Red Blood Cell Development Research Articles

The Super-Enhancer-Derived alncRNA-EC7/Bloodlinc Potentiates Red Blood Cell Development in trans

Enhancer-derived RNAs are thought to act locally bycontributing to their parent enhancer function. Whether large domains of clustered enhancers (super-enhancers) also produce cis-acting RNAs, however, remains unclear. Unlike typical enhancers, super-enhancers form large spans of robustly transcribed chromatin, amassing capped and polyadenylated RNAs that are sufficiently abundant to sustain trans functions. Here, we show that one such RNA, alncRNA-EC7/Bloodlinc, is transcribed from asuper-enhancer of the erythroid membrane transporter SLC4A1/BAND3 but diffuses beyond this site. Bloodlinc localizes to trans-chromosomal lociencoding critical regulators and effectors of terminal erythropoiesis and directly binds chromatin-organizingand transcription factors, including the chromatin attachment factor HNRNPU. Inhibiting Bloodlinc or Hnrnpu compromises the terminal erythropoiesis gene program, blocking red cell production, whereas expressing Bloodlinc ectopically stimulates this program and can promote erythroblast proliferation and enucleation in the absence ofdifferentiation stimuli. Thus, Bloodlinc is a trans-acting super-enhancer RNA that potentiates red blood cell development.

A regulatory function of long non-coding RNAs in red blood cell development.

In recent years it has been discovered that long non-coding RNAs are important regulators in many biological processes. In this review, we summarize the role of lncRNA in erythropoiesis. lncRNA are crucial for regulation of gene expression during both, proliferation and differentiation stages of red blood cell development. Many are regulated by erythroid-specific transcription factors and some are expressed in a developmental stage-specific manner. The majority of individually studied lncRNAs are involved in regulating the terminal maturation stages of red cell differentiation. Their regulatory function is accomplished by various mechanisms, including direct regulation in cis or trans by the lncRNA product or by the cis-localized presence of the lncRNA transcription itself. These add additional levels of regulation of gene expression during erythropoiesis.

Widespread and dynamic translational control of red blood cell development

AbstractCell development requires tight yet dynamic control of protein production. Here, we use parallel RNA and ribosome profiling to study translational regulatory dynamics during murine terminal erythropoiesis. Our results uncover pervasive translational control of protein synthesis, with widespread alternative translation initiation and termination, robust discrimination of long noncoding from micropeptide-encoding RNAs, and dynamic use of upstream open reading frames. Further, we identify hundreds of messenger RNAs (mRNAs) whose translation efficiency is dynamically controlled during erythropoiesis and that enrich for target sites of RNA-binding proteins that are specific to hematopoietic cells, thus unraveling potential regulators of erythroid translational programs. A major such program involves enhanced decoding of specific mRNAs that are depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. We find that RBM38, an erythroid-specific RNA-binding protein previously implicated in splicing, interacts with the general translation initiation factor eIF4G and promotes translation of a subset of these irreplaceable mRNAs. Inhibition of RBM38 compromises translation in erythroblasts and impairs their maturation, highlighting a key function for this protein during erythropoiesis. These findings thus reveal critical roles for dynamic translational control in supporting specialized mammalian cell formation.

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation.

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation.

Widespread and Dynamic Translational Control of Red Blood Cell Development

Cell development requires tight yet dynamic control of protein production. Here, we use murine erythropoiesis as a model to study translational regulatory dynamics during mammalian cell differentiation. We uncover pervasive translational control of protein synthesis, including widespread alternative translation initiation and termination, stoichiometric synthesis, and dynamic use of upstream reading frames. In addition, we identify hundreds of mRNAs with dynamic translation efficiencies during erythropoiesis. Interestingly, these mRNAs have significantly enriched binding motifs of many mRNA-binding proteins that are expressed with high specificity in hematopoietic cells, suggesting that those mRNA-binding proteins are important players in the translational regulatory programs in erythropoiesis. A major such program involves enhanced decoding of specific mRNAs depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. We functionally characterized one such protein, Rbm38, which was previously identified as a splicing factor in terminal erythropoiesis, can enhance the translation of a subset of those mRNAs by interacting with the general translation initiation factor eIF4G in erythroid cells. Altogether, our findings reveal widespread and dynamic translational control in supporting specialized mammalian cell formation. DisclosuresNo relevant conflicts of interest to declare.

