Taiju Utsugisawa

Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond‐Blackfan Anemia

Diamond‐Blackfan anemia (DBA) is a congenital red‐cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19‐deficient DBA, we analyzed the effects of RPS19 deficiency on erythropoietin (EPO)‐induced signal transduction, cell cycle, and apoptosis in RPS19‐deficient TF‐1 cells. We did not find any abnormality in EPO‐induced signal transduction. However, RPS19‐deficient TF‐1 cells showed G0/G1 arrest (82% vs. 58%; p < .05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin V analysis also increased compared with control cells (13% vs. 3.1%; p < .05). Western blot analysis of apoptosis‐related proteins showed that the level of bcl‐2 and Bad was decreased and Bax was increased in RPS19‐deficient TF‐1 cells. Moreover, primary CD34‐positive cells from DBA patients detected by Annexin V analysis also generated a higher number of apoptotic cells compared with normal CD34‐positive cells during in vitro culture (38% vs. 8.9%; n = 5; p < .001). Finally, we show that although RPS19 silencing reduces EPO‐induced development of erythroid progenitors expressing glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19‐deficient DBA.

A road map toward defining the role of smad signaling in hematopoietic stem cells

The transforming growth factor‐β (TGF‐β) superfamily encompasses the ligands and receptors for TGF‐β, bone morphogenic proteins (BMPs), and Activins. Cellular response to ligand is context‐dependent and may be controlled by specificity and/or redundancy of expression of these superfamily members. Several pathways within this family have been implicated in the proliferation, differentiation, and renewal of hematopoietic stem cells (HSCs); however, their roles and redundancies at the molecular level are poorly understood in the rare HSC. Here we have characterized the expression of TGF‐β superfamily ligands, receptors, and Smads in murine HSCs and in the Lhx2‐hematopoietic progenitor cell (Lhx2‐HPC) line. We demonstrate a remarkable likeness between these two cell types with regard to expression of the majority of receptors and Smads necessary for the transduction of signals from TGF‐β, BMP, and Activin. We have also evaluated the response of these two cell types to various ligands in proliferation assays. In this regard, primary cells and the Lhx2‐HPC line behave similarly, revealing a suppressive effect of Activin‐A that is similar to that of TGF‐β in bulk cultures and no effect of BMP‐4 on proliferation. Signaling studies that verify the phosphorylation of Smad2 (Activin and TGF‐β) and Smad1/5 (BMP) confirm cytosolic responses to these ligands. In addition to providing a thorough characterization of TGF‐β superfamily expression in HSCs, our results define the Lhx2‐HPC line as an appropriate model for molecular characterization of Smad signaling.

ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia

Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca2+ concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient’s senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca2+-activated scrambling in both control erythrocytes and the patient’s erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.