KLF2 Primes the Antioxidant Transcription Factor Nrf2 for Activation in Endothelial Cells

Objective—Atheroprotective blood flow induces expression of anti-inflammatory Krüppel-like factor 2 (KLF2) and activates antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in vascular endothelium. Previously, we obtained KLF2-induced gene expression profiles in ECs, containing several Nrf2 target genes. Our aim was to investigate the role of KLF2 in shear stress–mediated activation of Nrf2 in human umbilical vein endothelial cells (HUVECs). Methods and Results—Expression of Nrf2 and its targets NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase (HO-1) was elevated by shear and KLF2. KLF2 knockdown showed that shear-induced expression of NQO1 but not Nrf2 was dependent on KLF2. KLF2 overexpression in absence of flow resulted in more efficient activation of Nrf2 by tert-butyl hydroquinone (tBHQ) through enhanced nuclear localization, and promoted expression of a large panel of Nrf2-dependent genes resulting in superior protection against oxidative stress. Comparison of shear-, KLF2-, and Nrf2-induced transcriptomes showed that the majority of shear-modulated gene sets is influenced by KLF2 or Nrf2. Conclusions—We report that KLF2 substantially enhances antioxidant activity of Nrf2 by increasing its nuclear localization and activation. The synergistic activity of these two transcription factors forms a major contribution to the shear stress–elicited transcriptome in endothelial cells.

[1]  J. Abe,et al.  Fluid Shear Stress Activates Proline-Rich Tyrosine Kinase via Reactive Oxygen Species–Dependent Pathway , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[2]  R. Nerem,et al.  Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. , 1998, Circulation research.

[3]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  C. Gélinas,et al.  To be, or not to be: NF-κB is the answer – role of Rel/NF-κB in the regulation of apoptosis , 2003, Oncogene.

[5]  S. Ylä-Herttuala,et al.  Nrf2 Gene Transfer Induces Antioxidant Enzymes and Suppresses Smooth Muscle Cell Growth In Vitro and Reduces Oxidative Stress in Rabbit Aorta In Vivo , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[6]  Takahiro Shibata,et al.  Differential Responses of the Nrf2-Keap1 System to Laminar and Oscillatory Shear Stresses in Endothelial Cells* , 2005, Journal of Biological Chemistry.

[7]  P. D. de Groot,et al.  Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2). , 2002, Blood.

[8]  A. I. Rojo,et al.  Regulation of Heme Oxygenase-1 Expression through the Phosphatidylinositol 3-Kinase/Akt Pathway and the Nrf2 Transcription Factor in Response to the Antioxidant Phytochemical Carnosol* , 2004, Journal of Biological Chemistry.

[9]  Yuzhi Zhang,et al.  Biomechanical Forces in Atherosclerosis-Resistant Vascular Regions Regulate Endothelial Redox Balance via Phosphoinositol 3-Kinase/Akt-Dependent Activation of Nrf2 , 2007, Circulation research.

[10]  T. Kodama,et al.  Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species. , 2007, Free radical biology & medicine.

[11]  F. Luscinskas,et al.  KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation , 2004, The Journal of experimental medicine.

[12]  M. Rondaij,et al.  KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. , 2006, Blood.

[13]  G. Garcı́a-Cardeña,et al.  Kruppel-Like Factor 2 (KLF2) Regulates Endothelial Thrombotic Function , 2005, Circulation research.

[14]  Thomas Werner,et al.  MatInspector and beyond: promoter analysis based on transcription factor binding sites , 2005, Bioinform..

[15]  B. Rovin,et al.  Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression. , 2006, American journal of physiology. Heart and circulatory physiology.

[16]  Anil K. Jaiswal,et al.  Bach1 Competes with Nrf2 Leading to Negative Regulation of the Antioxidant Response Element (ARE)-mediated NAD(P)H:Quinone Oxidoreductase 1 Gene Expression and Induction in Response to Antioxidants* , 2005, Journal of Biological Chemistry.

[17]  Johan Kuiper,et al.  Prolonged shear stress and KLF2 suppress constitutive proinflammatory transcription through inhibition of ATF2. , 2007, Blood.

[18]  Jurgen Seppen,et al.  Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. , 2005, The American journal of pathology.

[19]  DelindaA . Johnson,et al.  Nuclear Factor E2-Related Factor 2-Dependent Antioxidant Response Element Activation by tert-Butylhydroquinone and Sulforaphane Occurring Preferentially in Astrocytes Conditions Neurons against Oxidative Insult , 2004, The Journal of Neuroscience.

[20]  J. Kuiper,et al.  KLF2 Suppresses TGF-&bgr; Signaling in Endothelium Through Induction of Smad7 and Inhibition of AP-1 , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[21]  Céline Gélinas,et al.  To be, or not to be: NF-kappaB is the answer--role of Rel/NF-kappaB in the regulation of apoptosis. , 2003, Oncogene.

[22]  B. Rovin,et al.  Activation of the Nrf2/antioxidant response pathway increases IL‐8 expression , 2005, European journal of immunology.

[23]  Y. Kan,et al.  Identification of the NF-E2-related Factor-2-dependent Genes Conferring Protection against Oxidative Stress in Primary Cortical Astrocytes Using Oligonucleotide Microarray Analysis* , 2003, The Journal of Biological Chemistry.

[24]  Shyam Biswal,et al.  Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. , 2007, Annual review of pharmacology and toxicology.

[25]  H. Huang,et al.  Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[26]  C. Kunsch,et al.  Laminar Flow Induction of Antioxidant Response Element-mediated Genes in Endothelial Cells , 2003, The Journal of Biological Chemistry.

[27]  L. Pachter,et al.  rVista for comparative sequence-based discovery of functional transcription factor binding sites. , 2002, Genome research.

[28]  C. Stoeckert,et al.  Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Masayuki Yamamoto,et al.  Nrf2-Keap1 defines a physiologically important stress response mechanism. , 2004, Trends in molecular medicine.

[30]  M. Cybulsky,et al.  Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis , 2006, The Journal of experimental medicine.

[31]  T. van der Poll,et al.  Nuclear Receptors Nur77, Nurr1, and NOR-1 Expressed in Atherosclerotic Lesion Macrophages Reduce Lipid Loading and Inflammatory Responses , 2006, Arteriosclerosis, thrombosis, and vascular biology.

[32]  Hong Zhu,et al.  Regulation of Antioxidants and Phase 2 Enzymes by Shear-Induced Reactive Oxygen Species in Endothelial Cells , 2007, Annals of Biomedical Engineering.

[33]  Yuzhi Zhang,et al.  Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. , 2005, The Journal of clinical investigation.