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. 2019 Jul;37(7):937-947.
doi: 10.1002/stem.3015. Epub 2019 May 3.

p53-TP53-Induced Glycolysis Regulator Mediated Glycolytic Suppression Attenuates DNA Damage and Genomic Instability in Fanconi Anemia Hematopoietic Stem Cells

Xue Li  1   2 , Limei Wu  1 , Morgan Zopp  1 , Shaina Kopelov  1 , Wei Du  1   3
Affiliations

p53-TP53-Induced Glycolysis Regulator Mediated Glycolytic Suppression Attenuates DNA Damage and Genomic Instability in Fanconi Anemia Hematopoietic Stem Cells

Xue Li et al. Stem Cells. 2019 Jul.

Abstract

Emerging evidence has shown that resting quiescent hematopoietic stem cells (HSCs) prefer to utilize anaerobic glycolysis rather than mitochondrial respiration for energy production. Compelling evidence has also revealed that altered metabolic energetics in HSCs underlies the onset of certain blood diseases; however, the mechanisms responsible for energetic reprogramming remain elusive. We recently found that Fanconi anemia (FA) HSCs in their resting state are more dependent on mitochondrial respiration for energy metabolism than on glycolysis. In the present study, we investigated the role of deficient glycolysis in FA HSC maintenance. We observed significantly reduced glucose consumption, lactate production, and ATP production in HSCs but not in the less primitive multipotent progenitors or restricted hematopoietic progenitors of Fanca-/- and Fancc-/- mice compared with that of wild-type mice, which was associated with an overactivated p53 and TP53-induced glycolysis regulator, the TIGAR-mediated metabolic axis. We utilized Fanca-/- HSCs deficient for p53 to show that the p53-TIGAR axis suppressed glycolysis in FA HSCs, leading to enhanced pentose phosphate pathway and cellular antioxidant function and, consequently, reduced DNA damage and attenuated HSC exhaustion. Furthermore, by using Fanca-/- HSCs carrying the separation-of-function mutant p53R172P transgene that selectively impairs the p53 function in apoptosis but not cell-cycle control, we demonstrated that the cell-cycle function of p53 was not required for glycolytic suppression in FA HSCs. Finally, ectopic expression of the glycolytic rate-limiting enzyme PFKFB3 specifically antagonized p53-TIGAR-mediated metabolic reprogramming in FA HSCs. Together, our results suggest that p53-TIGAR metabolic axis-mediated glycolytic suppression may play a compensatory role in attenuating DNA damage and proliferative exhaustion in FA HSCs. Stem Cells 2019;37:937-947.

Keywords: Fanconi anemia; Glycolysis; Hematopoietic stem cells; TP53-inducible glycolysis and apoptosis regulator; p53.

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Conflict of interest statement

The authors indicated no potential conflicts of interest.