Csk-binding protein controls red blood cell development via regulation of Lyn tyrosine kinase activity.

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells invitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment invivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp-/- mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp-/- mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp-/- mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp-/- mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal.

Characterizing the hypomethylated DNA methylation profile of nucleated red blood cells from cord blood.

To provide insight into fetal nucleated red blood cell (nRBC) development using genome-wide DNA methylation (DNAm) profiling. The DNAm profile (Illumina 450K array) of cord blood (n = 7) derived nRBCs was compared with B cells, CD4 and CD8 T cells, natural killer cells, granulocytes, monocytes and placenta (n = 5). nRBCs and placenta had similarly low array-wide DNAm compared with white blood cells, but their patterns of hypomethylation differed at biologically relevant subsets of the array. High interindividual variability in nRBC DNAm was driven by a negative association between DNAm and nRBC count. nRBC hypomethylation is likely an epigenetic signature of erythropoiesis rather than of early development. Variability in nRBC DNAm may stem from differences in the cell population's maturity or hematopoietic source.

The splice factor PTBP1 regulates hematopoietic stem cell function and red blood cell development

The splice factor PTBP1 regulates hematopoietic stem cell function and red blood cell development

Defining the minimal factors required for erythropoiesis through direct lineage conversion

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Utilization Analysis of Neonatal Red Blood Cell Aliquots and Development of Electronic Issuing System

Utilization Analysis of Neonatal Red Blood Cell Aliquots and Development of Electronic Issuing System Minjeong Nam, Mi Jung Kim, Soon He Choi, Yun Ji Hong, Hyungsuk Kim, Sang Mee Hwang, Jeong Su Park, Chang Won Choi, Kyoung Un Park, Junghan Song, Kyou Sup Han Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Departments of Laboratory Medicine and Pediatrics, Seoul National University Bundang Hospital, Seongnam, Korea

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.

SummaryErythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis.

This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5′ untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

Parsing Physiological Functions of Erythropoietin One Domain at a Time.

A domain of erythropoietin (EPO), separate from the domain involved in red blood cell development, has been identified. This region of EPO has anti-inflammatory and neuroprotective effects. Use of a peptide sequence from this region provides the potential for an effective therapeutic without effects on erythropoiesis.

The sorting of blood group active proteins during enucleation.

Enucleation represents the critical stage during red blood cell development when the nucleus is extruded from an orthochromatic erythroblast in order to generate a nascent immature reticulocyte. Extrusion of the nucleus results in loss of a proportion of the erythroblast plasma membrane, which surrounds the nucleus, the bulk of the endoplasmic reticulum and a small region of cytoplasm. For this reason enucleation provides an important point in erythroblast differentiation at which proteins not required for the function of the erythrocyte can be lost, whilst those that are important for the structure-function properties of the mature erythrocyte must be efficiently retained in the reticulocyte plasma membrane. Disturbances in protein distribution during enucleation are envisaged to occur during human diseases such as Hereditary Spherocytosis. This article will discuss the current knowledge of erythroblast enucleation in the context of retention and loss of proteins that display antigenic blood group sites and that exist within multiprotein complexes within the erythrocyte membrane.

Multi-omic signature of body weight change: results from a population-based cohort study.

