Localization And Function Of KLF4in Cytoplasm Of Vascular Smooth Muscle Cells

Objective: The phenotypic modulation of vascular smooth muscle cells(VSMCs) is known to be critical in blood vessel formation duringembryogenesis and in pathological states such as atherogenesis, restenosis,and hypertension. The Krüppel-like factors (KLFs) are DNA-bindingtranscriptional regulators that regulate a diverse array of cellular processes,including development, differentiation, proliferation, and apoptosis. As one ofthe KLFs, KLF4mainly distributes in the nucleus and induces VSMCdifferentiation by activating VSMC differentiation markers, including SMα-actin and SM22α. Many chemokines, such as PDGF-BB, can regulateVSMC phenotype by inducing the expression of KLF4. The previous studiesabout KLF4functions mainly focused on its role as a transcription factor, itsfunctions in the cytoplasm are still unknown. In this study, we observed thedistribution of KLF4in the cytoplasm and investigated its functions regulatingthe cytoskeleton remodeling.Methods: Immunofluorescence staining analysis and Western blotanalysis were done to detect the interaction between KLF4and cytoskeleton.Confocal microscopy was used to detect KLF4distribution in the nucleus andcytoplasm. GST pull-down analysis was used to detect the interaction betweenKLF4and actin. Reporter gene analysis was used to detect the activation ofthe SM22α promoter by KLF4.Results:1CRM1mediated KLF4nuclear exportVSMCs were pretreated with LMB (20ng/mL) for8h and then treatedwith PDGF-BB (20ng/mL) for2h, cytoplasmic and nuclear proteins wereextracted for Western blot analysis. The results showed that compared with thecontrol group, the LMB inhibited KLF4nuclear export induced by PDGF-BB. Immunofluorescence staining also showed that PDGF-BB increaseddistribution of KLF4in the cytoplasm. After LMB pretreatment, KLF4distribution in the cytoplasm could almost be detected, suggesting thatPDGF-BB induces KLF4nuclear export through the CRM1-mediatedpathway.2KLF4participated in cytoskeleton organization by stabilizingcytoskeletonVSMCs were transfected with GFP-KLF4expression vector, and thentreated with PDGF-BB, cytochalasin (CB), and cytoskeleton polymerizationagent (JPK). Immunofluorescence analysis showed that actin filament wasdistributed as thick and long actin bundles in VSMCs. After treated withPDGF-BB treatment, stress fibers significantly reduced. After CB treatment,the stress fibers disappeared, the microfilament depolymerizated. On thecontrast, JPK could make the stress fibers stronger and thicker than the control.Compared with the control, the stress fibers did not decrease obviously inKLF4-overexpressioned VSMCs after PDGF-BB treatment; the stress fibersdepolymerizated partly in KLF4-overexpressioned VSMCs upon CB treatment,suggesting that KLF4participates in cytoskeleton organization by stabilizingthe cytoskeleton.3KLF4interacted with actinThe interaction between KLF4and actin was detected byimmunofluorescence analysis after cells were treated with PDGF-BB, CB, andJPK for2h. After PDGF-BB treatment, KLF4transferred from the nucleus tothe cytoplasm, and co-localized with α-actin. CB led to depolymerization ofmyofilament, KLF4labeled by TRITC in the cytoplasm had no fluorescentoverlap with actin. JPK made filaments polymerized into bundles and KLF4colocalized with α-actin. These results indicated that KLF4associates withα-actin in the cytoplasm and participates in cytoskeleton organization.4The amino acid residues117to181in KLF4molecule were necessaryfor the interaction of KLF4with actin In order to further identify the structural basis of KLF4interaction withactin, KLF4cDNA was truncated, and expression plasmids for KLF4wereconstructed and transformed into bacteria. GST-KLF4-2, GST-KLF4-3,GST-KLF4-4, GST-KLF4-full-length fusion proteins with different molecularsizes were obtained through GST affinity chromatography. GST-Sepharose4Bcombined with five kinds of fusion protein was incubated with VSMC lysatesand precipitated by centrifugation Western blot analysis showed thatGST-KLF4-3interacted with actin. These results confirmed that the aminoacid residues117to181were the structural domain of KLF4interaction withactin.5Phosphorylation and SUMOylation of KLF4affected the cytoskeletonremodelingVSMCs were transfected with expression vectors for KLF4phosphorylation-deficient mutants and sumoylation-deficient mutants and thentreated with PDGF-BB. Immunofluorescence staining was used to obsere theeffect of different mutants on the cytoskeleton remodeling in VSMCs. Theresults showed that the cytoskeleton of all transfected groups had to significantdifference in quiescent VSMCs. However, upon PDGF-BB treatment,phosphorylation-deficient mutants made stress fiber gathered to the marginalarea of the cells. At the same time, the cell morphology was changed fromlong spindle shape to polygonal shape. After SUMOylation-deficient mutantswere transfected, stress fibers were still thickened, but arrangement wasapparently disordered. These results prompted us to believe thatPDGF-BB-induced KLF4phosphorylation and SUMOylation were involvedin the regulation of cytoskeleton remodeling of VSMCs.Conclusions:1CRM1mediated KLF4nuclear export2KLF4participated in the cytoskeleton organization by stabilizingcytoskeleton.3KLF4interacted with actin. 4The amino acid residues117to181in KLF4molecule were necessary forits interaction with actin.5Phosphorylation and SUMOylation of KLF4induced by PDGF-BB thecytoskeleton remodeling of VSMCs.