PERSPECTIVE
AP Endonucleases and the Many Functions of Ref-1

Dawn M. Flaherty, Martha M. Monick, and Gary W. Hunninghake

Department of Internal Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa

APE-1/Ref-1 is a ubiquitous multifunctional protein that possesses both DNA repair activity and redox regulatory activity. Originally named apurinic/apyrimidinic endonuclease (APE-1) and HAP-1 (Human APE-1) for its endonuclease activity, APE-1/Ref-1 is a major enzyme in the base excision repair pathway (1) and is involved in repair of spontaneous and oxidative damage to cellular DNA (2). In signal transduction, APE-1/Ref-1 is important in mediating DNA binding of a number of transcription factors including AP-1, nuclear factor (NF)-B, Pax-5, Pax-8, HIF-1, and HLF (Table 1) (2). Although the nomenclature is not standardized, many authors refer to the protein as APE-1/ Ref-1 to reflect its dual function. APE-1/Ref-1 is ubiquitously expressed, though there are complex patterns of distribution within cells and between different cell types (9). These patterns of cellular distribution may be related to the varying functions of APE-1/Ref-1. Based on its multiple functions, APE-1/Ref-1 likely plays an important role in maintaining genomic integrity and in regulating gene expression via redox activation of a variety of transcription factors.

Human cells in vitro are estimated to undergo spontaneous depurination of DNA at a rate of approximately 10,000 bases/day/cell (14), with the greatest number of apurinic/apyrimidinic (AP) sites occurring in the brain, heart, and colon (15). In addition to spontaneous loss of nucleotides, DNA can also be damaged via oxidant stress that results in oxidation of bases and sugars (16). To protect the integrity of the genome, all cells have developed a repair system to excise and replace the damaged nucleotides (AP sites) in DNA. In the base excision repair pathway, damaged bases are excised by a DNA glycosylase, creating an AP site. APE-1/Ref-1 cleaves the DNA backbone 5' to the AP site and repair is completed by DNA polymerase and DNA ligase (17).

There are two families of endonucleases that are differentiated based on their functions and homology to Escherichia coli exonucleases (20). APE-1/Ref-1 belongs to the first of two families of AP endonucleases and shares sequence homology with E. coli exonuclease III. It accounts for approximately 95% of DNA repair capabilities in humans. In the yeast Saccharomyces cerevisiae, AP endonuclease-1 (APN-1) is the primary AP endonuclease and accounts for the majority of the yeast DNA repair activity. In contrast to APE-1/Ref-1, APN-1 belongs to the second family of AP endonucleases and shares homology with E. coli exonuclease IV (21). Though both enzymes have comparable 5' endonuclease activity and the ability to repair AP sites, APN-1 has much greater 3'-diesterase activity, and no known redox capability. In APE-1/Ref-1, the DNA repair activity resides in the C-terminal portion of the protein, while the N-terminal domain is necessary for redox regulation of transcription (22). In addition to the > 90% homology between various mammalian AP endonucleases, there is a highly conserved cysteine at position 65 in human, mouse, and rat AP endonucleases that is thought to be important in redox function of these proteins.

APE-1/Ref-1 functions as a regulatory protein for redox activation of a number of transcription factors (2). Xanthoudakis and Curran (2) originally identified the redox function of APE-1/Ref-1 when they studied the in vitro activation of AP-1 DNA binding in HeLa cells. APE-1/ Ref-1 acts via a post-translational mechanism in which conserved cysteine residues in the DNA-binding domains of Fos and Jun proteins are reduced, allowing DNA binding to occur. We have demonstrated a direct correlation between the amounts of nuclear APE-1/Ref-1 and AP-1 activity in alveolar macrophages, suggesting an important role for APE-1/Ref-1 in the inflammatory response in the lung (23). AP-1 is one of many transcription factors under redox control of APE-1/Ref-1. Table 1 lists the transcription factors shown to exhibit redox regulation by APE-1/ Ref-1. For example, Jayaraman and colleagues (24) demonstrated that cotransfection of p53, a transcription factor that is involved in the response to oxidative stress and apoptosis, with APE-1/Ref-1, increases expression of the p53-dependent genes, cyclin G, p21, and BAX. Additionally, the Pax family of genes, important for normal development, cellular differentiation, and thyroid function, has been shown to exhibit transcriptional activation of specific promotors in cotransfection experiments with APE-1/Ref-1 (7, 8). These and other transcription factors under redox control of APE-1/Ref-1 are involved in regulation of a number of important cellular functions and these studies suggest a complex, multifunctional role for APE-1/Ref-1 in cellular response to stress.

