【原】MDH1介导苹果酸-天冬氨酸NADH穿梭维持胎儿肝造血干细胞的活性水平。(Sonar signal)

GCTA 2022-06-11 发布于贵州

展开全文

MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

Visual Abstract

|核心内容：

造血干细胞在不同发育阶段的能量代谢和干细胞之间的关系仍然很大程度上是未知的。

我们为 NADH/NAD+ 传感器(SoNar)构建了一个转基因小鼠系，并根据 SoNar 荧光比率分析了3个不同的胎肝造血细胞群。

Sonar 值低的细胞线粒体呼吸水平增强，但糖酵解水平与 sonar 值高的细胞相似。

有趣的是，10% 的 SoNar-low 细胞含有65% 的免疫表型胎儿肝造血干细胞(fl-hsc) ，并且比 SoNar-high 细胞含有大约五倍的功能性造血干细胞。

Sonar 能够敏感地监测体内外造血干细胞能量代谢的动态变化。

STAT3通过反式激活 MDH1维持苹果酸-天冬氨酸 NADH 的穿梭活性和 HSC 的自我更新和分化。

我们揭示了一个意想不到的 fl-hsc 代谢程序，为造血干细胞或其他类型干细胞的代谢研究提供了一个强有力的遗传工具。

原文摘要：

The connections between energy metabolism and stemness of hematopoietic stem cells (HSCs) at different developmental stages remain largely unknown.

We generated a transgenic mouse line for the genetically encoded NADH/NAD⁺ sensor (SoNar) and demonstrate that there are 3 distinct fetal liver hematopoietic cell populations according to the ratios of SoNar fluorescence.

SoNar-low cells had an enhanced level of mitochondrial respiration but a glycolytic level similar to that of SoNar-high cells. Interestingly, 10% of SoNar-low cells were enriched for 65% of total immunophenotypic fetal liver HSCs (FL-HSCs) and contained approximately fivefold more functional HSCs than their SoNar-high counterparts.

SoNar was able to monitor sensitively the dynamic changes of energy metabolism in HSCs both in vitro and in vivo.

Mechanistically, STAT3 transactivated MDH1 to sustain the malate-aspartate NADH shuttle activity and HSC self-renewal and differentiation.

We reveal an unexpected metabolic program of FL-HSCs and provide a powerful genetic tool for metabolic studies of HSCs or other types of stem cells.

Key Points

FL-HSCs mainly use oxidative phosphorylation but with normal glycolysis, as indicated by a highly responsive NADH/NAD+ sensor.
FL-HSC activities are tightly regulated by the STAT3/MDH1-mediated malate-aspartate NADH shuttle.

Subjects:

Hematopoiesis and Stem Cells

Topics:

aspartate, fetus, fluorescence, liver, malates, mice, transgenic, mitochondria, nicotinamide adenine dinucleotide (nad), stat3 protein, hematopoietic stem cells

Introduction

Hematopoietic stem cells (HSCs) originate from the aorta-gonad-mesonephros region¹ and migrate into the fetal liver (FL) and undergo dramatic expansion,^2,3 gradually localizing to and residing in the bone marrow niche after birth.⁴

HSCs can self-renew to maintain the stem cell pool and generate all downstream progenitors and terminally differentiate into multiple lineages.^5,6

Increasing evidence indicates that the metabolic state is tightly connected to HSC activity.^7-9

Adult HSCs preferentially undergo glycolysis, rather than oxidative phosphorylation, in the hypoxic niche,^7,10,11 which is extensively regulated by several signaling pathways, including HIF1A,¹² MYC,¹³ PDK,¹⁴ DLK-GTL2,¹⁵ and vitamin A–retinoic acid signaling.¹⁶

We have also shown that both murine and human HSCs adopt a glycolytic metabolic profile under certain conditions and that this profile is fine-tuned by MEIS1/PBX1/HOXA9/HIF1A signaling pathways.^17-19

Interestingly, recent studies have suggested that adult HSCs also have high mitochondrial mass and enhanced dye efflux but possess limited respiratory and turnover capacity,²⁰ which indicates that mitochondria are likely required for the function of adult HSCs, as evidenced by the fact that FOXO3 serves as a regulator to couple mitochondrial metabolism with HSC homeostasis.²¹

The metabolic profiles of FL-HSCs and the effects of metabolism on HSC function, however, remain largely unknown.

