Activation of KLF8 Transcription by Focal Adhesion Kinase in Human Ovarian Epithelial and Cancer Cells

Xianhui Wang,Alison M Urvalek,Jinsong Liu,Jihe Zhao

doi:10.1074/jbc.m709300200

Abstract

KLF8 (Krüppel-like factor 8) is a transcription factor downstream of focal adhesion kinase (FAK) important in the regulation of the cell cycle and also plays a critical role in oncogenic transformation and epithelial to mesenchymal transition. Here we report the mechanisms by which FAK regulates KLF8 expression in human ovarian epithelial and cancer cells. We show that the overexpression of both KLF8 and FAK in the human ovarian cancer cells as compared with the normal human ovarian surface epithelial cells is critical for cell growth. Using promoter luciferase reporter assays, we demonstrate that exogenous FAK strongly promotes the activity of the KLF8 promoter, and knockdown of FAK inhibits it. KLF8 promoter activity and mRNA levels are induced by expression of constitutively active (CA) phosphatidylinositol 3-kinase (PI3K) or CA-Akt but are repressed by dominant negative Akt or the PI3K inhibitor LY294002. Disruption of an Sp1 binding site in the KLF8 promoter abolishes the FAK- or Sp1-mediated promoter activation. Sp1 knockdown prevents the KLF8 promoter from being activated by Sp1 or CA-Akt, and expression of CA-Akt enhances Sp1 expression in SKOV3ip1 cells. Chromatin immunoprecipitation and oligonucleotide precipitation results show that Sp1 binds to the KLF8 promoter. Taken together, our data suggest that FAK induces KLF8 expression in human ovarian cancer cells by activating the PI3K-Akt signaling pathway, leading to the activation of KLF8 promoter by Sp1.

Highlights

focal adhesion kinase (FAK) is an important protein-tyrosine kinase downstream of integrins and growth factors in the regulation of diverse cellular events, including cell adhesion, cell cycle, and migration (8 –10), and plays critical roles in skin tumor initiation [11] and breast tumor growth and metastasis [12,13,14]
We show that FAK signaling through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway plays a major role in the activation of KLF8 transcription in the ovarian cancer cells, and this regulation is mediated by increased expression of Sp1 transcription factor that binds to and activates KLF8 gene promoter
FAK Modulates KLF8 Expression via Transcriptional Activation—We previously reported that inducible expression of FAK regulates cell cycle progression in NIH3T3 cells [17], and using microarray techniques, we identified KLF8 as a downstream target of FAK in this regulation [25]

Summary

Introduction

FAK is an important protein-tyrosine kinase downstream of integrins and growth factors in the regulation of diverse cellular events, including cell adhesion, cell cycle, and migration (8 –10), and plays critical roles in skin tumor initiation [11] and breast tumor growth and metastasis [12,13,14]. It has been reported that Akt is frequently activated in ovarian cancer, and introduction of Akt along with either c-Myc or K-Ras into p53-null ovarian surface epithelial cells was sufficient to induce ovarian tumor formation [22] These studies suggest that the FAK-PI3K-AKT signaling pathway is likely to be involved in the initiation and progression of ovarian cancer. We report that FAK regulates KLF8 expression at the transcriptional level in human ovarian epithelial and cancer cells. We show that FAK signaling through the PI3K-AKT pathway plays a major role in the activation of KLF8 transcription in the ovarian cancer cells, and this regulation is mediated by increased expression of Sp1 transcription factor that binds to and activates KLF8 gene promoter. Tin immunoprecipitation; BOP, biotinylated oligonucleotide precipitation; BrdUrd, 5-bromodeoxyuridine; RT, reverse transcription; qRT, quantitative real time; HA, hemagglutinin; WT, wild type

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