BackgroundExcess body weight is a major risk factor for cardiometabolic diseases. The complex molecular mechanisms of body weight change-induced metabolic perturbations are not fully understood. Specifically, in-depth molecular characterization of long-term body weight change in the general population is lacking. Here, we pursued a multi-omic approach to comprehensively study metabolic consequences of body weight change during a seven-year follow-up in a large prospective study.MethodsWe used data from the population-based Cooperative Health Research in the Region of Augsburg (KORA) S4/F4 cohort. At follow-up (F4), two-platform serum metabolomics and whole blood gene expression measurements were obtained for 1,631 and 689 participants, respectively. Using weighted correlation network analysis, omics data were clustered into modules of closely connected molecules, followed by the formation of a partial correlation network from the modules. Association of the omics modules with previous annual percentage weight change was then determined using linear models. In addition, we performed pathway enrichment analyses, stability analyses, and assessed the relation of the omics modules with clinical traits.ResultsFour metabolite and two gene expression modules were significantly and stably associated with body weight change (P-values ranging from 1.9 × 10−4 to 1.2 × 10−24). The four metabolite modules covered major branches of metabolism, with VLDL, LDL and large HDL subclasses, triglycerides, branched-chain amino acids and markers of energy metabolism among the main representative molecules. One gene expression module suggests a role of weight change in red blood cell development. The other gene expression module largely overlaps with the lipid-leukocyte (LL) module previously reported to interact with serum metabolites, for which we identify additional co-expressed genes. The omics modules were interrelated and showed cross-sectional associations with clinical traits. Moreover, weight gain and weight loss showed largely opposing associations with the omics modules.ConclusionsLong-term weight change in the general population globally associates with serum metabolite concentrations. An integrated metabolomics and transcriptomics approach improved the understanding of molecular mechanisms underlying the association of weight gain with changes in lipid and amino acid metabolism, insulin sensitivity, mitochondrial function as well as blood cell development and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0282-y) contains supplementary material, which is available to authorized users.

Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells has limited their use for modeling of hematological diseases. We previously reported a strategy to respecify lineage-restricted CD34+hematopoietic precursors derived from iPSCs into multilineage progenitors that can be expanded in vitro and transplanted in vivo. Five transcription factors, HOXA9, ERG, RORA, SOX4 and MYB, enable expansion and maintenance of primitive CD34+CD38- cells, and allow their differentiation upon transgene silencing. Respecified iPSC-derived hematopoietic progenitors give rise to robust short-term engraftment with myeloid and erythroid lineages. Notably, the erythrocytes undergo definitive maturation and hemoglobin switching to express β-globin in vivo. This system presents a useful platform for modeling hematological disorders due to its capacity to generate large numbers of engraftable disease cells for in vitro and in vivo screens.Congenital anemias, such as Diamond Blackfan anemia (DBA) represent a defined system for understanding red blood cell development and more common idiopathic anemias. To model this disease in vitro, we combined factor-induced respecification with stepwise erythroid maturation. We show that respecified iPSCs from DBA patients recapitulate the defect in erythroid differentiation. Consistent with clinical observations, early erythroblasts show impaired proliferation marked by increased apoptosis and p21 expression, while terminal maturation is unaffected. Interestingly, while early passage iPSC lines display a profound erythroid defect, continuous passage restores a near-normal capacity for erythroid differentiation. Furthermore, transplanted DBA iPSC-derived progenitors give rise to normal myeloid, but fail to generate erythroid, engraftment. This validates this system as a powerful tool for disease modeling and drug discovery. Mice transplanted with DBA iPSC-derived progenitors treated with conventional anemia drugs, such as dexamethasone, show a modest improvement in human erythroid output suggesting the need for novel treatments. Using high-throughput chemical screens, we have identified several signaling pathways which may be attractive therapeutic targets in DBA and other anemias. Further characterization is currently underway to better target defective erythropoiesis and dissect underlying disease mechanisms. DisclosuresZon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Direct Lineage Reprogramming of Murine Fibroblasts to Erythroid Progenitor Cells By Defined Factors