FL-HSCs undergo rapid division/expansion, conceivably through an increased demand on energy sources compared with that needed by adult HSCs, which are usually maintained in a relatively quiescent state.

It is also possible that distinct microenvironments in different hematopoietic organs may affect the metabolism of HSCs.

Interestingly, a recent report showed that loss of Rieske iron-sulfur protein, a mitochondrial complex III subunit, impairs the quiescent status of adult HSCs and the differentiation capacity of FL-HSCs.²²

FL-HSCs seem to have increased expression levels of many mitochondrial respiration–related genes, although whether metabolic status determines the cell fate of FL-HSCs remains unknown.²³

Results from previous studies indicate that mitochondrial activity may play a role in HSCs in the FL stage, although the detailed metabolic profiles and their underlying mechanisms await further investigation.

Because of limitations in the availability of HSCs, most studies related to the nutrient metabolism of HSCs have depended heavily on flow cytometric analysis with MitoTracker dyes, TMRE, and DCFDA to determine mitochondrial mass, membrane potential, and ROS level, respectively.

Improved techniques have been used to measure several metabolic features of HSCs, such as oxygen consumption and lactate generation^9,24; however, these studies may not directly reflect the true extent of glycolysis, oxidative phosphorylation, or other metabolic processes in HSCs.

Recent studies have provided interesting evidence showing that it is feasible to perform a metabolomic analysis with fewer than 10⁴ HSCs to explore the metabolic networks of different types of nutrients.²⁵

Nevertheless, it remains difficult to detect all of the metabolites sensitively with a limited number of HSCs using conventional metabolomic analysis.

Few tools are available for real-time imaging of metabolic states in live HSCs, either in vitro or in vivo. Therefore, alternative approaches, such as metabolite biosensors, are required for the direct, precise, and real-time detection of subtle changes in nutrient metabolism in HSCs.

Recently, we developed a highly responsive NADH/NAD⁺ sensor, called SoNar,²⁶ which was designed by inserting cpYFP into the NAD(H)-binding domain of T-Rex.

SoNar shows distinct fluorescence responses to NADH and NAD⁺.

Inside the cell under physiological conditions, the total intracellular pool of NAD⁺ and NADH in the range of hundreds of micromolars^27-30 far exceeds the dissociation constants of SoNar for NAD⁺ (K_d, 5.0 μM) and NADH (K_d, 0.2 μM);

thus, the sensor would be occupied by either NAD⁺ or NADH molecules, and its steady-state fluorescence would report the NAD⁺/NADH ratio rather than the absolute concentrations of either of the 2 nucleotides according to equilibrium thermodynamics.²⁶

In addition, SoNar fluorescence is intrinsically ratiometric（比率计）, with 2 excitation wavelengths, and its fluorescence excited at 420 (or 405) and 485 nm shows opposing responses to ligand binding.^26,31

This ratiometric property of a sensor is highly desired for quantitative imaging in live cells and in vivo,^32,33 because it eliminates the differences in instrumental efficiency, environmental effects, and probe concentration, enabling it to be widely used in different biological samples.

The SoNar sensor has a 15-fold (or 1500%) dynamic range, enabling us to measure the cytosolic NAD⁺/NADH ratio from 0.8 to 2000.^26,31,34

Interestingly, SoNar has many desirable properties that make it an ideal sensor; it has a rapid response, high sensitivity, intense fluorescence, and large dynamic range, and it is capable of reporting subtle perturbations in many pathways affecting energy metabolism, including glycolysis and mitochondrial respiration.

We generated SoNar transgenic mice and examined the metabolic profiles of FL-HSCs and their contributions/connections to cell fate determinations, as well as the underlying mechanisms governing FL-HSC function.

