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. 2010 Jan 21;115(3):677-86.
doi: 10.1182/blood-2009-05-223107. Epub 2009 Nov 9.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

Hozumi Motohashi  1 , Momoko Kimura, Rie Fujita, Ai Inoue, Xiaoqing Pan, Mariko Takayama, Fumiki Katsuoka, Hiroyuki Aburatani, Emery H Bresnick, Masayuki Yamamoto
Affiliations

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

Hozumi Motohashi et al. Blood. .

Abstract

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

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Figures

Figure 1
Figure 1
p45 competitively inhibits Nrf2-mediated transcriptional activation. (A) Quantitative RT-PCR of platelet genes and cytoprotective genes in p45-null megakaryocytes. The relative values were calculated against the values of WT megakaryocytes. (B) Immunoblot analysis of nuclear extracts of p45-null and WT megakaryocytes cultured from fetal livers. Anti-Nrf2 and anti–lamin B antibodies were used. (C) Comparison of protein abundance of p45 and Nrf2. FLAG-tagged p45 and FLAG-tagged Nrf2 were transiently overexpressed in 293T cells. Whole-cell extracts were prepared and subjected to immunoblot analysis with anti-FLAG or anti–lamin B antibodies. formula image and ◀ indicate FLAG-Nrf2 and FLAG-p45, respectively. (D) Comparison of transcriptional activation abilities of p45 and Nrf2. Equal amount of the expression vectors were introduced into 293T cells with a luciferase gene driven by MARE of the Nqo1 promoter in triplicate as a reporter gene. (E) Reporter assay in 293T cells with the same reporter gene used in panel D. Expression vectors of Nrf2 and/or p45 without tags were added as effector molecules. (F) Comparison of transcriptional activation abilities of FLAG-tagged Nrf2 and FLAG-tagged fusion protein of the N-terminal half of Nrf2 and C-terminal half of p45 with the same reporter gene used in panel D. The protein expression detected by anti-FLAG antibody is shown below. The relative luciferase activities were calculated against the activity generated by the reporter gene alone (D-F). The average values of triplicate experiments are presented, and the error bars represent SD (A,D-F). The Student t test was used to calculate statistical significance. *P < .05; **P < .005 (A,D-F).
Figure 2
Figure 2
Competitive regulation of cytoprotective genes by p45 and Nrf2. (A) ChIP assays were performed with primary megakaryocytes derived from WT fetal livers using anti-p45 and anti–trimethyl-histone H3 (Lys4) antibodies. (B-C) ChIP assays were performed with primary megakaryocytes derived from p45-null and control fetal livers using anti-p45 and anti-Nrf2 antibodies (B) and anti–trimethyl-histone H3K4, anti–dimethyl-histone H3K4, anti–acetyl-histone H3, and anti–acetyl-histone H4 antibodies (C). Enrichment of the promoter regions containing MAREs of the Txas and Nqo1 genes is shown. The third intron of the Txas gene was used as a negative control. Quantitative analysis was performed, and the average values and SD were calculated from the triplicate samples (B-C). The Student t test was used to calculate statistical significance. *P < .02; **P < .002 (B-C). (D-E) Effect of p45 deletion on the expression of platelet genes (D) and cytoprotective genes (E) in the presence (top panels) and in the absence (bottom panels) of Nrf2. Quantitative RT-PCR was performed, and the relative values were calculated against the values of WT (D top panel), Nrf2−/− (D bottom panel), the mixture of WT and p45+/− (E top panel), and the mixture of Nrf2−/− and Nrf2−/−:p45+/− (E bottom panel) megakaryocytes. The Student t test was used to calculate statistical significance. *P < .002; **P < .05; ***P < .1 (D-E).
Figure 3
Figure 3
