Small Rab GTPase Rab7b Promotes Megakaryocytic Differentiation by Enhancing IL-6 Production and STAT3-GATA-1 Association.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1549-1549
Author(s):  
Donghua He ◽  
Taoyong Chen ◽  
Mingjing Yang ◽  
Xuhui Zhu ◽  
Xuetao Cao ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Small Gtpase ◽  
Rab Gtpase ◽  
Differentiation Markers ◽  
Megakaryocytic Differentiation ◽  
Morphological Alterations

Abstract Abstract 1549 Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA-1 (GATA binding protein 1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. However, the mechanisms for IL-6-induced megakaryocytic differentiation and regulation of GATA-1 have not been fully elucidated. By using phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation of K562 cells as a model, we investigated the roles of Rab7b, a late endosome/lysosome-localized myeloid small GTPase, in megakaryocytic differentiation. We find that Rab7b can promote PMA-induced megakaryocytic differentiation, as evidenced by typical morphological alterations, increased fibronectin-specific adhesion, increased polyploidy formation, and increased expression of CD41a. The GTP-bound status and lysosomal localization of Rab7b are required for promotion of K562 megakaryocytic differentiation, which can be blocked by inhibitor of nuclear factor κB (NF-κB) and neutralizing antibodies for IL-6 and gp130. In Rab7b-silenced K562 cells, PMA-induced activation of NF-κB, IL-6 production and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may favor megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rab5a Promotes Cytolethal Distending Toxin B-Induced Cytotoxicity and Inflammation

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Ming-xian Chen ◽  
Yu Chen ◽  
Rui Fu ◽  
Guo-qun Mao ◽  
Sai-yue Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proinflammatory Cytokines ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Small Gtpase ◽  
Cytolethal Distending Toxin ◽  
Colonic Epithelial Cells ◽  
Toxin B ◽  
Proinflammatory Responses ◽  
Underlying Mechanisms

ABSTRACT The cytolethal distending toxin B subunit (CdtB) induces significant cytotoxicity and inflammation in many cell types that are involved in the pathogenesis of postinfectious irritable bowel syndrome (PI-IBS). However, the underlying mechanisms remain unclear. This study tested the potential role of Rab small GTPase 5a (Rab5a) in the process. We tested mRNA and protein expression of proinflammatory cytokines (interleukin-1β [IL-1β] and IL-6) in THP-1 macrophages by quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs), respectively. In the primary colonic epithelial cells, Cdt treatment induced a CdtB-Rab5a-cellugyrin association. Rab5a silencing, by target small hairpin RNAs (shRNAs), largely inhibited CdtB-induced cytotoxicity and apoptosis in colon epithelial cells. CRISPR/Cas9-mediated Rab5a knockout also attenuated CdtB-induced colon epithelial cell death. Conversely, forced overexpression of Rab5a intensified CdtB-induced cytotoxicity. In THP-1 human macrophages, Rab5a shRNA or knockout significantly inhibited CdtB-induced mRNA expression and production of proinflammatory cytokines (IL-1β and IL-6). Rab5a depletion inhibited activation of nuclear factor-κB (NF-κB) and Jun N-terminal protein kinase (JNK) signaling in CdtB-treated THP-1 macrophages. Rab5a appears essential for CdtB-induced cytotoxicity in colonic epithelial cells and proinflammatory responses in THP-1 macrophages.

Hypoxia-Inducible Factor 1-Mediated Human GATA1 Induction Promotes Erythroid Differentiation Under Hypoxic Conditions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4794-4794
Author(s):  
Jun-Wu Zhang ◽  
Feng-Lin Zhang ◽  
Guo-Min Shen ◽  
Xiao-Ling Liu ◽  
Fang Wang
Keyword(s):  
Rna Interference ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Flanking Sequence ◽  
Differentiation Markers ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions

Abstract Abstract 4794 The stimulation of red blood cell (RBC) production is one of the systemic adaptions to hypoxia. Hypoxia-inducible factor (HIF) promotes erythropoiesis through coordinated cell type-specific hypoxia responses. Hematopoietic transcription factor GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells (HPCs) derived from human cord blood. Enforced HIF1Á expression increased GATA1 expression, while HIF1Á knock-down by RNA interference decreased GATA1 expression in K562 cells. We searched the human GATA1 gene sequence on NCBI and identified a putative HRE in the 3'-flanking sequence of the gene. The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1Á or GATA1. Flow cytometry analysis also indicated that hypoxia or desferrioxamine or CoCl2 induced expression of erythroid surface marker CD71 and CD235a, while expression repression of HIF1Á or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 upregulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. Disclosures: No relevant conflicts of interest to declare.

