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Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34

The Gluthation peroxidase (GPX) enzymes are part of the protective system against lipid peroxydation that includes prevention of oxydation and reduction of already oxidized lipid through enzymatic reactions catalyzed by GSH. GPX4 is one of the five GPX able to incorporate selenium. It is also the only GPX able to directly reduce in the membrane the oxidized fatty acids and cholesterol. Recent reports identified GPX4 as the central inhibitor of ferroptosis, a process during which iron-induced peroxydation of membrane lipids causes a specific cell death that can be reverted by lipophilic antioxydants or by iron chelators. GPX4 has recently been involved during mice erythropoiesis: GPX4-/-mice present a hemolytic anemia and a high apoptotic rate in spleen erythroid progenitors. Although transcriptomics and proteomics found it expressed in human erythroid precursors, its role during human erythropoiesis has not been described. Using an in-vitroerythroid differentiation protocol from CD34+cells obtained from apheresis, we confirmed that GPX4 expression was induced at RNA and protein level during differentiation. RSL3, a specific GPX4 inhibitor, didn't affect early steps of erythropoiesis (i.eclonogenic potential and progenitor amplification) nor the early maturation of erythroid precursors (assessed by sequential CD49d/CD235/CD71 staining) but led to a significant decrease in the enucleation rate as assessed by Hoechst staining using flow cytometry (74%±9 DMSO versus 35%±6 RSL3, p Using Western Blot, we observed that RSL3 exposure induced a strong GPX4 depletion in erythroid progenitors while it didn't affect GPX1, another member of the GPX family expressed in erythroid cells. In order to confirm that enucleation defect was related to GPX4 knockdown, we used an Sh-RNA strategy that allowed a 62%±6 GPX4 decrease at RNA level and a 46%±5 at protein level. We observed a significant defect in terminal enucleation in the cells transduced with shGPX4-lentiviruses in comparison with sh-Scramble (59%±5 Sh-Scr versus 39%±6 Sh-Gpx4, p We investigated then whether GPX4-knock-down affected quantitatively or qualitatively the membrane lipid content, which was shown to be involved in the enucleation process. Addition of Cholesterol to RSL3 in the medium partially restored the enucleation rate. However, lipidomics failed to show any significant difference in the total membrane lipid content (and particularly in the cholesterol content) after RSL3 exposure in comparison to DMSO. Since cholesterol is particularly abundant in the lipid rafts, we investigated whether the lipid distribution was qualitatively altered within the cell membrane. We observed a disruption of membrane lipid rafts when cells were exposed to RSL3, as shown by a 60%±12 decrease in the mean cholera toxin fluorescence intensity. GPX4 presence in lipid rafts was confirmed using immunofluorescence showing their co-localization at cell surface in human primary erythroblasts. Since lipid rafts play a role in the contractile ring that separates pyrenocyte from reticulocyte, we evaluated the myosin-light chain phosphorylation using flow cytometry and Western Blot and found it drastically decreased in GPX4-knockdown conditions. In summary, we identified GPX4 as a new actor of human terminal erythroid differentiation, independently to its function in ferroptosis control. We described its interaction at cell surface with lipid rafts that are required for the assembly of the contractile ring and cytokinesis leading to the nucleus extrusion. Disclosures No relevant conflicts of interest to declare.

Bone tissue engineering goes beyond the limitations of conventional methods of treating bone loss, such as autograft-induced morbidity and a lack of integration for large grafts. Novel biomimicry approaches (using three-dimensional [3D] electrospinning and printing techniques) have been designed to offer the most appropriate environment for cells and thus promote bone regeneration. In the present study, we assessed the bone regeneration properties of a composite 3D honeycomb structure from the electrostatic template assisted deposition (ETAD) process by an alternate deposition of electrospun polycaprolactone (PCL) nanofibers and electrosprayed hydroxyapatite nanoparticles (nHA) on a honeycomb micropatterned substrate. We first confirmed the cytocompatibility of this honeycomb PCL-nHA scaffold in culture with bone-marrow-derived mesenchymal stem cells (BM-MSCs). The scaffold was then implanted (alone or with seeded MSCs) for 2 months in a rat critical-sized calvarial defect model. The observation of new bone synthesis in situ (monitored using micro-computed tomography every 2 weeks and a histological assessment upon extraction) demonstrated that the honeycomb PCL-nHA scaffold was osteoconductive. Moreover, the combination of the scaffold with BM-MSCs was associated with significantly greater bone volume and mineralized regeneration during the 2-month experiment. The combination of the biomimetic honeycomb PCL-nHA scaffold with patient mesenchymal stem cells might therefore have great potential for clinical applications and specifically in maxillofacial surgery.

