Abstract
Alveolar macrophage (AM) immune function depends on the activation of the transcription factor PU.1 by granulocyte macrophage colony-stimulating factor. We have determined that chronic alcohol ingestion dampens PU.1 signaling via an unknown zinc-dependent mechanism; specifically, although PU.1 is not known to be a zinc-dependent transcription factor, zinc treatment reversed alcohol-mediated dampening of PU.1 signaling. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a zinc-dependent basic leucine zipper protein essential for antioxidant defenses, is also impaired by chronic alcohol ingestion and enhanced by zinc treatment. We hypothesized that the response of PU.1 to zinc treatment may result from the action of Nrf2 on PU.1. We first performed Nrf2/PU.1 protein coimmunoprecipitation on a rat AM cell line (NR8383) and found no evidence of protein-protein interactions. We then found evidence of increased Nrf2 binding to the PU.1 promoter region by chromatin immunoprecipitation. We next activated Nrf2 using either sulforaphane or an overexpression vector and inhibited Nrf2 with silencing RNA to determine whether Nrf2 could actively regulate PU.1. Nrf2 activation increased protein expression of both factors as well as gene expression of their respective downstream effectors, NAD(P)H dehydrogenase[quinone] 1 (NQO1) and cluster of differentiation antigen-14 (CD14). In contrast, Nrf2 silencing decreased the expression of both proteins, as well as gene expression of their effectors. Activating and inhibiting Nrf2 in primary rat AMs resulted in similar effects. Taken together, these findings suggest that Nrf2 regulates the expression and activity of PU.1 and that antioxidant response and immune activation are coordinately regulated within the AM.
Keywords: nuclear factor (erythroid-derived 2)-like 2, PU.1, alveolar macrophage
the alveolar macrophage plays a critical role in innate immune function within the airways. Maturation and activation of alveolar macrophage innate immune functions are absolutely dependent on stimulation by granulocyte macrophage colony-stimulating factor (GM-CSF). When GM-CSF binds its receptor, it initiates an intracellular signaling cascade that culminates in activation of its master transcription factor, PU.1, a member of the E26 transformation-specific family. Its targets are myriad, but tend to fall into two major categories: myeloid lineage growth and differentiation and the cellular response to inflammation (16). In the early stages of cellular development, PU.1 regulates the development of myeloid precursors into macrophages and B cells, with the absolute amount of PU.1 signaling determining the ultimate fate of the cell (24). Several factors have been identified that regulate PU.1 during the myeloid differentiation phase, including C/EBPα (6) and NF-κB (2). Of note, the only mature tissue macrophage expressing PU.1 is the alveolar macrophage (19). There, PU.1 induces a program of genes that play critical roles in a number of cellular functions, including phagocytosis, Toll-like receptor (TLR) signaling, and surfactant clearance (23).
Previously, we determined that chronic alcohol ingestion decreases GM-CSF receptor expression and PU.1 signaling (11). We also determined that chronic alcohol ingestion decreased zinc transport into the alveolar space in an experimental animal model and that dietary supplementation with high doses of zinc restored GM-CSF signaling and alveolar macrophage immune function. This finding was somewhat surprising, since PU.1 is not known to be a zinc-dependent protein (17). In parallel, we identified in experimental and clinical studies that chronic alcohol ingestion causes profound and previously unrecognized depletion of glutathione in the alveolar space (9, 18, 25). A mechanism for the severe oxidative stress in the alcoholic lung remained mysterious until we more recently determined that it also inhibits the expression and activity of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master transcription factor that activates a program of genes that protect the cell from oxidative stress via a consensus-binding sequence called the antioxidant response element (ARE). (10). Interestingly, alcohol-induced inhibition of Nrf2-ARE signaling is also mediated in a zinc-dependent manner, and dietary zinc supplementation likewise restores antioxidant defenses within the airway (17). Furthermore, even though Nrf2-ARE signaling is stereotypically linked to antioxidant defenses, evidence suggests that it also plays a role in innate immune function in the alveolar macrophage (7, 22). Taken together, these findings led us to speculate that the salutary effects of zinc treatment in restoring PU.1 function in the setting of chronic alcohol abuse could be explained by an as yet unrecognized interaction between Nrf2 and PU.1. We therefore hypothesized that Nrf2 may in fact modulate PU.1 signaling in the alveolar macrophage and tested this hypothesis by manipulating Nrf2 expression and activity in an alveolar macrophage cell line and determining if this in turn modulated the expression and activity of PU.1.
