Study On The Mechanisms Of Angâ…¡-induced Angiotensinogen Gene Expression And VSMC Proliferation

The proliferation of vascular smooth muscle cells (VSMC) is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. It has been known that a number of the growth factors and vasoactive substances can induce the proliferation of VSMC, AngII play a central role in VSMC proliferation and a variety of pathological processes of vascular remodeling diseases including atherosclerosis, hypertension and restenosis after coronary angioplasty. Not only does VSMC as a source of AngII, but it also serves an important target tissue of AngII-regulating vascular function. Therefore, to investigate mechanisms of AngII-induced VSMC proliferation and regulating its precursor gene expression is more important to clarify the molecular mechanisms of proliferative vascular disease.To elucidate the molecular mechanism of angiotensinogen gene expression and VSMC proliferation induced by AngII, we detected the effect of AngII on activator protein-1 (AP-1) expression and activation, and on angiotensinogen gene expression and VSMC proliferation. We also investigated the interaction between AP-1 and STAT5 in the transcriptional activation of angiotensinogen gene. Additionally, signal transduction pathways of AngII-induced VSMC proliferation and the potential targets of the inhibition of VSMC proliferation by roscovitine were studied.1 Molecular mechanisms of angiotensinogen gene expression induced by AngII in VSMCAP-1 was identified as a transcription factor involved in regulation of the expression of genes responsible for cell proliferation. It has been demonstrated that expression of angiotensinogen gene induced by AngII is related with the increase in the activity of AP-1 binding to angiotensinogen promoter in VSMC. To determine the molecular mechanism of AP-1 binding to its cis-element, we investigated the effect of AngII on expression of angiotensinogen gene and interaction between AP-1 and its cis-element by using cycloheximide (CHX) as an inhibitor of inhibiting c-Jun phosphorylation in this part. The results were as follows:1.1 Effects of different concentrations of CHX on viability of the VSMCTo assess whether CHX has cytotoxic effect on VSMC, the cells were treated with different concentrations of CHX, and then the viability of cells was analyzed by MTT method. The results showed that there was no difference in viability of the VSMC at the range of 15~45μmol/L of CHX. The results suggested that CHX has no cytotoxic effect on VSMC at examined concentration ranges.1.2 AngII induces c-Jun proteins expression and phosphorylationWestern blot results showed that the level of c-Jun in VSMC was significantly increased after the cells were treated with AngII for 0.5 h, and then kept to 3 h at this level. Immunocytochemistry analysis indicated that majority of c-Jun proteins were located in the nucleus. The phosphorylated c-Jun levels were assessed by immunoblotting with anti-c-Jun antibody after the nuclear extracts were immunoprecipitated by anti-phosphoserine antibody. The results confirmed that AngII could induce serine phosphorylation of c-Jun. VSMC was pretreated with CHX, and then stimulated by AngII, the levels of c-Jun protein were not altered, but the phosphorylated c-Jun levels were significantly decreased. These results indicated that the c-Jun expression and phosphorylation induced by AngII is one of the important mechanisms whereby AngII regulates its precursor gene expression in feedback manner. It is found that CHX is an inhibitor of AP-1 phosphorylation.1.3 CHX inhibits angiotensinogen gene expression induced by AngIIThe effect of CHX on angiotensinogen gene expression was detected by RT-PCR. The results showed that angiotensinogen gene expression activity was significantly increased after the cells were treated with AngII for 3 h. There was a reduction of angiotensinogen expression induced by AngII in VSMC pretreated with CHX. The results suggested that CHX downregulates the expression of angiotensinogen gene through inhibiting c-Jun phosphorylation. It indicated that the phosphorylation activation of AP-1 is necessary for angiotensinogen gene expression.1.4 AngII promotes AP-1 binding to angiotensinogen gene promoterTo determine the relationship between the c-Jun phosphorylation and angiotensinogen gene expression induced by AngII, the effect of AngII on the binding activity of AP-1 to its cis-acting elements was detected by EMSA. The results showed that the binding activity of AP-1 to the probes was increased significantly after the cells were treated by AngII for 30 min, and