Exacerbation of mitochondrial fission in human CD34+ cells halts erythropoiesis and hemoglobin biosynthesis

AbstractErythropoiesis is the most powerful cellular differentiation and proliferation system, with a production of 1011 cells per day. In this fine-tuned process, the hematopoietic stem cells (HSCs) generate erythroid progenitors, which proliferate and mature into erythrocytes. During erythropoiesis, mitochondria are reprogrammed to drive the differentiation process before finally being eliminated by mitophagy. In erythropoiesis, mitochondrial dynamics (MtDy) is expected to be a regulatory key point that has not been described previously. We described that a specific MtDy pattern is occurring in human erythropoiesis from EPO-induced human CD34+ cells, characterized by a predominant mitochondrial fusion at early stages followed by predominant fission at late stages. The fusion protein MFN1 and the fission protein FIS1 are shown to play a key role in the accurate progression of erythropoiesis. Fragmentation of the mitochondrial web by the overexpression of FIS1 (gain of fission) resulted in both the inhibition of hemoglobin biosynthesis and the arrest of erythroid differentiation, keeping cells in immature differentiation stages. These cells showed specific mitochondrial features as compared with control cells, such as an increase in round and large mitochondria morphology, low mitochondrial membrane potential and a drop in the expression of the respiratory complexes II and IV. Interestingly, treatment with the mitochondrial permeability transition pore (mPTP) inhibitor cyclosporin A, rescued mitochondrial morphology, hemoglobin biosynthesis and erythropoiesis. Studies presented in this work revealed MtDy as a hot spot in the regulation of erythroid differentiation which might be signaling downstream for metabolic reprogramming through the aperture/close of the mPTP.Key Points-. Excessive fission disrupts erythroid progression, heme biosynthesis and mitochondrial function, keeping cells mostly in progenitors and proerythroblast stage.-. Mitochondrial Dynamics signaling for erythroid differentiation involves FIS1 and the mPTP

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Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.592035 ◽

2020 ◽

Vol 8 ◽

Author(s):

Alvaro M. Gonzalez-Ibanez ◽

Lina M. Ruiz ◽

Erik Jensen ◽

Cesar A. Echeverria ◽

Valentina Romero ◽

...

Keyword(s):

Mitochondrial Permeability Transition ◽

Mitochondrial Dynamics ◽

Hot Spot ◽

Erythroid Differentiation ◽

Mitochondrial Fusion ◽

Metabolic Reprogramming ◽

Mitochondrial Morphology ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Hemoglobin Biosynthesis

Erythropoiesis is the most robust cellular differentiation and proliferation system, with a production of ∼2 × 1011 cells per day. In this fine-tuned process, the hematopoietic stem cells (HSCs) generate erythroid progenitors, which proliferate and mature into erythrocytes. During erythropoiesis, mitochondria are reprogrammed to drive the differentiation process before finally being eliminated by mitophagy. In erythropoiesis, mitochondrial dynamics (MtDy) are expected to be a key regulatory point that has not been described previously. We described that a specific MtDy pattern occurs in human erythropoiesis from EPO-induced human CD34+ cells, characterized predominantly by mitochondrial fusion at early stages followed by fission at late stages. The fusion protein MFN1 and the fission protein FIS1 are shown to play a key role in the progression of erythropoiesis. Fragmentation of the mitochondrial web by the overexpression of FIS1 (gain of fission) resulted in both the inhibition of hemoglobin biosynthesis and the arrest of erythroid differentiation, keeping cells in immature differentiation stages. These cells showed specific mitochondrial features as compared with control cells, such as an increase in round and large mitochondrial morphology, low mitochondrial membrane potential, a drop in the expression of the respiratory complexes II and IV and increased ROS. Interestingly, treatment with the mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporin A, rescued mitochondrial morphology, hemoglobin biosynthesis and erythropoiesis. Studies presented in this work reveal MtDy as a hot spot in the control of erythroid differentiation, which might signal downstream for metabolic reprogramming through regulation of the mPTP.

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Mitochondrial morphology transitions and functions: implications for retrograde signaling?

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00584.2012 ◽

2013 ◽

Vol 304 (6) ◽

pp. R393-R406 ◽

Cited By ~ 125

Author(s):

Martin Picard ◽

Orian S. Shirihai ◽

Benoit J. Gentil ◽

Yan Burelle

Keyword(s):

Cell Function ◽

Mitochondrial Permeability Transition ◽

Mitochondrial Dynamics ◽

Permeability Transition ◽

Retrograde Signaling ◽

Mitochondrial Morphology ◽

Transport Chain ◽

Dynamic Interface ◽

Morphological Transitions ◽

Species Production

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment.

