HBP1 repression of the p47phox gene: cell cycle regulation via the NADPH oxidase

Abstract

Several studies have linked the production of reactive oxygen species (ROS) by the NADPH oxidase to cellular growth control. In many cases, activation of the NADPH oxidase and subsequent ROS generation is required for growth factor signaling and mitogenesis in nonimmune cells. In this study, we demonstrate that the transcriptional repressor HBP1 (HMG box-containing protein 1) regulates the gene for the p47phox regulatory subunit of the NADPH oxidase. HBP1 represses growth regulatory genes (e.g., N-Myc, c-Myc, and cyclin D1) and is an inhibitor of G(1) progression. The promoter of the p47phox gene contains six tandem high-affinity HBP1 DNA-binding elements at positions -1243 to -1318 bp from the transcriptional start site which were required for repression. Furthermore, HBP1 repressed the expression of the endogenous p47phox gene through sequence-specific binding. With HBP1 expression and the subsequent reduction in p47phox gene expression, intracellular superoxide production was correspondingly reduced. Using both the wild type and a dominant-negative mutant of HBP1, we demonstrated that the repression of superoxide production through the NADPH oxidase contributed to the observed cell cycle inhibition by HBP1. Together, these results indicate that HBP1 may contribute to the regulation of NADPH oxidase-dependent superoxide production through transcriptional repression of the p47phox gene. This study defines a transcriptional mechanism for regulating intracellular ROS levels and has implications in cell cycle regulation.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 1.

HBP1 regulates the p47phox promoter through specific binding to the HBP1 DNA elements. (A) Schematic diagram of the p47phox promoter and associated reporter constructs. The top part depicts the array and sequence of the HBP1 sites in the p47 phox promoter at positions −1392 to −1217 from the transcriptional start site (a generous gift of Robert Clark). Letters representing the 76 bp encompassing the HBP1 sites are underlined and italicized. A schematic of the reporters used in this paper is also shown. (B) The high-affinity HBP1 sites confer transcriptional repression. HEK-293T cells were transfected with 0.5 μg of the indicated reporter and 2 μg of pRSV-β-Gal (β-gal) (to normalize for transfection efficiency). (C) Schematic diagram of wild-type HBP1 and associated mutants. MAPK, mitogen-activated protein kinase. (D) Relative activity of HBP1 and associated mutants on the native p47 phox promoter. Transfections were performed as in panel B and included 5 μg of the indicated HBP1 expression plasmid. The results are normalized for transfection efficiency and are expressed as relative transcriptional activity within each set. As shown in panel B, the activity of each reporter varies, but for comparison, the relative activity in the control (Cont.) was set to a value of 1. Each data set represents three to five experiments. (E) Relative activity of HBP1 and associated mutants on heterologous high-affinity HBP1 site reporter. Experiments were performed as described for panel D. (F) Expression of wild-type and mutant HBP1 proteins. HA-tagged proteins from experiments shown in panels D and E were detected by immunoblots (IB) with anti-HA antisera. A representative blot is shown.

FIG. 2.

HBP1 occupies its high-affinity sites in the endogenous p47phox promoter. (A) ChIPs were used to test the binding of HBP1 to the endogenous p47phox gene. HEK-293T cells were transfected with pEFBOS-HBP1 or the indicated mutant plasmids. The region from position −1474 to position −1146 contains the HBP1 element and was analyzed by specific PCR. Either anti-HA or control (anti-DP1) antisera were used in the indicated lanes. Numbers representing the test and control lanes are underlined. Lanes 1 and 2 show the PCR results when no chromatin is included in the absence and presence of anti-HA, respectively. Lane 11 shows the PCR on total isolated chromatin from HEK-293T cells. Only lanes with HBP1 proteins that contain intact HMG boxes show binding to the p47phox promoter (lanes 3 and 7). At the bottom, anti-HA Western immunoblotting (IB) for HBP1 protein expression is shown. It should be noted that normal HBP1 migrates aberrantly at a molecular weight of ∼80,000, though the mRNA sequence specifies a 64,000-molecular-weight protein. (B) EMSA assays were performed by using a radiolabeled double-stranded probe consisting of one HBP1 high-affinity site. Ten-microgram amounts of nuclear extracts from HEK-293T cells or cells transfected with the indicated HBP1 expression plasmids were used. Cold competitors were included in the indicated lanes at 100-fold excess. The presence of specific complexes, including supershifted HA-HBP1 in the complexes, is indicated with arrows. WT, wild type.

