Ref‐1/Ape is critical for formation of the hypoxia‐inducible transcriptional complex on the hypoxic response element of the rat pulmonary artery endothelial cell VEGF gene

The co‐transcription factor and DNA repair enzyme, Redox effector factor‐1/apurinic/apyrimidinic endonuclease (Ref‐1/Ape), facilitates DNA binding and transcriptional activity of a number of transactivating factors, including those governing hypoxia‐induced gene expression HIF‐1. It is not known, however, whether Ref‐1/Ape is a component of the hypoxic transcriptional complex. Electrophoretic mobility shift assays failed to detect direct DNA binding of Ref‐1/Ape to either the HIF‐1 or AP1 DNA recognition sequences present in the hypoxic response element of the VEGF gene. However, immunodepletion of Ref‐1/Ape from nuclear extract prevented DNA binding of ATF/CREB and HIF‐1 to the HIF‐1 DNA recognition sequence. DNA affinity‐precipitation analyses showed that Ref‐1/Ape was part of the multiprotein transcriptional complex forming on a 64‐mer sequence encompassing a minimal hypoxic response element. Immunodepletion of Ref‐1/Ape prevented probe association with HIF‐1, p300, ATF, and CREB. Co‐immunoprecipitation experiments indicated that Ref‐1/Ape present in nuclear extract interacted with HIF‐1 and p300 but not ATF/CREB. However, when Ref‐1/Ape was immunoprecipitated from the oligonucleotide probe, both HIF‐1 and p300 remained probe‐associated while ATF/CREB co‐immunoprecipitated. These findings suggest that Ref‐1/Ape is a critical component of the hypoxia‐inducible transcriptional complex forming on the VEGF gene's hypoxic response element and that the presence of Ref‐1/Ape in the complex is required for the apparent high affinity association between HIF‐1 and its DNA recognition sequence.

[1]  L. Oberley,et al.  Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells. , 2003, American journal of physiology. Heart and circulatory physiology.

[2]  F. Pinet,et al.  Implication of Ref-1 in the repression of renin gene transcription by intracellular calcium , 2003, Journal of hypertension.

[3]  K. Bhakat,et al.  Acetylation of human AP-endonuclease 1, a critical enzyme in DNA repair and transcription regulation. , 2003, Methods in enzymology.

[4]  P. Babál,et al.  Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells. , 2002, American journal of physiology. Lung cellular and molecular physiology.

[5]  S. Mitra,et al.  Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter. , 2002, Nucleic acids research.

[6]  D. Killilea,et al.  Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  C. Lundby,et al.  Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  M. Kelley,et al.  Going APE over ref-1. , 2000, Mutation research.

[9]  P. Babál,et al.  Free radical production in hypoxic pulmonary artery smooth muscle cells. , 2000, American journal of physiology. Lung cellular and molecular physiology.

[10]  L. Poellinger,et al.  A Redox Mechanism Controls Differential DNA Binding Activities of Hypoxia-inducible Factor (HIF) 1α and the HIF-like Factor* , 2000, The Journal of Biological Chemistry.

[11]  L. Poellinger,et al.  Redox-Regulated Recruitment of the Transcriptional Coactivators CREB-Binding Protein and SRC-1 to Hypoxia-Inducible Factor 1α , 2000, Molecular and Cellular Biology.

[12]  G. Greeley,et al.  Hypoxia‐induced mitochondrial and nuclear DNA damage in the rat brain , 1999, Journal of neuroscience research.

[13]  N. Chandel,et al.  Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  B. Ebert,et al.  Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein , 1998, Molecular and Cellular Biology.

[15]  N. Chandel,et al.  Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes* , 1998, The Journal of Biological Chemistry.

[16]  D. Livingston,et al.  Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit* , 1996, The Journal of Biological Chemistry.

[17]  S. Bhattacharya,et al.  An essential role for p300/CBP in the cellular response to hypoxia. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  F. C. Lucibello,et al.  Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression. , 1995, The EMBO journal.

[19]  M. Goldberg,et al.  Transcriptional Regulation of the Rat Vascular Endothelial Growth Factor Gene by Hypoxia (*) , 1995, The Journal of Biological Chemistry.

[20]  M. Gassmann,et al.  The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site. , 1995, Nucleic acids research.

[21]  T. Okazaki,et al.  A redox factor protein, ref1, is involved in negative gene regulation by extracellular calcium. , 1994, The Journal of biological chemistry.

[22]  G. Semenza,et al.  Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. , 1993, The Journal of biological chemistry.