Regulation of APE-1/Ref-1 occurs at both the transcriptional and post-translational level. Transcriptional regulation of APE-1/Ref-1 has been shown to occur via a number of stimuli, including oxidant stress, hormones, and asbestos (25). This appears to involve both new protein synthesis (30) and increased nuclear translocation (8). We have recently shown that GM-CSF increases amounts of APE-1/Ref-1 nuclear protein in human alveolar macrophages resulting in increased AP-1 DNA binding (23).

Post-translational modification of APE-1/Ref-1 occurs via phosphorylation and redox modification. There are several potential phosphorylation sites contained in APE-1/Ref-1, including consensus sequences for protein kinase C (PKC) and casein kinase I and II (CKI and CKII). There is some disagreement with regard to the location of these sites in the protein and to the functional effects of phosphorylation, with some investigators demonstrating alterations of repair and redox function with phosphorylation at these different sites (34, 35). Recently, Hsieh and colleagues showed that the in vitro redox activity of APE-1/Ref-1 is stimulated by PKC phosphorylation. They postulate that in vivo redox regulation is correlated with susceptibility of cells to the PKC-induced phosphorylation of APE-1/Ref-1 that occurs in response to oxidant stress (33). Finally, APE-1/Ref-1 is altered via redox modification through a direct interaction with thioredoxin (TRX), a ubiquitous thiol-reducing enzyme that is important in many cellular signaling pathways (36). TRX acts as a hydrogen donor via association of APE-1/Ref-1 with Cys-32 and Cys-35 at the catalytic center of TRX. The interaction between TRX and APE-1/Ref-1 is essential for DNA binding of the AP-1 complex to occur and requires that TRX be translocated from the cytoplasm to nucleus (37). Stimuli that induce TRX translocation include oxidants (38), phorbol esters (37), cytokines (39), and UV irradiation (40).

In this issue of the journal, He and colleagues show that lung epithelial cells transduced with APN-1 (a yeast endonuclease) exhibit decreased DNA damage and cytotoxicity when treated with bleomycin (41). Bleomycin induces DNA damage via oxidation of the deoxyribose moiety, leaving an AP site, or by excising the base along with a portion of the sugar, resulting in a strand break with a 3'-phosphoglycolate terminus. This latter lesion blocks the ability of DNA polymerase to initiate repair (42). Though APE-1/Ref-1 has comparable AP site repair activity, its ability to excise 3' blocking fragments is limited. Because APN-1 is capable of repairing both these lesions, the authors hypothesized that expression of this protein in mammalian cells might reduce the damaging effects of bleomycin. Indeed, they illustrate through DNA repair and clonal survival assays that cells overexpressing APN-1 exhibit less bleomycin-related toxicity as compared with controls. They propose that this is due to the repair capabilities of the APN-1; however, levels of the enzyme do not strictly correlate with levels of protection. This suggests that a small increase in amounts of APN-1 has profound effects on protection of cells against damage by bleomycin. Alternatively, there may be a complex set of interactions between APN-1 and other protective enzymes that account for this observation.