参考文献：https:///10.1182/blood.2019003940

HEMATOPOIESIS AND STEM CELLS| JULY 30, 2020

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Methods

Mice

SoNar DNA consists of the sequence of cpYFP, truncated T-Rex (78-211), and the linkers between them.²⁶ Its coding gene (1.2 kbp) is much smaller than those of the first-generation NADH sensors Peredox (2.8 kbp) and Frex³⁵ (1.8 kbp). For transgenic studies, smaller is better for the expression of the sensor in cells and in vivo.³¹ To generate SoNar transgenic mice, SoNar DNA was cloned into the pCAG vector with chicken β-actin promoter. The targeting construct was linearized, purified, and microinjected into FVB blastocysts. SoNar DNA was randomly incorporated into the genome and determined by polymerase chain reaction (PCR) assay. Messenger RNA (mRNA) and protein expressions of SoNar in different tissues of SoNar mice were further evaluated by both reverse transcription PCR (RT-PCR) and fluorescence microscopy. The resulting chimeric mice were bred with FVB mice to obtain germ line transmission. These mice were next backcrossed with a C57BL/6 CD45.2 background, and germ line transmission was checked by PCR and flow cytometry. Heterozygote transgenic SoNar mice were used for most of the experiments in the current study. C57BL/6 CD45.2 mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. CD45.1 mice were provided by Dr Jiang Zhu at Shanghai Jiao Tong University School of Medicine. All animal experiments were conducted according to the Guidelines for Animal Care at Shanghai Jiao Tong University School of Medicine. All these materials, including SoNar sensor, are available upon request.

In vivo imaging of SoNar transgenic mice

Genotyping, mRNA expression, and histology of SoNar transgenic mice

Competitive reconstitution assay

Metabolic imaging and quantification of cytosolic NAD⁺/NADH ratio in living cells

Real-time metabolic imaging in the BM niche

Flow cytometry

Ultrahigh-performance LC–qTOF–MS analysis

Microarray and quantitative RT-PCR

Metabolic analysis

Oxygen consumption rate (OCR) and extracellular acidification rate were determined in CD45.2⁺ SoNar-high and -low FL hematopoietic cells with the XF Cell Mito Stress Test Kit (Seahorse #103015-100) and XF Glycolysis Stress Test Kit (Seahorse #103020-100) according to the manufacturer’s instructions using a Seahorse XF96 analyzer. In brief, for the OCR analysis, 3 × 10⁵ SoNar-high and -low FL hematopoietic cells were incubated in the 37°C carbon dioxide–free incubator in 175 μL of assay medium (XF Base Medium with 2 mM of glutamine, 1 mM of pyruvate, and 10 mM of glucose [pH, 7.4]; 37°C); 1.5 μM of oligomycin, 2 μM of FCCP, and 0.5 μM of rotenone/antimycin A were loaded in injection ports A, B, and C, respectively. For the detection of extracellular acidification rate, 3 × 10⁵ CD45.2⁺ SoNar-high and -low FL hematopoietic cells were incubated in the 37°C carbon dioxide–free incubator in 175 μL of assay medium (XF Base Medium with 1 mM of glutamine [pH, 7.4]; 37°C); 10 mM of glucose, 1.5 μM of oligomycin, and 100 mM of 2-DG were loaded into injection ports A, B, and C, respectively. ATP level was analyzed using the ATP Bioluminescence Assay Kit HS II (Roche) according to the manufacturer’s protocol, and data were normalized to cell count. To analyze the mitochondrial DNA (mtDNA) copy numbers, total genomic DNA was extracted from the indicated cells for comparing the copies of the mitochondrial-specific mt-ND4 gene with those of the nuclear B2m gene. The primer sequences used are shown in supplemental Table 1.