MATERIALS AND METHODS
Cell culture.
A rat alveolar macrophage cell line (NR8383; ATCC, Manassas, VA) was cultured in F12K (ATCC) with 10% FBS (Atlanta Biologicals, Lawrenceville, GA) with penicillin, streptomycin, and amphotericin B (Sigma-Aldrich, St. Louis, MO) at 37°C in 5% CO2. Plating of cells for treatment with sulforaphane (SFP; LKT Laboratories, St. Paul, MN) was done with F12K + 5% FBS. Primary macrophages were obtained by lavage from 6-wk-old Fischer rats and cultured for 24 h in DMEM/F12K (Mediatech, Manassas, VA) + 10% FBS before treatment as described below. All procedures were approved by the Institutional Animal Care and Use Committee at the Atlanta Veterans Affairs and Emory University.
Protein coimmunoprecipitation.
Total protein was isolated from NR8383 cells using immunoprecipitation lysis/washing buffer containing 0.025 M Tris (pH 7.4), 0.15 M NaCl, 0.001 M EDTA, 1% Nonidet P-40, 5% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL). Coimmunoprecipitation (co-IP) was performed with antibody to rabbit PU.1 (Santa Cruz, Santa Cruz, CA) or nonrelative control antibody according to the Santa Cruz Biotechnologies protocol using protein A/G plus-agarose (Santa Cruz Biotechnologies). Protein was eluted using Nu-PAGE sample buffer (Invitrogen). Western blotting was then performed as described below with monoclonal Nrf2 antibody (Abcam, Cambridge, MA) and goat antibody to PU-1 (Santa Cruz).
Chromatin immunoprecipitation.
Five million NR8383 cells were plated in 10 ml F12K + 5% FBS on each 100-mm culture dish. Cells were then treated with or without 5 μM SFP for 24 h. Cells were lysed, and samples were prepared according to the Millipore chromatin immunoprecipitation (ChIP) protocol (Temecula, CA). Each sample was divided into portions for no antibody, anti-Nrf2, anti-H3, and nonimmune IgG (Cell Signaling Technology, Danvers, MA). Real-time PCR was carried out using 2 μl purified DNA from each sample (1:25 dilution for input DNA, no dilution for immunoprecipitated fractions) with commercially available primers as outlined in Table 1.
Table 1.
Primers used in RT-PCR
| Primer | Forward | Reverse |
|---|---|---|
| Rat PU.1 | 5′-TGGAGACAGCCATAGCGATCA-3′ | 5′-CAGCTCCGTGAAGTGGTTCT-3′ |
| Rat CD14 | 5′-ACTGAAGCCTTTCTCGGAGC-3′ | 5′-AGCATAAGCTTCATGGTCGGT-3′ |
| ChIP Primers | Company | Catalog/Part No. | Lot No. |
|---|---|---|---|
| PU.1 | Qiagen (Valencia, CA) | GPR1062946(-)14A | 20131202008 |
| Negative control | Active Motif (Carlsbad, CA) | 103788 | 25611001 |
CD14, cluster of differentiation antigen-14; ChIP, chromatin immunoprecipitation.
Nrf2 silencing RNA.
Nrf2 Stealth Select RNAi [set of 3 small-interfering RNA (siRNA)] were obtained from Invitrogen (Nfe2l2, oligo ID: RSS343557, RSS343558, RSS343559; Carlsbad, CA). All three siRNA for Nrf2 were tested. RSS343557 was more effective and chosen for this study. NR8383 cells (200,000 cells/well in a 24-well plate for PCR and 1 × 106 cells/well in a 6-well plate for protein) were plated in OptiMEM (Life Technologies, Grand Island, NY) and transfected with 10 nM of stealth RNAi for rat Nrf2 or negative control using the Lipofectamine transfection reagent. mRNA expression was quantified by RT-qPCR 24 h posttransfection, and protein expression was quantified by Western blotting 48 h posttransfection.
Nrf2 overexpression.
