Stages Of Terminal Differentiation Research Articles

Temporal Differences in Terminal Erythroid Differentiation of Human CD34+ Cells Derived From Cord Blood and Adult Peripheral Blood

Abstract 3200Purified CD34+ cells derived from either cord blood (CB) or peripheral blood (PB) are currently being used to further our molecular and mechanistic understanding of human terminal erythroid differentiation. What is unclear is whether there are differences in the kinetics of terminal erythroid differentiation of CD34+ cells from these two sources. In the present study, we document that terminal differentiation in cultured CD34+ cells purified from peripheral blood is faster than that of CD34+ cells from cord blood. For these studies, we optimized an 18 day, three phase, in vitro culture system using CD34+ cells to obtain enucleated reticulocytes. In this system, proerythroblasts are generated starting at day 6 which further differentiate during the duration of culture to eventually generate reticulocytes. Based on the expression of various membrane surface markers, we used flow cytometry to quantitatively monitor terminal erythroid differentiation from proerythroblasts to enucleated reticulocytes during culture. The three surface markers, alpha-4 integrin, band 3 and CD36 enabled us to clearly distinguish between all distinct stages of terminal erythroid differentiation – proerythroblasts, early- and late- basophilic erythroblasts, polychromatic and orthochromatic erythroblasts. These analyses enabled us to show that CD34+ cells purified and cultured from peripheral blood underwent terminal erythroid differentiation at a faster rate than CD34+ cells from cord blood. Terminal erythroid differentiation in cord blood cultures was delayed on an average of 2 to 3 days compared to peripheral blood. For example, the surface protein expression pattern seen on days 11–12 of cell culture of peripheral CD34+ cells was not achieved in cord blood cultures until day 14. This delay in terminal differentiation was also reflected by increased extents of enucleation in peripheral blood cultures compared to cord blood (culture day 12: 33% enucleation in PB and 7% in CB and on day 14: 45% enucleation in PB and 19% in CB). These findings have enabled us to document significant differences between the kinetics of terminal erythroid differentiation of CD34+ cells derived from fetal cord and adult peripheral blood. While at the present time we do not have a mechanistic understanding for this difference, we are currently exploring if the observed differences may be related to differences in cell cycle dynamics between fetal and adult erythropopiesis. Disclosures:No relevant conflicts of interest to declare.

Mitochondria Superoxide Is an Early Trigger of Apoptosis in an Ex Vivo Model of Human Beta Thalassemia.

