212 research outputs found
Erythropoietin Receptor Signaling Is Membrane Raft Dependent
Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units
Erythropoietin in the intensive care unit: beyond treatment of anemia
Erythropoietin (EPO) is the major hormone stimulating the production and differentiation of red blood cells. EPO is used widely for treating anemia of critical illness or anemia induced by chemotherapy. EPO at pharmacological doses is used in this setting to raise hemoglobin levels (by preventing the apoptosis of erythroid progenitor cells) and is designed to reduce patient exposure to allogenic blood through transfusions. Stroke, heart failure, and acute kidney injury are a frequently encountered clinical problem. Unfortunately, in the intensive care unit advances in supportive interventions have done little to reduce the high mortality associated with these conditions. Tissue protection with EPO at high, nonpharmacological doses after injury has been found in the brain, heart, and kidney of several animal models. It is now well known that EPO has anti-apoptotic effects in cells other than erythroid progenitor cells, which is considered to be independent of EPOs erythropoietic activities. This review article summarizes what is known in preclinical models of critical illness and discusses why this does not correlate with randomized, controlled clinical trials
Sox6 Is Necessary for Efficient Erythropoiesis in Adult Mice under Physiological and Anemia-Induced Stress Conditions
BACKGROUND: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. METHODOLOGY/PRINCIPAL FINDINGS: Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1
Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases
The production of peroxide and superoxide is an inevitable consequence of
aerobic metabolism, and while these particular "reactive oxygen species" (ROSs)
can exhibit a number of biological effects, they are not of themselves
excessively reactive and thus they are not especially damaging at physiological
concentrations. However, their reactions with poorly liganded iron species can
lead to the catalytic production of the very reactive and dangerous hydroxyl
radical, which is exceptionally damaging, and a major cause of chronic
inflammation. We review the considerable and wide-ranging evidence for the
involvement of this combination of (su)peroxide and poorly liganded iron in a
large number of physiological and indeed pathological processes and
inflammatory disorders, especially those involving the progressive degradation
of cellular and organismal performance. These diseases share a great many
similarities and thus might be considered to have a common cause (i.e.
iron-catalysed free radical and especially hydroxyl radical generation). The
studies reviewed include those focused on a series of cardiovascular, metabolic
and neurological diseases, where iron can be found at the sites of plaques and
lesions, as well as studies showing the significance of iron to aging and
longevity. The effective chelation of iron by natural or synthetic ligands is
thus of major physiological (and potentially therapeutic) importance. As
systems properties, we need to recognise that physiological observables have
multiple molecular causes, and studying them in isolation leads to inconsistent
patterns of apparent causality when it is the simultaneous combination of
multiple factors that is responsible. This explains, for instance, the
decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference
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VITELLOGENINS OF THE COCKROACH FAT BODY: STRUCTURE, BIOSYNTHESIS, SECRETION AND A POSSIBLE ROLE FOR FAT BODY CYTOSOLIC JUVENILE HORMONE BINDING PROTEINS IN VITELLOGENESIS
Cockroach fat body vitellogenesis has been employed as a model for studies concerning the juvenile hormone (JH) induction and biosynthetic processing of insect vitellogenins (VGs). Fat body of Blaberus discoidalis was shown to synthesize and secrete two discrete VGs in response to JH. Oocyte-bound vitellins (VTs) corresponding to each VG were purified by salt fractionation and gel filtration. The native VTs were of similar size (17S, 8.8 nm radii, 680K Mr), yet (i) separated in NaBr density gradients, (ii) possessed unique peptide substructures (6 and 8 peptides of 36K to 192K Mr), and (iii) were immunologically unrelated. Using antiserum to purified VT, precursor VGs for each VT were demonstrated to be synthesized and secreted by fat body cultured in vitro. In fat body cytosol, JH binding components potentially mediating JH induction of VG were indicated by a developmental concomitancy in the onset of vitellogenesis, and the expression of a saturable, specific, high-affinity JH-III binding protein (Kd (TURN) 10-9M, 5.5nm radius, 7.5S, 180K Mr). Prior to equilibrium analyses, JH-degradative, low Mr fat body esterases were separated from JH binding activity on Biogel A-1.5. The JH analogue ZR-515 competed for saturable JH-III sites (Kd (TURN) 2 x 10-6) while farnesal did not, indicating that both the epoxy and terminal ester functions of JH were required for efficient binding. Additionally, maximum likelihood nonlinear regression analysis of equilibrium data, JH-III dissociation kinetics, and DEAE-Cellulose fractionation of JH binding activity resolved a second JH binder (Kd (TURN) 10-8 M) constitutively expressed by fat body. In Blattella germanica, efficient in vivo radiolabeling of VG allowed an examination of the role of glycosylation in its biosynthetic processing and secretion from fat body. The antibiotic tunicamycin inhibited VG glycosylation and the subsequent cleavage of a pro-VG peptide into the 160K and 100K peptides of controls. Glycosylation was also required for the efficient secretion of VG from fat body. Mapping of VG and VT peptides helped to define the normal sequence of VG processing during its secretion and ovarian storage
Erythroid mRNA processing: a splice of life .
In this issue of Blood, Cheng and colleagues report first on intriguingly complex pre–messenger RNA (pre-mRNA) global alternate splicing (AS) events that occur in primary erythroblasts. Predominant regulatory roles then are implicated for a Muscleblind-like (MBNL) splicing factor, Mbnl1 (an AS regulator in pluripotent embryonic stem [ES] cells and myoblasts), and for nuclear distribution element like-1 (Ndel1) as a Mbnl1 target. Via loss-of-function (LOF) analyses, Mbnl1 and Ndel1 further are evidenced to support erythroblast development.(1
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Developmental Heterogeneity of Juvenile-Hormone Binding-Sites in Insect Fat-Body Cytosol
Biotinylated recombinant human erythropoietins: bioactivity and utility as receptor ligand [see comments]
Emerging EPO and EPO receptor regulators and signal transducers.
As essential mediators of red cell production, erythropoietin (EPO) and its cell surface receptor (EPO receptor [EPOR]) have been intensely studied. Early investigations defined basic mechanisms for hypoxia-inducible factor induction of EPO expression, and within erythroid progenitors EPOR engagement of canonical Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5), rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/MEK/ERK), and phosphatidylinositol 3-kinase (PI3K) pathways. Contemporary genetic, bioinformatic, and proteomic approaches continue to uncover new clinically relevant modulators of EPO and EPOR expression, and EPO\u27s biological effects. This Spotlight review highlights such factors and their emerging roles during erythropoiesis and anemia
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