FIG. 3.

Expression of HBP1 and Δ393 affects endogenous P47phox mRNA and protein expression. (A) p47 phox mRNA expression is decreased in a stable cell line that constitutively expresses HBP1. RT-PCR for the p47phox gene was performed with 2 μg of total RNA from the control C2C12 muscle cell line and a C2C12 line overexpressing HBP1 (B2 [24]). 18S RNA was used as an internal control, as indicated in Materials and Methods. The reactions were analyzed at the indicated cycle number. MW, molecular weight. (B) Transient expression of HBP1 represses endogenous p47 phox mRNA. 293T cells were cotransfected with 2 μg of F-GFP and 5 μg of HBP1 or control plasmids. The cells were selected by FACS 36 h after transfection. The mRNA was immediately isolated from the GFP-selected cells and analyzed by RT-PCR for the p47phox gene. One microgram of RNA was used with 18S RNA as an internal control with 18S competimers (Ambion; see Materials and Methods). Amplified products were analyzed at the indicated cycle numbers. (C) Transient expression of Δ393 increases p47phox mRNA. Cells were transfected with 2 μg of F-GFP or cotransfected with 5 μg of Δ393. The purification of transfected cells and RT-PCR analysis are as described for panel B. (D) Expression of Δ393 results in increased p47phox protein levels. HEK-293T cells were transfected with either 5 μg of the pmΔ393 (lanes 2 and 3) plasmid or Δ393 (lanes 4 and 5). Western blotting was performed on protein lysates using anti-human p47 phox antisera (a generous gift of Tom Leto) (top). Lane 1 contains untransfected 293T lysate. The blot was stripped and immunoblotted (IB) with anti-HA to show expression of the Δ393 and pmΔ393 proteins.

FIG. 4.

HBP1 regulates endogenous superoxide levels. (A) Effects on superoxide production depend upon specific DNA binding. HEK-293T cells were cotransfected with 5 μg of HBP1, Δ393, pmHMG, or pmΔ393 and with F-GFP. After 36 h, the relative superoxide production level was determined by using lucigenin as a substrate. Superoxide levels from F-GFP-transfected samples were set to 100%. A total of 2 × 10⁶ cells were used for each sample. (B) The addition of exogenous p47phox partially rescues HBP1-mediated superoxide reduction. HEK-293T cells were cotransfected with 2 μg of F-GFP and the indicated plasmids. The p47phox protein was expressed from either 1, 3, or 5 μg of pREP10-p47 phox with or without 5 μg of HBP1. The relative superoxide levels were determined as described for panel A and in Materials and Methods. The level in the control F-GFP expressing cells was set to a value of 1. (C) GSH levels in control C2C12 cells and in HBP1-expressing C2C12 lines (B1 and B2). GSH was measured three times independently as described in Materials and Methods. GSH levels in HBP1 cells lines B1 and B2 were significantly lower than that in the corresponding control C2C12 line, as determined by Student's t test (P < 0.05). (D) GSH levels in the livers of nontransgenic (non-tg) and HBP1 transgenic mouse strains. GSH levels in the livers of 1Z1 and HBP4 transgenic mice was significantly lower than those in the corresponding livers of nontransgenic mice, as determined by Student's t test (P < 0.05).

FIG. 5.