AbstractAbstract 2131Apoptotic cell death is a central feature of beta-thalassemia and other anemias caused by ineffective erythropoiesis. In this study, markers of apoptosis were examined using an ex vivo model of human beta-thalassemia to explore the cause(s) and timing of erythroblast cell death. Beta-globin gene and protein expression were knocked down by lentiviral transduction of beta-globin shRNA (beta-KD) in adult human CD34+ cells. Beta-globin mRNA (Control=4.0E+07 ± 1.4E+06 copies/ng, vs. beta-KD=2.5E+06 ± 1.6E+06 copies/ng, p=0.014) and protein were reduced in beta-KD cells by 90% compared to controls while alpha globin expression was maintained. The effects of imbalanced globin chain synthesis were therefore studied according to the stage of erythroblast maturation. Erythroid progenitor cell commitment and proliferation occur over the first two weeks in culture. During the third week of culture (days 14–21), the proerythroblasts undergo terminal differentiation with the characteristic loss of CD71 from the cell surface.Prior to culture day 14, phenotypic analyses demonstrated low levels of apoptosis in beta-KD and control cultures. On culture day 14, a small but significant increase in active caspase 3 was detected in the beta-KD cells compared to controls (beta-KD=4.0±1.0%, Control=0.7±0.3%, p=0.02) suggesting that apoptosis was initiated during the early stages of terminal maturation. Increases in annexin V staining did not achieve statistical significance on day 14 beta-KD cells (beta-KD=13.1±3.9%, Control=7.6±2.2%, p=0.16). By culture day 18, when orthochromic normoblasts are the most prevalent population in control cultures, a large population of apoptotic cells was detected in the beta-KD. The beta-KD erythroblasts demonstrated further increases in active caspase 3 (beta-KD=11.4±2.2% vs. Control=1.1±0.1%, p=0.014), as well as significant increases in surface annexin V (beta-KD=75.8±3.3%, vs. Control=35.9±12.7%, p=0.024). Western analysis of culture day 18 beta-KD membranes demonstrated a marked increase in alpha-chains compared with culture day 14.Since cleaved caspase 3 was increased in beta-KD cells near the beginning of their terminal maturation and prior to the accumulation of alpha chains in the cell membranes, other triggers of apoptosis were investigated. Mitochondrial superoxide is a reactive species that can be generated by iron or other mitochondrial toxins. Increased levels of mitochondrial superoxide cause apoptosis. The cultured erythroblasts were stained with MitoSOX, a cell-permeable dye that specifically detects mitochondrial superoxide. On culture day 11, mitochondrial superoxide was barely detectable in beta-KD and control cell populations (beta-KD=5.2±3.3% vs. Control=3.6±2.9%, p=0.051). Thereafter, the superoxide detection was increased significantly in beta-KD cells on culture day 14 (beta-KD=54.2±6.7% vs. Control=9.1±2.9%, p=0.003), and culture day 18 (beta-KD=81.1±3.2% vs. Control=34.6±3.8%, p=0.007).Oxidation of cellular membranes by hemichromes and free alpha chains damages thalassemic erythrocytes and their precursors. These data suggest that imbalanced globin chain synthesis also triggers apoptosis during the early stages of terminal differentiation by increasing superoxide formation in the mitochondria. Disclosures:No relevant conflicts of interest to declare.

DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

BackgroundMedullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.ResultsWe identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.ConclusionsThese results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

Survivin: a new player during erythroblast maturation

Enucleation of erythroblasts is a phenomenon unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. They complete their terminal differentiation and enucleation in “erythroid niches” composed of erythroblastic islands nested

A novel role for survivin in erythroblast enucleation

Nucleus free red blood cells are unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. We previously found that survivin, a member of the chromosomal passenger complex that is required for cytokinesis, is highly expressed in late non-dividing cells. The role of survivin in enucleating erythroblasts is not known. In order to identify the role of survivin in these late erythroblasts, we performed proteomic analysis on survivin-bound protein complexes purified from murine erythroleukemia cells. Various molecular and cell biological techniques were used to confirm the presence and function of this novel complex. Furthermore, we used survivin(fl/fl) mice to study the effect of loss of survivin in enucleating erythroblasts. We found that survivin failed to co-localize with its known partners' inner centromere protein or Aurora-B in enucleating erythroblasts but rather exists in a multi-protein complex with epidermal growth factor receptor substrate15 and clathrin, two proteins that mediate endocytic vesicle trafficking. As evidence for a direct role of this latter complex in enucleation, we found that knockdown of the genes reduced the efficiency of enucleation of primary human erythroblasts. We also observed that loss of survivin in murine erythroblasts inhibited enucleation and that survivin-deficient cells harbored smaller cytoplasmic vacuoles. Interestingly, vacuolin-1, a small molecule that induces vacuole fusion, rescued the defective enucleation caused by survivin deficiency. This study identified a novel role for survivin in erythroblast enucleation through previously unknown protein partners.