Enrichment and Trafficking of BCR-ABL Oncoprotein in Exosomes Promotes the IL-3 Independent Proliferation of Bystander Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3751-3751
Author(s):  
Michael Layoun ◽  
Jianya Huan ◽  
Amy M. Skinner ◽  
Peter Kurre
Keyword(s):  
Disease Progression ◽  
Reverse Transcription ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Cell Types ◽  
Leukemic Cells ◽  
Cell Contact ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Bystander Cells

Abstract Abstract 3751 Chronic Myeloid Leukemia (CML) accounts for 15% of all adult leukemias worldwide and is characterized by the BCR-ABL t(9;22)(q34;q11) translocation. Experimental overexpression of BCR-ABL also provides evidence for oncoprotein-mediated induction of instability phenotypes and rescue from apoptosis in non-leukemic cells. Recent work with Chronic Lymphoid Leukemia (CLL) reveals the circulation of cell membrane-derived vesicles in the serum of patients and suggests in vitro signaling effects in the CLL microenvironment. Such lipid-enclosed vesicles, termed exosomes (30–100nm), are released by normal and malignant cell types and have been shown to traffic RNA and proteins between cells, presumably contributing to disease progression. These studies prompted us to evaluate a potential role for vesicles in transferring BCR-ABL oncoprotein to bystander cells and test the hypothesis that oncoprotein transfer may contribute to signaling in the leukemic microenvironment. K562 cells, a BCR-ABL-positive human CML cell line, have been shown to constitutively release membrane-derived vesicles and serve as a model for understanding their biogenesis. Using electron microscopy, we have confirmed exosome release from K562 cells, with >90% of vesicles ranging from 30–100nm in size (n=291). Comparison of cell of origin and exosome content confirmed the previously reported differential global protein profiles and showed colocalization of the exosomal marker CD63 with BCR-ABL by immunofluorescent imaging. Further, quantitative reverse transcription PCR indicated a 35-fold enrichment of BCR-ABL transcript in exosomes compared to the K562 cytosol. Because BCR-ABL is known to rescue certain cytokine-starved cell types from apoptosis, we next tested whether K562 cells could transfer BCR-ABL-enriched exosomes to bystander cells and subsequently foster their proliferation. To demonstrate trafficking of the BCR-ABL-enriched exosomes to bystander cells, a 0.4uM Boyden transwell chamber was used to coculture K562 and recipient cells while preventing direct cell-to-cell contact. Through semi-quantitative reverse transcription PCR we have successfully shown transfer of BCR-ABL transcript to two murine-derived cell lines, BaF3 (pro-B) and OP9 (stromal) cells, resembling cell types present in the leukemic microenvironment. Furthermore, transwell coculture with exosome-producing K562 cells promoted the canonical survival of BaF3 cells following IL-3-deprivation. This is consistent with previous studies where BCR-ABL overexpression rescues IL-3-deprived BaF3 cells from cell cycle arrest and apoptosis. Our work demonstrates the potential of exosome trafficking in affecting the phenotype of bystander cells in the CML microenvironment. Additionally, exosome enrichment encourages further research toward utilizing BCR-ABL-enriched vesicles as a minimally invasive serum biomarker for disease progression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nobiletin Promotes Megakaryocytic Differentiation through the MAPK/ERK-Dependent EGR1 Expression and Exerts Anti-Leukemic Effects in Human Chronic Myeloid Leukemia (CML) K562 Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 877 ◽  
Author(s):  
Jui-Hung Yen ◽  
Ching-Yen Lin ◽  
Chin-Hsien Chuang ◽  
Hsien-Kuo Chin ◽  
Ming-Jiuan Wu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Chronic Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Cell Cycle Regulators ◽  
Differentiation Therapy ◽  
Differentiation Markers ◽  
Small Interference Rna ◽  
Megakaryocytic Differentiation