Transcription factor-based direct lineage reprogramming is a powerful tool to discover and study factors determining cell lineage fate. We propose that this methodology can be used to define the core transcriptional program directing red blood cell development. The aim of this study was therefore to identify the minimal set of transcription factors that allows direct lineage reprogramming of murine fibroblasts to erythroid progenitor cells.A retrovirus library was created expressing the coding region of 63 transcription factors known to be involved in erythroid and blood development. Adult tail tip fibroblasts were obtained from erythroid lineage tracing mice, which express yellow fluorescent protein (YFP) in cells that have once expressed the erythropoietin receptor (EpoR) gene at any time of their development. Fibroblasts were depleted for hematopoietic lineage markers and passaged at least 3 times prior to transduction with different combinations of reprogramming factors. The readout for erythroid lineage conversion was formation of colonies of round YFP+ (EpoR+) cells, which were further subjected to extensive analyses to determine their resemblance with primary erythroid progenitor cells. Factor-subtraction experiments revealed a combination of 4 transcription factors, Gata1, Tal1, Lmo2 and c-Myc (collectively referred to as GTLM), capable of directly converting fibroblasts to erythroid progenitors in vitro. These induced erythroid progenitors (iEPs) emerged 5 to 8 days after transduction and displayed an erythroid progenitor-like morphology, featuring a characteristic central nucleus, coarse chromatin and deep blue cytoplasm after May Grünwald-Giemsa staining. When cultured in erythroid-promoting conditions, iEPs differentiated to Benzidine-positive normoblast-like cells. Flow cytometric analysis revealed that 28.9% ± 2.2 of live YFP+ cells collected at day 7 co-expressed CD71 and Ter119, and did not express CD45.Bulk GTLM-transduced fibroblasts formed two types of colonies in methylcellulose assays, one with visibly red hemoglobinized cells and one with blast-like cells. Global gene expression analyses showed GTLM expression was generally higher in the hemoglobinized colonies, suggesting that complete reprogramming only occurs when each factor is expressed at a sufficient level. Hierarchical clustering and pairwise comparisons of gene expression data showed that iEP-derived hemoglobinized colonies correlated tightly with definitive erythroid (BFU-E) colonies from bone marrow and fetal liver. As expected, iEP-derived hemoglobinized colonies displayed large-scale downregulation of the fibroblast-specific program and extensive upregulation of genes specific to the erythroid lineage. In contrast to definitive erythroid colonies, iEP-derived hemoglobinized colonies did not upregulate Sox6 and Bcl11a; and predominantly expressed embryonic hemoglobin, similar to primitive erythroid cells. This could mean that additional factors are required to induce definitive erythropoiesis, which we are currently investigating.Importantly, reprogramming was never successful using any combination of three of the GTLM factors, clearly demonstrating that all four factors are needed and that this is the minimal combination of required factors. Reprogramming of p53-null fibroblasts enhanced efficiency, but did not allow reprogramming without c-Myc. Additional experiments demonstrated that other factors known to be important for red cell development, including Klf1, Nfe2 and Myb, were not required and could not substitute for any of the 4 factors to induce erythroid fate.To our knowledge this is the first successful direct conversion of non-hematopoietic cells to the erythroid lineage. Our results suggest that GTLM constitute the core of the erythroid program, capable of inducing expression of other transcriptional regulators such as Klf1, Zfpm1, Gfi1b, Nfe2 and Myb, which are necessary for normal red cell development. We anticipate that GTLM-induced direct erythroid reprogramming can be used as a new platform for understanding, controlling and studying erythroid lineage development and disease. Furthermore, this knowledge could potentially be applied to enhance methods for in vitro production of erythrocytes for personalized transfusion medicine. DisclosuresNo relevant conflicts of interest to declare.

Mmu-Mir-181a Regulates Terminal Erythroid Nuclear Maturation By Regulating Levels of Xpo7