Cis-9, trans-11, trans-13-conjugated linolenic acid induces apoptosis and sustained ERK phosphorylation in 3T3-L1 preadipocytes

ObjectiveThe aim of this study was to investigate the adipogenic effect of cis-9, trans-11, trans-13-conjugated linolenic acid (c9,t11,t13-CLN), a fatty acid naturally present in bitter melon. MethodsThe 3T3-L1 murine preadipocyte cell line was used to test the effect of saponifiables from whole bitter melon and of commercially prepared pure c9,t11,t13-CLN on adipocyte differentiation. The effect of c9,t11,t13-CLN on 3T3-L1 cell viability was also tested at proliferation, mitotic clonal expansion, and terminal differentiation stages. ResultsCompared to the free fatty acid control mixture, the proadipogenic effect on 3T3-L1 was less potent using saponifiables obtained from bitter melon. C9,t11,t13-CLN, unlike its non-conjugated counterpart linolenic acid (LN) or other common fatty acids such as oleic acid or linoleic acid, exerted no proadipogenic effect on 3T3-L1. In contrast to LN displaying no cytotoxic effect at a concentration ≤100 μM, c9,t11,t13-CLN caused a dose-dependent reduction in the viability of pre- and postconfluent preadipocytes associated with apoptosis. Sustained ERK/MAPK activation, accompanied by increased peroxisome proliferator-activated receptor γ phosphorylation, was seen in c9,t11,t13-CLN-treated cells at initiation of differentiation. ConclusionC9,t11,t13-CLN is less adipogenic for 3T3-L1 cells than LN and this is partly due to its apoptotic effect on proliferating preadipocytes and to the sustained ERK phosphorylation seen during mitotic clonal expansion.

TIS21/BTG2/PC3 enhances downregulation of c-Myc during differentiation of HL-60 cells by activating Erk1/2 and inhibiting Akt in response to all-trans-retinoic acid

Mouse 12-O-tetradecanoyl phorbol-13-acetate inducible sequence 21 (TIS21), an orthologue of human B-cell translocation gene 2 (BTG2) and rat PC3, is a tumour suppressor that belongs to the antiproliferative gene family, and is implicated in a variety of biological processes. c-Myc is a transcription factor and its deregulation is common in leukaemia and lymphomas; the tumours are highly proliferative and often blocked at an earlier phase than the terminal stage of differentiation. The interrelation and the functional interplay of these two different proteins are not defined yet. We have shown here that the tumour suppressor TIS21 negatively regulated c-Myc expression during all-trans-retinoic acid (ATRA)-induced differentiation that accelerated differentiation and reduced proliferation of acute promyelocytic leukaemia (APL) HL-60 cells. TIS21 downregulated c-Myc mRNA and additionally decreased c-Myc protein stability by increasing its phosphorylation at S62 and T58 residues via activation of Erk1/2 and inhibition of PI3K/Akt along with the subsequent activation of GSK-3β in response to ATRA treatment. HL-60 cells treated with GSK-3β or proteosome inhibitors revealed marked accumulation of c-Myc both in the presence and absence of ATRA plus TIS21, confirming ATRA plus TIS21 mediated c-Myc phosphorylation and its consequent degradation in proteosome. Immunoprecipitation assay revealed that TIS21 hindered the interaction of p-Erk1/2 with Akt, thus directly regulating MAPK and Akt activities without interaction with c-Myc. These findings exhibit anticarcinogenic potential of TIS21 via downregulation of c-Myc expression during ATRA induced differentiation of HL-60 cells involving activation and deactivation of two major c-Myc regulators, Erk1/2 and Akt, respectively.

Tr17: A Unique Subset of T Regulatory Cells with Distinct Molecular and Functional Properties