The role of APE-1/Ref-1 in maintaining genomic and functional integrity in the cell is complex and is yet to be fully understood. A number of investigators have examined the functional relevance of APE-1/Ref-1 through overexpression and antisense studies. Knockout mice for APEX-1 (the mouse homolog of APE-1/Ref-1) are embryonic lethal (43) and hence many studies have used techniques to either deplete or increase APE-1/Ref-1 in cultured human and other mammalian cells. In experiments that restore normal APE-1/Ref-1 levels to deficient cells, protection against DNA-damaging agents is seen (44). However, increasing APE-1/Ref-1 above normal levels has not been shown to be clearly protective (45). Alternately, antisense studies show that decreasing levels of APE-1/Ref-1 sensitize cells to the effects of numerous toxic agents (27, 49). It is unclear whether these effects are due to alterations in redox or DNA repair functions or both. These observations may be analogous to the findings of He and colleagues. Perhaps a small increase in the AP endonuclease pushes levels of the enzyme over a threshold that protects the cells from a strong oxidant stress. Recently, heterozygous mice (APEX^+/) have been created and shown to be abnormally sensitive to oxidative stress (50). Certainly, the implications of overexpression of APE-1/Ref-1 in a cellular system are far-reaching and complex. Though increasing APE-1/Ref-1 in situations of oxidant stress could potentially reduce DNA damage and cytotoxicity, it is unknown how changes in redox regulation with induction of transcription factors involved in cell cycle regulation and differentiation could affect an organism. Numerous investigators have shown the presence of increased APE-1/Ref-1 in a variety of malignancies, including colon, ovarian, cervical, germ cell, and prostate (51). Alteration in APE-1/Ref-1 expression and mutations in the APE-1/Ref-1 gene have been found in patients with a variety of neurodegenerative diseases (54, 55). Despite the role of APE-1/Ref-1 in regulation of transcription factors involved in cell cycle control, differentiation and signal transduction, a relationship between APE-1/ Ref-1 and the development of pulmonary fibrosis has yet to be demonstrated.

Expression of APE-1/Ref-1, and other DNA-repair enzymes, may be related to fibrotic lung diseases, like that induced by bleomycin. We have previously shown that normal alveolar macrophages express decreased AP-1 DNA binding compared with blood monocytes. This appears to be due to a decrease in the amount of APE-1/Ref-1 (56). Although relevance of this observation to pulmonary fibrosis is unknown, we postulate that decreased amounts of APE-1/Ref-1 may also decrease expression of profibrotic genes that are driven by AP-1. AP-1 DNA binding is important for expression of a number of genes whose proteins have been implicated in the development of fibrosis (57), including GM-CSF, alveolar macrophage-derived collagenase (MMP-1), and TGF-. We also believe that certain stimuli increase APE-1/Ref-1 in alveolar macrophages and other types of lung cells and this may play a role in the initiation/development of fibrosis in the lung. Alveolar macrophages are chronically exposed to cytokines, growth factors, and other fibrotic stimuli during the course of various lung diseases. If some of these factors increase APE-1/Ref-1, it would suggest a mechanism for maintaining AP-1 DNA binding activity in human alveolar macrophages from patients with chronic lung diseases. Recent data from our laboratory (Flaherty, unpublished observations) has shown that nuclear APE-1/Ref-1 amounts increase minutes after treatment of alveolar macrophages with crocidolite asbestos, and that this response can be inhibited by treatment with the NADPH inhibitor, diphenyleneiodonium chloride (DPI). This increase in APE-1/Ref-1 is accompanied by an increase in AP-1 DNA binding which is also inhibited by DPI. We postulate that macrophages respond to fibrogenic stimuli such as asbestos by increasing APE-1/Ref-1 and AP-1 DNA binding. This appears to be mediated by reactive oxygen species representing an initial response to stress that may activate pathways leading to alterations of normal cell growth and differentiation.

APE-1/Ref-1 is a multifunctional protein with a number of complex roles in maintenance of cellular health and genomic integrity (Figure 1). It is involved in repair of DNA damage, in regulation of a variety of transcription factors, cell cycle control, hematopoesis, and response to environmental stress. Many studies also support a role for APE-1/Ref-1 in disease including cancer, neurodegenerative disease, and aging. Though much has been learned about APE-1/Ref-1 since it was initially identified as a DNA repair enzyme, there are still more questions to be answered about its regulation and role in disease.

View larger version (38K): [in this window] [in a new window]   Figure 1.   The role of AP endonucleases in cellular function and disease.

	Footnotes

Address correspondence to: Dawn M. Flaherty, M.D., Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa Hospitals and Clinics, C-33 GH, Iowa City, Iowa 52242. E-mail: flahertydm{at}mail.medicine.uiowa.edu

(Received in original form September 22, 2001).

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