NR8383 cells (200,000 cells/well in a 24-well plate for PCR and 1 × 106 cells/well in a 6-well plate for protein) were plated and transfected with 1.0 μg DNA of rat pCMV-SPORT6.Nrf2 or pCMV-SPORT empty vector (ATCC) for the 24-well plate or 2.5 μg of the same vectors for the 6-well plate using Lipofectamine as described in the Life Technologies protocol. mRNA expression was quantified by RT-qPCR 24 h posttransfection, and protein expression was quantified by Western blotting 48 h posttransfection.
RNA isolation and RT-PCR.
Total RNA was extracted using the Qiagen RNeasy Mini kit (Valencia, CA). Reverse transcription and real-time PCR (Bio-Rad Lab, Hercules, CA) were performed as described previously (12) using primer pairs either previously published (5) or shown in Table 1. PCR products were normalized to 9S from the same RT sample.
Western blotting.
Cells were lysed using Laemmli Sample Buffer (Bio-Rad). Protein electrophoresis was then performed as described previously (4), and membranes were probed with primary antibodies to Nrf2 and PU.1 obtained from Santa Cruz Biotechnologies, and secondary antibodies from GE Healthcare Life Sciences (Piscataway, NJ) before capture of immunoreactive bands with the ChemiDoc XRS system (Bio-Rad).
Statistical analyses.
Student's t-test was used for single comparisons (Microsoft Excel, Redmond, WA). Data are presented as means ± SE. Significance was accepted at P < 0.05.
RESULTS
PU.1 and Nrf2 proteins do not interact at basal conditions in vitro, but activation of Nrf2 enhances the binding of Nrf2 to the promoter region of PU.1.
To determine whether Nrf2 could regulate PU.1, we began by exploring the possibility of a protein-protein interaction using a co-IP assay. As shown in Fig. 1, the PU.1 band indicated by the arrow appears both in the non-IP lysate sample and in the immunoprecipitated sample (lanes 1 and 2), demonstrating appropriate pull down of PU.1. Nrf2, in contrast, is seen in the non-IP lysate sample but not in the immunoprecipitated sample (lanes 4 and 5), suggesting that Nrf2 and PU.1 do not bind together in steady-state conditions in vitro. We employed a variety of lysis buffers to ensure that even a weak protein-protein interaction would be maintained, including lysis with PBS and shearing only (data not shown), but under none of these various conditions did we detect any significant binding between Nrf2 and PU.1.
Fig. 1.
Protein coimmunoprecipitation of PU.1 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). NR8383 whole cell lysate confirms the presence of both Nrf2 and PU.1 proteins (lanes 1 and 4). NR8383 whole cell lysate immunoprecipitated (IP) with antibody to PU.1 resulted in precipitation of PU.1 but not Nrf2 (lanes 2 and 5). In contrast, lysate with nonspecific IgG did not result in precipitation of Nrf2 or PU.1 (lanes 3 and 6). Multiple iterations of the same experiment were performed with similar results. WB, Western blot.
We next explored the possibility of a protein-DNA interaction in which activated Nrf2 might directly bind to the PU.1 promoter region. To test our hypothesis, we employed a ChIP assay with predesigned and validated qPCR primers in rat Chromosome 3-NC_005102.2 from Qiagen targeted around a putative Nrf2-binding site (ACAGGAAGCG at position 75443066) in the PU.1 promoter region, 14 kb upstream from the transcription start site of PU.1. We used SFP in the assay because it is known to cause nuclear translocation of Nrf2 and would enable us to see differential binding of the PU.1 promoter due to Nrf2 activation. As shown in Fig. 2, relative to the nonspecific IgG antibody sample, immunoprecipitation with Nrf2 antibody resulted in over a 100-fold increase in binding of Nrf2 to the PU.1 promoter region with SFP treatment in NR8383 cells, as detected by qPCR. Of note, an earlier iteration of the ChIP protocol resulted in a 12-fold increase in Nrf2 binding to the PU.1 promoter region (data not shown), but the protocol was adjusted to reduce the amount of nonspecific binding by tightening the bead pellets during the precleaning and washing steps with slightly faster centrifugation speeds to produce the data shown in Fig. 2.
Fig. 2.
Chromatin immunoprecipitation of Nrf2 targeting PU.1 promoter. NR8383 cells were treated with 5 μM sulforaphane (SFP) for 24 h. Chromatin immunoprecipitation was performed using Nrf2, H3, and nonspecific IgG. After treatment with SFP, qPCR demonstrated a marked increase in binding of Nrf2 to the promoter region of PU.1, as detected by qPCR. Data are presented as fold change compared with untreated cells.
