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. 2011 Dec 15;118(25):6572-9.
doi: 10.1182/blood-2011-05-355362. Epub 2011 Oct 28.

The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels

Akil A Merchant  1 , Anju Singh, William Matsui, Shyam Biswal
Affiliations

The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels

Akil A Merchant et al. Blood. .

Abstract

Several studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2(-/-)) BM were not elevated compared with wild-type. Nrf2(-/-) HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2(-/-) BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2(-/-) HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2(-/-) cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

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Figures

Figure 1
Figure 1
Nrf2−/− mice have abnormalities in BM and HSPC function. (A) Nrf2 and target gene expression is highest in HSPCs. Expression of the Nrf2 target genes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and heme oxygenase 1 (HO-1) were quantified in pooled BM from 5 Nrf2+/+ mice. Cells from the KSL, myeloid (c-Kit+Sca1−Lin−), and lymphoid (c-KitdimSca1+Lin−) progenitor compartments were isolated by FACS and quantitative RT-PCR performed in triplicate. Gene expression was normalized to β-actin and quantified relative to KSL cells. (B) Whole BM from Nrf2−/− mice has decreased stem cell activity. Whole BM cells (666 000) from Nrf2+/+ and Nrf2−/− were mixed with 333 000 CD45.1 competitor normal BM cells (2:1 ratio) and injected into lethality irradiated CD45.1 hosts. Peripheral blood was analyzed for donor chimerism at the indicated time points. Data are the combined averages from 6 total mice per genotype from 2 separate experiments. *P < .05. (C) Phenotypic HSPCs from Nrf2−/− show defective transplantation. Five hundred KSL cells from Nrf2+/+ and Nrf2−/− mice were mixed with 250 000 CD45.1 competitor normal BM cells and injected into lethally irradiated CD45.1 hosts. Peripheral blood was analyzed for donor chimerism at the indicated time points. Data represent the average peripheral blood engraftment from 10 total recipient mice per genotype in 2 separate experiments.*P < .05. (D) BM cellularity in Nrf2+/+ and Nrf2−/− mice. Average number of cells per tibia. n = 6 mice each group. *P = .019. (E) Progenitor and stem cell numbers are increased in Nrf2−/− mice. Relative frequency of KSL, LT-HSC (CD34− c-Kit+Sca1+Lin−), and ST-HSC (CD34+ c-Kit+Sca1+Lin−) cells. (F) HSPCs in Nrf2−/− mice are more proliferative as shown by BrdU uptake in vivo. Mice were injected with 100 mg/kg of BrdU to mark actively proliferating cells, and BM was harvested 14 hours later and assessed for BrdU labeling by flow cytometry. Data represent the average number of BrdU-positive KSL cells for each genotype. P = .06. (G) Greater rates of spontaneous apoptosis in Nrf2−/− HSPCs. Freshly isolated BM cells were analyzed by flow cytometry for annexin V positivity to identify apoptotic cells. Data are average values of annexin V+ cells as a percentage of the KSL compartment. *P < .01. (H) Myeloid progenitor frequency. Nrf2−/− BM cells were examined by flow cytometry. Increased common myeloid progenitors (FcRγlowCD34+c-Kit+Sca1−Lin−) and decreased granulocyte-monocyte progenitors (FcRγhighCD34+c-Kit+Sca1−Lin−) were observed, with a trend (P < .1) toward statistical significance. (I) Early myeloid engraftment is impaired in mice transplanted with Nrf2−/− BM. Percentage of CD45.2+ cells in peripheral blood expressing myeloid lineage markers Gr1+/Mac1+ from mice transplanted with Nrf2+/+ and Nrf2−/− BM as in Figure 1C. *P = .007. (J) In vitro myeloid colony formation is impaired in Nrf2−/− mice. One hundred KSL cells were plated in methylcellulose medium supplemented with cytokines, and the colonies were scored at 14 days. *P < .05
Figure 2
Figure 2
Nrf2−/− HSPCs have no increase in basal ROS but are more susceptible to oxidative stress. (A) ROS levels in Nrf2+/+ and Nrf2−/− BM cells. BM isolated from Nrf2+/+ and Nrf2−/− mice was incubated with H2-CM-DCFDA to detect intracellular ROS by flow cytometry. Mean fluorescence intensity was measured in LT-HSC (CD34− c-Kit+Sca1+Lin−), ST-HSC (CD34+ c-Kit+Sca1+Lin−), and Lin+ cells. Relative MFIs are normalized to wild-type LT-HSCs. Data represent mean values from 13 mice of each genotype in 4 different experiments. (B) Induction of intracellular ROS in Nrf2+/+ and Nrf2−/− BM cells by increasing doses of H2O2. BM cultured in normoxia for 30 minutes with H2O2 (50-200μM) to induce oxidative stress. BM from 3 mice per genotype was pooled for each concentration of H2O2. Data are mean fluorescence intensity values of H2-CM-DCFDA (arbitrary units). (C) Nrf2−/− progenitors show increased sensitivity to oxidative stress in vitro. Whole BM cells were treated with 50μM H2O2 for 30 minutes, plated in methylcellulose medium supplemented with cytokines, and colonies were scored at 14 days. Data are mean values from 3 individual mice plated in duplicated. *P = .012. (D) Schema for testing in vivo sensitivity of Nrf2−/− LT-HSCs to radiation injury. Mice were transplanted with Nrf2 +/+ or Nrf2−/− and allowed to form stable chimeras. Mice were then treated with a sublethal dose of radiation (400 rads) and their chimerism compared 4 months later to assess the effect of radiation on LT-HSCs. (E) Nrf2−/− LT-HSCs show increased radiosensitivity. Four months after radiation, peripheral blood chimerism returned to baseline in mice transplanted with Nrf2+/+ BM, but remained much lower in Nrf2−/− chimeras, suggesting a selective loss of Nrf2−/− LT-HSCs. *P = .018
