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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 14, 2004 as doi:10.1096/fj.03-1160fje. |
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Departments of Pharmacology and of
* Cell Biology and Neuroscience, University of South Alabama College of Medicine, Mobile, Alabama, USA
2Correspondence: Department of Pharmacology, College of Medicine, University of South Alabama, MSB 3344, Mobile, Al 36688, USA. E-mail: mgillesp{at}jaguar1.usouthal.edu
SPECIFIC AIMS
Redox effector factor-1/apurinic/apyrimidinic endonuclease (Ref-1/Ape) is a ubiquitous bifunctional protein. In hypoxia-induced gene expression it serves as a transcriptional coactivator by promoting redox-dependent interactions between HIF-1 and other proteins forming the transcriptional complex. Ref-1/Ape also serves as a critical component of the base excision pathway repairing oxidative DNA damage. The question has often been raised as to why a single molecule would be endowed with functions that, at least on the surface, appear to be disparate and unrelated.
We recently reported that in main pulmonary artery endothelial cells, hypoxia caused an oxidative base modification at the guanine located at the extreme 3' end of the HIF-1 DNA binding sequence of the vascular endothelial cell growth factor (VEGF) gene. That an oxidative base modification was introduced by hypoxia in a key sequence involved in transcriptional complex formation suggests a potential link between the two functions of Ref-1/Ape; perhaps the DNA repair domain of the molecule is required for removal of the hypoxia-induced base oxidation product while the redox effector domain plays an important role in transcriptional complex assembly. To explore this possibility, we determined whether Ref-1/Ape is a component of the transcriptional complex binding to the hypoxic response element of the PAEC VEGF gene and assessed its interactions with other protein components of the complex.
PRINCIPAL FINDINGS
1. Binding of ATF/CREB and HIF-1 to the HIF-1 DNA recognition sequence requires Ref-1/Ape
EMSAs were used to assess protein-DNA binding in two 26-mer oligonucleotide probes spanning a functionally active sequence of the VEGF gene’s hypoxic response element. One probe encompassed the AP1 DNA recognition sequence. Constitutive nuclear protein binding to this probe was detected, but no changes were evoked by hypoxia. No bands were shifted or deleted with antibodies to p300, HIF-1, ATF/CREB, or AP-1. The second probe harbored the HIF-1 DNA recognition sequence. A protein-DNA complex binding constitutively to this probe was supershifted by an antibody to ATF/CREB; another complex, observed only in hypoxia, was supershifted with an antibody to HIF-1. Antibodies to p300 failed to shift any protein-DNA complex. Application of an antibody to Ref-1/Ape abolished and attenuated the protein-DNA complexes identified as HIF-1 and ATF/CREB, respectively, forming on the HIF-1 oligonucleotide but failed to affect the protein-DNA complexes binding constitutively to the AP-1 oligonucleotide probe. Although Ref-1/Ape was readily detectable in normoxic nuclear extract and increased appreciably in hypoxia, we were unable to detect any evidence for direct DNA binding of Ref-1/Ape. Application of exogenous Ref-1/Ape to either oligonucleotide probe, incubated in the absence or presence of nuclear extract under a variety of experimental conditions, failed to engender formation of a protein-DNA complex or to intensify any complex already present.
2. Ref-1/Ape is part of the transcriptional complex binding to the hypoxic response element of the PAEC VEGF gene
DNA affinity precipitation studies using a 64-mer oligonucleotide sequence of the VEGF hypoxic response element incorporating HIF-1 and the AP1 protein binding motifs were used to confirm the presence of HIF-1 and ATF/CREB in the hypoxia-inducible transcriptional complex and to search for other proteins that may be present in the complex but did not interact directly with either specific protein-DNA recognition sequence. SDS-PAGE and Western analyses of proteins associating with the oligonucleotide probe indicated the presence of p300, CREB, ATF, and Ref-1/Ape in nuclear extract from normoxic PAECs (Fig. 1
). Along with HIF-1, these same proteins were associated with the oligonucleotide probe when it was incubated with nuclear extract from PAECs cultured for 4 h in hypoxia. The abundance of probe-associated p300 and, to a lesser extent, Ref-1/Ape appeared to increase when incubated with hypoxic nuclear extract. When Ref-1/Ape was immunodepleted from nuclear extract before DNAP analysis, none of the transcriptional proteins bound to the oligonucleotide probe.
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The significance of the above studies on DNA-protein interactions with the VEGF gene’s hypoxic response element require that the sequence used for the binding analyses is functionally active. To verify that this is the case in rat PAECs, cells transfected with a construct consisting of the 64-mer sequence of the VEGF gene’s hypoxic response element and a luciferase reporter were cultured for 24 h in normoxia or hypoxia. Hypoxia increased luciferase activity by 
twofold compared with normoxic PAECs.
3. Interactions between Ref-1/Ape and other proteins binding to the hypoxic response element
We wondered whether Ref-1/Ape was associated with HIF-1 and other transcriptional coactivators in the nuclear extract in the absence of the oligonucleotide probe. We immunoprecipitated Ref-1/Ape from nuclear extract derived from PAECs cultured in hypoxia for 4 h and determined whether HIF-1, p300, and ATF/CREB co-immunoprecipitated with Ref-1/Ape. As shown in Fig. 2
, HIF-1 and p300 immunoreactivity, but not ATF/CREB, could be readily detected in the pellet containing Ref-1/Ape. ATF/CREB and some p300 remained in the supernatant.