Activation of the Nrf2-ARE signaling pathway increased expression of PU.1 and its downstream effector cluster of differentiation antigen-14 (CD14) in a rat alveolar macrophage cell line.
To determine whether Nrf2 was able to regulate PU.1 expression and activity by binding to its promoter, we stimulated Nrf2 activity by using SFP, a Nrf2 activator, and a Nrf2 overexpression vector. First, a rat alveolar macrophage cell line (NR8383) was cultured in the presence or absence of SFP. Forty-eight hours later, we measured protein expression by Western blotting and found a significant increase in both Nrf2 and PU.1 proteins when normalized to a loading control (229 and 178%, P < 0.05, respectively) as shown in Fig. 3A. Of note, as discussed in more detail below, Nrf2 has been described at both ∼100 and 60 kDa. To determine which band was most relevant in our particular system, we assessed both bands throughout our experiments. As shown in Fig. 3A, no 100-kDa band was seen with or without SFP treatment. In parallel, we assessed downstream gene activity of both Nrf2 and PU.1 by RT-PCR and found a marked increase in NAD(P)H dehydrogenase[quinone] 1 (NQO1), a downstream effector of Nrf2 (542 ± 136%, P < 0.05), suggesting increased Nrf2/ARE activity, and a marked increase in CD14 (250 ± 53% P < 0.05), a downstream effector of PU.1, thereby suggesting increased PU.1 activity as well (Fig. 3B).
Fig. 3.
Treatment of a rat alveolar macrophage cell line with SFP increases Nrf2 and PU.1 expression and activity. A: 48 h after treatment of a rat alveolar macrophage cell line (NR8383 cells) with and without 5 μM SFP, protein expression was assessed by Western blotting. Expression of Nrf2 and PU.1 was both increased compared with untreated control when normalized to a loading control. Data are presented as means ± SE, n = 3 experiments. *P < 0.05 compared with untreated cells. B: 24 h after treatment of a rat alveolar macrophage cell line (NR8383 cells) with and without 5 μM SFP, gene expression was assessed by RT-PCR. Expression of the Nrf2 downstream effector NAD(P)H dehydrogenase[quinone] 1 (NQO1) and the PU.1 downstream effector cluster of differentiation antigen-14 (CD14) was both increased compared with untreated control. Data presented as means ± SE, n = 4. *P < 0.05 compared with untreated cells.
To further confirm the effect of Nrf2 activation on PU.1 in a more specific fashion, we transfected NR8383 cells with either pCMV SPORT6-Nrf2 vector or an empty pCMV SPORT6 vector. Forty-eight hours posttransfection, cells transfected with the Nrf2 overexpression vector had a twofold increase (P < 0.05) in Nrf2 protein and threefold increase (P < 0.05) in PU.1 protein than cells transfected with empty vector (Fig. 4A). No significant changes were seen in the 100-kDa band on the Nrf2 immunoblot in response to Nrf2 overexpression (100% control vs. 134% Nrf2 overexpression, P = 0.09), again suggesting that the 57-kDa band is the more relevant under the conditions we studied. In parallel, an increase in both NQO1 and CD14 mRNA expression (230 ± 60 and 330 ± 12%, P < 0.05, respectively) was also found, as shown in Fig. 4B.
Fig. 4.
Selective overexpression of Nrf2 gene increases Nrf2 and PU.1 expression and activity. A: NR8383 cells were transfected with pCMV-SPORT6 containing either empty vector or Nrf2. Forty-eight hours posttransfection, Western blotting showed significant increases in both Nrf2 at 57 kDa and PU.1 when normalized to a loading control. The band at ∼100 kDa on the Nrf2 blot showed no significant differences compared with control (100 vs. 134%, P = 0.09). Data are presented as means ± SE, n = 3. *P < 0.05 compared with untreated cells. B: NR8383 cells were transfected with pCMV-SPORT6 containing either empty vector or Nrf2. Twenty-four hours posttransfection, gene expression of NQO1, a downstream effector of Nrf2, and CD14, a downstream effector of PU.1, were both increased significantly. Data are presented as means ± SE, n = 4. *P < 0.05 compared with untreated cells.