p45 promotes ROS accumulation and increased ROS enhances platelet gene activation. (A) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes from p45-null and WT livers (top left panel), Nrf2−/− and Nrf2−/−:p45−/− fetal livers (top middle panel), and Nrf2-null and WT livers (top right panel). The intensities of DCFDA in the CD41+CD61+ cells are displayed in histograms. Average ROS levels obtained from the histograms of p45-null, Nrf2−/−:p45−/−, and Nrf2-null cells were changed to relative values against those from the WT, Nrf2-null, and WT cells, respectively (bottom panel). Average values are shown with SD. Statistical significance of the relative ROS levels was calculated using paired t test. (B) Quantitative RT-PCR of platelet genes in primary megakaryocytes after supplementation with 10mM NAC for scavenging ROS. The relative values were calculated against the values of the vehicle-treated samples. (C) Primary megakaryocytes were fractionated into either DCFDA high or DCFDA low populations according to the fluoroprobe intensity in flow cytometry. The gates are depicted in bold rectangles. (D) Quantitative RT-PCR of platelet genes (left panel) and of p45 and Nrf2 (right panel). The CD41+DCFDAhigh and CD41+DCFDAlow populations were compared. The relative values were calculated against the values of the CD41+DCFDAlow fraction. (E) Primary megakaryocytes cultured from p45-null and WT fetal livers were fractionated as in panel C. The intensities of DCFDA of CD41+ cells contained in the gate indicated with broken lines in the left panel are displayed in histograms (right panel). L and H in the right panel correspond to the gates depicted in bold rectangles in the left panel. (F) Quantitative RT-PCR of platelet genes. The CD41+DCFDAhigh and CD41+DCFDAlow populations from p45-null and WT fetal livers were compared. The relative values were calculated against the values of the CD41+DCFDAlow fraction of WT cells. The average values of triplicate experiments are presented, and the error bars represent SD (B,D,F). The Student t test was used to calculate statistical significance. *P < .001 (B,D). **P < .05 (B,D). **P < .005 (F).
Figure 4
Figure 4
Nrf2 activation reduces ROS levels and platelet gene expression. (A) Nuclear accumulation of Nrf2 in primary megakaryocytes by DEM treatment. DEM was added to a final concentration of 100 μM every 24 hours. CD41+ cells were isolated from day 1 and day 3 cultures, and nuclear extracts were prepared. Anti-Nrf2 and anti–lamin B antibodies were used. (B) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes treated with DEM. The intensities of DCFDA in the CD41+CD61+ cells are displayed in histograms. Average ROS levels obtained from the histograms of DEM-treated cells were changed to relative values against those from vehicle-treated cells. (C) Quantitative RT-PCR of platelet genes (top panel) and of cytoprotective genes (bottom panel) in DEM-treated megakaryocytes. The relative values were calculated against the values of vehicle-treated megakaryocytes. (D) Nuclear accumulation of Nrf2 in primary megakaryocytes cultured from Keap1-null fetal livers. CD41+ cells were isolated, and nuclear extracts were prepared. Anti-Nrf2 and anti–lamin B antibodies were used. (E) Flow cytometric analysis of intracellular ROS levels in Keap1-null megakaryocytes. The intensities of DCFDA in the CD41+CD61+ cells are displayed in histograms. Average ROS levels obtained from the histograms of Keap1-null megakaryocytes were changed to relative values of those from WT cells. Average values are shown with SD (B,E). Statistical significance of the relative ROS levels was calculated using paired t test (B,E). (F) Quantitative RT-PCR of platelet genes (top panel) and of cytoprotective genes (bottom panel) in Keap1-null megakaryocytes. The relative values were calculated against the values of WT megakaryocytes. The average values of triplicate experiments are presented, and the error bars represent SD (C,F). The Student t test was used to calculate statistical significance. *P < .002 (C,F).
Figure 5
Figure 5
Increased p45 and ROS levels correlate with megakaryocytic maturation. (A) Expression of p45 and Nrf2 during megakaryocytic maturation. mRNA levels (left panel) and protein levels in nuclei (right panel) are shown. CD41+ cells were isolated from day 1 and day 3 cultures, and total RNA and nuclear extracts were prepared. The relative values were calculated against the values of day 1 megakaryocytes (left panel). Anti-Nrf2, anti-p45, and anti–lamin B antibodies were used (right panel). (B) Flow cytometric analysis showing megakaryocytic differentiation in day 1 and day 3 cultures. Frequencies of CD41+CD61+ cells are shown in the figure (top panel). Relative frequencies of CD41+CD61+ cells were calculated against the value of day 1 culture (bottom panel). Average values are shown with SD. Statistical significance of