Abstract 1116 A major challenge of the immune system is to fight pathogens and abnormal cell growth while preserving immune tolerance to self-antigen. Active immune suppression by T regulatory (Treg) cells is one of the mechanisms that maintain peripheral T cell tolerance and homeostasis. Recent findings suggest that Treg cells are heterogeneous in functions and phenotypes. Moreover, Treg cells might not be at a terminal stage of differentiation but rather display a significant degree of plasticity particularly under inflammatory conditions. We have previously determined that co-culture of conventional CD4+ T cells and Treg cells in the presence of antigen presenting cells (APC) during T cell receptor mediated stimulation, induced Treg cells producing IL-17 (thereafter named Tr17 cells). IL-1β and IL-2, endogenous products of the co-culture, were essential for the conversion of Treg cells into Tr17 cells. In the present study we sought to identify the phenotypic and functional properties of Tr17 cells. First, we examined the role of IL-2 and IL-1β in the generation of Tr17 cells. Treg cells from Foxp3.GFP knock-in mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-1β-plus-IL-2. A fraction of purified Foxp3+ Treg cells produced significant amounts of IL-17 as determined by ELISA and by intracellular cytokine staining. Very few Tr17 cells were observed during T cell activation in the absence of either IL-1β or IL-2, suggesting that the combination of IL-1β and IL-2 was required for the development of Tr17 cells. Compared to IL-17-Treg, Tr17 cells expressed increased levels of RORγt, the key transcription factor for Th17 cell differentiation. Expression of CCR6 is regulated by RORγt and is one of the features of Th17 cells. To determine whether expression of CCR6+ correlates with the ability of Treg to produce IL-17, we depleted CCR6+ Treg from Foxp3.GFP+ populations and examined the ability of the resulting Treg to produce IL-17. Depletion of CCR6+ Treg resulted in significantly reduced IL-17 production compared with total Foxp3+ Treg cells, suggesting that CCR6+ Treg cells might represent Tr17 cells. Because inducible costimulatory molecule (ICOS) was recently shown to be critical for the differentiation, expansion and function of Th17 cells we examined its role in Tr17 cells. We determined that Tr17 cells expressed strikingly elevated levels of ICOS compared to IL-17− Treg obtained from the same cultures. These results indicate that Tr17 cells acquire phenotypic properties of Th17 cells, which involve RORγt-dependent and independent mechanisms. To determine whether besides obtaining features of Th17 cells, Tr17 cells also retained properties of Treg we first assessed the expression of Foxp3. Compared to IL-17− Treg, Tr17 cells displayed lower levels of Foxp3. However, expression of CTLA-4 and GITR, two other signature makers of Treg cells, was comparable in IL-17− Treg and Tr17 cells. Similarly, both Tr17 and IL-17− Treg cells displayed suppression capacity. Taken together, our results suggest that Tr17 is a unique subset of Treg cells that is differentiated under inflammatory conditions and displays combined phenotypic and functional properties of both Th17 and Treg cells. Because Treg use canonical CD4+ effector cell-associated transcription factors to regulate the immune responses of lineage-specific effectors, our findings suggest that Tr17 cells may have a unique role in regulating Th17-mediated inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

Cytoplasmic Regulators of β-Globin mRNA Are Structurally Modified During Erythroid Terminal Differentiation