Figures

Figure 1
Figure 1
Reduced glycolysis in Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Gating strategy for FACS sorting. (B): Reduced glucose consumption in FA HSCs. SLAM (Lin−Sca‐1+c‐kit+CD150+CD48−) cells from the bone marrow of Fanca −/−, Fancc −/−, and wild‐type (WT) mice were isolated by FACS sorting and subjected to analysis for glucose consumption analysis. (C): Reduced lactate production in FA HSCs. SLAM cells described in (B) were analyzed for lactate production (C). (D): Lower ATP levels in FA HSCs. Relative intracellular ATP concentrations were measured in cells described in (A). Results depicted in (B)–(D) are means ± SD of three independent experiments (n = 6–9 per group). (E): Schematic presentation of glycolytic pathway. (F): Expression of the genes encoded for the key enzymes of the glycolysis pathway in WT and FA HSCs. RNA was extracted from SLAM cells described in (A) followed by quantitative polymerase chain reaction analysis for the indicated genes using primers listed in Supporting Information Table S1. Samples were normalized to the level of GAPDH mRNA (n = 6–9 per group). (G): Increased TP53‐induced glycolysis regulator (TIGAR) protein in FA HSPCs. Whole‐cell lysates were extracted from 30,000 Lin−Sca1+c‐kit+ cells isolated from WT, Fanca −/−, and Fancc −/− mice followed by immunoblotting using antibodies against TIGAR or β‐actin; *, p < .05; **, p < .01.
Figure 2
Figure 2
p53‐Dependent suppression of glycolysis in Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Increased p53 and TP53‐induced glycolysis regulator (TIGAR) proteins in FA HSPCs. Whole‐cell lysates were extracted from 30,000 Lineage−Sca‐1+c‐kit+ isolated from wild‐type (WT), Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice followed by immunoblotting using antibodies against p53, TIGAR, or β‐actin. (B): Deletion of p53 increases F2,6BP level in Fanca −/− HSCs. SLAM cells isolated from WT, Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice were subjected to MicroAssay for F26BP. (C, D): Deletion of p53 increases glucose consumption and lactate production in FA HSCs. SLAM cells isolated from WT, Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice were subjected to glucose uptake (C) and lactate production analysis (D). Results depicted in (B)–(D) are means ± SD of three independent experiments (n = 6–9 per group); *, p < .05; **, p < .01.
Figure 3
Figure 3
p53‐TP53‐induced glycolysis regulator (TIGAR)‐mediated glycolytic suppression attenuates reactive oxygen species (ROS), DNA damage, and chromosomal instability in Fanconi anemia (FA) HSPCs. (A): Overactivation of p53 in FA HSPCs. Whole cell lysates (WCLs) were extracted from Lineage−Sca‐1+c‐kit+ (LSK) cells isolated from wild‐type (WT), Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice followed by Western blotting using antibodies against total p53, p53ser18, and β‐actin. (B): Lentiviral transduction and FACS sorting. LSK cells isolated from WT, Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice were transduced with lentiviral vector expressing mCherry or mCherry‐TIGAR. Flow plots after FACS sorting for mCherry are shown. The sorted mCherry+ cells were then subjected to ex vivo expansion for 72 hours followed by the analyses described below. (C): Expression of TIGAR in transduced HSCs. RNA was extracted from mCherry+ SLAM cells described in (B) were subjected to quantitative polymerase chain reaction analysis for TIGAR expression. Samples were normalized to the level of GAPDH mRNA. (D): Overexpression of TIGAR reduces ROS level in HSCs. Cells described in (B) were gated for mCherry+ SLAM population and subjected to flow cytometry analysis for ROS. Representative plots (left) and quantification (right) are shown. (E): Overexpression of TIGAR reduces DNA damage in FA HSPCs. WCLs were extracted from mCherry+ cells described in (B) followed by Western blotting using antibodies against γ‐H2AX and β‐actin. (F): Overexpression of TIGAR reduces spontaneous chromosomal breakage in FA HSPCs. mCherry+ cells described in (B) were subjected to chromosomal breakage assay. Representative images (left) and quantification of 50 cells in random fields (right) are shown. Arrowheads point to chromosome breaks or radial structures. (G, H): Overexpression of TIGAR enhances the pentose phosphate pathway and cellular antioxidant function in FA HSCs. Cells described in (B) were gated for mCherry+ population and subjected to assays for NADPH/NADP+ ratio (F) or intracellular GSH analysis. Results depicted in (C)–(H) are means ± SD of three independent experiments; *, p < .05; **, p < .01; ***, p < .001.
Figure 4
Figure 4
