Krüppel-like factor 4 regulates blood-tumor barrier permeability via ZO-1, occludin and claudin-5

Blood-tumor barrier (BTB) constitutes an efficient organization of tight junctions which significantly reduce permeability for chemotherapy drugs. Krüppel-like factor 4 (KLF4), a member of the Krüppel-like family, has been documented in endothelial cells and may serve as an essential regulator of endothelial barrier function. However, our knowledge about the expression and function of KLF4 in the endothelial cells of BTB still remains unclear. In this study, we sought to investigate the role of KLF4 in regulation of BTB function as well as the potential molecular mechanisms. Quantitative RT-PCR, Western blot, and immunofluorescence assays demonstrated that KLF4 was down-regulated in the glioma endothelial cells (GECs) which were obtained through endothelial cells co-cultured with glioma cells. Short hairpin RNA targeting KLF4 impaired the integrity of BTB detected by trans-endothelial electric resistance assay, and meanwhile reduced the expression of ZO-1, occludin and claudin-5, demonstrated by quantitative RT-PCR, Western blot, and immunofluorescence assays. Depletion of KLF4 increased BTB permeability to small molecules detected by permeability assays. Furthermore, luciferase assays and chromatin immunoprecipitation assays showed that KLF4 up-regulated the promoter activities and interacted with "CACCC" DNA sequence presented in the promoters of ZO-1, occludin, and claudin-5. GATA-1, GATA-6, Sp1, and Sp3 factors participated in KLF4 regulation of promoter activities through binding to the promoters of tight junctions related proteins. Collectively, our results indicated that KLF4 is a key transcriptional regulator of BTB function by regulating expressions of tight junction related proteins, which would draw growing attention to KLF4 as a potential target for glioma therapy.