reached to preak at 3 h. The supershift analysis using antibody to c-Jun or STAT5b showed that there was appearance of supershifted band. The binding activity of AP-1 to its cis-acting elements was declined significantly in VSMC pretreated with CHX. To explore the mechanism of the inhibition of AP-1 binding activity by CHX, the nuclear protein at same condition was detected by Western blot. The results showed that there was no relationship between c-Jun protein level and AP-1 binding activity. It was results of inhibition of AP-1 phosphorylation by CHX, which suggested that AngII-induced AP-1 phosphorylation was one of the mechanisms of AngII regulating angiotensinogen gene expression.2 Roscovitine inhibits VSMC proliferationRoscovitine is a potent and specific inhibitor of the CDKs that inhibits cell proliferation and induces apoptosis. However, it was not clear whether roscovitine has same antiproliferative effect in VSMC and other type cells. In this part, we compared the effect of roscovitine on VSMC proliferation with several cell lines from different tissues and the mechanism of inhibition of cell proliferation by roscovitine.2.1 Effect of roscovitine on proliferation of different cellsThe cell proliferation activity was determined by cell counting. After the cells were stimulated by 10 % FBS and angiotensin II (10-6 mol/L), the cell number was significantly increased by 2.63 and 1.68 times compared with control, respectively. VSMC was pretreated with different concentration of roscovitine (15, 30, 45μmol/L) for 15 h, the AngII-induced VSMC proliferation was inhibited by roscovitine. The inhibitive rate was 37.3 %, 46.7 % and 51.8 %, respectively. Roscovitine also inhibited FBS- induced VSMC proliferation. The results suggested that roscovitine significantly inhibited VSMC proliferation in vitro.Roscovitine also inhibited HeLa, COS-7 and M17 proliferation induced by FBS. The inhibitive rate of roscovitine (30μmol/L) on HeLa, COS-7 and M17 proliferation was 61.8 %, 60.4 % and 54.8 % respectively, which was higher than that of VSMC. These data indicated that the different cell types exhibit variable sensitivity to roscovitine.2.2 The effect of roscovitine on c-Jun protein expressionImmunocytochemistry staining results showed that stimulation with AngII markedly increased expression of c-Jun in VSMC. There was a reduction of AngII-stimulated c-Jun expression in VSMC pretreated with roscovitine. Western blot analysis also confirmed that the levels of c-Jun were significantly increased after the cells were stimulated by AngII or FBS, and the levels of c-Jun were decreased significantly by pretreatment with roscovitine for 15 h. However, in COS-7, HeLa, M17 cell lines, the c-Jun expression was not affected by roscovitine. The STAT5b expression was not affected by roscovitine in HeLa, COS-7, M17 and VSMC. These results suggested that inhibition of c-jun expression was involved in antiproliferative effect of roscovitine and was not related with the inhibition of HeLa, COS-7 and M17. It indicated that there were different mechanisms in roscovitine-inhibiting cell proliferation at different cells.2.3 Roscovitine inhibits c-jun gene expression in VSMC at transcriptional levelThe VSMC was pretreated with roscovitine, and then c-jun mRNA level was determined by RT-PCR. We demonstrated that roscovitine caused a down-regulation of c-Jun mRNA in VSMC, but it did not affect c-jun mRNA expression in HeLa and M17 cells. The results indicated that roscovitine down-regulates c-Jun protein expression by inhibiting c-jun gene transcription.3 Signal transduction pathways of AngII-induced VSMC proliferation and the targets of the inhibition of VSMC proliferation by roscovitineAngII has been shown to induce proliferation of VSMC and through AT1 receptor. Once AngII binds to the AT1R, it activates a series of signaling cascades and this led to the gene expression involved in cell proliferation. