicated by interactions with other signal pathway. To provide the evidences of JAK2-STAT5 pathway participated in Ang II signal transduction, cultured VSMCs were stimulatedwith Ang II after pretreatment with AG490, the specific inhibitor of JAK2. Immunoprecipitation results showed that AG490 inhibited Ang II-induced phosphorylation of STAT5. Laser scanning confocal microscopy results showed that anti-STAT5 fluorescence stain distribution to VSMC nuclear space was decreased after AG490 pretreatment. Thus the JAK2-STAT5 cascade participated in transduction Ang II extracelluar signal into nuclear, Ang II-induced phosphorylation and nuclear translocation of STAT5 was inhibited by JAK2 inhibitor AG490. 2.4 Ang II enhances the binding activity of STAT5 to cis-acting elements located in the vasoactive peptide gene promoter To determine the relationship between the trans-activation of vasoactive peptide genes and STAT5 nuclear translocation, the binding activity of STAT5 to cis-acting elements located in the vasoactive peptide gene promoter was detected by EMSA. After Ang II treatment for 30 min, the binding activity of STAT5 to cis-acting elements located in the angiotensinogen gene promoter was increased significantly; whereas the binding activity of STAT5 to cis-acting elements located in the ANF gene promoter was declined significantly. Stimulation of VSMCs with Ang II led to the induction of DNA-binding protein complex, and the observed band was specific since it could be inhibited by unlabeled oligonucleotide. The presence of STAT5 in the observed complex was tested by using anti-STAT5 antibody in supershifted assay. We observed that the DNA-binding protein complex containing STAT5 was abrogated by AG490, which suggested that STAT5 participated in trans-activation of vasodilative and vasoconstrictive peptide genes. 3 AP-1 and STAT5 mediate Ang II-induced trans-activation of angiotensinogen gene in VSMC Previously, we have shown that AP-1 and STAT5 were present in the binding complexes within the angiotensinogen promoter, and the two transcription factors also participated in trans-activation of angiotensinogen gene. In the present study, we investigated the role of interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene.3.1 AG490 inhibites the expression of angiotensinogen gene RT-PCR results showed that Ang II regulated the expression of its prohormone-angiotensinogen gene by way of positive feedback. The angiotensinogen expression kept low level in quiescent VSMCs treated with serum deprivation, and the gene expression increased dramatically after stimulated by Ang II for 3 h. AG490 abrogated the Ang II-induced increasing of gene expression at the same time periods. It suggested that JAK2-STAT5 cascade was an important pathway in trans-activation of angiotensinogen gene. 3.2 AG490 inhibites the interaction between AP-1 and STAT5 To determine the role of interaction between AP-1 and STAT5 in trans-activation of angiotensinogen gene, AP-1 levels were assessed on the basis of anti-c-Jun immunoblotting using anti-STAT5 immunoprecipitates of nuclear extracts in VSMC. A band corresponding AP-1 protein was found in the anti-STAT5 immunoprecipitates, and the band became deeper with prolongating Ang II stimulus duration, it suggested that Ang II stimulation induced the interaction between AP-1 and STAT5 in VSMC. AG490 inhibited the interaction by abrogating the Ang II-induced phosphorylation of STAT5. It suggested that the interaction between AP-1 and STAT5 depended on the level of phosphorylated STAT5. 3.3 AG490 inhibites the binding activity of AP-1 EMSA results showed that AG490 abrogated the Ang II-induced increasing binding activity of AP-1 to the promoter of angiotensinogen gene. It suggested that JAK2 kinase pathway played an important role in activation of AP-1, and AP-1 and STAT5 were necessary for trans-activation of angiotensinogen gene. 3.4 AG490 inhibites chromatin remodeling at STAT5 binding site located in the promoter of angiotensinogen gene To determine the molecular mechanism of the inhibition of AG490 to angiotensinogen gene, we investigated the relationship between the expression of angiotensinogen gene and chromatin remodeling at STAT5 binding site... |
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