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

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.

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

Erythropoiesis 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

BAP1 deubiquitinase is a potent repressor of fetal hemoglobin biosynthesis

SummaryHuman globin gene production transcriptionally “switches” from fetal to adult synthesis shortly after birth, and is controlled by macromolecular complexes that enhance or suppress transcription by cis-elements scattered throughout the locus. The DRED repressor is recruited to the ε- and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA binding and epigenetic enzyme components (e.g. DNMT1 and LSD1) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: the deubiquitinase BAP1 is a component of the repressor complex whose activity maintains NCoR1 at sites in the β-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.

Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation

Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as α-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

Imatinib (Glivec) and Nilotinib (AMN107), Two Selective Inhibitors of Bcr-Abl Tyrosine Kinase, Induce Hemoglobin Production in Human K-562 CML Erythroleukemia Cells in Addition To Promoting Apoptosis.

Imatinib (Glivec) and Nilotinib (AMN107) have been developed as selective targeted inhibitors of Bcr-Abl chimaeric tyrosine kinase activated in CML and other malignancies characterized by the t(9;22) translocation (Manley P.W., et al. Biochim Biophys Acta 1754(1–2):3–13, 2005). Both agents are considered innovative targeted cell death promoting agents designed for treatment of human leukemias. During the course of their evaluation as apoptosis-promoting agents in human K-562 leukemia cells, we observed that both agents in addition to blocking Bcr-Abl tyrosine kinase, promoted production of hemoglobin but to different extent. Imatinib caused hemoglobin production in 6–7% of cells at concentration of 0.01 μM to 0.5 μM. This proportion reached 54% in the presence of 90 μM of Hemin. Nilotinib alone, on the other hand, caused hemoglobin production in 66% of cells at a concentration of 0.01 μM. This proportion increased to 75% in the presence of 30 μM of Hemin. These findings indicate that: Imatinib and Nilotinib are weak and potent inducers of hemoglobin biosynthesis, respectively. Treatment of K-562 cells with either Imatinib (0.5 μM) and Nilotinib (0.01 μM) enhanced the ability of Hemin to promote erythroid differentiation. This ability of Nilotinib to induce hemoglobin accumulation at concentrations lower than those causing apoptosis, suggest that this agent may be clinically useful by promoting hemoglobin biosynthesis in diseases characterized by hemoglobin deficiency (hemoglobinopathies). We are currently investigating the mechanism whereby Nilotinib can selective activate the globin gene expression in K-562 leukemia cells.