Mammalian terminal erythroid differentiation involves cell cycle arrest, termination of transcription, and finally progressive chromatin condensation culminating in enucleation. The mechanisms underlying these final events are not completely understood. We have previously published that an erythroid-specific variant of the protein Exportin 7 (Xpo7) plays an important role in terminal erythroid nuclear maturation. Xpo7 is highly induced and highly abundant in late erythroblasts. Xpo7 knockdown in mouse erythroblasts inhibits the final stages of normal erythroid development such as chromatin condensation and enucleation without affecting such important processes as cell surface Ter119 acquisition or hemoglobin accumulation. Migration of nuclear proteins such as core histones into the cytoplasm during normal erythropoiesis is also inhibited by Xpo7 knockdown (1).In order to interrogate the function of Xpo7 in erythropoiesis, we next investigated Xpo7’s genomic structure and how it is regulated during mouse red blood cell development. Xpo7 exerts its effect on the erythroid nucleus after erythroblasts have left the cell cycle and transcription has ended so the timing of its induction must be tightly regulated.We have previously published that Xpo7 has an erythroid–specific start site and first exon (1). Using 5’ RACE, we determined that the erythroid variant of Xpo7 has a different 5’ UTR than the non-erythroid transcript. In contrast, the 3’ UTR is identical in both erythroid and non-erythroid transcripts, and contains a miR-181a binding site. MiR-181a has been previously shown to be abundant in hematopoietic stem cells and to regulate normal B-cell (2) and megakaryocyte development (3). However, in erythropoiesis, miR-181a decreases in expression just as Xpo7 increases, with lowest levels of miR-181a when Xpo7 is most abundant and presumably exerting its effect on erythroid nuclear maturation. Through luciferase reporter assay experiments, we have found that miR-181a directly binds the 3-UTR binding site of Xpo7. Moreover, miR-181a overexpression results in decreased Xpo7 transcript levels via qPCR and decreased enucleation via flow cytometry. Based on these results, we conclude that miR-181a regulates enucleation by repressing Xpo7 expression while the nucleus is active during terminal erythroid development. Our next step is to examine how miR-181a itself is downregulated during erythroid development, such as perhaps through the expression of a complementary long-noncoding RNA.1. Hattangadi, SM, et. al. Histones to the cytosol: Exportin 7 plays an important role in terminal erythroid nuclear maturation. Blood 2014, in press.2. Chen,CZ; Li, L; Lodish, HF; Bartel,DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303: 83–86 3. Li, X; Zhang J; Gao, L; McClellan, S; Finan, MA; Butler TW; Owen, BL; Piazza, GA; Xi, Y. MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit. Cell Death and Differentiation (2012) 19, 378–386. DisclosuresNo relevant conflicts of interest to declare.

Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme

AbstractHeme is essential for the function of all aerobic cells. However, it can be toxic when it occurs in a non–protein-bound form; cells maintain a fine balance between heme synthesis and catabolism. The only physiological mechanism of heme degradation is by heme oxygenases (HOs). The heme-inducible isoform, HO-1, has been extensively studied in numerous nonerythroid cells, but virtually nothing is known about the expression and potential significance of HO-1 in developing red blood cells. We have demonstrated that HO-1 is present in erythroid cells and that its expression is upregulated during erythroid differentiation. Overexpression of HO-1 in erythroid cells impairs hemoglobin synthesis, whereas HO-1 absence enhances hemoglobinization in cultured erythroid cells. Based on these results, we conclude that HO-1 controls the regulatory heme pool at appropriate levels for any given stage of erythroid differentiation. In summary, our study brings to light the importance of HO-1 expression for erythroid development and expands our knowledge about the fine regulation of hemoglobin synthesis in erythroid cells. Our results indicate that HO-1 plays an important role as a coregulator of the erythroid differentiation process. Moreover, HO-1 expression must be tightly regulated during red blood cell development.

Mechanism of Erythropoietin Regulation by Angiotensin II

Erythropoietin (EPO) is the primary regulator of red blood cell development. Although hypoxic regulation of EPO has been extensively studied, the mechanism(s) for basal regulation of EPO are not well understood. In vivo studies in healthy human volunteers and animal models indicated that angiotensin II (Ang II) and angiotensin converting enzyme inhibitors regulated blood EPO levels. In the current study, we found that Ang II induced EPO expression in situ in murine kidney slices and in 786-O kidney cells in culture as determined by reverse transcription polymerase chain reaction. We further investigated the signaling mechanism of Ang II regulation of EPO in 786-O cells. Pharmacological inhibitors of Ang II type 1 receptor (AT1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) suppressed Ang II transcriptional activation of EPO. Inhibitors of AT2R or Src homology 2 domain-containing tyrosine phosphatase had no effect. Coimmunoprecipiation experiments demonstrated that p21Ras was constitutively bound to the AT1R; this association was increased by Ang II but was reduced by the AT1R inhibitor telmisartan. Transmembrane domain (TM) 2 of AT1R is important for G protein-dependent ERK1/2 activation, and mutant D74E in TM2 blocked Ang II activation of ERK1/2. Ang II signaling induced the nuclear translocation of the Egr-1 transcription factor, and overexpression of dominant-negative Egr-1 blocked EPO promoter activation by Ang II. These data identify a novel pathway for basal regulation of EPO via AT1R-mediated Egr-1 activation by p21Ras-mitogen-activated protein kinase/ERK kinase-ERK1/2. Our current data suggest that Ang II, in addition to regulating blood volume and pressure, may be a master regulator of erythropoiesis.