p53‐TP53‐induced glycolysis regulator (TIGAR) ameliorates exhaustion of Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Limited dilution assay. Various doses of sorted transduced Lineage−Sca‐1+c‐kit+ (LSK) cells (25, 75, 150, and 250 mCherry+ cells) expressing mCherry or mCherry‐TIGAR were mixed with 2 × 105 protector bone marrow (BM) cells (CD45.1+) and transplanted into irradiated congenic recipients (n = 8–10 per group). Plotted are the percentages of recipient mice containing <1% CD45.2+ blood nucleated cells (negative) at 8 weeks after transplantation. Frequency of functional HSCs was calculated according to Poisson statistics. (B): Overexpression of TIGAR improves hematopoiesis reconstitution capacity of FA HSPCs. One thousand sorted mCherry or mCherry‐TIGAR lentiviral vector transduced LSK cells were transplanted into lethally irradiated BoyJ mice. The recipients were subjected to flow cytometry analysis for donor‐derived cells 16 weeks after BM transplantation. Representative flow plots (left) and quantification (right) are shown (n = 9 per group). (C): Overexpression of TIGAR improves long‐term repopulating activity of FA HSCs. One million BM cells from the primary recipient mice described in (B) were transplanted into sublethally irradiated secondary CD45.1+ recipient mice and donor‐derived chimera in secondary recipients were determined by flow cytometry 16 weeks later (n = 9 per group). (D): Overexpression of TIGAR reduces DNA damage in donor cells. CD45.2+ LSK cells from the recipients described in (B) were subjected to flow cytometry analysis for γ‐H2AX. Representative plots (upper) and quantification (lower) are shown. (E): Overexpression of TIGAR increases donor HSC quiescence. Percentage of Pyronin Y+ cells in donor HSC compartment (CD45.2+ SLAM) was analyzed by flow cytometry. Representative flow plots (left) and quantification (right) are shown (n = 6 per group); *, p < .05; **, p < .01; ***, p < .001.
Figure 5
Figure 5
p53‐Dependent cell‐cycle control is not required for TP53‐induced glycolysis regulator (TIGAR)‐mediated metabolic reprograming. (A): Overexpression of TIGAR reduces glucose consumption in p53 R172P hematopoietic stem cells (HSCs). Bone marrow Lineage−Sca‐1+c‐kit+ cells from wild‐type, Fanca −/−, p53 R172P, and Fanca −/− p53 R172P mice were transduced with lentivirus expressing mCherry‐TIGAR or mCherry alone. The sorted mCherry+ cells were expanded for 72 hours. The transduced cells were gated for mCherry+ SLAM population and assayed for glucose consumption. (B): Overexpression of TIGAR reduces PFK1 activity in p53 R172P HSPCs. Cells described in (A) were gated for mCherry+ population and assayed for PFK1 activity. (C): Overexpression of TIGAR reduces ROS production in p53 R172P HSPCs. Cells described in (A) were gated for mCherry+ SLAM population and analyzed for intracellular ROS by flow cytometry (n = 6 per group). (D): Overexpression of TIGAR reduces DNA damage in p53 R172P HSPCs. WCLs were extracted from mCherry+ cells described in (A) followed by Western blotting using antibodies against γ‐H2AX and β‐actin. Results depicted in (A) and (B) are means ± SD of three independent experiments; *, p < .05; **, p < .01.
Figure 6
Figure 6
PFKFB3 specifically antagonizes p53‐TP53‐induced glycolysis regulator metabolic function in Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Lentiviral mCherry‐PFKFB3 transduction and FACS sorting. LSK cells isolated from wild‐type, Fanca −/−, p53 −/−, or p53 −/− Fanca −/− mice were transduced with lentiviral vector expressing mCherry or mCherry‐PFKFB3. Flow plots after FACS sorting for mCherry are shown. The sorted mCherry+ cells were then subjected to ex vivo expansion for 72 hours followed by the analyses described below. (B): Expression of PFKFB3 in transduced HSCs. Cells described in (A) were gated for mCherry+ SLAM population and RNA was extracted for q‐PCR analysis for PFKFB3 expression. Samples were normalized to the level of GAPDH mRNA (n = 6 per group). (C): Overexpression of PFKFB3 increases Fru‐2,6‐BP production. Cells described in (A) were gated for mCherry+ SLAM population and analyzed for F26BP by MicroAssay. (D): Overexpression of PFKFB3 increases PFK1 activity. Cells described in (A) were gated for mCherry+ population and subjected to analysis for PFK1 activity. (E): Overexpression of PFKFB3 increases intracellular GSH. Cells described in (A) were gated for mCherry+ population and subjected to GSH assay. (F): Overexpression of PFKFB3 increases ROS. Cells described in (A) were gated for mCherry+ population and subjected to analysis for ROS by flow cytometry. Representative plots (left) and quantification (right) are shown (n = 6 per group); *, p < .05; **, p < .01.
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