scholarly journals ALKBH5 Modulates Hematopoietic Stem and Progenitor Cell Energy Metabolism through m 6a Modification-Mediated RNA Stability

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 298-298
Author(s):  
Yimeng Gao ◽  
Joshua T Zimmer ◽  
Radovan Vasic ◽  
Chengyang Liu ◽  
Rana Gbyli ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Energy Metabolism ◽  
Mrna Stability ◽  
Rna Stability ◽  
Hematopoietic Cells ◽  
Tca Cycle ◽  
Translational Efficiency ◽  
Metabolic Shift ◽  
Hematopoietic Stem ◽  
Hematopoietic Development

Abstract During hematopoietic differentiation from hematopoietic stem cells (HSCs) to mature blood cells, cells undergo a metabolic shift from glycolysis to mitochondrial respiration. The mechanisms by which hematopoietic cells adjust their energy metabolism are still under investigation. N6-mehyladenosine (m 6A) mRNA modification has been reported to regulate numerous fundamental cellular processes through control of RNA stability or translational efficiency. The fat mass and obesity-associated protein (FTO), an m 6A m and m 6A mRNA demethylase, has been reported to affect cellular metabolism in acute myeloid leukemia (AML). ALKBH5, the specific RNA m 6A demethylase, controls oncogene expression in AML. ALKBH5 becomes highly expressed in hematopoietic progenitors during hematopoietic development but the physiological role of RNA m 6A demethylase during hematopoiesis remains unknown. To investigate the function of the RNA m 6A demethylase ALKBH5 in hematopoiesis, we generated Vav-iCre +; Alkbh5fl/fl (vcAlkbh5-/-) mice, resulting in deletion of Alkbh5 specifically in the hematopoietic system. vcAlkbh5-/-mice showed no hematopoietic defects at steady states up to 12 months of age. We applied TimeLapse-seq on lineage-depleted bone marrow cells of WT and vcAlkbh5-/- mice to determine whether loss of ALKBH5 perturbed mRNA stability and/or RNA turnover. Ogdh mRNA was the most destabilized transcript resulting in significantly reduced OGDH protein levels. OGDH is the rate-limiting enzyme in the tricarboxylic acid (TCA) cycle. Inhibition of OGDH subsequently induces production of L-2-hydroxyglutarate (L-2-HG), whose metabolism is closely coupled to energy metabolism through inhibition of oxygen consumption. L-2-HG, the enantiomer of D-2-HG, inhibits the function of a-ketoglutarate (a-KG)-dependent enzymes, including TET and KDM enzymes. We measured L- and D-2-HG in the plasma of WT and vcAlkbh5-/- mice by chiral derivatization to distinguish the two enantiomers. Although D-2-HG levels were similar in the plasma of WT and vcAlkbh5-/- mice, L-2-HG levels were significantly increased in the plasma of vcAlkbh5-/- mice. We therefore determined the function of Jumonji C-domain lysine demethylases (JmjC-KDMs) by measuring histone methylation: H3K9me3, H3K27me3 and H3K36me3 modifications were all significantly increased in Alkbh5-deficient hematopoietic cells. We next sought to understand whether reduction of OGDH expression and resulting increased L-2-HG levels production could impair energy metabolism via perturbation of the TCA cycle and oxidative phosphorylation (OXPHOS) in the mitochondria. We isolated lineage negative hematopoietic stem and progenitor cells (HSPCs) from WT and vcAlkbh5-/- mice and subjected these to the Seahorse ATP Rate Assay. Comparing oxygen consumption rate (OCR) data and the kinetics of the Extracellular Acidification Rate (ECAR) of both groups, we found that less ATP was produced by mitochondria of the vcAlkbh5-/- cells, while ATP produced by glycolysis showed no difference between the two groups. In the meantime, the ultrastructure of mitochondria in the Alkbh5-deficient cells remains normal. We next determined whether the attenuated energy metabolism of Alkbh5-deficient HSPCs was functionally relevant by testing HSPC function in competitive transplantation assays. Interestingly, vcAlkbh5-/- cells showed a significant competitive defect at all differentiation stages except in phenotypic long-term HSCs (LT-HSCs). This suggests that LT-HSCs, thought to preferentially rely on glycolysis as opposed to OXPHOS for their energy source, are protected from loss of ALKBH5 and OGDH. In conclusion, our study demonstrates that ALKBH5 modulates energy metabolism by regulating mRNA stability of metabolic enzymes through its m 6A demethylation activity during hematopoiesis. This finding links Alkbh5 expression kinetics to the metabolic shift from glycolysis to mitochondrial OXPHOS during hematopoietic development. Disclosures No relevant conflicts of interest to declare.

scholarly journals ZRSR1 cooperates with ZRSR2 in regulating splicing of U12-type introns in murine hematopoietic cells

Haematologica ◽  
2021 ◽  
Author(s):  
Vikas Madan ◽  
Zeya Cao ◽  
Weoi Woon Teoh ◽  
Pushkar Dakle ◽  
Lin Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Cells ◽  
Myeloid Cells ◽  
Myeloid Differentiation ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Functional Components ◽  
Deficient Mice

