CBP knockdown inhibits angiotensin II-induced vascular smooth muscle cells proliferation through downregulating NF-kB transcriptional activity
- 412 Downloads
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-α 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.
KeywordsCBP Nuclear factor kappa-B Vascular smooth muscle cells RNA interference Gene therapy
This work was supported by a grant from the National Natural Science Foundation of China (No. 30770849).
- 3.Jia G, Cheng G, Agrawal DK (2006) Differential effects of insulin-like growth factor-1 and atheroma-associated cytokines on cell proliferation and apoptosis in plaque smooth muscle cells of symptomatic and asymptomatic patients with carotid stenosis. Immunol Cell Biol 84:422–429CrossRefPubMedGoogle Scholar
- 8.Chen Y, Zheng J, Zhang Y, Wang J, Liu Q, Huang Z et al (2009) N-4-tert-butyl benzyl haloperidol chloride suppresses Ca2+-dependent Egr-1 expression and subsequently inhibits vascular smooth muscle cell proliferation induced by angiotensin II. Cell Physiol Biochem 23:295–304CrossRefPubMedGoogle Scholar
- 12.Hernández-Presa M, Bustos C, Ortego M, Tuñon J, Renedo G, Ruiz-Ortega M et al (1997) Angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 95:1532–1541PubMedGoogle Scholar
- 13.Yamamoto K, Shioi T, Uchiyama K, Miyamoto T, Sasayama S, Matsumori A (2003) Attenuation of virus-induced myocardial injury by inhibition of the angiotensin II type 1 receptor signal and decreased nuclear factor-kappa B activation in knockout mice. J Am Coll Cardiol 42:2000–2006CrossRefPubMedGoogle Scholar
- 30.Shah RD, Gonzales F, Golez E, Augustin D, Caudillo S, Abbott A et al (2005) The antidiabetic agent rosiglitazone upregulates SERCA2 and enhances TNF-alpha- and LPS-induced NF-kappaB-dependent transcription and TNF-alpha-induced IL-6 secretion in ventricular myocytes. Cell Physiol Biochem 15:41–50CrossRefPubMedGoogle Scholar