Differentiation therapy is an alternative strategy used to induce the differentiation of blast cells toward mature cells and to inhibit tumor cell proliferation for cancer treatment. Nobiletin (NOB), a polymethoxyflavone phytochemical, is present abundantly in citrus peels and has been reported to possess anti-cancer activity. In this study, we investigated the anti-leukemic effects of NOB on cell differentiation and its underlying mechanisms in human chronic myeloid leukemia (CML) K562 cells. NOB (100 μM) treatment for 24 and 48 h significantly decreased viability of K562 cells to 54.4 ± 5.3% and 46.2 ± 9.9%, respectively. NOB (10–100 μM) significantly inhibited cell growth in K562 cells. Flow cytometry analysis and immunoblotting data showed that NOB (40 and 80 μM) could modulate the cell cycle regulators including p21, p27, and cyclin D2, and induce G1 phase arrest. NOB also increased the messenger RNA (mRNA) and protein expression of megakaryocytic differentiation markers, such as CD61, CD41, and CD42 as well as the formation of large cells with multi-lobulated nuclei in K562 cells. These results suggested that NOB facilitated K562 cells toward megakaryocytic differentiation. Furthermore, microarray analysis showed that expression of EGR1, a gene associated with promotion of megakaryocytic differentiation, was markedly elevated in NOB-treated K562 cells. The knockdown of EGR1 expression by small interference RNA (siRNA) could significantly attenuate NOB-mediated cell differentiation. We further elucidated that NOB induced EGR1 expression and CD61 expression through increases in MAPK/ERK phosphorylation in K562 cells. These results indicate that NOB promotes megakaryocytic differentiation through the MAPK/ERK pathway-dependent EGR1 expression in human CML cells. In addition, NOB when combined with imatinib could synergistically reduce the viability of K562 cells. Our findings suggest that NOB may serve as a beneficial anti-leukemic agent for differentiation therapy.

scholarly journals ARHGAP21 Is Upregulated and Triggers the Modulation of Rho Gtpase Signaling Pathways during Megakaryocytic Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4760-4760
Author(s):  
Vanessa Aline Bernusso ◽  
Mariana Lazarini ◽  
João Agostinho Machado-Neto ◽  
Karin Spat Albino Barcellos ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Conflicts Of Interest ◽  
Morphological Changes ◽  
Fold Increase ◽  
Cytoskeletal Proteins ◽  
Membrane Receptors ◽  
Differentiation Markers ◽  
Megakaryocytic Differentiation ◽  
Hel Cells