Abstract 1093The high-level accumulation of β globin in mature erythrocytes requires a correspondingly high level of its encoding mRNA in terminally differentiating erythroid progenitors. We recently identified two RNA-binding proteins–AUF1 and YB1–that appear to regulate levels of β-globin mRNA in these cells by assembling a cytoplasm-restricted RNA-protein ’β-complex’ on its 3’UTR. The function of the β-complex was predicted by in vitro analyses mapping it to a cis-acting determinant of β-globin mRNA stability, and subsequently validated by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF1 and YB1 ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. Although both AUF1 and YB1 are ubiquitously expressed, studies in cultured cells and in Epo-induced CD34+ primary cells indicated that their β-globin mRNA-specific regulatory properties are restricted to erythroid cells during later stages of terminal differentiation. Based upon these observations, we reasoned that AUF1 and YB1 undergo erythroid and differentiation stage-restricted alterations that permit their assembly into the mRNA-regulatory β-complex.Our analyses of AUF1 focused on three structural isoforms, observed in K562 cells, resulting from alternative pre-mRNA processing events that retain or exclude exon 7. GST-AUF1 fusion isoforms that retain exon 7 fail to bind the β-globin 3’UTR in vitro, while related isoforms that exclude exon 7 bind the 3’UTR with high efficiency. These results, which implicate the importance of exon 7 exclusion to AUF1 function, were subsequently validated in intact K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these in vivo experiments, a reduction in exon 7-excluded AUF1 effected a two-fold decrease in steady-state β-globin mRNA, while similar reductions in exon 7-retained AUF1 isoforms had no measurable effect. The isotype-specific mRNA-binding characteristics of AUF1 may be particularly important during terminal differentiation: Epo-induced CD34+ cells display an increase in exon 7-excluded AUF1, paralleling their capacity to assemble a regulatory β-complex in vitro. Among several possible mechanisms, we asked whether the isoform-specific function of AUF1 might relate to the unusually high number (20) of phosphorylation-capable residues encoded by exon 7. In vitro analyses were fully consistent with this possibility, demonstrating that the β-globin mRNA-binding activity of exon 7-retained AUF1 could be restored by prior dephosphorylation. These experiments suggest that post-transcriptional regulation of β-globin mRNA during erythroid differentiation is likely to be effected by alternative splicing of AUF1 pre-mRNA that eliminates phosphorylation-active exon 7 amino-acids in the translated protein.Based upon these results, we reasoned that related post-translational processes might similarly regulate the β-globin mRNA-binding specificity of YB1. Our analyses focused on a specific residue (Ser102) that is a known target for regulatory phosphorylation and can be experimentally identified using a Ser102 phospho-specific YB1 antibody. In in vitro studies with K562 cytoplasmic extract, which contains both phospho- and dephospho- forms of YB1, we observed that only dephospho-YB1 adheres to the β-globin 3’UTR; likewise, in in vivo studies of CD34+ cells we noted a substantial increase in the ratio of dephospho:phospho-YB1 following Epo induction. Both experiments indicate the likely importance of this post-translational process to the function of YB1 during terminal differentiation. Confirmatory studies are currently being conducted in vivo using an epitope-tagged YB1 containing a position 102 Ser->Ala substitution.Collectively, our analyses indicate that the β-globin mRNA-binding specificities of AUF1 and YB1–and, hence, their corresponding regulatory activities–are determined by post-transcriptional and -translational events. This work suggests mechanisms through which erythroid progenitors can maintain dynamic regulatory control during an interval when transcriptional processes are beginning to silence, and identifies new pathways that can be therapeutically targeted in patients with congenital disorders of β-globin gene expression. Disclosures:No relevant conflicts of interest to declare.

Increased Transferrin Concentration Ameliorates Anemia in Beta-Thalassemic Mice Through Changes in Iron Uptake and TfR1 Trafficking

