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Objective: Vitamin A derivative of all-trans retinoic acid (all-trans retinoic acid, ATRA) in a variety of cells has a role in regulation of cell growth and induction of differentiation. Retinoic acid receptor (retinoic acid receptor, RAR) is an important class of nuclear receptors, RARα, RARβ, and RARγ three subtypes. Type of receptor by its ligand ATRA combination, activate or inhibit the expression of many genes, resulting in cell proliferation, differentiation and apoptosis play an important role. Krüppel-like factor 4 (krüppel-like factor, GKLF/KLF4) is a class of zinc finger nuclear transcription factor can inhibit the proliferation of vascular smooth muscle cells (vascular smooth muscle cell, VSMC) induced differentiation role. The large number of experimental results show that, in VSMC, ATRA can significantly KLF4 gene expression induced by ATRA-induced KLF4 expression but the molecular mechanism is unclear. In this study, ATRA activate KLF4 gene expression mechanism, research RARα and related signaling pathway in ATRA-induced KLF4 expression process to clear the molecular basis of ATRA-induced VSMC differentiation. Methods: attached method separation cultured rat VSMC, trypsin digestion and passage, take 3 to 5 cells experiments. Western blot analysis detected ATRA the KLF4 and RARα expression of, affect the application of different signaling pathway inhibitors mediated KLF4 expression signal transduction pathways. Results: 1 ATRA induced KLF4 and RAR alpha expression 10μM ATRA stimulate VSMC 0, 6, 12, 24 h, total protein was extracted and RNA, Western blot analysis and RT-PCR. The results showed that ATRA stimulation time, the the KLF4 expression level was time-dependent increase in RARα expression levels significantly higher, suggesting that RAR alpha may be involved in ATRA-induced expression of KLF4. 2 the RAR alpha mediated guide ATRA treatment KLF4 expression applications RAR antagonist Ro 41-5253 (20μM) pretreatment of VSMC 1 h after ATRA (10μM) stimulate VSMC 24 h, respectively, total protein was extracted and RNA, Western blot analysis and RT -PCR. The results show that the application RARα inhibitor pretreatment, ATRA increase the KLF4 expression of the role of inhibition. To further verify the RAR alpha whether or not mediated KLF4 expression and ATRA, RARα specific small interfering RNA (RARalpha-siRNA) import VSMC after blocking within borne RAR expression detected KLF4 expression changes. The results show that the RARα-siRNA transfected cells compared to the control group of NS-siRNA transfection, ATRA induced KLF4 expression was inhibited, indicating that the the RAR alpha mediated guide ATRA KLF4 expression induced. 3 ATRA activation of p38 MAPK inhibition of ERK signaling pathway of ATRA by which signaling pathways activated KLF4 gene expression, the application of ATRA (10μM) stimulated VSMC 0,15,30,60 min, respectively, to detect ERK, p38 MAPK, Akt and beta -catenin phosphorylation levels. The results showed that ATRA stimulation 15 min, the level of ERK phosphorylation is reduced p38 MAPK phosphorylation levels were significantly increased, ATRA stimulation does not affect the level of Akt and beta-catenin phosphorylation. Showed that ATRA can inhibit ERK signaling pathway, activation of the p38 MAPK pathway. The 4 RAR alpha through ERK and p38 MAPK signal conditioning KLF4 gene expression in order to further investigate the role of RARα in ATRA-induced KLF4 gene expression, the application of RARα antagonist Ro 41-5253 pretreatment VSMC 1 h after ATRA stimulation VSMC 1 h. Western blot analysis showed that the level of ERK phosphorylation significantly increased compared with the control group, p38 phosphorylation levels were significantly lower. In addition, the application of RAR alpha-siRNA transfected stained VSMC after knockdown of endogenous RARα expression, ERK phosphorylation level increased compared with the control group, p38 MAPK phosphorylation levels were significantly lower, indicating that RARα gene KLF4 ERK and p38 MARK signal conditioning expression. P38 MAPK signal mediated ATRA-induced KLF4 expression in order to further prove that ATRA induced the KLF4 expression of p38 MAPK and ERK signaling pathway, with p38 MAPK specific inhibitor SB203580 (20μM), ERK inhibitor PD98059 (20μM) and Akt inhibitor LY294002 (20μM) pre-incubated VSMC after 2 h, given ATRA stimulation for 24 h, cells were collected for Western blot analysis. The results showed that the inhibition of p38 MAPK signaling pathways ATRA-induced KLF4 expression can be inhibited, inhibition of ERK signaling pathway increases KLF4 expression, inhibition of Akt signaling pathway does not affect ATRA induced KLF4 expression. To further validate the ERK signaling pathway in ATRA-induced KLF4 expression, the application of the continuous activation of ERK plasmid pCMV-MEKca transfer dye VSMC, ATRA to stimulate the cells for 24 h, cells were collected to detect expression of KLF4. Western blot analysis showed that the transfection the pCMV-MEKca the cells compared to cells transfected with control plasmid (pCMV), ATRA induced KLF4 expression is suppressed. These results suggest that p38 MAPK and ERK signaling pathway in ATRA regulation KLF4 gene expression play an important role. Conclusion: 1 in VSMC, ATRA induced KLF4 and RARα expression. 2 RAR alpha-mediated ATRA KLF4 gene expression induced. 3 RAR alpha through ERK and p38 MAPK signaling pathways regulating KLF4 ATRA-induced gene expression.
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