Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
Mol Cell Biochem. 2010 Jul;340(1-2):55-62. doi: 10.1007/s11010-010-0400-2. Epub 2010 Feb 16.
CREB binding protein (CBP), a powerful transcriptional co-activator for various transcriptional factors, regulates cell behavior in many cell types. Angiotensin II (Ang II) contributes to vascular lesion by promoting vascular smooth muscle cells (VSMCs) proliferation and migration. Therefore, we examined whether CBP knockdown could suppress Ang II-induced VSMCs proliferation, and elucidated its underlying molecular mechanism. We constructed lentiviral vector expressing CBP-specific short hairpin RNAs (shRNAs) that efficiently silenced CBP. VSMCs proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assay. Protein and mRNA expression of CBP and relevant cytokines were examined by Western blot, ELISA, and real-time PCR, respectively. We also used luciferase reporter gene and electrophoretic mobility shift assay (EMSA) to detect Nuclear factor kappaB (NF-kB) transcriptional activity and DNA binding. Meanwhile, NF-kB p65 subunit nuclear translocation was confirmed by immunoblotting. Lentiviral-mediated CBP-shRNAs at different multiplicities of infection (MOI = 100, 150) both significantly suppressed Ang II-induced CBP expression. Knockdown of CBP markedly inhibited Ang II-stimulated VSMCs proliferation and cytokines (TNF-alpha and IL-6) production. However, this inhibitory effect was not enhanced at MOI of 150 compared with MOI of 100 (P > 0.05). CBP siRNA showed the potent inhibition on Ang II-induced NF-kB transcriptional activity. Similarly, no significant difference was found between CBP siRNA lentivirus treatment groups. Furthermore, CBP gene silencing had no effect on NF-kB nuclear translocation and DNA binding. These findings suggest that CBP knockdown inhibits Ang II-induced VSMCs proliferation and the mechanism is involved with downregulation of NF-kB transcriptional activity, not through reduction in NF-kB nuclear translocation or DNA binding. Maintaining proper CBP level may be a potential therapeutic target for Ang II-induced cardiovascular disorders.
CREB 结合蛋白（CBP）是多种转录因子的有力转录共激活因子，调节多种细胞类型的细胞行为。血管紧张素 II（Ang II）通过促进血管平滑肌细胞（VSMCs）增殖和迁移促进血管病变。因此，我们研究了 CBP 敲低是否可以抑制 Ang II 诱导的 VSMCs 增殖，并阐明了其潜在的分子机制。我们构建了表达 CBP 特异性短发夹 RNA（shRNA）的慢病毒载体，该载体能够有效地沉默 CBP。通过溴脱氧尿苷（BrdU）掺入测定评估 VSMCs 的增殖。通过 Western blot、ELISA 和实时 PCR 分别检测 CBP 和相关细胞因子的蛋白和 mRNA 表达。我们还使用荧光素酶报告基因和电泳迁移率变动分析（EMSA）检测核因子 kappaB（NF-kB）转录活性和 DNA 结合。同时，通过免疫印迹法证实 NF-kB p65 亚基核转位。不同感染复数（MOI=100、150）的慢病毒介导的 CBP-shRNA 均显著抑制 Ang II 诱导的 CBP 表达。CBP 敲低显著抑制 Ang II 刺激的 VSMCs 增殖和细胞因子（TNF-α和 IL-6）的产生。然而，与 MOI 为 100 相比，MOI 为 150 时这种抑制作用没有增强（P>0.05）。CBP siRNA 对 Ang II 诱导的 NF-kB 转录活性具有强大的抑制作用。同样，在 CBP siRNA 慢病毒处理组之间也未发现明显差异。此外，CBP 基因沉默对 NF-kB 核转位和 DNA 结合没有影响。这些发现表明，CBP 敲低抑制 Ang II 诱导的 VSMCs 增殖，其机制涉及 NF-kB 转录活性的下调，而不是通过减少 NF-kB 核转位或 DNA 结合。维持适当的 CBP 水平可能是治疗 Ang II 诱导的心血管疾病的潜在治疗靶点。
