The Role And Mechanism Of KLF4 In ATRA-Induced VSMC Differentiation

In response to pathogenic vascular injuries, such as atherosclerosis and restenosis after angioplasty, vascular smooth muscle cells (VSMCs) can revert from differentiated phenotype to dedifferentiated phenotype, and then proliferate and migrate toward intima, subsequently, result in stenosis. Studies have shown that retinoids inhibit proliferation and induce differentiation in VSMC, however the exact mechanism is not clear. Therefore, exploring the regulatory mechanisms of VSMC phenotype is helpful for therapy of these remodeling cardiovascular diseases.All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to regulate growth and induce differentiation in malignant cells. The gut enriched krüpple-like factor (GKLF/KLF4) is a member of KLF family which plays important roles in cell growth, proliferation, differentiation, and embryogenesis as well as carcinogenesis. Studies show that KLF4 is implicated in development of pathological vascular processes. It is known that ATRA inhibits VSMC proliferation and induces VSMC differentiation, and that KLF4 is involved in VSMC phenotypic switching.To elucidate the molecular mechanisms of VSMC phenotype modulation induced by ATRA, we detected the effects of ATRA on VSMC proliferation, migration, contractibility and the expression of marker genes. We further investigated the effects of KLF4 overexpression or knockdown on the phenotype modulation in VSMC. Based upon KLF4 close relation with KLF5, we also detected the effects of KLF4 and/or KLF5 overexpression on VSMC phenotype modulation by infecting VSMC with KLF4 or KLF5 adenovirus expression vectors.1 The effects of ATRA on VSMC differentiationTo profile the effects of ATRA on VSMC phenotype modulation, we treated VSMC with ATRA at different concentrations (5,10,20μmol/L) for 48 h or 10μmol/L for different times (24,48,72 h), and then detected the changes of proliferation, migration, contractibility and the expression of marker genes. The results were as follows:1.1 The effects of ATRA on VSMC morphology and biological behaviours The results of MTT and migration assays showed that treatment of VSMCs with ATRA resulted in a significant reduction of cell proliferation and migration in a dose-dependent (5,10,20μmol/L) and time-dependent (24,48,72 h) manner. A significant inhibiting effect was observed by treating VSMCs with 10μmol/L of ATRA for 48 h.VSMCs treated with 10μmol/L ATRA for 48 h changed from spindle-shaped to highly elongated cells, resembling the organization of VSMCs in blood vessels in vivo. In addition, compared with control cells, VSMCs treated with ATRA produced significant contraction elicited by acetylcholine (Ach) (100μmol/L).1.2 The effects of ATRA on VSMC marker gene expressionSM22αand SMα-actin (α-SMA) which are highly expressed in differentiated VSMCs are the differentiated marker genes, while embryonic vascular smooth muscle myosin heavy chain-B (SMemb) is the VSMC-dedifferentiated marker. The results of RT-PCR, Western blot and immunocytochemistry assays showed that treatment with a range of ATRA concentrations from 0 to 20μmol/L for 72 h resulted in increased level of SM22αandα-SMA in a dose-dependent manner, and decreased level of SMemb in VSMCs. In addition, when VSMCs were treated with 10μmol/L of ATRA from 0 to 72 h, the level of SM22αandα-SMA was increased in a time-dependent manner. At the same time, the level of SMemb was decreased, in contrast with SM22αandα-SMA.2 The role of KLF4 in VSMC differentiation induced by ATRAMany studies have revealed that KLF4 plays important roles in cell growth, proliferation and differentiation in many cell lines. However, whether KLF4 is involved in VSMC differentiation induced by ATRA is not clear. In this section, we detected the KLF4 level in the neointima after balloon injury, and the effects of KLF4 overexpression or knockdown on VSMC phenotype and marker gene expression.2.1 The change of KLF4 expression in the