the relative ratio of CD41+CD61+ cells was calculated using paired t test. (C) Flow cytometric analysis of intracellular ROS levels in primary megakaryocytes at day 1 and day 3. The intensities of DCFDA of CD41+CD61+ cells in panel B (gates depicted in bold rectangles) are shown in the histogram. Average ROS levels obtained from the histograms of day 3 megakaryocytes were changed to relative values against those from day 1 cells. Average values are shown with SD. Statistical significance of the relative ROS levels was calculated using paired t test. (D) Quantitative RT-PCR of platelet genes in day 1 and day 3 megakaryocytes. The relative values were calculated against the values of the day 1 sample. The average values of triplicate experiments are presented, and the error bars represent SD (A,D). The Student t test was used to calculate statistical significance. *P < .05 (A). *P < .001 (D).
Figure 6
Figure 6
Nrf2 cooperates with p45 to promote megakaryocytic proliferation but does not activate platelet genes. (A) DNA content of primary megakaryocytes cultured from Keap1-null and control fetal livers (left panel). Frequencies of CD41+ cells containing DNA contents of 2n and between 2n and 4n are indicated. A relative number of CD41+ cells of each ploidy against the total CD41+ cells generated in the WT culture is shown in the right panel. Representative data from 2 independent experiments are shown. (B) Flow cytometric analysis of fetal liver cultures. Cells were stained with FITC-conjugated CD41 and PE-conjugated CD61. Keap1-null cells (i), Nrf2-null cells (ii), and p45-null cells (iii) were compared with control WT cells derived from their corresponding littermates. Nrf2−/−:p45−/− cells were compared with Nrf2-null cells (iv). Representative data are presented from more than 3 independent experiments. Frequencies of CD41+CD61+ cells are shown in the figure. Relative frequencies of CD41+CD61+ cells were calculated against the value of the WT culture (for Keap1-null, Nrf2-null, and p45-null cells) and Nrf2-null culture (for Nrf2−/−:p45−/− cells) (bottom panel). (C) Flow cytometric analysis showing megakaryocytic differentiation in fetal liver cultures from WT, p45-null, and p45−/−:Keap1−/− mice obtained from the same litter. Cells were stained with FITC-conjugated CD41 and PE-conjugated CD61. Frequencies of CD41+CD61+ cells are shown in the figure (left panel). Relative frequencies of CD41+CD61+ cells were calculated against the value of the mixture of WT and Keap1+/− culture (right panel). Average values are shown with SD (B-C). Statistical significance of the relative ratio of CD41+CD61+ cells was calculated using paired t test (B-C). (D) Quantitative RT-PCR of platelet genes and cytoprotective genes in primary megakaryocytes cultured from Keap1+/−, p45−/−:Keap1+/−, and p45−/−:Keap1−/− fetal livers obtained in the same litter. The relative values were calculated against the values of Keap1+/− megakaryocytes. The average values of triplicate experiments are presented, and the error bars represent SD. The Student t test was used to calculate statistical significance. *P < .001. **P < .005.
Figure 7
Figure 7
Model for p45 and Nrf2-regulated megakaryocytic maturation. (A) In WT megakaryocytes at an immature stage (WT, left panel), Nrf2 seems to make a major contribution to cytoprotective gene regulation, which could promote cell proliferation. In mature megakaryocytes (WT, right panel), p45, as a modest activator, seems to maintain the moderate expression of cytoprotective genes by competing with Nrf2 and to enhance ROS accumulation. Increased ROS cooperates with p45 to promote platelet gene expression. (B) In p45-null cells, platelet genes are dramatically repressed and cytoprotective genes are induced by Nrf2, resulting in the reduction of ROS levels. In Nrf2-null cells, neither gene expressions nor ROS levels are changed. In both cases, proliferation of megakaryocytes is impaired (right panels). In Keap1-null cells, constitutively activated Nrf2 strongly activates cytoprotective genes but not platelet genes. ROS accumulation is inhibited, and proliferation of immature megakaryocytes is promoted (left panel). Solid and broken black arrows indicate explicit and ineffective interaction, respectively.
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