Presentation
# 2
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6th Internet World Congress for Biomedical Sciences |
S. Dhakshinamoorthy(1), D. Bloom(2), Anil Jaiswal(3)
(1)(2)Department of Pharmacology. Baylor College of Medicine. - Houston. United States
(3)Baylor College of Medicine - Houston. United States
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Nuclear transcription factors c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1, Nrf2, YABP, ARE-BP1, Ah (aromatic hydrocarbon) receptor and the estrogen receptor have been reported to bind to the AREs from various genes (46-48, 63; 54-55, 62, 64-67). Among these transcription factors, c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1 and Nrf2 bind to the human NQO1 gene ARE (46-48; 63) (Fig. 4). Nrf1 and Nrf2 have been shown to positively regulate the ARE-mediated expression and induction of NQO1 gene in response to antioxidants and xenobiotics (63; Fig. 5). Nrf1 and Nrf2 are b-zip (leucine zipper) proteins that do not heterodimerize with each other and require another leucine zipper protein for its activity (68-69). Recently, Nrf2-/- mice lacking the expression of Nrf2 were generated (70). These mice showed significantly reduced expression and induction of NQO1 gene indicating a physiological (in vivo) role of transcription factor Nrf2 in the regulation of expression of NQO1 gene (70). More recently, nuclear transcription factors Nrf1 and Nrf2 have been demonstrated to heterodimerize with c-Jun, Jun-B and Jun-D proteins to upregulate ARE-mediated expression and coordinated induction of detoxifying enzymes in response to antioxidants and xenobiotics (71; Fig. 6). Nrf2 containing one mutated leucine in its leucine zipper region was more efficient in upregulation of ARE-mediated gene expression, as compared to Nrf1 with two mutated leucines (71). Interestingly, Nrf-Jun association/heterodimerization and binding to the ARE required unknown cytosolic factor(s) (71). The identity of cytosolic factor(s) remains unknown.
Alignment of DNA binding regions of Nrf1 and Nrf2 with the Jun (c-Jun, Jun-B and Jun-D) proteins revealed the presence of highly conserved, redox labile, cysteine residue in the ´Basic region´. The alignment also revealed several conserved leucines in the leucine zipper regions (Fig.7). Of particular interest are two leucines (# 4 and 5) in the leucine zipper region of Nrf1 and one leucine (#4) in Nrf2. When compared to Jun proteins, these leucines were found to be mutated. These differences may be responsible for the variation in their capacity to mediate ARE-regulated gene expression (63).
It has been suggested that overexpression of c-Fos and Fra1 in transfected Hep-G2 cells repressed the ARE-mediated gene expression (63; Fig. 8). The negative role of c-Fos in ARE-mediated expression has also been observed in c-Fos-/- mice (72; Fig. 8). c-Fos-/- mice showed increased expression of NQO1 and GST Ya as compared to wild type c-Fos+/+ mice (72; Fig. 8). Therefore, it appears that ARE-mediated expression of detoxifying enzyme genes is balanced by positive and negative regulatory factors. One hypothesis is that small amounts of superoxide and related reactive species are consistently required for keeping cellular defenses active. Since activation of detoxifying enzymes and other defensive proteins leads to significant reduction in the levels of superoxide and other free radicals, the cell may require negative regulatory factors like c-Fos to keep the expression of detoxifying enzymes and other defensive genes "in check".
Recently, a 74.3 kDa protein designated as YABP has been shown to bind to the GST Ya gene ARE (64). At present it is not clear if YABP is related to the 65 kDa Nrf1 and 68 kDa Nrf2 proteins, or represents a new class of protein(s).
Recent studies in our and other laboratories have identified a inhibitory protein that retains the Nrf2 in the cytoplasm (S. Dhakshinamoorthy and A. K. Jaiswal, Unpublished; reference 73). We have cloned this protein from rat liver cytosol. This protein was designated by us as ´Inhibitor of Nrf2 or INrf2´. The peptide sequence of INrf2 is shown in Fig. 9. INrf2 is highly homologous to mouse and human Keap1 and Drosophila kelch protein. The various results have shown that INrf2 retains Nrf2 in the cytoplasm. The treatment of cells with xenobiotics and antioxidants releases Nrf2 from Inrf2 (data not shown). The free Nrf2 moves into the nucleus, heterodimerizes with c-Jun and binds to ARE that regulates expression and induction of NQO1 and other detoxifying enzyme genes. The mechanism of dissociation of Nrf2 from Inrf2 remains unknown. Rat, mouse and human INrf2/Keap1 are cysteine rich proteins (Fig. 9). The various cysteins were found highly conserved among the three proteins.