Dampening of Nrf2-ARE signaling decreased gene expression of PU.1 and CD14.
We next employed Nrf2 RNA silencing to determine if dampening Nrf2 expression in turn decreases expression of PU.1. To do so, we introduced Nrf2 silencing RNA into NR8383 cells. Forty-eight hours posttransfection, we measured protein expression of Nrf2 and PU.1 and found significant decreases in both when normalized to a loading control (to 35 ± 7 and 25 ± 8%, P < 0.05, respectively), as shown in Fig. 5A. Again, the 100-kDa bands on the Nrf2 immunoblot were not significantly different in response to Nrf2 silencing [100% silencing (si)-Control vs. 107% si-Nrf2, P = 0.13]. In parallel, we assessed downstream gene activity 24 h posttransfection by RT-PCR and found significant decreases in both NQO1 and CD14 (to 65 ± 8 and 35 ± 7%, P < 0.05, respectively), as shown in Fig. 5B.
Fig. 5.
Nrf2 RNA interference decreases Nrf2 and PU.1 expression and activity in NR8383 cells. A: 48 h after Nrf2 silencing (si)-RNA was transfected into NR8383 cells, protein expression by Western blotting showed significant decreases in both Nrf2 at 57 kDa and PU.1 when normalized to a loading control. Data are presented as means ± SE, n = 3. *P < 0.05 compared with untreated cells. The band at ∼100 kDa on the Nrf2 blot showed no significant differences compared with control (100 vs. 107%, P = 0.13). B: in a parallel experiment performed 24 h posttransfection, NQO1 and CD14 gene expression was both significantly decreased by RT-PCR. Data are presented as means ± SE, n = 6. *P < 0.05 compared with untreated cells.
Activation and silencing of Nrf2 in primary rat alveolar macrophages replicates the effects seen in NR8383 cells.
Alveolar macrophages were isolated from 6-wk-old Fischer wild-type rats. The cells were cultured in DMEM/F12K + 10% FBS for 24 h on a 24-well plate. Cells were then treated with or without SFP or transfected with si-Nrf2 or si-Control as described above. Twenty-four hours later, we assessed gene expression by RT-PCR. As shown in Fig. 6A, SFP results in significant increases in both glutamate-cysteine ligase, catalytic subunit (GCLC, a glutathione precursor and downstream effector of Nrf2), and PU.1 (to 264 ± 24 and 193 ± 18%, P < 0.05, respectively). As shown in Fig. 6B, silencing of Nrf2 results in a significant decrease in both Nrf2 and PU.1 gene expression (to 60 ± 10 and 27 ± 8%, P < 0.05, respectively).
Fig. 6.
Activation and silencing of Nrf2 in primary rat alveolar macrophages replicates the effects seen in NR8383 cells. A: 24 h after treatment of primary rat alveolar macrophages with SFP, cells showed significant increases in both glutamate-cysteine ligase, catalytic subunit (GCLC) and PU.1 relative to untreated cells. Data are presented as means ± SE, n = 3. *P < 0.05 compared with untreated cells. B: 24 h after transfection of primary rat alveolar macrophages with si-Nrf2, gene expression of both Nrf2 and PU.1 was significantly decreased. Data are presented as means ± SE, n = 3. *P < 0.05 compared with untreated cells.
DISCUSSION
This study provides evidence of a previously unrecognized mechanism by which Nrf2 regulates PU.1, a key player in innate immune function in the alveolar macrophage, and thereby reveals that these two fundamental defense systems are coordinately controlled. Specifically, although there was no evidence by co-IP for protein-protein interaction between Nrf2 and PU.1, ChIP analysis detected Nrf2 binding to the promoter region of PU.1, thereby suggesting a protein-DNA interaction through which Nrf2 could modulate PU.1 expression. We then determined that this protein-DNA interaction resulted in active regulation by activating Nrf2 with SFP and overexpressing Nrf2. Both methods of Nrf2 stimulation resulted in increases in Nrf2 and PU.1 protein expression, as well as gene expression of NQO1 and CD14, their respective downstream effectors. Of note, in addition to being downstream of PU.1, CD14 is known to be an important component of TLR4 signaling in response to LPS and is constitutively absent in a GM-CSF-deficient system (1). It is thus an important surrogate for innate immune function and PU.1 signaling in the alveolar macrophage more generally. Next, we performed Nrf2 RNA silencing, which diminished Nrf2 and PU.1 proteins, as well as gene expression of NQO1 and CD14. Treatment of primary cells with SFP or transfection with si-Nrf2 showed the same activation and suppression, respectively, of Nrf2 and PU.1 seen in NR8383 cells. Taken together, these findings provide novel evidence that Nrf2 directly influences the expression and subsequent activity of PU.1 and thereby coordinately regulates both antioxidant defenses and immune functions in the alveolar macrophage.