Figure 3
Figure 3
NAC protects HSPCs from exogenous ROS, but does not rescue transplantation defect in Nrf2−/− cells. (A) Experimental schema for NAC experiments. (B) Treatment with NAC protects Nrf2+/+ and Nrf2−/− HPSCs from induction of ROS. Mice were treated with daily IP injection of NAC (100 μg/kg/d) for 3 weeks and their BM harvested. Cells were labeled with Abs, treated with 50μM H2O2 for 30 minutes, and then stained with H2-CM-DCFDA to measure intracellular ROS. Data are average MFI values in the KSL population (arbitrary units). Three mice were analyzed in each group. (C) Treatment with NAC does not rescue transplantation defect in Nrf2−/− HPSCs. Mice were treated with NAC for 3 weeks, BM was harvested, and KSL cells were isolated by FACS. KSL cells (500/mouse) were transplanted, along with 250 000 CD45.1 competitor normal BM cells, and injected into lethally irradiated CD45.1 hosts. Peripheral blood was analyzed for donor chimerism at the indicated time points. Data are average peripheral blood engraftments from 5 total recipient mice per genotype. *P = .03 at 20 weeks.
Figure 4
Figure 4
G-CSF restores cytokine signaling and promotes survival of Nrf2−/− HSPCs. (A) Gene expression of inflammatory cytokines, cytokine receptors, and prosurvival molecules is down-regulated in Nrf2−/− KSL cells. BM was harvested from 5 mice/group and KSL cells were isolated by FACS. RNA was isolated from sorted cells and quantitative RT-PCR performed for specific genes. Data are average values from 2 separate experiments and are normalized to expression levels in Nrf2+/+ KSL cells (represented by the dotted line). BCL2A1 inidicates BCL2-related protein A1; CCL2, chemokine (C-C motif) ligand 2; CCL5, chemokine (C-C motif) ligand 5; CSF1, colony-stimulating factor 1 (macrophage); CSF1R, colony-stimulating factor 1 receptor; CXCL10, chemokine (C-X-C motif) ligand 10; IL1B, interleukin 1 beta; IL1R, interleukin 1 receptor type II; IL10, interleukin 10; and TREM1, triggering receptor expressed on myeloid cells 1. (B) G-CSF treatment leads to expansion of KSL, LT-HSC, and ST-HSC compartments in both Nrf2+/+ and Nrf2−/− mice. Mice were treated with 100 μg/kg of G-CSF daily or vehicle control for 1 week, and BM was isolated and labeled with Abs and analyzed by flow cytometry. Data are averages from 3 mice per group. (C) G-CSF treatment enhances survival of Nrf2+/+ and Nrf2−/− HSPCs. Mice were treated with 100 μg/kg of G-CSF daily or vehicle control for 1 week. BM from 3 mice in each group was isolated, pooled, and treated with H2O2 (50μm for 30 minutes) or control and cultured in serum-free medium at an atmospheric O2 concentration for 6 hours. Viable cells were annexin V−/propidium iodide−. Data are averages from 3 replicates. (D) G-CSF treatment corrects many of the gene-expression differences seen between Nrf2+/+ and Nrf2−/− KSL cells. Mice were treated with 100 μg/kg of G-CSF daily or control for 1 week, and KSL cells were isolated from BM. Quantitative RT-PCR was performed and analyzed as described in panel A.
Figure 5
Figure 5
G-CSF treatment induces ROS in HSPCs and rescues Nrf2−/− HSPC transplantation. (A) G-CSF treatment induces ROS in KSL cells. Nrf2+/+ and Nrf2−/− mice were treated with 100 μg/kg of G-CSF daily for 1 week and their BM was examined for ROS using H2-CM-DCFDA. G-CSF induces a 3-fold increase in ROS in the KSL compartment. Data are mean values from 3 mice in each group. *P < .05. (B) G-CSF treatment rescued the hematopoietic transplantation defect in the Nrf2−/− HSPCs. Nrf2+/+ and Nrf2−/− mice were treated with 100 μg/kg of G-CSF daily for 1 week. CD45.2 BM cells (250 000) from each genotype were transplanted with 250 000 CD45.1 competitor cells, and peripheral blood was examined for chimerism at the indicated time points. Peripheral blood chimerism was higher mice transplanted with Nrf2−/− cells, but this was not statistically significant (P = .19), at 20 weeks after transplantation (n = 5 in each group). (C) G-CSF treatment does not improve in vitro myeloid colony formation of Nrf2−/− BM. Nrf2+/+ and Nrf2−/− mice were treated with 100 μg/kg of G-CSF daily for 1 week, 20 000 unsorted BM cells were plated in methylcellulose medium supplemented with cytokines, and colonies were scored at 14 days. Data represent mean colony number from 3 separate mice each plated in duplicate (normalized to untreated wild-type). *P < .03.
Figure 6
Figure 6
Model demonstrating role of Nrf2 in HSPCs. (A) In Nrf2+/+ BM, normal levels of Nrf2 are associated with low levels of ROS in HSPCs and intact cytokine signaling. (B) In Nrf2−/− BM, basal levels ROS are not increased in HSPCs, but HSPC survival is decreased due to reduced cytokine signaling levels. Nrf2−/− stem/progenitor cells show increased sensitivity to induced ROS. (C) G-CSF induces ROS in both Nrf2+/+ and Nrf2−/− HSPCs. Although Nrf2 loss is associated with increased sensitivity to ROS, G-CSF is able to restore HSPC survival despite an increase in ROS.
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