Role And Mechanism Of PPAR-γ Agonist Pioglitazone In Regulating KLF4Expression In Vascular Smooth Muscle Cells

Objective: Krüppel-like factor4(KLF4), a zinc-finger containingtranscription factor, regulates a variety of cellular processes, including cellproliferation, differentiation, apoptosis, as well as maintenance of tissuehomeostasis by controlling the expression of a large number of genes withGC/GT-rich promoters. In vascular smooth muscle cell (VSMC), KLF4playsa key role in cell phenotypic switching by regulating a variety of VSMCmarker gene and cell-cycle-regulatory gene expression.The peroxisome proliferator activated receptors (PPARs) are a subgroupof ligand-activated nuclear receptor superfamily and three isoforms have beenidentified: PPARα, PPARβ, and PPARγ. The most widely studied form amongthe three known forms of PPARs is PPARγ, which is expressed in a widevariety of cell types, including adiposities, macrophages, endothelial cell, andVSMC. PPARγ can inhibit cell proliferation and promote cell differentiation.In addition, evidence has demonstrated potential anti-inflammatory andanti-atherogenic properties of PPARγ in monocyte/macrophages, endothelialcells, and VSMC. Pioglitazone, a PPARγ agonist, has been widely used inclinical treatment of atherosclerosis, myocardial infarction and othercardiovascular diseases.Both KLF4and PPARγ are involved in VSMC proliferation anddifferentiation. Our previous study also demonstrated that KLF4and PPARγcould inhibit cell proliferation synergistically. However, whether KLF4isregulated by PPARγ in VSMC is unknown. In the present study, we sought toelucidate whether and how PPARγ regulates KLF4expression in VSMC.Methods: VSMC was isolated from the thoracic aorta of Sprague-Dawle-y rats. The expression of KLF4was detected by Western blotting and real-timePCR analysis. siRNA targeting PPARγ was used to knock down endogenous PPARγ expression. Reporter gene analysis was used to detect the promoteractivities of KLF4. Confocal microscopy was used to detect the expression ofKLF4.Results:1PPAR-γ agonist Pioglitazone up-regulates KLF4protein expression inVSMCsTo examine whether PPAR-γ influences KLF4expression, VSMC wastreated with Pioglitazone and then western blot analysis was performed. Wefound that Pioglitazone significantly induced KLF4expression in a time-anddose-dependent manner. In addition, laser scanning confocal microscope wasused to detect KLF4expression and similar results were obtained.2The induction of KLF4expression by PPAR-γ agonist is PPAR-γdependentTo clarify whether Pioglitazone-induced KLF4expression isPPAR-γ-dependent, VSMC transfected with PPAR-γ-specific siRNA (siPPAR-γ) was stimulated with or without Pioglitazone and western blotanalysis was performed to detect KLF4expression. We found that in cellswhere PPAR-γ was knocked down by transfecting si-PPAR-γ, KLF4expression was reduced and no longer affected by Pioglitazone. This resultindicated that Pioglitazone induced KLF4expression through aPPAR-γ-dependent manner.3PPAR-γ agonist Pioglitazone does not affect KLF4gene transcriptionNext, we sought to determine whether Pioglitazone increases thetranscription of KLF4. VSMC was stimulated with Pioglitazone and real-timePCR analysis was performed. Interestingly, we found that the expression ofKLF4mRNA was not affected by Pioglitazone treatment. Furthermore, weperformed luciferase reporter gene assays and found that Pioglitazone did notaffect KLF4gene promoter activity in NIH3T3cells transfected without orwith PPAR-γ expression plasmids. These results demonstrated that PPAR-γdid not regulate KLF4gene transcription. 4PPAR-γ agonist Pioglitazone enhances KLF4protein stabilityThe fact that Pioglitazone can induce KLF4protein expression, but notinfluence its mRNA expression indicated that Pioglitazone might enhanceKLF4protein stability. We performed experiment using CHX to block de novoprotein translation to study the expression of KLF4in VSMC. As a result,treatment of VSMC with CHX significantly reduced KLF4expression.However, Pioglitazone-induced KLF4expression was not influenced by CHXtreatment, suggesting that Pioglitazone increases KLF4protein level throughenhancing its protein stability.5PPAR-γ agonist Pioglitazone stabilizes KLF4protein via activation ofAkt signalingTo test which signal pathway mediates the KLF4expression in responseto Pioglitazone, we measured the effect of Pioglitazone on the phosphorylationof ERK, p38, and Akt by western blotting. The result showed that the levels ofp-p38and p-Akt were increased30min after Pioglitazone treatment, but thelevel of p-ERK had no change. We pretreated VSMC with signal pathwayinhibitors for2h before exposure to Pioglitazone. Pharmacological inhibitionof Akt blocked Pioglitazone-induced expression of KLF4, whereas inhibitionof ERK and p38had no effect on the expression of KLF4induced byPioglitazone.Conclusions:1PPAR-γ agonist Pioglitazone up-regulates KLF4protein expression inVSMCs.2The induction of KLF4expression by PPAR-γ agonist is PPAR-γdependent.3PPAR-γ agonist Pioglitazone does not affect KLF4gene transcription.4PPAR-γ agonist Pioglitazone enhances KLF4protein stability.5PPAR-γ agonist Pioglitazone stabilizes KLF4! protein via activation ofAkt signaling.