Importantly, a controversy currently exists in the literature regarding the true molecular mass of Nrf2. While many authors, including our group and the Santa Cruz and Abcam antibody manufacturers (e.g., Refs. 5, 14, and 21), identify it between 55 and 70 kDa, others point to a molecular mass of ∼100 kDa as the more active form (e.g., Refs. 13 and 15). The differences in size may be due to changes in phosphorylation (20) or ubiquitination under different conditions. Furthermore, Nrf2 may be modified differently in different cell types as well. Specifically, the immunoblots analyzed in this study were made using NR8383 cells, but others who have found a molecular weight of ∼100 kDa have used, for example, MDA-MB-231, HEK293, HeLa, and COS1 cells (13). Finally, we note that many authors do not specifically note the molecular weight of Nrf2 assessed on their immunoblots, and a consensus on the true size is therefore difficult to discern. Given the fact that only the 57-kDa bands changed significantly in our Nrf2 immunoblots in response to activation or suppression of Nrf2, we believe that, under the conditions we studied and the cell type we were using, those bands are the most relevant. The changes in NQO1 gene expression seen in the associated PCR assays suggest that Nrf2 activity was indeed changing coordinately with the 57-kDa band. Of note, neither the 100-kDa band nor the 68-kDa band were pulled down with PU.1 in our co-IP assay.
We mainly worked with NR8383 cells in this study, a rat alveolar macrophage cell line that has the phenotype and functional characteristics of alveolar macrophages (8), to manipulate Nrf2 expression in a controlled fashion, but our experiments with primary cells show similar responses. However, these findings will ultimately need to be confirmed in human alveolar macrophages to assess their clinical significance. In addition, our mechanistic assays were performed under basal conditions (co-IP) and with SFP treatment (ChIP) in vitro, and these experimental conditions do not replicate the state of the alveolar space in vivo. Even though we could only identify a protein-DNA interaction in our current study, we cannot rule out the possibility of a protein-protein interaction in other contexts.
Our findings have important implications for potential therapeutic interventions given that SFP, a compound found in cruciferous vegetables such as broccoli, can activate both Nrf2-ARE antioxidant defense systems and PU.1-mediated immune defense systems. SFP is thought to act by releasing Nrf2 from its chaperone protein (Kelch-like ECH-associated protein 1 or Keap1) in the cytosol, allowing it to translocate to the nucleus and bind to the ARE (26). The binding of Nrf2 to the ARE thereby initiates the transcription of a range of genes that defend the cell against oxidative stress, including genes necessary for the synthesis and maintenance of glutathione. However, this study suggests that SFP can also induce the PU.1-dependent immune defenses indirectly through its activation of Nrf2. Therefore, if these findings are borne out in human subjects, SFP and/or other similar phytochemicals could prove to be effective in promoting and/or restoring a healthier alveolar environment in individuals with underlying alcohol abuse or human immunodeficiency virus (HIV) infection, which at least experimentally are now recognized to inhibit both the antioxidant and immune defense systems in the airways (10, 12).
In addition, although SFP is known to have Nrf2-independent effects, none of these would explain the differential site occupancy of Nrf2 on the PU.1 promoter that we identified in the ChIP assay. Both our recognition of a non-ARE Nrf2-binding site on a Nrf2-regulated gene as well as our use of SFP to discover novel Nrf2-regulated genes are consistent with the findings and techniques of other investigators (3), although our specific promoter site appears to be farther away than that used for prior studies. In this context, it appears that PU.1 can be added to the list of “novel Nrf2-regulated genes” that are not classically considered to be directly involved in antioxidant defenses. However, since the alveolar macrophage must respond quickly and robustly to stresses that involve both “immune” and “oxidant” challenges, it seems logical that there would be coordinated regulation of these complementary defense pathways.
