KLF4 Participates In Remoding Of The Actin Cytoskeleton In Vascular Smooth Muscle Cells

Objective: The cytoskeleton of eukaryotic cells is a network structure composed of many kinds of structural and contractile proteins and plays an important role in maintaining cell shape and contraction. There are three types of cytoskeleton: microfilaments of actin, microtubules and intermediate filaments 0f various molecules. The microfilaments are a complex, plastic, dynamic network structure, which is associated with the shape and function changes of vascular smooth muscle cells (VSMCs). Many proteins binding to actin play a regulatory role in microfilament remodeling progress. The gut enriched krüppel-like factor (GKLF/KLF4) is a member of KLF family transcription factors which plays important roles in cell growth, proliferation, differentiation, and embryogenesis as well as carcinogenesis. As a transcription factor of eukaryotic organism, KLF4 distributes mainly in the nuclear, which is one of the important inducing factors for reprogramming differentiated somatic cells into pluripotent stem cells. Recently, the studies about KLF4 mainly focused on its functions in the nucleus, its functions in the cytoplasm have not been well characterized. In the present study, we observed the interaction between KLF4 and the cytoskeleton and investigated its molecular mechanism where by KLF4 regulates the cytoskeleton remodeling.Methods: VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats. In all experiments, only cell passages 3~5 were used. Expression and localization of KLF4 in VSMCs were determined by cell immunofluorescence analysis; Co-immunoprecipitation assay was done to examine the interaction between KLF4 and SMα-actin; Immunofluorescence and confocal scanning microscopy were done to examine the interaction between KLF4 and the cytoskeleton. TRITC-phalloidin was used to detect the remodeling of the cytoskeleton.Results:1 PDGF-BB promotes the nuclear export of KLF4Cell immunofluorescence analysis indicated that KLF4 was located mainly in the nucleus in quiescent VSMCs, but location in the cytoplasm was increased in VSMCs treated with PDGF-BB for 30 min to 2 h. Western blot analysis also showed that PDGF-BB increased the distribution of KLF4 in the cytoplasm of VSMCs with a time-dependent manner. These results suggest that PDGF-BB promotes the nuclear export of KLF4.2 KLF4 colocalizes withα-actin in the cytoplasmIn quiescent VSMCs, KLF4 tagged by FITC was located mainly in the nucleus, and there was no fluorescence overlapping withα-actin tagged by TRITC. After VSMCs were treated with PDGF-BB for 60 min, KLF4 transfered from the nucleus to the cytoplasm, and colocalized withα-actin tagged by TRITC, represented as yellow fluorescence. Co-immunoprecipitation analysis showed that immuno precipitates pulled down with anti-KLF4 antibody could delectedα-actin via Western blotting using anti-actin antibody. Ater VSMCs were treated with PDGF-BB,α-actin in the precipitates increased obviously. These results suggest that KLF4 associates withα-actin in the cytoplasm.3 KLF4 participates in remodeling of the cytoskeletonWestern blot analysis showed that JPK could increase the distribution of KLF4 in the cytoplasm and CB could dscrease the distribution of KLF4 in the cytoplasm,. suggesting that, the subcellular localization of KLF4 could be affected by the remodeling of the cytoskeleton.NIH3T3 cells were transfected with GFP-KLF4 expression vector, and then treated with PDGF-BB, CB, PMA and JPK. Cell immunofluorescence analysis indicated that actin filament stress fibers were distributed as thick and long actin bundles in NIH3T3 cells in the absexce of stimulation . After cells were treated with PDGF-BB, the stress fibers decreased; CB could make the stress fiber disappearsd and the microfilament depolymerizated; PMA could couse the stress fibers disappeared and the microfilament depolymerizated; PMA could make the stress fibers shaped as podosome body; JPK could make the stress fibers stronger. Compared with control groups, the stress fibers did not decrease not obviously in NIH3T3 cells transferred with KLF4 and treated with PDGF-BB; the stress fibers depolymerizated only partly in NIH3T3 cells transferred with KLF4 and treated with CB ; the podosome body decreased obviously in NIH3T3 cells transferred with KLF4 and treated with PMA. These results suggest that KLF4 participates in the remodeling of the cytoskeleton4 KLF4 regulates the remodeling of the cytoskeleton which m through the interaction with hhLIMco-immunoprecipitation assay showed, that hhLIM could be detected in the immunoprecipitates pulled down with anti-KLF4 antibody, and that PDGF-BB increased the interaction between KLF4 and hhLIM. These results suggest that KLF4 promotes the remodeling of the cytoskeleton mediated by PDGF-BB through the interaction with hhLIMConclusions:1 PDGF-Bbpromotes the nuclear export of KLF4, and KLF4 colocalizes with the cytoskeleton in the cytoplasm.2 KLF4 stimulates the remodeling of the cytoskeleton mediated by PDGF-BB through the interaction with hhLIM.