Δ393 expression increases cell cycle progression in an NADPH oxidase-dependent manner. (A) Expression of HBP1 and Δ393 oppositely regulate the cell cycle. NIH3T3 cells were transfected with 0.5 μg of F-GFP and 5 μg of the indicated plasmid DNAs with Lipofectamine 2000 (Promega) in DMEM with 10% fetal calf serum. After 36 h, the cells were pulsed with BrdU for 2 h and were then stained with mouse anti-BrdU (Roche) (see Materials and Methods). GFP expression was used to mark the transfected cells. The percentage of BrdU-positive cells that are also GFP positive is a measure of the S-phase population. Approximately 400 GFP-positive cells were counted in each of three individual experiments. (B) Δ393 expression increases superoxide production that is inhibited by the addition of DPI. Superoxide assays with lucigenin were performed as described in the legend to Fig. 4. DPI is a specific inhibitor of NADPH oxidase and similar flavoprotein enzymes. DPI in dimethylsulfoxide was added at 24 h after transfection at a concentration of 5 μM. (C) DPI inhibits the Δ393 increase in S-phase cells. BrdU incorporation assays were performed as described for panel B with the addition of DPI as in the experiment presented in panel A. Veh., vehicle.

FIG. 5.

Δ393 expression increases cell cycle progression in an NADPH oxidase-dependent manner. (A) Expression of HBP1 and Δ393 oppositely regulate the cell cycle. NIH3T3 cells were transfected with 0.5 μg of F-GFP and 5 μg of the indicated plasmid DNAs with Lipofectamine 2000 (Promega) in DMEM with 10% fetal calf serum. After 36 h, the cells were pulsed with BrdU for 2 h and were then stained with mouse anti-BrdU (Roche) (see Materials and Methods). GFP expression was used to mark the transfected cells. The percentage of BrdU-positive cells that are also GFP positive is a measure of the S-phase population. Approximately 400 GFP-positive cells were counted in each of three individual experiments. (B) Δ393 expression increases superoxide production that is inhibited by the addition of DPI. Superoxide assays with lucigenin were performed as described in the legend to Fig. 4. DPI is a specific inhibitor of NADPH oxidase and similar flavoprotein enzymes. DPI in dimethylsulfoxide was added at 24 h after transfection at a concentration of 5 μM. (C) DPI inhibits the Δ393 increase in S-phase cells. BrdU incorporation assays were performed as described for panel B with the addition of DPI as in the experiment presented in panel A. Veh., vehicle.

FIG. 5.

Δ393 expression increases cell cycle progression in an NADPH oxidase-dependent manner. (A) Expression of HBP1 and Δ393 oppositely regulate the cell cycle. NIH3T3 cells were transfected with 0.5 μg of F-GFP and 5 μg of the indicated plasmid DNAs with Lipofectamine 2000 (Promega) in DMEM with 10% fetal calf serum. After 36 h, the cells were pulsed with BrdU for 2 h and were then stained with mouse anti-BrdU (Roche) (see Materials and Methods). GFP expression was used to mark the transfected cells. The percentage of BrdU-positive cells that are also GFP positive is a measure of the S-phase population. Approximately 400 GFP-positive cells were counted in each of three individual experiments. (B) Δ393 expression increases superoxide production that is inhibited by the addition of DPI. Superoxide assays with lucigenin were performed as described in the legend to Fig. 4. DPI is a specific inhibitor of NADPH oxidase and similar flavoprotein enzymes. DPI in dimethylsulfoxide was added at 24 h after transfection at a concentration of 5 μM. (C) DPI inhibits the Δ393 increase in S-phase cells. BrdU incorporation assays were performed as described for panel B with the addition of DPI as in the experiment presented in panel A. Veh., vehicle.

FIG. 6.

Model of HBP1 repression and ROS regulation. The results of this paper are summarized in this schematic diagram. The repression of the endogenous p47phox gene results in decreased superoxide levels and the inhibition of proliferation. Some aspects of Ras and small G protein signaling utilize the NADPH oxidase and ROS levels as part of the signaling mechanisms. A prediction is that cells with high HBP1 expression may be refractory to growth signals that emanate from tyrosine kinase receptors and the Ras/Rac/Rho signaling networks.