Abstract 906Hemoglobin (Hb) synthesis during terminal erythroid differentiation is iron dependent and iron delivery requires transferrin (Tf) to transferrin receptor 1 (TfR1) binding. After binding of Tf to TfR1, the ligand-receptor complex is endocytosed through a clathrin dependent mechanism, results in iron delivery via the endosomal compartment, and ends with TfR1 recycling back to the plasma membrane. During erythroid differentiation, TfR1 expression is downregulated and is completely absent from the mature red blood cells (RBCs). Reticulocyte TfR1 is rerouted from the endosomal recycling pathway by sorting to exosomes where it is shed from the cell. Exosomes are small membrane vesicles originating from the fusion of a multi-vesicular endosome compartment with the plasma membrane, leading to the secretion of intraluminal vesicles into circulation. Cell surface TfR1 expression is proportional to the concentration of soluble TfR1 found in circulation as a consequence of exosomal secretion and is increased both in iron deficiency and expanded erythropoiesis.Increased soluble TfR1 is observed in disease of expanded and ineffective erythropoiesis (IE) such as beta-thalassemia, a disease associated with anemia, extramedullary hematopoiesis (EMH), and splenomegaly. We previously demonstrated that apoTf-treated beta-thalassemic mice have more circulating RBCs, increased Hb, reversed splenomegaly and EMH, with fewer reticulocytes and erythroid precursors in the bone marrow and spleen. Furthermore, in both beta-thalassemic and C57BL/6 mice treated with apoTf, we observed a significant reduction in mean cellular hemoglobin (MCH). We hypothesize that TfR1 trafficking is impaired in beta-thalassemia and that exogenous apoTf reduces cellular iron uptake and normalizes TfR1 trafficking pathways, resulting in reduced heme synthesis and a lower MCH observed in apoTf-treated mice.To test this hypothesis, we evaluate 1) cellular iron uptake in cell culture and 2) TfR1 mRNA expression, cell surface expression, and endosomal/exosomal trafficking pathways in C57BL/6 and beta-thalassemic mice. Cell culture experiments were performed using K562 cells +/− 50- and 250-fold excess apoTf relative to holoTf. Mice were evaluated after 20 days of 10 mg human apoTf IP injections (compared with PBS injection). Using a calcein fluorescence quenching approach, we demonstrate that exogenous apoTf decreases iron uptake in culture in a dose response manner. Furthermore, in mouse bone marrow samples sorted using CD44/TER119, we show that TfR1 mRNA expression is higher in beta-thalassemic relative to C57BL/6 mice and increases further in apoTf treated mice in all stages of terminal erythroid differentiation. In addition, although western blot experiments show an increase in cellular TfR1 in beta-thalassemic relative to C57BL/6 mice, cell fractionation experiments demonstrate a proportional shift from the plasma membrane to the endosomal compartment in apoTf-treated mice and reticulocytes of apoTf treated beta-thalassemic mice exhibit significantly reduced TfR1 expression per cell. Finally, reticulocyte TfR1 sorting into exosomes is impaired in beta-thalassemic relative to C57BL/6 mice with a proportional increase in exosomal TfR1 clearance and a reduction in soluble TfR1 in the serum in apoTf treated beta-thalassemic mice.Taken together, our findings demonstrate for the first time that TfR1 trafficking is important for RBC physiology separately from its role in iron uptake and that exogenous apoTf enhances TfR1 endosomal trafficking, normalizes TfR1 sorting into exosomes, and reduces cellular iron uptake. Finally, our results elucidate mechanisms by which MCH is reduced in apoTf-treated mice and provide evidence for multiple consequences of Tf:TfR1 binding on erythroid differentiation and proliferation that characterize diseases of IE. Disclosures:No relevant conflicts of interest to declare.

Mice Lacking the Sodium-Dependent Phosphate Import Protein, Pit-1, Have a Severe Defect in Terminal Erythroid Differentiation