Overall, the evidence of PU.1 regulation by Nrf2 is both novel and provocative. Nrf2 is a critical player in the defense against oxidative stress, and its link to PU.1 brings with it a host of previously unrecognized innate immune functions as well. In addition, the connection between these two critical transcription factors ties immune function together with oxidative stress by a novel mechanism. Importantly, our findings also provide a Nrf2-dependent mechanism for our previous observations on the effects of zinc supplementation on PU.1 function.
In summary, we identified that Nrf2 regulates the expression and activity of PU.1, likely via direct binding to the promoter region of the PU.1 gene. In retrospect, the link between the two is not surprising since prior work in our lab has shown that both Nrf2 and PU.1 are functionally diminished in the alveolar macrophage by chronic alcohol ingestion in a zinc-dependent manner (17). Furthermore, these new findings offer a mechanism by which these two impairments that were previously thought to be separate may in fact be linked. As a potential translation to the clinical setting, this study suggests that enhancing Nrf2-ARE activity and antioxidant defenses in disease states such as chronic alcohol abuse or HIV with treatments such as SFP could also increase PU.1 activity and thereby also enhance innate immune function within the airway.
GRANTS
B. Staitieh and W. Neveu are supported by training grants from the National Institutes of Health (NIH) (T32-HL-076118-09 and T32-HL-116271-01A1, respectively). D. Guidot is supported by NIH Grant R01-AA-017627 and by a Veterans Affairs Merit Review.
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: B.S.S., X.F., and D.M.G. conception and design of research; B.S.S. and X.F. performed experiments; B.S.S., X.F., and W.N. analyzed data; B.S.S., X.F., W.N., and D.M.G. interpreted results of experiments; B.S.S. and D.M.G. prepared figures; B.S.S. drafted manuscript; B.S.S., X.F., W.N., and D.M.G. edited and revised manuscript; B.S.S., X.F., W.N., and D.M.G. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Dr. Viranuj Sueblinvong for assistance in optimizing assays and troubleshooting experiments, Drs. Michael Koval and Samuel Molina for valuable scientific discussions, Tierra Smith for help preparing samples for Western Blotting, and Robert Raynor and S. Todd Mills for invaluable technical support.
REFERENCES
- 1.Berclaz PY, Carey B, Fillipi MD, Wernke-Dollries K, Geraci N, Cush S, Richardson T, Kitzmiller J, O'Connor M, Hermoyian C, Korfhagen T, Whitsett JA, Trapnell BC. GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS. Am J Respir Cell Mol Biol 36: 114–121, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Bonadies N, Neururer C, Steege A, Vallabhapurapu S, Pabst T, Mueller BUPU. is regulated by NF-kappaB through a novel binding site in a 17 kb upstream enhancer element. Oncogene 29: 1062–1072, 2010. [DOI] [PubMed] [Google Scholar]
- 3.Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, Bangura F, Xue P, Pi J, Kleeberger SR, Bell DA. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha. Nucleic Acids Res 40: 7416–7429, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Fan X, Joshi PC, Koval M, Guidot DM. Chronic alcohol ingestion exacerbates lung epithelial barrier dysfunction in HIV-1 transgenic rats. Alcohol Clin Exp Res 35: 1866–1875, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fan X, Staitieh BS, Jensen JS, Mould KJ, Greenberg JA, Joshi PC, Koval M, Guidot DM. Activating the Nrf2-mediated antioxidant response element restores barrier function in the alveolar epithelium of HIV-1 transgenic rats. Am J Physiol Lung Cell Mol Physiol 305: L267–L277, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Friedman AD. C/EBPalpha induces PU.1 and interacts with AP-1 and NF-kappaB to regulate myeloid development. Blood Cells Mol Dis 39: 340–343, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Harvey CJ, Thimmulappa RK, Sethi S, Kong X, Yarmus L, Brown RH, Feller-Kopman D, Wise R, Biswal S. Targeting Nrf2 signaling improves bacterial clearance by alveolar macrophages in patients with COPD and in a mouse model. Sci Transl Med 3: 32–78, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Helmke RJ, Boyd RL, German VF, Mangos JA. From growth factor dependence to growth factor responsiveness: the genesis of an alveolar macrophage cell line. In Vitro Cell Dev Biol 23: 567–574, 1987. [DOI] [PubMed] [Google Scholar]
