The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera
2017; Elsevier BV; Volume: 50; Linguagem: Inglês
10.1016/j.exphem.2017.02.001
ISSN1873-2399
AutoresMario Falchi, Lilian Varricchio, Fabrizio Martelli, Manuela Marra, Orietta Picconi, Agostino Tafuri, G. Girelli, Vladimir N. Uversky, Anna Ritá Migliaccio,
Tópico(s)Kruppel-like factors research
Resumo•CALR mediates the nuclear export of GR in normal human erythroid cells.•This function is exerted by the C-terminal domain of CALR and is regulated by Ca2+.•JAK2V617F impairs the Ca2+ regulation of C-CALR nuclear export function in PV. 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. 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. Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident protein that regulates the functions of numerous proteins by chaperoning them to their active sites in response to Ca2+ [1Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Calreticulin: one protein, one gene, many functions.Biochem J. 1999; 344: 281-292Crossref PubMed Scopus (657) Google Scholar, 2Jiang Y. Dey S. Matsunami H. Calreticulin: roles in cell-surface protein expression.Membranes (Basel). 2014; 4: 630-641Crossref PubMed Scopus (26) Google Scholar]. Structurally and functionally, mature human CALR (residues 18-417; UniProt ID: P27797) can be divided into three domains, a globular N-terminal domain (N-domain, N-CARL, residues 18–197), an extended proline-rich P-domain (residues 198–308), and an acidic C-terminal domain (C-domain, C-CARL, residues 309–417) [3Michalak M. Groenendyk J. Szabo E. Gold L.I. Opas M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.Biochem J. 2009; 417: 651-666Crossref PubMed Scopus (502) Google Scholar, 4Chouquet A. Paidassi H. Ling W.L. et al.X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism.PLoS One. 2011; 6: e17886Crossref PubMed Scopus (67) Google Scholar]. The chaperone function of this protein requires both N-CALR, which binds other proteins, and C-CALR, which contains the KDEL motif required for translocation of the protein and its complexes across membranes. CALR does not contain a transmembrane domain and is anchored to the membrane by C-CALR binding to specific adaptors such as CD59 in neutrophils [5Ghiran I. Klickstein L.B. Nicholson-Weller A. Calreticulin is at the surface of circulating neutrophils and uses CD59 as an adaptor molecule.J Biol Chem. 2003; 278: 21024-21031Crossref PubMed Scopus (80) Google Scholar]. On the plasma membrane, CALR acts as a receptor for orphan ligands such as thrombospondin [6Goicoechea S. Pallero M.A. Eggleton P. Michalak M. Murphy-Ullrich J.E. The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin.J Biol Chem. 2002; 277: 37219-37228Crossref PubMed Scopus (90) Google Scholar]. In the cytoplasm, CALR shuttles other proteins between Golgi and ER [1Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Calreticulin: one protein, one gene, many functions.Biochem J. 1999; 344: 281-292Crossref PubMed Scopus (657) Google Scholar, 7Williams D.B. Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum.J Cell Sci. 2006; 119: 615-623Crossref PubMed Scopus (363) Google Scholar]. In murine cells, it is also present in the nucleus, where it mediates the export of all nuclear receptors, including the glucocorticoid receptor (GRα) [8Dedhar S. Rennie P.S. Shago M. et al.Inhibition of nuclear hormone receptor activity by calreticulin.Nature. 1994; 367: 480-483Crossref PubMed Scopus (304) Google Scholar, 9Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. Calreticulin is a receptor for nuclear export.J Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (222) Google Scholar]. CALR is an intrinsically disordered protein, the function of which is exquisitely dependent on its tertiary structure [10Shivarov V. Ivanova M. Tiu R.V. Mutated calreticulin retains structurally disordered C terminus that cannot bind Ca(2+): some mechanistic and therapeutic implications.Blood Cancer J. 2014; 4: e185Crossref PubMed Scopus (26) Google Scholar, 11Wijeyesakere S.J. Gafni A.A. Raghavan M. Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments.J Biol Chem. 2011; 286: 8771-8785Crossref PubMed Scopus (30) Google Scholar]. Biophysical studies on recombinant constructs containing the globular domain and C-CALR revealed that Ca2+ alters the conformation of the protein by affecting the levels of C-CALR being exposed [12Villamil Giraldo A.M. Lopez Medus M. Gonzalez Lebrero M. et al.The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.J Biol Chem. 2010; 285: 4544-4553Crossref PubMed Scopus (34) Google Scholar]. In the absence of Ca2+ or at low Ca2+ concentrations (0.01–100 nmol/L), C-CALR is deeply buried in the globular domain. It becomes loosely bound to the globular domain, and therefore susceptible to modulation, at Ca2+ concentrations of 50–500 μmol/L, whereas it is completely exposed in the presence of a 5 mmol/L Ca2+ concentration [12Villamil Giraldo A.M. Lopez Medus M. Gonzalez Lebrero M. et al.The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.J Biol Chem. 2010; 285: 4544-4553Crossref PubMed