Molecular Mechanism Of ATRA-induced KLF4 Expression In Vascular Smooth Muscle Cells

Objective: All-trans retinoic acid (ATRA), a derivative of vitamin A, is involved in regulating growth and inducing differentiation in a variety of cell types. RAR is an important nuclear receptor, which has three isoforms including RARα, RARβand RARγ. RAR interacts with its ligands, and subsequently activates or suppresses a variety of gene expression, which plays a key role in regulating cell growth, proliferation, differentiation, and apoptosis. It has been identified that the Krüppel-like factor 4 (KLF4/GKLF) is a zinc finger transcription factor. In vascular smooth muscle cells (VSMCs), KLF4 plays a critical role in suppressing proliferation, promoting differentiation and regulating cardiovascular remodeling.We have recently demonstrated that ATRA induced the expression of KLF4 in VSMCs. However, the molecular mechanism of ATRA-induced KLF4 expression has not been well characterized. Here, we explore the molecular mechanism of ATRA-induced KLF4 expression and study the function of RARαin ATRA-induced KLF4 expression.Methods: VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats. Cell passages 3~5 were used in all experiments. The expression of KLF4 and RARαand phosphorylation of various signaling molecules were examined by Western blotting.Results:1 ATRA induces the expression of KLF4 and RARαin VSMCsAfter treating cells with ATRA (10μM) for different times (0, 6, 12 and 24 h), the expression of KLF4 and RARαsignificantly increased in time-dependent manners, indicating that ATRA significantly induces the expression of KLF4 and RARαin VSMCs. 2 RARαmediates ATRA-induced expression of KLF4In order to identify the role of RARαin ATRA-induced KLF4 expression, we treated VSMCs with RARαinhibitor (Ro 41-5253), to block the endogenous RARαexpression, and determined the effects of ATRA on KLF4 expression. VSMCs were pretreated for 1 h with RARαinhibitor (Ro 41-5253), and then stimulated with ATRA for 24 h. The results show that the level of KLF4 was reduced when RARαwas inhibited. To further examine the role of RARαin ATRA-induced expression of KLF4, VSMCs were transfected with RARα-specific small interfering RNA (RARα-siRNA) to silence the expression of endogenous RARα. The expression of KLF4 and RARαwas detected by Western blot analysis. The results showed that the level of KLF4 was reduced in the cells transfected with RARα-siRNA when compared with those transfected with NS-siRNA, suggesting that RARαmediates the expression of KLF4 induced by ATRA.3 The effect of ATRA on the signaling activation in VSMCsTo test which signaling mediates ATRA-induced expression of KLF4, we treated VSMCs with ATRA (10μM) for the different times (0, 15, 30 and 60 min), and measured the effect of ATRA on the phosphorylation of ERK,p38,Akt andβ-catenin by Western blotting. The results showed that the phosphorylation of p38 increased in a time-dependent manner, and the phosphorylation of ERK decreased without affecting the levels of total ERK and total p38. However, the phosphorylation of Akt andβ-catenin was not changed after ATRA treatment. These results demonstrate that ATRA can inhibit ERK1/2 and stimulate p38 MAPK activation in VSMCs.4 RARαmediates ATRA-induced expression of KLF4 via ERK and p38 MAPK signalingsTo further investigate the role of RARαin ATRA-induced expression of KLF4, we treated VSMCs with RARαantagonist (Ro 41-5253) to block the endogenous RARαexpression. VSMCs were pretreated for 1 h with 20μM RARαinhibitor (Ro 41-5253), and then stimulated with 10μM ATRA for 1 h. The results showed that phospho-ERK level was increased and phospho-p38 level was decreased. Cells were transfected with RARα-siRNA to silence the expression of endogenous RARα. The levels of phospho-ERK and phospho-p38 were detected by Western blot analysis. ATRA increased phospho-ERK level but decreased phospho-p38 level in the cells transfected with RARα-siRNA when compared with those transfected with NS-siRNA. These results demonstrate that RARαmediates KLF4 expression induced by ATRA via ERK and p38 MAPK signalings.5 The effect of ERK and p38 MAPK signalings on ATRA-induced expression of KLF4To further determine whether ERK and p38 MAPK signalings mediate ATRA-induced expression of KLF4, we pretreated VSMCs with 20μM of the ERK inhibitor PD98059, 20μM of the p38 MAPK inhibitor SB203580, or 20μM of the Akt inhibitor LY294002 for 2 h before application of ATRA (10μM) for 24 h. Cells were collected and analyzed by Western blotting for KLF4 expression. The results showed that inhibiting p38 phosphorylation by SB203580 abrogated ATRA-induced KLF4 expression, inhibition of ERK by PD98059 enhanced ATRA-induced KLF4, the Akt inhibitor LY294002 had no effect on KLF4 expression induced by ATRA, suggesting that activation of p38 MAPK pathway is involved in ATRA-induced KLF4 expression. Cultured VSMCs were transfected with constitutively active ERK pCMV-MEKca for 24 h, and then stimulated with ATRA (10μM) for 1 h. The empty vector (pCMV) was used as a control. Transfection of VSMCs with constitutively active ERK pCMV-MEKca prevented the expression of KLF4 induced by ATRA. Taken together, these findings indicate that the ERK and p38 MAPK signalings play key roles in ATRA-induced KLF4 expression in VSMCs. The activation of p38 MAPK signaling and the inhibition of ERK signaling are responsible for the expression of KLF4 induced by ATRA.Conclusions:1 ATRA induces the expression of KLF4 and RARαin VSMCs.2 RARαmediates ATRA-induced expression of KLF4 via ERK and p38 MAPK signalings.3 The activation of p38 MAPK and the inhibition of ERK are responsible for the expression of KLF4 induced by ATRA.