Molecular Mechanism Of Am80-reduced Interaction Of KLF5 With RARα In Vascular Smooth Muscle Cells

Objective: Proliferation and differentiation of vascular smooth muscle cells (VSMC) are governed by the activity of a regulator network, in which functional interactions between pro-proliferation factors, such as Krüppel-like factor 5 (KLF5) and pro-differentiation factors, such as retinoic acid receptor (RAR) are involved in regulating cell growth, proliferation, differentiation, and apoptosis. It has been identified that KLF5 has growth-promoting functions in a variety of cell types through its activation of key cell-cycle-promoting genes. KLF5 plays a critical role in regulating cardiovascular remodeling through activating many genes inducible during cardiovascular remodeling. RAR is an important nuclear receptor and plays a key role in inducing cell differentiation through interacting with its ligands, subsequently activating or suppressing a variety of gene expression. A total of 3 RAR isoforms have been identified (designated RARα, RARβand RARγ). Among them, RARαplays a critical role in mediating the effects of retinoid on cell differentiation and proliferation inhibition in VSMC.Nagai proved that Am80, a synthetic RARα-specific agonist, reduced in-stent neointima formation via inhibiting the interaction between KLF5 and RARα. However, the molecular mechanism of this inhibitory effect by Am80 is unclear. In the present studies, we analyzed the mechanism by which Am80 inhibits the interaction between KLF5 and RARα.Methods: VSMC was isolated from the thoracic aorta of Sprague-Dawley rats. In all experiments, only cell passages 3~5 were used. Co-immunoprecipitation assay was done to examine the interaction between KLF5 and RARα. The expression of KLF5 and RARαand phosphorylation of various signaling molecules were examined by Western blotting.Results:1 Am80 induces RARαexpression and inhibits KLF5 expression in VSMCExposure of VSMC to Am80 (2μM) for 12 h significantly induced RARαexpression, and the level of RARαincreased by 2-fold at 24 h. In contrast, the expression of KLF5 was inhibited by Am80 in dose- and time-dependent manners.2 Am80 inhibits the interaction between KLF5 and RARαTreating cells with Am80 for 0.5 and 1 h significantly reduced RARαlevels in precipitates pulled down with anti-KLF5 antibody. Likewise, Am80 also reduced levels of co-immunoprecipitated KLF5 pulled down with anti-RARαantibody.The injured arteries caused by balloon were harvested at 3 and 14 days following injury, and protein extracts were prepared and subjected to Co-IP assay. The Am80-administered group showed significantly less interaction between KLF5 and RARα than the control and model groups. Apparently, Am80 inhibits the association of KLF5 with RARαat the cellular and tissue levels.3 Am80 inhibits the interaction between KLF5 and RARαby inducing KLF5 dephosphorylationAm80 stimulation rapidly induced dephosphorylation of KLF5 within 0.5 h. KLF5 phosphorylation reached a minimum between 0.5 h and 1 h and returned to nearly basal levels after 2 h.Am80 time-dependently increased phospho-Akt level but decreased phospho-p38 level without affecting the levels of total Akt and total p38. These results demonstrate that the PI3K/Akt and p38 MAPK signaling play a key role in Am80-induced dephosphorylation of KLF5 in VSMC.4 Akt inhibitor LY294002 prevents Am80-induced dephosphorylation of KLF5Since Am80 treatment leads to Akt phosphorylation, we decided to test whether activated Akt is involved in Am80-induced KLF5 dephosphorylation that subsequently leads to the suppression of the interaction between KLF5 and RARαin VSMC. The effects of Akt inhibitor LY294002 on Am80-induced KLF5 dephosphorylation and the interaction between KLF5 and RARαin VSMC were detected. LY294002 almost completely blocked Am80-induced Akt phosphorylation and Am80-induced KLF5 dephosphorylation. Am80-induced suppression of interaction between KLF5 and RARαwas reversed by treatment with LY294002, suggesting that activation of PI3K/Akt pathway is involved in Am80-induced KLF5 dephosphorylation.5 Am80 inhibits KLF5 phosphorylation via p38 pathwayInfection of VSMC with constitutively active p38 kinase MKK6b substantially prevented the KLF5 dephosphorylation induced by Am80, strongly suggesting that the activation of p38 MAPK pathway stimulates KLF5 phosphorylation in VSMC. VSMC was pretreated for 2 h with p38 kinase inhibitor SB203580 and then stimulated with Am80 for 1 h. SB203580 further attenuated the interaction between KLF5 and RARα. Taken together, these findings clearly indicate that Am80 inhibits KLF5 phosphorylation and its interaction with RARαby blocking activation of p38 pathway.6 Activation of the PI3K/Akt pathway is involved in Am80-triggered p38 inhibitionInhibiting Akt phosphorylation by LY294002 abrogated Am80-induced inhibition of p38 phosphorylation. In contrast, inhibition of p38 by the p38 kinase inhibitor SB203580 had no effect on Akt phosphorylation induced by Am80. These data suggest that the activation of PI3K/Akt signaling is responsible for the inhibition of the p38 MAPK signaling by Am80, and that p38 MAPK is a downstream target of PI3K/Akt pathway in response to Am80 in VSMC.Conclusions:1 Am80 induces RARαexpression and inhibits KLF5 expression in VSMC.2 Am80 inhibits the interaction between KLF5 and RARα. 3 Am80 inhibits the interaction of KLF5 with RARαvia inducing KLF5 dephosphorylation mediated by the PI3K/Akt signaling.4 There are crosstalks between the PI3K/Akt pathway and the p38 MAPK pathway, and p38 MAPK is a downstream target of PI3K/Akt pathway in response to Am80 in VSMC.