Alcohol-related liver disease is the most prevalent chronic liver disease worldwide, accounting for 30% of hepatocellular carcinoma (HCC) cases and HCC-specific deaths. However, the knowledge on mechanisms by which alcohol consumption leads to cancer progression and its aggressiveness is limited. Better understanding of the clinical features and the mechanisms of alcohol-induced HCC are of critical importance for prevention and the development of novel treatments.Early stage Huh-7 and advanced SNU449 liver cancer cell lines were subjected to Chronic Alcohol Exposure (CAE), at different doses for 6 months followed by one-month alcohol withdrawal period. ADH activity and ALDH expression were much lower in SNU449 compared with Huh-7 cells and at the 270mM dose, CAE decreased cell viability by about 50% and 80%, respectively in Huh-7 and SNU449 cells but induced mortality only in Huh-7 cells. Thus, Huh-7 may be more vulnerable to ethanol toxicity because of the higher levels of acetaldehyde. CAE induced a dose-dependent increase in cell migration and invasion and also in the expression of Cancer Stem Cells markers (CD133, CD44, CD90). CAE in Huh-7 cells selectively activated ERK1/2 and inhibited GSK3β signaling pathways. Most of the changes induced by CAE were reversed after alcohol withdrawal. Interestingly we confirmed the increase in CD133 mRNA levels in the tumoral tissue of patients with ethanol-related HCC compared to other HCC etiologies.Our results may explain the benefits observed in epidemiological studies showing a significant increase of overall survival in abstinent compared with non-abstinent patients.

Alcohol-related liver disease is the most prevalent chronic liver disease worldwide, accounting for 30% of hepatocellular carcinoma (HCC) cases and HCC-specific deaths. However, the knowledge on mechanisms by which alcohol consumption leads to cancer progression and its aggressiveness is limited. Better understanding of the clinical features and the mechanisms of alcohol-induced HCC are of critical importance for prevention and the development of novel treatments. Early stage Huh-7 and advanced SNU449 liver cancer cell lines were subjected to chronic alcohol exposure (CAE), at different doses for 6 months followed by 1-month alcohol withdrawal period. ADH activity and ALDH expression were much lower in SNU449 compared with Huh-7 cells and at the 270 mM dose, CAE decreased cell viability by about 50% and 80%, respectively, in Huh-7 and SNU449 cells but induced mortality only in Huh-7 cells. Thus, Huh-7 may be more vulnerable to ethanol toxicity because of the higher levels of acetaldehyde. CAE induced a dose-dependent increase in cell migration and invasion and also in the expression of cancer stem cells markers (CD133, CD44, CD90). CAE in Huh-7 cells selectively activated ERK1/2 and inhibited GSK3β signaling pathways. Most of the changes induced by CAE were reversed after alcohol withdrawal. Interestingly, we confirmed the increase in CD133 mRNA levels in the tumoral tissue of patients with ethanol-related HCC compared to other HCC etiologies. Our results may explain the benefits observed in epidemiological studies showing a significant increase of overall survival in abstinent compared with non-abstinent patients.

International audience; Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATAI ratio and decreased alpha/beta-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STATS and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

Background and Aims Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of renal function due to high serum levels of uremic toxins. Anemia is a major trouble in CKD patients that contributes to a faster deterioration of renal failure, leading to cardiovascular disease and increasing morbimortality. Erythropoietin (EPO) is known to contribute to CKD-associated anemia. Thus, accumulation of uremic toxins in blood impairs EPO synthesis, leading to a subsequent impairment of erythropoiesis in the bone marrow. Very few molecular clues explain why erythropoiesis is affected in CKD or explain why erythropoiesis-stimulating agents (ESA) are not efficient in some patients with CKD. The current study aims to characterize the impact of one of the most representative uremic toxins, Indoxyl Sulfate (IS), in CKD-related anemia. IS is a protein-bound uremic toxin derived from the tryptophan dietary metabolism which is difficult to remove by dialysis. Our study demonstrates the molecular effects of IS on the growth and the differentiation of red blood cells in an erythroid cell line and in primary cell cultures CD34+. Method Firstly, we examined in vitro the time-courses of IS under clinically relevant concentrations of IS (250 µM -1 mM) in a human leukemic cell line in which proliferation is induced by EPO, the UT7/EPO cell line. Cell apoptosis, proliferation, differentiation and cell cycle analysis were assessed by the MACSQuant flow cytometry. Erythroid gene expression analysis was assessed by RT-qPCR (Quantstudio 7 flex). The ratio A260/280 assessed the quality of nucleic acids. Western blotting experiments were performed to study protein expression. Human primary CD34+ cells were obtained from mobilized peripheral blood mononuclear cells (MNC) of healthy subjects and were isolated by magnetic microbeads separation on MACS columns. Results IS at 250 µM and 1 mM increased apoptosis of UT7/EPO cell line at 48h compared to control condition. On the other hand, we found no significant effect of IS on the phenotype of UT7/EPO, when using CD235a (Glycophorin A), as a marker for the detection of the erythroid cell lineage. Ki67 cellular levels, a cell proliferation marker, was not altered between control and IS experiments. This indicated that IS did not affect proliferation in UT7/EPO. At 48h, at the clinically relevant concentration of IS (250 µM), we observed an increase of the cell phase cycle Sub-G1. The analysis of erythropoiesis related genes shows that HIF2α was deregulated with IS (250 µM). Finally, in the Epo-EpoR signalling pathway, we studied the activation of the Jak2/Stat5 proteins. Results in human primary CD34+ cells confirmed the apoptotic effect of IS observed in UT7/EPO. Conclusion Our findings suggest that IS, a representative protein-bound uremic toxin, could affect cell viability, apoptosis and the cell cycle. This study suggests clues to develop new therapies for CKD-associated anemia.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases. PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets. This article is protected by copyright. All rights reserved.