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Interactions between Ref-1/Ape and other hypoxia-related transcriptional proteins appeared to differ when bound to the hypoxic response element. When Ref-1/Ape and proteins interacting with it were immunoprecipitated from the oligonucleotide probe, ATF/CREB co-immunoprecipitated with Ref-1/Ape while HIF-1 and p300 remained probe associated.
CONCLUSIONS AND SIGNIFICANCE
Results of the present study show that Ref-1/Ape is required for the constitutive as well as hypoxia-inducible binding of transcriptional proteins to the HIF-1 DNA recognition sequence in the rat PAEC VEGF gene. Results of DNAP analyses indicate that Ref-1/Ape interacts constitutively with the hypoxic response element of the VEGF gene and that its association with the hypoxic response element increases with formation of the hypoxia-inducible transcriptional complex. Co-immunoprecipitation studies indicate that Ref-1/Ape interacts differentially with the known components of the complex depending on whether the proteins are bound to the hypoxic response element.
Previous reports have shown that the DNA binding activities of ATF, CREB, and HIF-1, each of which is known to interact with the HIF-1 DNA recognition site, are stimulated by overexpression of Ref-1/Ape. The present observations add that the constitutive presence of Ref-1/Ape in nuclear extract is essential for DNA binding of these proteins. We found that Ref-1/Ape was detectable in nuclear extract from normoxic PAECs and that the abundance of Ref-1/Ape protein increased as a function of time in hypoxia. The obligatory involvement of Ref-1/Ape for DNA-protein interactions was seen in DNAPs. We found that HIF-1, ATF, CREB, p300 were not incorporated into the transcriptional complex unless Ref-1/Ape was present.
The mechanism by which Ref-1/Ape depletion prevented formation of the hypoxia-inducible transcriptional complex seems complicated. One possibility is that the known components of the hypoxia-inducible transcriptional complex interact directly with Ref-1/Ape such that immunoprecipitation of the protein from nuclear extract depletes the other complex components as well. This does not appear to be an adequate explanation since only HIF-1 and p300 co-immmunoprecipitated with Ref-1/Ape whereas ATF/CREB failed to do so. We found that when Ref-1/Ape was immunodepleted after its association with the oligonucleotide probe, HIF-1 and p300 remained probe associated while ATF/CREB was depleted from the probe along with Ref-1/Ape. These findings imply that Ref-1/Ape forms a complex with certain components of the hypoxia-inducible transcriptional complex that, in hypoxia, engenders high-affinity associations between HIF-1, p300, and the hypoxic response element of the VEGF gene
It has been known for several years that Ref-1/Ape binds to nCaRE elements in the parathyroid gene and its own promoter, where it may down-regulate expression of both genes. More recently, evidence for a similar negative regulatory role for Ref-1/Ape binding to the nCaRE element in the renin gene has been reported. In these cases it appears that Ref-1/Ape does not bind directly to the nCaRE, but requires the presence of another protein, notably the heterogeneous nuclear ribonucleoprotein-L (hnRNP-L). We could not detect direct DNA binding activity of Ref-1/Ape; in a manner similar to the nCaRE, perhaps its constitutive and hypoxia-inducible association with the VEGF hypoxic response element requires participation of other proteins. Bhakat and colleagues reported that p300, best known as a scaffolding protein that facilitates transcriptional complex assembly and for promoting chromatin remodeling through its intrinsic histone acetyltransferase activity, also acetylates specific lysine residues on Ref-1/Ape. This acetylation of Ref-1/Ape enhances its association with the nCaRE. Our data suggest that Ref-1/Ape is intimately associated with p300. Thus, p300-dependent acetylation of Ref-1/Ape, perhaps in concert with other proteins, may be key to incorporation of Ref-1/Ape in the constitutive and hypoxia-inducible transcriptional complex forming on the VEGF gene hypoxic response element. Additional studies are needed to address this possibility.
Why would Ref-1/Ape, a molecule exhibiting DNA repair activity and functioning as a transcriptional coactivator, be an important component of both the constitutive and hypoxia-inducible transcriptional complex forming on the VEGF hypoxic response element in PAECs? The answer is speculative, but clues in the literature and from the present study point to a model (Fig. 3
). AP endonuclease activity of Ref-1/Ape is involved with the base excision pathway repairing oxidative DNA damage. A prerequisite for the DNA repair function of Ref-1/Ape is a mechanism of surveillance for abasic sites. Perhaps the association between Ref-1/Ape and cotranscription factors bound constitutively to the hypoxic response element brings Ref-1/Ape in close proximity to DNA so that its surveillance function is facilitated. Hypoxia promotes an oxidant stress in many cell types that, in PAECs, is manifest as oxidative "modifications" in the VEGF promoter. Ligation-mediated PCR analysis of the VEGF gene’s hypoxic response element revealed that a guanine residing at the extreme 3' end of the HIF-1 DNA recognition sequence was one of the three most frequently modified nucleotides. The presence of an abasic site at that specific location thus could serve as a substrate for Ref-1/Ape binding and, as shown by results of the present study, Ref-1/Ape seems critical for assembly of the complex. We propose that the oxidative modification of the guanine residing at an extreme 3' end of the HIF-1 DNA recognition sequence serves to localize Ref-1/Ape in close proximity to HIF-1 and other key transcriptional coactivators, thereby facilitating assembly and/or activation of the transcriptional complex. Additional studies using Ref-1/Ape proteins with specific mutations in its two functional domains are required to address key elements of this hypothesis.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1160fje; 
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