Functional characterization of KLF10 signaling-modulated gene, COX-1, in endothelial cells

• Main Authors: DianeYang, 楊皓安
• Other Authors: Jiann-Ping Wang
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/74650266531941295571

碩士 === 國立成功大學 === 生命科學系碩博士班 === 100 === TGF-beta is a vital cytokine known to participate in vascular formation and angiogenesis processes by modulating its several downstream factors. One of the TGF-beta inducible gene, Krüppel-like factor 10 (KLF10), is a zinc-finger transcription factors which play key regulatory roles in cellular growth, development, differentiation etc. Recent study has shown that KLF10 is involved in the process of angiogenesis by acting as a key transcriptional regulator of TGF-beta1 in pro-angiogenic cells (PAC) differentiation and function. KLF10–/– mice also displayed impaired blood flow recovery after hindlimb ischemia. However, the mechanism of KLF10 induced angiogenesis is still not well understood. From our ChIP-chip result, which have been adopt to elucidate the novel target genes and signaling cascades of KLF10, cyclooxygenase 1 (COX-1; prostaglandin-endoperoxide synthase 1; PTGS1) was identified to be one of the target genes that may be regulated by KLF10 through direct promoter binding. In order to investigate the function of KLF10/COX-1 axis, promoter activity, EMSA, ChIP-PCR, tube formation assays and platelet aggregation assays were serially performed. Using luciferase reporter assays, KLF10 was found to activate COX-1 promoter activity when overexpressed KLF10 in both endothelial cells: bEnd-3 and HUVEC. In the same experiment, the expression levels of COX-1 mRNA and protein were up-regulated by KLF10 transfected. It is known that COX-1 is the key enzyme in prostaglandin biosynthesis which further regulated angiogenesis in endothelial cells. In tube formation assay, KLF10 overexpressed endothelial cells formed organized tube-like structure better than in contrast to the control group. This KLF10-induced tube formation could be repressed by COX-1 selected inhibitor- valeryl salicylate. Moreover, KLF10-deficient mice perform weaker arachidonic acid-induced platelet aggregation. In sum, these results indicate an important role for KLF10 in angiogenesis and platelet aggregation through the activation of COX-1.