Abstract 681Phosphate is the second most abundant mineral in the human body and plays an essential role in phospholipids, nucleoproteins and nucleic acids, bone mineralization, the storage and liberation of metabolic energy, and enzyme activity. While our understanding of the mechanisms of phosphate homeostasis has advanced, little is known about how the body initially senses changes in phosphate concentration or the downstream biological processes regulated by these concentration changes. Among the three families of membrane mammalian sodium-phosphate import proteins, PiT-1 and its family member, PiT-2, have the highest substrate affinity. PiT-1 is broadly expressed (Proc Nat Acad Sci, 91,7071-75;1994). Cell line models suggest PiT-1 functions in cellular phosphate homeostasis and vascular calcification (PLoS One, 5,e9148;2010). To determine its in vivo role, two groups have generated PiT-1-null mice. Constitutive deletion of PiT-1 results in embryonic lethality at midgestation and mutant embryos display pale livers (the site of fetal erythropoiesis) with increased apoptosis and reduced hematopoietic colony growth (PLoS One, 5, e9148:2010 & Genesis, 47,858-863;2009). These findings suggest that PiT-1 is required for normal erythropoiesis or hematopoiesis, and provided the rationale to characterize the hematopoietic phenotype of post-natal mice lacking PiT-1. We have discovered that mice lacking PiT-1 have markedly abnormal erythropoiesis which models low-grade myeodysplastic syndromes(MDS). MDS comprise a varied group of malignant stem cell disorders characterized by ineffective blood cell production resulting in increased apoptosis and dysplasia in bone marrow progenitor cells and peripheral blood cytopenias. The precise molecular basis of MDS remains unknown and its wide phenotype likely reflects multiple pathophysiologies. We bred mice expressing a conditional PiT-1 allele (PiT-1flox, gift from the Giachelli Lab) to mice expressing the Mx-Cre transgene to generate a viable null mouse for study. PiT-1-deleted mice develop a severe hypoproliferative, macrocytic anemia (HGB 4.5g/dL±0.3 vs. 13.3±1.0, p<1.0E10−4, MCV 53.1fL±0.9 vs. 49.1±0.6, p=4.6E−3, deleted n=9, control n=7, mean±SEM, Student’s t-test) with elevated erythropoietin levels (33,300pg/mL±1300 vs. 650±210, deleted n=7, control n=6, p<1.0E10−4). Their spleens (a site of stress erythropoiesis in mice) are markedly enlarged (spleen weight in mg/body weight in g: 44.1±4.0 vs. 4.8±0.6, deleted n=4, control n=4, p<1.0E10−4) due to a dramatic expansion of erythroid precursors. Bone marrow cytospins and sternal sections from PiT-1-deleted mice demonstrate erythroid hyperplasia, maturation arrest, and morphologic dyserythropoiesis. Flow cytometric analyses of marrow performed to quantitatively define the terminal stages of erythroid differentiation demonstrate a relative expansion of proerythroblasts and basophilic erythroblasts in deleted mice with increased apoptosis (annexinV+7AAD−/7AAD− cells: 17.9%±2.7 vs. 8.0±0.4, p=0.02, deleted n=3, control n=3) compared to control mice; splenic analyses show similar findings. We confirmed that the defect in erythroid differentiation in PiT-1-deleted mice is intrinsic to the hematopoietic system by hematopoietic colony assays and by demonstrating anemia in lethally irradiated mice transplanted with PiT-1flox/flox;Mx-cre marrow and then treated with poly(I)poly(C) to delete PiT-1 specifically in engrafted cells (HGB deleted 4.3 g/dl±1.0, n=5 vs. control 13.9±0.4, n=6, 7–9 weeks post-deletion, p < 1.0E10−4). Cell cycle analyses of Ter119+-erythroid cells reveal an expanded S-phase fraction in PiT-1-deleted cells compared to controls, which can be accounted for by the expanded proerythroblast and basophilic erythroblast populations in mice lacking PiT-1. Ongoing studies are aimed at determining how a lack of PiT-1 results in ineffective erythropoiesis and severe anemia. Our work may lend insight into the pathways linking erythroid differentiation with proliferation and potentially to cellular respiration. These are pathways implicated in the pathogenesis of myelodysplastic syndromes. This work may also contribute to the emerging and novel hypothesis that phosphate acts as a signaling molecule, which modulates cell proliferation via PiT-1 (J Bio Chem 284,31363-74,2009) potentially in a tissue-specific manner. Disclosures:No relevant conflicts of interest to declare.

Technical Advance: Immunophenotypical characterization of human neutrophil differentiation

The current study reports a flow cytometry-based protocol for the prospective purification of human BM populations representing six successive stages of terminal neutrophil differentiation, including early promyelocytes and late promyelocytes, myelocytes, metamyelocytes, band cells, and PMN neutrophilic granulocytes. Validation experiments revealed a high purity of each bone marrow population and biological meaningful expression profiles for marker genes of neutrophil differentiation at a hitherto unprecedented resolution. Hence, the present protocol should be useful for studying neutrophil differentiation in vivo in the human setting and constitutes an important alternative to models that are based on in vitro differentiation of myeloid cell lines and HPCs.