neointima after balloon injuryThe result of RT-PCR and Western blot assays showed that KLF4 was up-regulated drastically in neointima after balloon injury for 3 days.2.2 The effect of ATRA on the expression of KLF4 in cultured VSMCsThe result of RT-PCR and Western blot assays showed that KLF4 was also up-regulated markedly by ATRA stimulation followed the increase of concentration and time. A significant change was observed at concentration of 10μmol/L for 48 h.2.3 The effects of KLF4 overexpression on VSMC marker gene expressionWe detected high expression level of KLF4 in VSMCs infected with KLF4 adenovirus vector Ad-KLF4 for 48 h by Western blot assay. The VSMCs infected with Ad-KLF4 changed to elongated cells, and produced significant contraction elicited by Ach, compared with control cells infected with empty vector Ad.At same time, the results of RT-PCR, Western blot and immunocytochemistry assays showed that the VSMC differentiation marker genes SM22αandα-SMA were up-regulated, while the dedifferentiation maker gene SMemb was down-regulated in VSMCs infected with Ad-KLF4.The promoter region ofα-SMA and SM22αcontains KLF4 binding sites, we proved that KLF4 bound to both of them within intact chromatin by ChIP assays. To assess whether KLF4 induces the activity of the SM22αpromoter, we performed a luciferase reporter gene assay. The result showed that KLF4 significantly increased the SM22αpromoter activity.2.4 The effects of KLF4 knockdown on VSMC phenotype and the expression of differentiation and dedifferentiation markersThe results of RT-PCR and Western blot assays showed that KLF4 expression was suppressed by over 70% in VSMCs transfected with KLF4-specific siRNA, and that KLF4 knockdown attenuated the inducing effects of ATRA on SM22αandα-SMA expression and the inhibitory effects on SMemb expression. Even by ATRA stimulation, VSMCs transfected with KLF4-siRNA presented dedifferentiation morphology character, and the contractile response elicited by Ach weakened significantly.3 The role of KLF4 in VSMC migrationVSMC proliferation and migration to intima play a key role in restenosis. Studies show that VSMC migration is closely related with the degradation of extracellular matrix (ECM), while the matrix metalloproteases (MMPs) are the main proteases which degradate ECM. In artery, MMP-2 and MMP-9 are the main MMPs. In this part, we investigated the effects of KLF4 overexpression or knockdown on VSMC migration and the expression of MMP-2 and MMP-9.3.1 KLF4 plays a role in VSMC migrationWe found that ATRA inhibited VSMC migration. In order to determine whether KLF4 is involved in migration, we knockdowned KLF4 in VSMCs by KLF4-specific siRNA. The inhibitory effect of ATRA on VSMC migration was abolished by KLF4 siRNA, and KLF4 overexpression suppressed VSMC migration.3.2 The effects of ATRA on MMP-9 and MMP-2 expression and activities in VSMCsMMP-2 and MMP-9 are the key proteases for ECM degradation. The results of Western blot and RT-PCR showed that ATRA decreased the expression of them. The zymography showed that ATRA could decrease the activities of MMP-2 and MMP-9.3.3 The effects of KLF4 on MMP-9 and MMP-2 expression and activities in VSMCsThe results of Western blot, RT-PCR and zymography showed that KLF4 overexpression decreased the expression and activity of MMP-2 and MMP-9 in VSMCs. Similarly, the inhibitory effect of ATRA on MMP-2 and MMP-9 expression and activities was abrogated by knockdown of KLF4 in VSMCs. These results suggested that ATRA-inhibiting VSMC migration was due to the induction of KLF4, and the subsequent suppression of MMP-2 and MMP-9.4 The role of KLF4 in VSMC proliferationIn response to stimulation of growth factors, differentiated VSMCs enter into cell cycle and proliferate by activating cyclin dependent kinase (CDKs). CDKs play a central role in cell cycle control, while P53 is a key mediator in G1 arrest. Therefore, in this part, we investigated effects of KLF4 on VSMC proliferation and the expression of P53 and