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. 2008 Dec 1;112(12):4494-502.
doi: 10.1182/blood-2007-12-127621. Epub 2008 May 28.

Heme oxygenase-1 Deficiency Leads to Disrupted Response to Acute Stress in Stem Cells and Progenitors

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Heme oxygenase-1 Deficiency Leads to Disrupted Response to Acute Stress in Stem Cells and Progenitors

Yu-An Cao et al. Blood. .
Free PMC article

Abstract

An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies.

Figures

Figure 1
Figure 1
Differential expression profiles of HO-1 during different stages of hematopoietic development and changes in levels of intracellular ROS, p38MAPK phosphorylation, and p21 expression in HO-1–deficient mice under steady-state conditions. Representative FACS plots of the levels of intracellular HO-1 protein (A), ROS (B), and p38MAPK phosphorylation (C) in whole BM, HSCs, MPPs, and restricted progenitors. BM cells from HO-1+/+ and HO-1+/− mice (n = 6 for each group) were harvested and stained with antibodies for phenotypically defined HSC (CD150+CD48CD244), MPP (CD244+CD48CD150), and more restricted progenitor (CD48+CD244+CD150) populations. To assess intracellular ROS levels, stained cells were incubated with DCF, a fluorescent dye for detection of intracellular ROS. For measurement of intracellular HO-1 protein, and p38MAPK phosphorylation, stained cells were fixed, permeabilized, and then stained with antibodies against HO-1, and phosphorylated p38MAPK, respectively. The broken line in the first panel of Figure 1A is the isotope control with FITC-conjugated anti–mouse IgG antibody. Values are the mean percentages indicating the gated proportion of cells. Please note that some of the histograms were skewed to the left due to the number of events on the y-axis. p21 expression was assessed by real-time PCR in HO-1–deficient cells. KTLS HSCs or Lin BM cells were isolated from HO-1+/− or HO-1+/+ mice at steady state, total RNA was prepared from these cells for p21 mRNA quantitation using real-time PCR. p21 mRNA levels were accumulated in purified KTLS HSCs (displayed is an average fold change of four experiments) (D) but decreased in HO-1+/− Lin BM cells (mean ± SD, n = 4) (E). The frequency of KTLS HSCs was analyzed from nucleated BM cells and are displayed as the mean (n = 3) (F).
Figure 2
Figure 2
Accelerated hematopoietic recovery in HO-1+/− mice treated with 5-FU and reconstitution in recipients of large numbers of HO-1+/− BM cells. (A) Leukocyte counts of HO-1+/− mice after myelotoxic injury. HO-1+/− or HO-1+/+ mice (n = 10) were treated with a single dose of 5-FU (150 mg/kg intraperitoneally), and leukocyte counts in the peripheral blood were performed at different time points after treatment and are shown as a mean (± SD) percentage of normal leukocyte count. (B,C) Hematopoietic engraftment from HO-1–deficient BM cells in lethally irradiated recipients. BM cells (5 × 106) from luc+HO-1+/− or luc+HO-1+/+ mice were transferred into lethally irradiated recipients (n = 4) and hematopoietic engraftment was monitored by BLI. Representative BLI images of the recipients of luc+HO-1+/− or luc+HO-1+/+ cells at day 18 are displayed at the same scale (B). Time course of hematopoietic reconstitution, as measured by whole-body bioluminescent intensity (mean ± SD, photons/s) over time (C). (D) Representative FACS plots and (E) relative trend of CFSE+LinSca-1+ cell distribution versus number of cell division in vivo. Nucleated BM cells (1 × 107) from either HO-1+/− or HO-1+/+ mice were labeled by CFSE (1 μM) and transferred into lethally irradiated recipients. Cells recovered from the BM of the recipients 48 hours after transfer were pooled (3 mice/group) and costained by antibodies against lineage markers and Sca-1. The number of cell divisions and cell distribution were analyzed by CFSE intensity after gating for the LinSca-1+ population. Values are absolute (D) or relative (E) mean percentages (n = 4) of cells undergoing different numbers of cell divisions from 2 different experiments.
Figure 3
Figure 3