- 9.Holguin F, Moss I, Brown LA, Guidot DM. Chronic ethanol ingestion impairs alveolar type II cell glutathione homeostasis and function and predisposes to endotoxin-mediated acute edematous lung injury in rats. J Clin Invest 101: 761–768, 1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Jensen JS, Fan X, Guidot DM. Alcohol causes alveolar epithelial oxidative stress by inhibiting the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling pathway. Am J Respir Cell Mol Biol 48: 511–517, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Joshi PC, Applewhite L, Ritzenthaler JD, Roman J, Fernandez AL, Eaton DC, Brown LA, Guidot DM. Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression and downstream signaling in the alveolar macrophage. J Immunol 175: 6837–6845, 2005. [DOI] [PubMed] [Google Scholar]
- 12.Lassiter C, Fan X, Joshi PC, Jacob BA, Sutliff RL, Jones DP, Koval M, Guidot DM. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction (Abstract). AIDS Res Ther 6: 1, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Lau A, Tian W, Whitman SA, Zhang DD. The predicted molecular weight of Nrf2: it is what it is not. Antioxid Redox Signal 18: 91–93, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lisk C, McCord J, Bose S, Sullivan T, Loomis Z, Nozik-Grayck E, Schroeder T, Hamilton K, Irwin DC. Nrf2 activation: a potential strategy for the prevention of acute mountain sickness. Free Rad Biol Med 63: 264–273, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Macoch M, Morzadec C, Genard R, Pallardy M, Kerdine-Romer S, Fardel O, Vernhet L. Nrf2-dependent repression of interleukin-12 expression in human dendritic cells exposed to inorganic arsenic. Free Radic Biol Med In press. [DOI] [PubMed] [Google Scholar]
- 16.Marecki S, Fenton MJ. PU1/interferon regulatory factor interactions: mechanisms of transcriptional regulation. Cell Biochem Biophys 33: 127–148, 2000. [DOI] [PubMed] [Google Scholar]
- 17.Mehta AJ, Joshi PC, Fan X, Brown LA, Ritzenthaler JD, Roman J, Guidot DM. Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats. Alcohol Clin Exp Res 35: 1519–1528, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Moss M, Guidot DM, Wong-Lambertina M, Ten Hoor T, Perez RL, Brown LA. The effects of chronic alcohol abuse on pulmonary glutathione homeostasis. Am J Respir Crit Care Med 161: 414–419, 2000. [DOI] [PubMed] [Google Scholar]
- 19.Nakata K, Kanazawa H, Watanabe M. Why does the autoantibody against granulocyte-macrophage colony-stimulating factor cause lesions only in the lung? Respirology Suppl 11: S65–S69, 2006. [DOI] [PubMed] [Google Scholar]
- 20.Pi J, Bai Y, Reece JM, Williams J, Liu D, Freeman ML, Fahl WE, Shugar D, Liu J, Qu W, Collins S, Waalkes MP. Molecular mechanism of human Nrf2 activation and degradation: role of sequential phosphorylation by protein kinase CK2. Free Rad Biol Med 42: 1797–1806, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ren J, Yang J, Xu Y, Huang Q, Yang M, Hu K. Lupiwighteone induces cell cycle arrest and apoptosis and activates the Nrf2/ARE pathway in human neuroblastoma cells. Biomed Pharmacother 69: 153–161, 2015. [DOI] [PubMed] [Google Scholar]
- 22.Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 116: 984–995, 2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Trapnell BC, Whitsett JA. Gm-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annu Rev Physiol 64: 775–802, 2002. [DOI] [PubMed] [Google Scholar]
- 24.Turkistany SA, DeKoter RP. The transcription factor PU.1 is a critical regulator of cellular communication in the immune system. Arch Immunol Ther Exp (Warsz) 59: 431–440, 2011. [DOI] [PubMed] [Google Scholar]
- 25.Velasquez A, Bechara RI, Lewis JF, Malloy J, McCaig L, Brown LA, Guidot DM. Glutathione replacement preserves the functional surfactant phospholipid pool size and decreases sepsis-mediated lung dysfunction in ethanol-fed rats. Alcohol Clin Exp Res 26: 1245–1251, 2002. [DOI] [PubMed] [Google Scholar]
- 26.Zhang DD, Hannink M. Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol Cell Biol 23: 8137–8151, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