Abstract Introduction: During differentiation, the megakaryocyte goes through profound changes in the cytoskeleton of actin and tubulin through Rho GTPase activity. Microtubules provide the elongation of proplatelets, whereas actin microfilaments mediate force to increase branching and release of platelets. ARHGAP21 is a RhoGAP for RhoA, RhoC and Cdc42, which has been shown to interact with α-tubulin in cancer cells. Moreover, arhgap21+/- mice exhibit significant reduction in platelet number and increased platelet volume. Aim: To evaluate ARHGAP21 function in the activity of Rho GTPase and their effectors during megakaryocytic differentiation. Materials and Methods: Megakaryocyte differentiation was stimulated in HEL cells through treatment with 20 nM of phorbol myristate acetate -13 -12 (PMA) for 4 days and was confirmed by the expression of CD41a, CD42b and CD61 and polyploidy using flow cytometry. Morphological changes were observed by optical microscopy. The localization of ARHGAP21, F-Actin and α-Tubulin cytoskeletal proteins was assessed by confocal microscopy. The expression of ARHGAP21, and the Rho GTPases RhoA, RhoC, Cdc42 and downstream proteins Rock1 e2, phospho-MLC2, MLC2, phospho-cofilin, cofilin and mDia1 were analyzed by western blotting. Rho GTPases activity was determined through pull down assays using Rhotekin-GST (RhoA, RhoB and RhoC) and WASP-GST (Cdc42) constructions. Tubulin polymerization was evaluated by soluble and insoluble tubulin precipitation assay. ARHGAP21 silencing was performed by siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of the expression and activity of Rho GTPases and their effectors, ploidy and differentiation markers. Results: Megakaryocytic differentiation of HEL cells was accompanied by intense rearrangement of the cytoskeleton, increased cell size, polyploidy and increased expression of the membrane receptors CD61, CD41a and CD42b. Interestingly, a gradual upregulation of ARHGAP21 was observed during differentiation, especially on days 2 and 3 of treatment (both 9.33-fold increase) and mainly in extracts containing polymerized tubulin. ARHGAP21 upregulation was concomitant with the reduction of RhoA and Cdc42 activities (92% decreased and 52% decreased, respectively), but not in RhoC. Silencing of ARHGAP21 by siRNA was confirmed by western blot. Downregulation of ARHGAP21 in HEL cells trigged increased phosphorylation on serine 19 of myosin light chain2 (MLC2) on the day 2. Moreover, mDia1, a common effector of RhoA and Cdc42, was also increased at the same point. ARHGAP21 silencing induced an increase in CD42b on day 3 (5% increased, P<0.015). No difference was observed in the expression of CD61 and CD41a and in the ploidy of ARHGAP21 silenced cells compared to control. Conclusion: Our results suggest that the upregulation of ARHGAP21 during megakaryocytic differentiation is important to control the dynamics of the cytoskeleton through the regulation of RhoA and Cdc42. Silencing of ARHGAP21 induces increased phosphorylation of MLC2 and the expression of mDia1, which may impair megakaryocytic differentiation. Furthermore, ARHGAP21 appears to regulate the acquisition of CD42b receptor, participating in the final stages of megakaryopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic

2008 ◽  
Vol 295 (1) ◽  
pp. E29-E37 ◽  
Author(s):  
Kei Sakamoto ◽  
Geoffrey D. Holman
Keyword(s):  
Glucose Transporter ◽  
Cell Types ◽  
Small Gtpase ◽  
Rab Gtpase ◽  
Rab Proteins ◽  
Energy Status ◽  
Vesicle Traffic ◽  
Vesicular Traffic ◽  
Gtpase Activating Proteins ◽  
Glucose Transporter Glut4

Vesicular traffic of the glucose transporter GLUT4 occurs in response to insulin, muscle contraction, and metabolic stimuli that lead to changes in the energy status of the cell. These stimuli are associated with linked kinase cascades that lead to changes in glucose uptake that meet the energy challenges imposed on the highly regulated cell types in insulin-responsive tissues. The need to mechanistically link these kinase-associated stimuli to identifiable intermediates in vesicular traffic has long been known but has been difficult to fulfill. The Rab-GTPase-activating proteins AS160 and TBC1D1 have now emerged as strong candidates to fill this void. Here we review the initial discovery of these proteins as phosphorylated substrates for Akt and the more recent emerging data that indicate that these proteins are substrates for additional kinases that are downstream of contraction and energy status signaling. The mechanism of coupling these phosphorylated proteins to vesicle traffic appears to be dependent on linking to small GTPase of the Rab family. We examine the current state of a hypothesis that suggests that phosphorylation of the Rab-GTPase-activating proteins leads to increased GTP loading of Rab proteins on GLUT4 vesicles and subsequently to increased interaction with Rab effectors that control GLUT4 vesicle translocation.

Dysregulation of PSTPIP2 Due to Loss of GATA-1 Contributes to Aberrant Megakaryopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 854-854
Author(s):  
Lu Liu ◽  
Shangqin Liu ◽  
Qiang Jeremy Wen ◽  
John Crispino ◽  
Zan Huang
Keyword(s):  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Src Kinase ◽  
K562 Cells ◽  
Negative Regulator ◽  
Mouse Bone Marrow ◽  
Megakaryocytic Differentiation ◽  
Erk Phosphorylation ◽  
Intron 1 ◽  
Development And Differentiation