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c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression

Blood ◽

10.1182/blood-2009-08-238311 ◽

2010 ◽

Vol 116 (22) ◽

pp. e99-e110 ◽

Cited By ~ 74

Author(s):

Elisa Bianchi ◽

Roberta Zini ◽

Simona Salati ◽

Elena Tenedini ◽

Ruggiero Norfo ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Luciferase Reporter ◽

Myb Transcription Factor ◽

Promoter Regions ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb–silenced CD34+ cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

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Mitochondrial Morphology Regulates Organellar Ca2+ Uptake and Changes Cellular Ca2+ Homeostasis

10.1101/624981 ◽

2019 ◽

Author(s):

Alicia J. Kowaltowski ◽

Sergio L. Menezes‐Filho ◽

Essam Assali ◽

Isabela G. Gonçalves ◽

Phablo Abreu ◽

...

Keyword(s):

Mitochondrial Permeability Transition ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Dominant Negative ◽

C2c12 Cells ◽

Mitochondrial Morphology ◽

Calcium Stores ◽

Intact Cells ◽

Cellular Calcium ◽

Cellular Ca

AbstractChanges in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1‐DN) markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion‐promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin 2 (MFN2) strongly reduced mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in inner membrane potentials or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium‐replenishing process of store‐operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1‐DN‐promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts on cellular Ca2+ homeostasis.

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Mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00028.2018 ◽

2018 ◽

Vol 315 (5) ◽

pp. H1341-H1352 ◽

Cited By ~ 15

Author(s):

Giuseppe Paradies ◽

Valeria Paradies ◽

Francesca Maria Ruggiero ◽

Giuseppe Petrosillo

Keyword(s):

Myocardial Ischemia ◽

Mitochondrial Dysfunction ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mitochondrial Morphology ◽

Myocardial Ischemia Reperfusion

Mitochondrial dysfunction plays a central role in myocardial ischemia-reperfusion (I/R) injury. Increased reactive oxygen species production, impaired electron transport chain activity, aberrant mitochondrial dynamics, Ca2+ overload, and opening of the mitochondrial permeability transition pore have been proposed as major contributory factors to mitochondrial dysfunction during myocardial I/R injury. Cardiolipin (CL), a mitochondria-specific phospholipid, plays a pivotal role in multiple mitochondrial bioenergetic processes, including respiration and energy conversion, in mitochondrial morphology and dynamics as well as in several steps of the apoptotic process. Changes in CL levels, species composition, and degree of oxidation may have deleterious consequences for mitochondrial function with important implications in a variety of pathophysiological conditions, including myocardial I/R injury. In this review, we focus on the role played by CL alterations in mitochondrial dysfunction in myocardial I/R injury. Pharmacological strategies to prevent myocardial injury during I/R targeting mitochondrial CL are also examined.

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Transcriptional regulation of HSCs in Aging and MDS reveals DDIT3 as a Potential Driver of Transformation

10.1101/2021.09.08.459384 ◽

2021 ◽

Author(s):

Nerea Berastegui ◽

Marina Ainciburu ◽

Juan P. Romero ◽

Ana Alfonso-Pierola ◽

Céline Philippe ◽

...

Keyword(s):

Transcriptional Profiling ◽

Erythroid Differentiation ◽

Genetic Alterations ◽

List Type ◽

Hematopoietic Stem ◽

Associated Lesions ◽

Key Points ◽

Transcriptional Rewiring ◽

Human Hscs ◽

Transcriptional State

ABSTRACTMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients.KEY POINTSHuman HSCs undergo a complex transcriptional rewiring in aging and MDS that may contribute to myeloid transformation.DDIT3 overexpression induces a failure in the activation of erythroid transcriptional programs, leading to inefficient erythropoiesis.

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The dynamic interactive network of long non-coding RNAs and chromatin accessibility facilitates erythroid differentiation

10.1101/2021.03.02.433076 ◽

2021 ◽

Author(s):

Yunxiao Ren ◽

Junwei Zhu ◽

Yuanyuan Han ◽

Pin Li ◽

Hongzhu Qu ◽

...

Keyword(s):

Progenitor Cells ◽

Specific Interaction ◽

Erythroid Differentiation ◽

Chromatin Accessibility ◽

List Type ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Interactive Network ◽

Non Coding Rnas ◽

Terminal Erythroid Differentiation

AbstractErythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility in erythropoiesis by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and LncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that coordinate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the interactive network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.Key PointsLncRNAs regulate erythroid differentiation through coordinating with chromatin accessibility.The integrative multi-omics analysis reveals stage-specific interaction network of LncRNAs and chromatin accessibility in erythropoiesis.