CDK4.4.1 The effect of KLF4 on VSMC proliferationCell counting and MTT assay showed that ATRA could inhibit VSMC proliferation. KLF4 knockdown in VSMCs abolished the inhibition of ATRA. On the contrary, KLF4 overexpression suppressed VSMC proliferation dramatically.4.2 KLF4 plays a key role in G1 arrestG1-S switch is a key point in cell proliferation, so we examined the alteration of VSMC cell cycle by flow cytometry. In response to ATRA stimulation, the number of VSMCs distributed in G0/G1 phase was increased, and in S phase decreased dramatically. The similar result was obtained in VSMCs infected with Ad-KLF4. KLF4 knockdown abolished the cell cycle inhibition by ATRA. The result suggests that induction of KLF4 is a main mechanism in VSMC cell cycle arrest induced by ATRA.4.3 P53 and CDK4 are involved in inhibition of VSMC proliferation by KLF4The results of Western blot and RT-PCR assays showed that ATRA could up-regulate the expression of P53 but down-regulate the expression of CDK4. The similar results were obtained in VSMCs infected with Ad-KLF4. While KLF4 knockdown attenuated the inducing effects of ATRA on P53 expression and the inhibitory effects on CDK4 expression.5 The interaction of KLF4 with KLF5 in VSMC phenotype modulationKLF4 and KLF5 are two closely related members of KLFs. They have high homology and similar tissue patterns of expression, and bind to a similar DNA sequence, but exert very different effects on gene transcription and cell proliferation. In general, KLF4 is an inhibitor of cell growth, and KLF5 stimulates proliferation. Both of them contribute to the balance of cell proliferation and differentiation by interaction. In this part, we investigated the changes of VSMC phenotype and biological behaviours after KLF4 and/or KLF5 overexpression by infecting VSMCs with Ad-KLF4 and /or Ad-KLF5.5.1 The effects of KLF4 and/or KLF5 overexpression on VSMC morphology and biological behavioursWe first verified, by Western blot assays, that the adenovirus expressing vectors Ad-KLF4 and Ad-KLF5 expressed stably products in VSMCs, respectively.VSMCs infected with Ad-KLF5 showed dedifferentiated state, and lost contraction after Ach stimulation. VSMCs co-infected with Ad-KLF4 and Ad-KLF5 did not change as above. VSMCs infected with Ad-KLF4 alone maintained differentiated state, changed to elongated cells, and produced significant contraction by Ach stimulation.5.2 The effect of KLF4 and/or KLF5 overexpression on MMP-9 and MMP-2 activities in VSMCThe result of zymography showed that KLF5 overexpression could increase the activities of MMP-2 and MMP-9, while co-overexpression with KLF4 decreased the gelatinase activities.5.3 The effects of KLF4 and/or KLF5 overexpression on the expression of VSMC markersThe results of Western blot and RT-PCR assays showed that the expression of VSMC differentiating marker genes SM22αandα-SMA was down-regulated by KLF5 overexpression, and retrieved by co-overexpression of KLF4 with KLF5, while the expression of dedifferentiation marker genes SMemb and PCNA was up-regulated by KLF5 overexpression, and supressed by co-overexpression of KLF4 with KLF5.Conclusion:1 ATRA promotes VSMC switching from dedifferentiated phenotype to differentiated phenotype, by up-regulating differentiation marker SM22α andα-SMA as well as down-regulating dedifferentiation marker SMemb, which was characterized as increased contraction in response to Ach stimulation and decreased proliferation and migration.2 KLF4 plays a key role in ATRA-induced VSMC differentiation by up-regulating differentiation marker genes and down-regulating dedifferentiation marker genes.3 ATRA inhibits VSMC migration via induction of KLF4 and the subsequent suppression of MMP-2 and MMP-9.4 Induction of KLF4 and P53 expression is an important mechanism by which ATRA inhibits VSMC proliferation.5 KLF4 antagonises KLF5 action which promotes VSMC proliferation, and induces VSMC differentiation by activating the expression of differentiation marker genes.