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to play a key role in transducting extracellular signals to nucleus. In this part, we observed the effect of roscovitine on MAPK signaling pathways and angiotensinogen gene expression.3.1 Effect of roscovitine on AngII-induced ERK1/2 and c-Jun phosphorylation AngII can rapidly induce ERK1/2 phosphorylation. After stimulation with AngII for 5 min, the phosphorylation of ERK1/2 reached to peak and still kept high level at 30 min. Pretreating the cells with roscovitine for 15 h, the level of ERK1/2 phosphorylation did not detected. The total levels of ERK1/2 were not changed. It showed that roscovitine completely inhibited ERK1/2 phosphorylation induced by AngII. To examine the effect of roscovitine on c-Jun phosphorylation, the cell lysates were immunoprecipitated with anti-phospho-Ser mAb, and immunoblotted using anti-c-Jun antibody, the results showed that phosphorylated c-Jun levels decreased following treatment with roscovitine. The results indicated that roscovitine significantly inhibited the AngII-induced c-Jun expression and phosphorylation, which was associated with the inhibition of ERK1/2 phosphorylation.3.2 Roscovitine inhibits AP-1-mediated angiotensinogen gene expression We further examined the effects of roscovitine on expression ofangiotensinogen, which was one of downstream gene of AP-1. RT-PCR results showed that roscovitine reduced AngII-induced angiotensinogen gene expression.To confirm the effect of c-Jun in vivo, ChIP assay was performed. After chromatin was immunoprecipitated with anti-c-Jun antibody, DNA fragments containing AP-1 binding site were subjected to PCR. The ChIP assay demonstrated that treatment with AngII could enhance binding of c-Jun to the angiotensinogen promoter, an effect that could be suppressed by roscovitine, which further demonstrated that roscovitine can block AngII-mediated expression of angiotensinogen by suppressing c-Jun expression and phosphorylation, and then inhibiting the binding of c-Jun to the angiotensinogen gene promoter.4 Interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen geneAP-1 and STAT5 phosphorylation activation is involved in AngII-induced its precursor gene expression, which suggested that AP-1 and STAT5 participated in trans-activation of angiotensinogen gene. In the present study, we investigated the interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene.4.1 AP-1 and STAT5 interaction is involved in AngII-induced its precursor gene expressionWe carried out co-immunoprecipitation (co-IP) experiments using VSMC lysates treated with or without AngII. The results showed that IP with anti-STAT5b antibody followed by Western blot analysis with anti-c-Jun antibody clearly demonstrated the presence of c-Jun among the immunoprecipitated proteins, and the AngII treatment caused increase in obviously the interaction between c-Jun and STAT5. These results confirmed that the physical interaction between c-Jun and STAT5b occurs in vivo. AngII can induce interaction between c-Jun and STAT5b.4.2 The complex formed by c-Jun and STAT5b interacts with cis-elements of angiotensinogen geneEMSA results showed that AngII enhanced the binding activity of AP-1 to the promoter of angiotensinogen gene. Supershift analysis was performed with anti-c-Jun or anti-STAT5b antibody. The results indicated that the DNA-protein complex contained both c-Jun and STAT5b. These data indicated that STAT5b and c-Jun can interact directly or indirectly with each other at the AP-1 binding site. ChIP assay results showed that DNA sequences contained AP-1 binding site could be amplified when DNA immunoprecipitated using anti-STAT5b antibody were subjected to PCR. DNA immunoprecipitated with anti-c-Jun antibody contained STAT5 binding site. The results demonstrated that there is presence of the interaction between STAT5 and c-Jun when they bind, respectively, to their cis-elements located in the angiotensinogen gene promoter.4.3 GST pull-down analysis for interaction between STAT5 and c-Jun in vitroGST pull-down results showed that STAT5b in whole cell lysates or nuclear extracts could not be pulled down by using GST-c-Jun fusion proteins. It indicated that c-Jun could not interact directly with STAT5b in vitro.4.4 STAT5 synergizes with c-Jun in transactivation of the expression of angiotensinogen geneThe reporter gene assay results showed that the relative luciferase activity was significantly enhanced, when 293A cells were co-transfected with c-Jun and STAT5b, These results suggested that interaction between AP-1 and STAT5 mediates the synergistic enhancement of angiotensinogen gene expression. Co-transfected c-Jun and STAT5b into HeLa cells could synergistically inhibit angiotensinogen gene expression. These results suggested that in different cells there were distinct transcriptional factors which mediate the angiotensinogen gene expression.Conclusion:1. AngII positive-feedback regulates its precursor gene expression by inducing AP-1 phosphorylation activation.2. Roscovitine exerts an antiproliferative effect on VSMC by specifically inhibiting c-Jun expression.3. ERK1/2 signal transduction pathway is involved in inhibition of VSMC proliferation by roscovitine.4. STAT5 and c-Jun have a synergistic effect in trans-activation of angiotensinogen gene.