Abstract Abstract 854 GATA-1-deficiency causes dysregulation of multiple genes and leads to aberrant megakaryopoiesis. How the dysregulated genes may contribute to abnormal megakaryocyte development and differentiation is largely unknown. In this study, we focused on Pstpip2, a gene that was upregulated in GATA-1low and GATA-1s mutant megakaryocytes. A strong GATA-1 binding site in the intron 1 of Pstpip2 gene was previously identified by GATA-1 ChIP-Seq studies. These observations strongly suggest that Pstpip2 is a GATA-1 target gene. By using ChIP-PCR we showed that GATA-1 indeed bound to Pstpip2 intron 1 region in megakaryocytes. We also observed that Pstpip2 was expressed in myeloid cells and hematopoietic progenitors, but not lymphocytes. Noticeably, two cell lines with GATA-1 deficiencies, G1ME and CMK, which approximate the MEP and megakaryoblast stages, respectively, showed moderate expression of Pstpip2. Moreover, K562 cells displayed significant upregulation of PSTPIP2 and concomitant downregulation of GATA-1 during TPA-induced megakaryocytic differentiation. To study its function in megakaryocytic differentiation, we modulated PSTPIP2 expression by overexpression or shRNA knockdown. Ectopic expression of PSTPIP2 in K562 or CMK cells impaired TPA-induced megakaryocytic differentiation as evidenced by decreased CD41 expression. In contrast, downregulation of PSTPIP2 by shRNA promoted CD41 expression and increased polyploidy. These findings suggest that failure to suppress PSTPIP2 due to loss of GATA-1 may contribute to abnormal megakaryopoiesis. To probe the mechanism by which PSTPIP2 inhibits megakaryocytic differentiation, we tested whether the interaction with its partner PEST-PTP or phosphorylation of key tyrosine residues in the protein by Src kinase is required. We created two mutants, W232A and Y323/333/F, which block its interaction with PEST-PTP or prevent tyrosine phosphorylation, respectively. Similar to WT PSTPIP2, both mutants retained the ability to inhibit megakaryocytic differentiation suggesting that inhibition of megakaryocytic differentiation does not depend on the PEST-PTP interaction or Src kinase phosphorylation. Separately, we have noted that PSTPIP2 also interacts with Fyn, Grb2, and Shc, suggesting that PSTPIP2 may affect signal transduction. Indeed, we found that ERK phosphorylation was downregulated by PSTPIP2. Since THPO/MPL signaling via JAK/STAT is the physiological pathway in megakaryocyte maturation rather than BCR-ABL signaling seen in K562 cells, we next analyzed the effect of PSTPIP2 on MPL signaling in G1ME cells. Consistent with our findings in K562 and CMK cells, both WT and mutant PSTPIP2 inhibited CD41 expression. Interestingly, ectopic expression of WT PSTPIP2 also caused cell cycle arrest at S phase in G1ME cells. PSTPIP2 also suppressed Erk and Shc phosphorylation. Considering the important role of MAPK/ERK signaling in megakaryocyte development, we believe that PSTPIP2 acts as a negative regulator of THPO-MPL signaling to suppress TPO-stimulated MAPK/ERK activation and subsequently inhibit megakaryopoiesis. Finally, we confirmed the function of PSTPIP2 in megakaryocyte differentiation using mouse bone marrow progenitor cells. Ectopic expression of PSTPIP2 significantly downregulated CD41 expression, reduced CFU-GM and CFU-Mk numbers, and reduced polyploidy, while knockdown of PSTPIP2 exhibited the opposite effects. Taken together, we have identified a new negative regulator of THPO-MPL signaling that is inhibited by GATA-1 in normal megakaryocyte development. Upregulation of PSTPIP2 may contribute to abnormal megakaryopoiesis observed in GATA-1-deficient megakaryocytes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hscb, a Mitochondrial Iron-Sulfur Cluster Assembly Co-Chaperone, Is a Novel Candidate Gene for Congenital Sideroblastic Anemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 79-79
Author(s):  
Andrew Crispin ◽  
Paul Schmidt ◽  
Dean Campagna ◽  
Chang Cao ◽  
Daniel Lichtenstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Iron Metabolism ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Mitochondrial Translation ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Mitochondrial Iron

Abstract Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins. To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C&gt;A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct. K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B). We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis. Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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