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Studies of Porphyrin Metabolism, 3. The Relation of Erythropoiesis to the Excretion of Coproporphyrin by Dogs and Rabbits, and to the Concentration of Coproporphyrin and Protoporphyrin in Rabbit Erythrocytes

Description: Report discussing studies regarding porphyrin excretion and hemoglobin metabolism in animals with phenylhydrazine anemia, and studies regarding the coproporphyrin and protoporphyrin in the red blood cells of rabbits.
Date: November 25, 1945
Creator: Schwartz, Samuel; Glickman, Marcia; Hunter, Rosie & Wallace, Jean
Partner: UNT Libraries Government Documents Department

Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions

Description: ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68 percent overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.
Date: February 18, 2003
Creator: Lee, Gloria; Spring, Frances A.; Parons, Stephen F.; Mankelow, Tosti J.; Peters, Luanne L.; Koury, Mark J. et al.
Partner: UNT Libraries Government Documents Department

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

Description: Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.
Date: March 1, 2006
Creator: Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C. et al.
Partner: UNT Libraries Government Documents Department

Erythroblastic Islands: Specialized Mircoenvironmental Niches forErythropoiesis

Description: This review focuses on current understanding of molecular mechanisms operating within erythroblastic islands including cell-cell adhesion, regulatory feedback, and central macrophage function. RECENT FINDINGS: Erythroblasts express a variety of adhesion molecules and recently two interactions have been identified that appear to be critical for island integrity. Erythroblast macrophage protein, expressed on erythroblasts and macrophages, mediates cell-cell attachments via homophilic binding. Erythroblast intercellular adhesion molecule-4 links erythroblasts to macrophages through interaction with macrophage alphav integrin. In intercellular adhesion molecule-4 knockout mice, erythroblastic islands are markedly reduced, whereas the erythroblast macrophage protein null phenotype is severely anemic and embryonic lethal. Retinoblastoma tumor suppressor (Rb) protein stimulates macrophage differentiation by counteracting inhibition of Id2 on PU.1, a transcription factor that is a crucial regulator of macrophage differentiation. Rb-deficient macrophages do not bind Rb null erythroblasts and the Rb null phenotype is anemic and embryonic lethal. Lastly, extruded nuclei rapidly expose phosphatidylserine on their surface, providing a recognition signal similar to apoptotic cells. SUMMARY: Although understanding of molecular mechanisms operating within islands is at an early stage, tantalizing evidence suggests that erythroblastic islands are specialized niches where intercellular interactions in concert with cytokines play critical roles in regulating erythropoiesis.
Date: January 6, 2006
Creator: Chasis, Joel Anne
Partner: UNT Libraries Government Documents Department

Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis

Description: Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.
Date: February 3, 2009
Creator: Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha et al.
Partner: UNT Libraries Government Documents Department

Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

Description: Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.
Date: August 21, 2008
Creator: Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan et al.
Partner: UNT Libraries Government Documents Department

Targeted Gene Deletion Demonstrates that Cell Adhesion MoleculeICAM-4 is Critical for Erythroblastic Island Formation

Description: Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule-4 (ICAM-4), animmunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha V integrins as ICAM-4 counter receptors. Since macrophages express alpha V, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knockout mice and developed quantitative, live cell techniques for harvesting intact islands and for reforming islands in vitro. We observed a 47 percent decrease in islands reconstituted from ICAM-4 null marrow compared to wild type. We also found a striking decrease in islands formed in vivo in knockout mice. Further, peptides that block ICAM-4 alpha V adhesion produced a 53-57 percent decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha V functions in island integrity. Importantly, we documented that alpha V integrin is expressed in macrophages isolated from erythro blastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.
Date: February 15, 2006
Creator: Lee, Gloria; Lo, Annie; Short, Sarah A.; Mankelow, Tosti J.; Spring, Frances; Parsons, Stephen F. et al.
Partner: UNT Libraries Government Documents Department

Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

Description: Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.
Date: April 6, 2005
Creator: Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla & Chasis, Joel Anne
Partner: UNT Libraries Government Documents Department

Hemopoietic cell precursor responses to erythropoietin in plasma clot cultures

Description: The time dependence of the response of mouse bone marrow cells to erythropoietin (Ep) in vitro was studied. Experiments include studies on the Ep response of marrow cells from normal, plethoric, or bled mice. Results with normal marrow reveal: (1) Not all erythroid precursors (CFU-E) are alike in their response to Ep. A significant number of the precursors develop to a mature erythroid colony after very short Ep exposures, but they account for only approx. 13% of the total colonies generated when Ep is active for 48 hrs. If Ep is active more than 6 hrs, a second population of erythroid colonies emerges at a nearly constant rate until the end of the culture. Full erythroid colony production requires prolonged exposure to erythropoietin. (2) The longer erythropoietin is actively present, the larger the number of erythroid colonies that reach 17 cells or more. Two distinct populations of immediate erythroid precursors are also present in marrow from plethoric mice. In these mice, total colony numbers are equal to or below those obtained from normal mice. However, the population of fast-responding CFU-E is consistently decreased to 10 to 20% of that found in normal marrow. The remaining colonies are formed from plethoric marrow at a rate equal to normal marrow. With increasing Ep exposures, the number of large colonies produced increases. From the marrow of bled mice, total erythroid colony production is equal to or above that of normal marrow. Two populations of colony-forming cells are again evident, with the fast-responding CFU-E being below normal levels. The lack of colonies from this group was compensated in bled mice by rapid colony production in the second population. A real increase in numbers of precursors present in this pool increased the rate of colony production in culture to twice that of normal marrow. The ...
Date: January 1, 1979
Creator: Kennedy, W.L.
Partner: UNT Libraries Government Documents Department