Ineffectiveness of primitive HO-1+/− cells in radioprotection of lethally irradiated recipients and in preservation of their adoptive reserve. (A) Kaplan-Meier plots showing a defect of HO-1+/− BM cells in providing radioprotection. BM cells (2 × 105) from either HO-1+/− or HO-1+/+ mice were transferred into lethally irradiated recipients (n = 30) and survival rates were compared. (B,C) Assessment of short-term hematopoiesis from limited number of HO-1+/− BM cells. BM (2 × 105) cells from either luc+HO-1+/− or luc+HO-1+/+ mice were transferred together with 2 × 106 unlabeled BM cells into lethally irradiated recipients and overall hematopoiesis was visualized over time by BLI. (B) Representative BLI images of the recipients at day 22 displayed at the same scale. (C) Kinetics of hematopoietic reconstitution from luc+ BM cells. (D,E) Frequency of HO-1+/− LinSca-1 + and LSK populations in hematopoietic cells recovered from recipients of 107 nucleated BM cells from either HO-1+/− or HO-1+/+ mice. CFSE-labeled nucleated BM cells were transferred into lethally irradiated recipients. Forty-eight hours after transfer, cells were recovered from BM of the recipients, pooled (3 mice/group), and costained by antibodies to lineage markers and Sca-1. The frequency of CFSE+ Lin Sca-1+ cells was analyzed between groups. Values are means of 2 experiments (D). In related experiments, a decreased frequency of the LSK population in hematopoietic tissues of recipients was observed. Because the levels of c-Kit expression are lower after transplantation and return to normal by approximately 2 weeks (A.J.W., unpublished observations), it enabled us to analyze LSK population in cells recovered from recipients. BM cells (2 × 106) from GFP+HO-1+/− or GFP+HO-1+/+ mice were transplanted into lethally irradiated recipients (n = 3) and donor-derived cells were then recovered for assessment of either donor-derived (GFP+) LSK population at day 20 after transfer (E).
Figure 4
Figure 4
Accelerated exhaustion of HO-1+/− HSCs provoked by stress of serial transplantation or repeated myelotoxic injuries in HO-1 deficiency. (A) Experimental design. Five thousand HSCs from luc+HO-1+/− or luc+HO-1+/+ mice were transferred into lethally irradiated primary recipients. Twelve weeks later, one-fifth of the BM recovered from these recipients was transplanted into secondary recipients, and so on, for a total of 4 transplantations. (B) Time course of hematopoietic reconstitution of serially transplanted recipients was measured by BLI. Values are shown as mean (± SD) whole-body BLI intensities (n = 10 for each group). (C,D) Time course of hematopoietic reconstitution from serially transplanted GFP+HO-1+/− or HO-1+/+ BM cells. Hematopoietic contribution from 2 × 106 BM cells to peripheral myeloid (Mac-1+) and lymphoid (CD3+ or B220+) subsets was analyzed by flow cytometry at days 49 (n = 6, P < .017), 98 (n = 5, P > .22), and 140 (n = 5, P > .35) in the primary recipients (C) and at days 49 (n = 5, P = .028) and 91 (n = 8, P = .0005) in the secondary recipients (D). (E) The adoptive (GFP+) HO-1+/− KTLS HSC compartment assessed at 12 weeks after secondary transplantation. Values displayed are mean (± SD) of 4 samples and P value is one-tailed. (F,G) Repeated myelotoxic injuries and accelerated exhaustion of HO-1+/− HSCs. HO-1+/− or HO-1+/+ mice were treated with doses of 5-FU (150 mg/kg, intraperitoneally) at weeks 1, 2, and 5, respectively. The frequency of KTLS HSC population in nucleated BM cells was analyzed at week 18, and mean (± SD); (n = 4) of KTLS numbers in LinThy1.1lo cells is displayed. P value is 1-tailed (F). In a separate experiment, mice were injected a fourth dose of 5-FU, followed by leukocyte counts in peripheral blood, and their relative WBC counts were compared with that of mice receiving a single dose of 5-FU. Values are mean (± SD) WBC count of HO-1+/− mice relative to that of HO-1+/+ after 4th dose (n = 6) of 5-FU versus that of 1st dose (n = 4) (G).
Figure 5
Figure 5
Differential p38MAPK phosphorylation and p21 induction upon acute stress in HO-1+/− HSCs and HPCs. (A) Mice were bled retro-orbitally to cause an approximately 10% blood loss, followed by heme challenge (50 μmol/kg, intraperiotoneally) to mimic a hemolysis. BM cells were prepared for immunostaining using antibodies against SLAM family members (CD150, CD48, and CD244) for measurement of levels of intracellular HO-1 protein (B) and ROS (C), phosphorylated p38MAPK (D), and p21 induction (E) within HSCs (CD150+CD48CD244), MPPs (CD244+CD48CD150), and more restricted progenitors (CD48+CD244+CD150). Values are the mean percentages indicating the gated proportion of cells. Please note that some of the histograms were skewed to the left because of the number of events on the y-axis.
Figure 6
Figure 6
Decreased total plasma bilirubin (milligrams per deciliter) levels in (A) 5-FU–treated HO-1+/− mice or (B) recipients of HO-1+/− BM cells. Levels of total plasma bilirubin in HO-1+/− or HO-1+/+ mice at day 8 after receiving a single dose of 5-FU (150 mg/kg, intraperitoneally) and in the recipients of 2 × 106 HO-1+/− or HO-1+/+ BM cells at day 22 day after BM transplantation. P values were calculated using one-tailed t test.

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