More recently, hMAF, a small human transcription factor was shown to homodimerize and heterodimerize with Nrf1 and Nrf2 (74). Although hMAF-hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the similar NF-E2 binding site, yet they exert functionally opposite effects. hMAF homodimers represses b-globin gene expression whereas hMAF heterodimerization with Nrf1 and Nrf2 activates b-globin gene expression. Since, hMAF heterodimerizes with Nrf1 and Nrf2, its role in the regulation of Nrf+c-Jun regulated hARE-mediated expression of detoxifying enzyme genes remains to be determined. Recently, an Nrf2-MafK heterodimer was shown to bind to the ARE of a gene encoding a subunit of GST Ya. This suggests that the Nrf2-MafK heterodimer may play a role in the ARE-mediated induction of GST Ya gene expression (70). However, the regulatory role of MafK in Nrf/Jun regulation of ARE-mediated expression of NQO1 gene remains unknown.
Mechanism of Signal Transduction for ARE-mediated expression and induction of NQO1 gene
The various steps in the mechanism of signal transduction from antioxidants and xenobiotics to the Nrf1, Nrf2, Jun and Fos proteins that bind to the ARE and regulate ARE-mediated basal expression and coordinated induction of the various detoxifying enzyme genes remains unknown. A hypothetical model showing ARE-mediated induction of NQO1 and other detoxifying enzyme genes expression in response to xenobiotics and antioxidants is shown (Fig. 10). Xenobiotics and antioxidants undergo metabolism to generate superoxide and electrophiles (75). The generation of superoxide and related species is a common phenomenon between antioxidants and xenobiotics. For this reason, superoxide is believed to serve as signal that activates a battery of defensive genes including NQO1 that protect cells against the adverse effects of oxidative stress. Recently, the quinone-mediated generation of hydroxyl radicals were reported to induce the ARE-mediated expression of the mouse glutathione S-transferase gene (76). This correlation remains unknown in the case of AREs from other genes including NQO1. Hydrogen peroxide, however, has been demonstrated to induce the ARE-mediated expression of rat GST Ya, rat NQO1 and human NQO1 genes (77-78). Hence, it may be an important intermediate in the induction of these genes by the antioxidants and xenobiotics. The role of electrophiles, if any, in the ARE mediated expression and induction of detoxifying enzyme genes remains unknown.
The superoxide signal presumably passes either directly or through unknown intermediary proteins, to the cytosolic factor(s) (Fig. 10). The cytosolic factor(s) catalyze modification of Nrf2 leading to the dissociation of Nrf2 from INrf2/Keap1. Nrf2 translocates in the nucleus. Alternatively, cytosolic factor(s) catalyze modification of INrf2/Keap1 resulting in dissociation of Nrf2 from INrf2/Keap1. This follows the translocation of Nrf2 in the nucleus. The modifications of Nrf2 and/or INrf2/Keap1 by cytosolic factor(s) are expected because the treatment of cells with xenobiotics and antioxidants do not alter the expression of genes encoding Nrf2 and Keap1 (S. Dhakshinamoorthy, V. Radjendirane and A.K. Jaiswal, unpublished). Nrf2 after translocation in the nucleus forms heterodimer with c-Jun which binds to the ARE resulting in the induction of NQO1 and other ARE-regulated genes expression. c-Jun may also undergo some modifications by cytosolic factor(s) before moving to the nucleus for heterodimerization with Nrf2. However, this is less likely because the transcription of c-Jun gene is significantly increased in response to xenobiotics and antioxidants. Nrf1 is expected to function in a similar manner as Nrf2. The Jun-B and Jun-D may function similar as c-Jun. This is because of their role in ARE-mediated expression and induction of NQO1 gene expression (70).
The identification of the unknown cytosolic factor(s) in the stimulation of Nrf+Jun binding to the ARE is an important step to our understanding of the signal transduction pathway from endogenous substances, xenobiotics and antioxidants to the Nrf+Jun proteins. However, the nature of the cytosolic factor(s) remains unknown. The unknown cytosolic factor(s) may be a kinase/phosphatase or redox protein(s) that catalyzes the necessary modification of Nrf and/or Jun proteins to stimulate their binding to the ARE. Previous studies have demonstrated increased binding of nuclear factors to the ARE and increased expression of ARE-mediated CAT gene expression in response to dithiothretol and b-mercaptoethanol (78). This suggests that the unknown cytosolic factor(s) may be redox protein(s). It is also supported by studies of transcription factors OxyR and SoxRS that protect bacterial cells against oxidative stress by inducing a group of defensive genes (79-80). The regulation of OxyR has been shown to involve oxidation and reduction of cysteine residues (81). However, the identification of the unknown cytosolic factor(s) as kinase(s)/phosphatase(s) could not be ruled out. The cytosolic factor(s) may serve as sensors of oxidative stress which receive signal from superoxide and related species and then pass it to the oxidative stress responsive factors, Nrf-Jun. This, in turn, leads to increased expression of ARE containing detoxifying and other defensive proteins.
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