For intracellular and extracellular pyruvate and lactate assays, the extracts were prepared from 5 × 10⁶ CD45.2⁺ FL hematopoietic cells using 300 μL of ice-cold 0.5-M perchloric acid for each sample. Extracts were centrifuged at 10 000 g for 5 minutes at 4°C, and the supernatant was neutralized with 5 M of KOH and centrifuged at 10 000 g for 5 minutes at 4°C. The supernatant was removed for assay. For the pyruvate assay, 180 μL of assay buffer (100 mM of potassium chloride [pH, 6.7], 1 mM of EDTA, 0.1% bovine serum albumin, 10 μM of flavin adenine dinucleotide, 0.2 mM of thiamine pyrophosphate, 0.5 U of pyruvate oxidase, 0.2 U of horseradish peroxidase, and 50 μM of AmplexRed) was added to a 96-well plate containing 20 μL of the cell extract or medium containing extracellular pyruvate. Changes in fluorescence were measured every 30 seconds for 15 minutes at 37°C by a Synergy 2 Multi-Mode Microplate Reader with an excitation filter of 530 BP 40 nm and emission filter of 590 BP 35 nm at 37°C. Calibration experiments were performed with 20 μL of pyruvate standards (0, 10, 20, 40, 60, 100, and 200 μM per well). For the lactate assay, 180 μL of assay buffer (PBS [pH, 7.4], 0.1% bovine serum albumin, 500 μM of NAD⁺, 0.5 U of lactate dehydrogenase (LDH), 0.2 U of diaphorase, and 10 μM of resazurin) was added to a 96-well plate containing 20 μL of the cell extract or medium containing extracellular lactate. Changes in fluorescence were measured every 30 seconds for 15 minutes at 37°C by a Synergy 2 Multi-Mode Microplate Reader with an excitation filter of 540 BP 25 nm and emission filter of 590 BP 35 nm at 37°C. Calibration experiments were performed with 20 μL of lactate standards (0, 10, 20, 40, 60, 100, and 200 μM per well). All samples were diluted to fit within the range of the standard curve and run in triplicate. For the evaluation of NADH/NAD⁺ ratios by a biochemical assay, 1 million SoNar-high and -low FL hematopoietic cells were sorted from E14.5 FLs and subjected to measurement of NADH/NAD⁺ level using a commercially available NADH/NAD⁺ assay kit (Sigma #MAK037) according to the manufacturer’s instructions.

Immunoblot analysis

Single cell colony forming

The single CD45.2⁺ SoNar-high or -low FL hematopoietic cell was freshly isolated and plated onto a 35-mm poly-D-lysine hydrobromide-coated glass-bottom dish (Cellvis) and cultured in Stemspan serum-free medium (Stemcell Technologies) containing 10 ng/mL of murine SCF and 10 ng/mL of murine TPO (Peprotech) for 96 hours. The ratios of daughter cells derived from a single parent cell were recorded with excitation at 405 and 488 nm using Nikon A1 confocal microscopy and analyzed with Image J software.

Luciferase reporter assays

The luciferase reporter vector pGL4.27 containing the Mdh1 promoter was constructed to identify transcriptional activation of Mdh1 by STAT3. Indicated doses of pLVX-Stat3 (or negative control vector) plasmid along with pGL4.27-mdh1 promoter vector were cotransfected into 293T cells. Luciferase activities were measured according to the manufacturer’s instructions (Promega #E1910) by using a luciferase reporter system (GloMax Multi Instrument) 24 hours after transfection.

ChIP assays

Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP Assay Kit (Beyotime #P2078). Briefly, 293T cells were overexpressed with pGL4.27-mdh1 promoter vector and STAT3 (with Strep II tag) crosslinked with 1% formaldehyde (Sigma) at 37°C for 10 minutes, and precleared DNA was then used for immunoprecipitation with 4 mL of anti–Strep II antibody (Genescript) or rabbit control immunoglobulin G (CST) at 4°C overnight. For the sample input, 1% of the sonicated pre-cleared DNA was purified at the same time with the precipitated immune complex. The ChIP samples were purified by the Gel and PCR Clean Up Kit (Necleospin). The STAT3-binding sequence was amplified by semiquantitative PCR using primers specific for the Mdh1 promoter region as listed in supplemental Table 1.

Methylation-specific PCR assay

Genomic DNA was extracted from 500 000 CD45.2⁺ SoNar-high and -low FL hematopoietic cells using a DNA extraction kit (Generay Biotech #GK0122). The promoter methylation status of MDH1 was determined by sodium bisulfate to convert unmethylated (but not methylated) cytosine to uracil, followed by analysis by methylation-specific PCR to amplify specifically either methylated or unmethylated DNA using the Zymoresearch kit (EZ DNA Methylation-Direct Kit D5020) according to the manufacturer’s instruction. The methylation-specific PCR primers are listed in supplemental Table 1.

Statistical analysis

Statistical analysis was performed using GraphPad and SPSS software (version 19.0). Data are represented as mean ± standard error of the mean. n represents the number of independent experiments or the number of cells or mice per group from independent experiments. All experiments were performed independently 3 to 5 times. Data were analyzed with a Student t test (2 tailed), 1-way analysis of variance with Tukey’s multiple comparison test, or 2-way analysis of variance with Sidak’s multiple comparison test according to the experimental design, and statistical significance was set at P < .05.