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. To investigate further the role of this splice factor in splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2- deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not human, contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to model accurately ZRSR2-mutant MDS in mice.

scholarly journals Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2046-2055 ◽  
Author(s):  
Marion Dorsch ◽  
Gang Zheng ◽  
David Yowe ◽  
Prakash Rao ◽  
Yanjun Wang ◽  
...  
Keyword(s):  
B Cells ◽  
Critical Role ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Lymphoproliferative Disease ◽  
Rapid Expansion ◽  
Soluble Form ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Cd8 Cells

Abstract Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4+CD8+ cells occurred shortly after transplantation. CD4+CD8+ cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4+CD8+cells were mainly non–Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre–CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.

scholarly journals Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2046-2055 ◽  
Author(s):  
Marion Dorsch ◽  
Gang Zheng ◽  
David Yowe ◽  
Prakash Rao ◽  
Yanjun Wang ◽  
...  
Keyword(s):  
B Cells ◽  
Critical Role ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Lymphoproliferative Disease ◽  
Rapid Expansion ◽  
Soluble Form ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Cd8 Cells

Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4+CD8+ cells occurred shortly after transplantation. CD4+CD8+ cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4+CD8+cells were mainly non–Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre–CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.

scholarly journals Regulation of Hematopoietic Development in the Aorta-Gonad-Mesonephros Region Mediated by Lnk Adaptor Protein

2003 ◽  
Vol 23 (23) ◽  
pp. 8486-8494 ◽  
Author(s):  
Ikuo Nobuhisa ◽  
Makiko Takizawa ◽  
Satoshi Takaki ◽  
Hirofumi Inoue ◽  
Keisuke Okita ◽  
...  
Keyword(s):  
Hematopoietic Cells ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Homozygous Mutant ◽  
Dependent Inhibition ◽  
Multistep Process ◽  
Homozygous Mutant Mouse

ABSTRACT Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.

scholarly journals Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Leukemia ◽  
2021 ◽  
Author(s):  
Melvin E. Thomas ◽  
Sherif Abdelhamed ◽  
Ryan Hiltenbrand ◽  
Jason R. Schwartz ◽  
Sadie Miki Sakurada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Transcriptional Profiling ◽  
Hematopoietic Cells ◽  
Protein Translation ◽  
Germline Mutations ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Cellular Environment ◽  
Primary Mouse

AbstractPediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Butyrate Prevents TGF-β1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 258
Author(s):  
Hyo Yeong Lee ◽  
Somi Nam ◽  
Mi Jeong Kim ◽  
Su Jung Kim ◽  
Sung Hoon Back ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Mitochondrial Dynamics ◽  
Tca Cycle ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Pulmonary Fibroblasts ◽  
Matrix Deposition ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

scholarly journals Metabolomic Analysis to Elucidate Mechanisms of Sunitinib Resistance in Renal Cell Carcinoma

Metabolites ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 1
Author(s):  
Tomonori Sato ◽  
Yoshihide Kawasaki ◽  
Masamitsu Maekawa ◽  
Shinya Takasaki ◽  
Kento Morozumi ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Energy Metabolism ◽  
Cell Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Progression ◽  
Renal Cell ◽  
Treatment Resistance ◽  
Tca Cycle ◽  
Glutamine Uptake

Metabolomics analysis possibly identifies new therapeutic targets in treatment resistance by measuring changes in metabolites accompanying cancer progression. We previously conducted a global metabolomics (G-Met) study of renal cell carcinoma (RCC) and identified metabolites that may be involved in sunitinib resistance in RCC. Here, we aimed to elucidate possible mechanisms of sunitinib resistance in RCC through intracellular metabolites. We established sunitinib-resistant and control RCC cell lines from tumor tissues of RCC cell (786-O)-injected mice. We also quantified characteristic metabolites identified in our G-Met study to compare intracellular metabolism between the two cell lines using liquid chromatography-mass spectrometry. The established sunitinib-resistant RCC cell line demonstrated significantly desuppressed protein kinase B (Akt) and mesenchymal-to-epithelial transition (MET) phosphorylation compared with the control RCC cell line under sunitinib exposure. Among identified metabolites, glutamine, glutamic acid, and α-KG (involved in glutamine uptake into the tricarboxylic acid (TCA) cycle for energy metabolism); fructose 6-phosphate, D-sedoheptulose 7-phosphate, and glucose 1-phosphate (involved in increased glycolysis and its intermediate metabolites); and glutathione and myoinositol (antioxidant effects) were significantly increased in the sunitinib-resistant RCC cell line. Particularly, glutamine transporter (SLC1A5) expression was significantly increased in sunitinib-resistant RCC cells compared with control cells. In this study, we demonstrated energy metabolism with glutamine uptake and glycolysis upregulation, as well as antioxidant activity, was also associated with sunitinib resistance in RCC cells.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

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