Transition of highly specific microRNA expression patterns in association with discrete maturation stages of human granulopoiesis

Transition of highly specific microRNA expression patterns in association with discrete maturation stages of human granulopoiesis

Conditional Tet-Regulated Over-Expression of Hoxa2 in CG4 Cells Increases Their Proliferation and Delays Their Differentiation into Oligodendrocyte-like Cells Expressing Myelin Basic Protein

Hoxa2 gene was reported to be expressed by oligodendrocytes (OLs) and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al. 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish a stable cell line (CG4-SHoxa2 [sense Hoxa2]), where the expression level of Hoxa2 gene could be up-regulated. The impact of Hoxa2 over-expression on the proliferation and differentiation of CG4-SHoxa2 cells was investigated. Up-regulation of Hoxa2 increased the proliferation of CG4-SHoxa2 cells. The mRNA levels of PDGFαR (platelet-derived growth factor [PDGF] alpha receptor), which is expressed by OL progenitor cells, were not different in CG4-SHoxa2 cells compared to wild-type CG4 cells. Semi-quantitative RT-PCR revealed that the mRNA levels of myelin basic protein (MBP) was lower in CG4-SHoxa2 cells than in wild-type CG4 cells indicating the differentiation of CG4-SHoxa2 cells was delayed when the Hoxa2 gene was up-regulated.

A large‐scale multi‐technique approach identifies forty‐nine new players of keratinocyte terminal differentiation in human epidermis

At the latest stage of terminal differentiation in the epidermis, granular keratinocytes (GKs) undergo cornification, a programmed cell death required for the establishment of a functional skin barrier. A complex genetic regulatory network orchestrates the underlying biochemical modifications, but very few transcription factors specific to this programme have been identified to date. Here, we describe a large-scale, multi-technique approach performed on cells purified from normal human epidermis, primarily focusing on the identification of regulators. We combined data from microarray analysis of cell fractions enriched in GKs or basal keratinocytes, from an expressed sequence tag (EST) library built from GKs and from an in silico promoter analysis of 52 differentiation-associated genes. Among 3576 genes potentially expressed in GK, 298 candidates were selected, and half were directly profiled for the first time in the different layers of the epidermis by quantitative real-time PCR. Forty-nine genes upregulated during terminal differentiation, associated with numerous function of GK including lipid synthesis and secretion, were identified. Of 94 transcription factors detected, 37 were found to be either positively or negatively regulated, suggesting their involvement as regulators of gene expression in the GKs. These results largely extend the number of genes known as involved in the latest step of the terminal differentiation of human epidermis as well as the number of transcription factors known to control the expression of these genes.

A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.

AbstractAbstract 1552 [Background]Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated. [Methods & Results]In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells. [Conclusion]P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions. Disclosures:No relevant conflicts of interest to declare.

RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

AbstractAbstract 2152 Background:CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods:Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results:We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions:RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures:No relevant conflicts of interest to declare.

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

Cytokines in bone diseases. Genetic defects of PTH/PTHrP receptor in chondrodysplasia

Parathyroid hormone-related protein (PTHrP) signaling plays important roles in regulating the differentiation of chondrocytes in endochondral bone development. PTHrP signaling functions as an inhibitory effect on chondrocyte hypertrophy which is a terminal stage of differentiation at a growth plate. Mutations of the PTH÷PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP and its receptor genes in mice have demonstrated the critical roles of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes.

MicroRNAs in benign and malignant hematopoiesis.

MicroRNAs (miRNAs) are a family of 19- to 24-nucleotide noncoding RNAs that regulate messenger RNA function at the posttranscriptional and translational level. Recent literature demonstrates a significant role of miRNAs in normal and malignant hematopoiesis. Specific miRNAs have been shown to regulate each step of hematopoiesis starting at the level of multipotent progenitors through terminal stages of myeloid and lymphoid differentiation. Similarly, individual miRNAs and miRNA signatures have been associated with specific hematologic malignancies. There is accumulating evidence that miRNAs can be used for diagnostic, prognostic, and therapeutic purposes. This review highlights the current status of knowledge on miRNA in normal and malignant hematopoiesis.