ATRA And TGF-β1Cooperatively Regulate KLF4Expression And Differentiation Of Vascular Smooth Muscle Cells

Objective: Vascular smooth muscle cell (VSMC) phenotypic switch isan important base of cardiovascular diseases, such as hypertension,atherosclerosis and restenosis. Krüppel-like factor4(KLF4/GKLF) is a zincfinger transcription factor, which plays a critical role in processes ofembryogenesis, proliferation and differentiation, cell reprogramming andcancer. In VSMCs, KLF4suppresses proliferation and promotesdifferentiation. All-trans retinoic acid (ATRA), one of the derivatives ofvitamin A, plays an important role in proliferation, differentiation, migrationand apoptosis in a variety of cell types by binding to retinoic acid receptors(RARs) and retinoid X receptors (RXRs). Transforming growth factor-β(TGF-β) binds to TGF-β type I receptor (TβR-I) or TGF-β type II receptor(TβR-II) to trigger signaling pathways and regulate proliferation,differentiation and apoptosis. In VSMCs, both ATRA and TGF-β1induce theexpression of KLF4and promote differentiation. However, it is not clear aboutthe relationship between ATRA, TGF-β1and KLF4in VSMC differentiation.Here, we explored the role of ATRA and TGF-β1on KLF4expression andinvestigated the molecular mechanisms whereby ATRA and TGF-β regulatethe expression of KLF4and VSMC differentiation.Methods: VSMCs were isolated from the thoracic aorta ofSprague-Dawley rats. In all experiments, only cells of passages3~5were used.The expressions of KLF4, SM22α, PCNA, cyclin D1and p21were examinedby Western blotting. Cell proliferation was examined by cell counting assayand bromodeoxyuridine (BrdU) incorporation assay. Hematoxylin-eosin(HE)and TRITC-phalloidin staining were used to detect the changing of cellmorphology and stress fibers. Wounding cell migration assay and Boydenchamber assay were used to evaluate the migration activity of VSMCs. Results：1ATRA and TGF-β1coordinately regulate the expression of KLF4inVSMCsVSMCs were treated with ATRA (10μmol/L) or9cRA (1μmol/L) for2h,and then stimulated with TGF-β1(2ng/mL) for24h. Total cell lysates werecollected for Western blot analysis. The results showed that the protein levelof KLF4significantly increased after ATRA or TGF-β1stimulation.Combination of ATRA with TGF-β1led to the higher increase in KLF4protein level, which indicates that ATRA and TGF-β1have a synergisticeffect on KLF4expression. However, there was not significant effect on KLF4expression when VSMCs were treated with9cRA alone or the combination of9cRA with TGF-β1. These results suggested that ATRA and TGF-β1cooperatively activate KLF4gene expression.2TGF-β1-induced KLF4expression can be blocked by inhibition ofretinoic acid receptor RARα activationRetinoid receptors include RXRs and RARs, both of which have α, β andγ forms. RARα plays an important role in VSMC differentiation. We haveindicated that ATRA and TGF-β1cooperatively activated KLF4geneexpression. In order to identify the link of ATRA and TGF-β1, we pretreatedVSMCs with RARα-specific antagonist (Ro41-5253) for2h to inhibit theactivity of RARα and then detected the effect of TGF-β1on KLF4expression.The results showed that TGF-β1treatment increased KLF4protein level,which was blocked when VSMCs were pretreated with RARα antagonist Ro41-5253. These results suggested that TGF-β1-induced KLF4expressiondepends on the activation of retinoic acid signaling.3ATRA and TGF-β1cooperatively inhibit VSMC proliferationTo further examine the biological effects of synergistic activation ofKLF4gene expression by ATRA and TGF-β1, we detected the expression ofproliferation-related gene PCNA and cyclin D1and p21. The results showedthat ATRA and TGF-β1inhibited the expression of PCNA and cyclin D1butincreased p21expression after ATRA and TGF-β1treatment. When VSMCs were pretreated with RARα antagonist Ro41-5253, the inhibitory effect ofTGF-β1on the expression of PCNA was blocked to some extent. Cellcounting assay and BrdU incorporation assay showed that cell number and therate of BrdU incorporation significantly reduced compared with control group,indicating the synergistic inhibition of ATRA and TGF-β1on VSMCproliferation.4ATRA and TGF-β1cooperatively induce VSMC differentiationTo test the effect of ATRA and TGF-β1on VSMC differentiation andbiological behaviors, we detected the protein level of VSMC differentiationmarker SM22α and the changes of VSMC morphology, cytoskeleton andmigration activity. The results showed that both ATRA and TGF-β1couldincrease SM22α expression. Combination of both stimulators led to furtherincrease in the level of SM22α than either one alone. TGF-β1-induced SM22αexpression could be blocked by RARα antagonist (Ro41-5253).Hematoxylin-eosin (HE) and TRITC-phalloidin staining indicated that ATRAand TGF-β1stimulation could alter cell morphology and stress fiberorganization. Wounding cell migration assay and Boyden chamber assayshowed that combination of both stimulators significantly inhibited VSMCmigration. These results suggest that ATRA and TGF-β1inhibit VSMCmigration by inducting the differentiation of VSMCs.Conclusions：1ATRA and TGF-β1coordinately regulate the expression of KLF4inVSMCs.2TGF-β1-induced KLF4expression can be blocked by inhibition of retinoicacid receptor RARα activation.3ATRA and TGF-β1cooperatively inhibit VSMC proliferation.4ATRA and TGF-β1cooperatively induce VSMC differentiation.