| Proliferation and differentiation of vascular smooth muscle cells (VSMC) are governed by a variety of regulatory factors, such as krüppel-like factor 5 (KLF5) that regulates gene expression. Recent study has shown that Am80, a synthetic RARα-specific agonist, removed the inhibitory effect of KLF5 p21 expression via inhibiting the interaction between KLF5 and RARα. Posttranslational modifications, such as acetylation, phosphorylation and ubiquitination, play a critical role in activation of KLF5. In the present studies, we investigated the effect of the interaction between KLF5 and HDAC2 induced by Am80 on p21 transcriptional activation, which may be contributes to the inhibition VSMC proliferation.Methods and results:1 Am80 blocks cell cycle progression in VSMCsThe effect of Am80 on cell cycle progression was detected by flow cytometry. The cell population of G0/G1 increased by 13 %, and S phase population displayed a 29 % decrease after treated by Am80, respectively, compared with control group. These results suggest that Am80 blocks progression of cell cycle through the G1 phase to the S phase in VSMCs.2 Am80 inhibits proliferation of VSMCsThe results of MTT assays showed that Am80 (12, 24, 48 h) treatment resulted in a significant reduction of VSMC proliferation in a time-dependent manner. The activity of VSMC proliferation decreased by 44 %, 35 %, and 26 %, respectively, compared with control group.3 Am80 induces p21 expression in VSMCsTo understand the mechanism of Am80 action, the effect of Am80 on the expression of p21 was detected by Western blot analysis. VSMCs were treated with Am80 at different doses (0-5μM) and used to examine the expression of p21. The results showed that the expression of p21 was up-regulated by Am80 in a dose-dependent manner.4 Am80 inhibits neointimal hyperplasia induced by balloon injuryThe rats injured with balloon were treatd by Am80, and then the intima to media area (I/M) ratio of carotid arteries was evaluated. The results showed that Am80 significantly reduced neointimal hyperplasia and I/M ratio (P < 0.05).5 KLF5 deacetylation induced by Am80 is associated with up-regulation p21 levelWestern blotting showed that Am80 treatment resulted in reduced acetylation of KLF5 in a dose-dependent manner in VSMCs. In vivo, the acetylated-KLF5 level in the Am80-administered group was lower than that in injured groups. These results suggest that Am80 induces the deacetylation of KLF5.6 The interaction of HDAC2 with KLF5 induced by Am80 is involved in the deacetylation of KLF5To examine whether HDAC2 mediates deacetylation of KLF5 induced by Am80, the interaction of HDAC2 with KLF5 was detected by Co-IP. The results revealed that the interaction between KLF5 and HDAC2 was significantly increased after Am80 treatment.7 KLF5 deacetylation activates the activity of p21 promoterp21 luciferase reportor analysis showed that a 3-fold increase in the p21 promoter activity was observed when the expression plasmids of KLF5 and HDAC2 were co-transfected. However, KLF5-K369R mutant that is acetylation-deficient plasmid was not able to enhance the promoter activity, suggesting that KLF5 deacetylation by HDAC2 is required for the activation of the p21 promoter.Conclusions:1 Am80 blocks the cell cycle progression in VSMCs;2 Am80 inhibits the proliferation of VSMCs; 3 Am80 induces the interaction between HDAC2 and KLF5, and subsequently leading to KLF5 deacetylation;4 The deacetylation of KLF5 is involved in activation of p21 transcription. |