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MicroRNA hsa-mir-155 Blocks Myeloid and Erythroid Differentiation of Human CD34+ Cells.

Blood ◽

10.1182/blood.v108.11.1337.1337 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1337-1337

Author(s):

Robert W. Georgantas ◽

Richard Hildreth ◽

Sebastien Morisot ◽

Jonathan Alder ◽

Curt I. Civin

Keyword(s):

K562 Cells ◽

Erythroid Differentiation ◽

Expression Data ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Human Cd34 ◽

Cd34 Cells ◽

Megakaryocyte Differentiation

Abstract In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data from a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published miR-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MircoRNA hsa-mir-155 was highly expressed in CD34+ HSPCs, and was predicted by our bioinformatics database to target several HSPC-expressed mRNAs (CREBBP, CXCR4, Jun, Meis-1, PU.1, AGTRI, AGTRII, Fos, and GATA3) that encode proteins known to be involved in myeloid and/or erythroid differentiation. We used luciferase-3′UTR reporter constructs to confirm that protein expression from these mRNAs were in fact down regulated by microRNA. As an initial test of mir-155′s effect on hematopoietic differentiation, K562 cells were transduced with hsa-mir-155 lentivirus and then exposed to TPA to induce megakaryocyte differentiation, or to hemin to induce erythroid differentiation. Compared to controls, miR-155 reduced K562 megakaryocyte differentiation by ~70%, and erythroid differentiation by >90%. Thus, mir-155 appears to be sufficient to inhibit both megakayrocyte and erythroid differentiation. K562 proliferation was not affected by mir-155, showing that the differentiation block was not due to cell cycle arrest. MicroRNA hsa-mir-155-transduced human mobilized blood CD34+ cells generated >70% fewer myeloid and erythroid colonies than controls in colony forming (CFC) assays, further indicating that mir-155 blocks both myeloid and erythroid differentiation. We are currently further testing the effects of mir-155 on differentiation of CD34+ cells in vitro, and also in vivo on their ability to engraft immunodeficient mice.

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ATG4A is a Novel Regulator of Mitophagy during Human Erythropoiesis

Blood ◽

10.1182/blood-2019-130567 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3522-3522

Author(s):

Massiel Chavez Stolla ◽

Neele Thom ◽

Andreea Reilly ◽

Courtnee Clough ◽

Janis L. Abkowitz ◽

...

Keyword(s):

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Critical Function ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Stem And Progenitor Cells ◽

Differentiation Gene ◽

Kinetics Of ◽

Late Stages

Autophagy is a highly conserved pathway that degrades and recycles intracellular components. While autophagy is activated in response to cellular stress, it also contributes to hematopoietic cell differentiation. Previous studies in mice lacking core autophagy proteins have identified mitochondrial clearance as a critical function of autophagy in erythroid differentiation and maturation. Impaired autophagy results in anemia, retained mitochondria and elevated levels of reactive oxygen species in mice. However, the kinetics and regulation of mitophagy in human erythropoiesis have not been investigated. To better understand the kinetics of mitophagy in human erythropoiesis we have developed a lentiviral mitophagy reporter (MT-Keima) and used it to monitor mitochondrial clearance during erythroid differentiation of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). Measurement of mitophagy during erythroid differentiation revealed active mitochondrial clearance during early and late stages of erythropoiesis culminating with total clearance of mitochondria at the terminal stages of differentiation. Gene expression analysis during human erythroid differentiation identified the upregulation of the core autophagy program including ATG4A encoding a cysteine protease, which was restricted to the erythroid lineage. Knockdown of ATG4A in primary human HSPCs significantly reduced mitophagy in early and late erythroid cells and resulted in increased mitochondrial mass in terminally differentiated reticulocytes. Furthermore decreased expression of ATG4A, but not its paralog ATG4B, reduced the (i) expansion of erythroid progenitors, (ii) total number of erythroblasts (iii) and significantly reduced enucleation relative to luciferase controls. Finally significantly fewer erythroid colonies were found in methylcellulose culture only in HSPCs with ATG4A KD when compared to luciferase controls, while numbers of myeloid colonies were preserved, supporting a role for ATG4A selectively in the human erythroid lineage. Together these results identify ATG4A as a novel erythroid-specific regulator of mitophagy and a new potential target for the therapeutic modulation of autophagy in human erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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