title: The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.
abstract: Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated in vitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.
authors.affiliation: National AIDS Center, Istituto Superiore Sanita, Rome, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Falchi
authors.affiliation: Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
authors.forename: Lilian
authors.initials: L
authors.lastname: Varricchio
authors.affiliation: Hematology/Oncology and Molecular Medicine, Istituto Superiore di Sanità, Roma, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Martelli
authors.affiliation: Hematology/Oncology and Molecular Medicine, Istituto Superiore di Sanità, Roma, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Marra
authors.affiliation: National AIDS Center, Istituto Superiore Sanita, Rome, Italy.
authors.forename: Orietta
authors.initials: O
authors.lastname: Picconi
authors.affiliation: Sant'Andrea Hospital-Sapienza, Department of Clinic and Molecular Medicine Sapienza University of Rome, Rome, Italy.
authors.forename: Agostino
authors.initials: A
authors.lastname: Tafuri
authors.affiliation: Immunohematology and Transfusion Medicine Unit, Sapienza University of Rome, Rome, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Girelli
authors.affiliation: Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.affiliation: Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Department of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy. Electronic address: annarita.migliaccio@mssm.edu.
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Migliaccio
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D037282:Calreticulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D003062:Codon; D004900:Erythroblasts / Q000378:metabolism; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism; D004920:Erythropoiesis / Q000235:genetics; D006801:Humans; D016130:Immunophenotyping; D053614:Janus Kinase 2 / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011087:Polycythemia Vera / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D037282:Calreticulin; D003062:Codon; D011965:Receptors, Glucocorticoid; D053614:Janus Kinase 2; D002118:Calcium