Scopus (34) Google Scholar]. However, because the constructs used in this study did not contain N-CALR, it is not known whether alterations in C-CALR exposure may affect exposure of N-CALR as well. This hypothesis is supported by X-ray crystallography of the truncated construct of human CALR containing the N-domain and the N-terminal half of the C-domain connected by a GSG tripeptide instead of the extended P-domain, indicating a tertiary structure in which C-CALR folds toward the N-domain [4Chouquet A. Paidassi H. Ling W.L. et al.X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism.PLoS One. 2011; 6: e17886Crossref PubMed Scopus (67) Google Scholar]. Binding of glucocorticoids to GRα initiates a JAK2-dependent signaling cascade that activates the stress response [13Zhou J. Cidlowski J.A. The human glucocorticoid receptor: one gene, multiple proteins and diverse responses.Steroids. 2005; 70: 407-417Crossref PubMed Scopus (295) Google Scholar, 14Bauer A. Tronche F. Wessely O. et al.The glucocorticoid receptor is required for stress erythropoiesis.Genes Dev. 1999; 13: 2996-3002Crossref PubMed Scopus (223) Google Scholar]. This cascade induces nuclear translocation of GRα and insures its binding to glucocorticoid-responsive elements regulating the expression of stress-responsive genes [13Zhou J. Cidlowski J.A. The human glucocorticoid receptor: one gene, multiple proteins and diverse responses.Steroids. 2005; 70: 407-417Crossref PubMed Scopus (295) Google Scholar]. Studies on murine fibroblasts transfected with green fluorescent protein-tagged proteins indicated that CALR resets the stress response by inducing nuclear export of GRα, thereby making the cells susceptible to novel ligand activation [9Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. Calreticulin is a receptor for nuclear export.J Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (222) Google Scholar]. In murine cells, this function requires N-CALR, which binds GRα [15Roderick H.L. Campbell A.K. Llewellyn D.H. Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors.FEBS Lett. 1997; 405: 181-185Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar] and is induced by Ca2+ [16Holaska J.M. Black B.E. Rastinejad F. Paschal B.M. Ca2+-dependent nuclear export mediated by calreticulin.Mol Cell Biol. 2002; 22: 6286-6297Crossref PubMed Scopus (91) Google Scholar], an indication that it may be regulated by C-CALR [12Villamil Giraldo A.M. Lopez Medus M. Gonzalez Lebrero M. et al.The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.J Biol Chem. 2010; 285: 4544-4553Crossref PubMed Scopus (34) Google Scholar]. In contrast, studies on CALR in human erythroid cells are sparse and its functions in stress erythropoiesis are still poorly defined. The need for studies on the function of CALR in human erythropoiesis was highlighted by the discovery of somatic mutations in exon 9 of CALR disrupting the C-terminal domain of CALR in patients with the Philadelphia-negative myeloproliferative neoplasms (MPNs) essential thrombocytopenia and primary myelofibrosis not harboring JAK2 mutations [17Klampfl T. Gisslinger H. Harutyunyan A.S. et al.Somatic mutations of calreticulin in myeloproliferative neoplasms.N Engl J Med. 2013; 369: 2379-2390Crossref PubMed Scopus (1368) Google Scholar, 18Nangalia J. Massie C.E. Baxter E.J. et al.Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.N Engl J Med. 2013; 369: 2391-2405Crossref PubMed Scopus (1274) Google Scholar, 19Nunes D.P. Lima L.T. Chauffaille Mde L. et al.CALR mutations screening in wild type JAK2(V617F) and MPL(W515K/L) Brazilian myeloproliferative neoplasm patients.Blood Cells Mol Dis. 2015; 55: 236-240Crossref PubMed Scopus (13) Google Scholar]. CALR mutations are instead not detectable in patients with the MPN polycythemia vera (PV), who harbor mostly (>90%) the gain-of-function JAK2V617F mutation [17Klampfl T. Gisslinger H. Harutyunyan A.S. et al.Somatic mutations of calreticulin in myeloproliferative neoplasms.N Engl J Med. 2013; 369: 2379-2390Crossref PubMed Scopus (1368) Google Scholar, 18Nangalia J. Massie C.E. Baxter E.J. et al.Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.N Engl J Med. 2013; 369: 2391-2405Crossref PubMed Scopus (1274) Google Scholar, 19Nunes D.P. Lima L.T. Chauffaille Mde L. et al.CALR mutations screening in wild type JAK2(V617F) and MPL(W515K/L) Brazilian myeloproliferative neoplasm patients.Blood Cells Mol Dis. 2015; 55: 236-240Crossref PubMed Scopus (13) Google Scholar]. Surprisingly, the phenotype of CALR+ MPNs includes constitutive activation of JAK2 [17Klampfl T. Gisslinger H. Harutyunyan A.S. et al.Somatic mutations of calreticulin in myeloproliferative neoplasms.N Engl J Med. 2013; 369: 2379-2390Crossref PubMed Scopus (1368) Google Scholar, 18Nangalia J. Massie C.E. Baxter E.J. et al.Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.N Engl J Med. 2013; 369: 2391-2405Crossref PubMed Scopus (1274) Google Scholar] and these patients respond to treatment with JAK inhibitors such as ruxolitinib [20Passamonti F. Caramazza D. Maffioli M. JAK inhibitor in CALR-mutant myelofibrosis.N Engl J Med. 2014; 370: 1168-1169Crossref PubMed Scopus (42) Google Scholar, 21Cervantes F. Vannucchi A.M. Kiladjian J.J. et al.COMFORT-II investigatorsThree-year efficacy, safety, and survival findings from COMFORT-II, a phase 3 study comparing ruxolitinib with best available therapy for myelofibrosis.Blood. 2013; 122: 4047-4053Crossref PubMed Scopus (337) Google Scholar]. Studies in human megakaryocytic–erythroleukemic cell lines and in mouse models have elucidated that CALR binds to the cytokine receptor homology domain of all the receptors of the hematopoietic superfamily, including the thrombopoietin (MPL) and erythropoietin (EPO-R) receptors [22Chachoua I. Pecquet C. El-Khoury M. et al.Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.Blood. 2016; 127: 1325-1335Crossref PubMed Scopus (211) Google Scholar, 23Araki M. Yang Y. Masubuchi N. et al.Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.Blood. 2016; 127: 1307-1316Crossref PubMed Scopus (176) Google Scholar]. In addition, binding of the mutated, but not of wild-type, CALR to MPL, but not to EPO-R, acts as a ligand-mimetic activating JAK2 [22Chachoua I. Pecquet C. El-Khoury M. et al.Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.Blood. 2016; 127: 1325-1335Crossref PubMed Scopus (211) Google Scholar, 23Araki M. Yang Y. Masubuchi N. et al.Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.Blood. 2016; 127: 1307-1316Crossref PubMed Scopus (176) Google Scholar], establishing an important link between the CALR/MPL/JAK2 axis and the pathobiology of CALR+ MPN. However, the biological functions exerted by the binding of wild-type CALR to the hematopoietic superfamily receptors in normal cells and whether these functions are impaired by JAK2V617F in PV have not yet been established. In cultures containing the synthetic GRα agonist dexamethasone (Dex), hematopoietic progenitors from normal sources (NS) activate the stress response, generating great numbers of the immature erythroid cells proerythroblasts (proErys) within 10–14 days [24von Lindern M. Zauner W. Mellitzer G. et al.The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro.Blood. 1999; 94: 550-559PubMed Google Scholar, 25Zhang L. Prak L. Rayon-Estrada V. et al.ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.Nature. 2013; 499: 92-96Crossref PubMed Scopus (71) Google Scholar]. These cultures, defined by us as human erythroid massive amplification (HEMA) cultures [26Migliaccio G. Di Pietro R. di Giacomo V. et al.In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients.Blood Cells Mol Dis. 2002; 28: 169-180Crossref PubMed Scopus (115) Google Scholar], are a model for human stress erythropoiesis. In this model, the combination of JAK2 and GR activation elicits a signal, which retains proErys in proliferation, allowing great expansion. In addition, hematopoietic progenitors from JAK2+-PV generate in culture large numbers of proErys, but they do so independently of Dex [27Varricchio L. Masselli E. Alfani E. et al.The dominant negative beta isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.Blood. 2011; 118: 425-436Crossref PubMed Scopus (32) Google Scholar]. In fact, in these cells, by mechanisms still not completely elucidated, GRα is constitutively retained in the nucleus in an active state [27Varricchio L. Masselli E. Alfani E. et al.The dominant negative beta isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.Blood. 2011; 118: 425-436Crossref PubMed Scopus (32) Google Scholar]. Using these observations as foundation, we aimed to generate a deeper understanding of the functions of CALR in human stress erythropoiesis and to explore whether these functions are altered by JAK2V617F in PV. Buffy coats from regular blood donations (NS) were provided by the Transfusion Center of La Sapienza University (Rome, Italy). Blood from 21 JAK2V617F-PV patients who underwent phlebotomy as part of their treatment was provided by Dr. A. Tafuri (GR-MPD/A, CE: Prot.530/12 Rif.2498/14.06.2012) and by the Myeloproliferative Disease Research Consortium Tissue Bank (protocol MPD-RC 116). Samples were collected according to guidelines established by local ethical committees for human subject studies in accordance with the 1975 Helsinki Declaration revised in 2000 and made available as de-identified material. The HEL cell line derived from a patient with Hodgkin's disease who later developed erythroleukemia [28Martin P. Papayannopoulou T. HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression.Science. 1982; 216: 1233-1235Crossref PubMed Scopus (433) Google Scholar] carries the JAK2V617F mutation and was provided by Dr. Papayannopoulou (Washington University, Seattle, WA, USA) in 2006 and maintained since then under good cell practice conditions. The JAK2V617F-negative K562 cell line was used as a negative control. Mononuclear cells (MNCs) were separated by Ficoll and cultured (106 cells/mL) under HEMA conditions with recombinant human stem cell factor (SCF, 100 ng/mL, Amgen, Thousand Oaks, CA, USA), EPO (5 U/mL, Janssen, Raritan, NJ), interleukin-3 (IL-3, 1 ng/mL, R&D Systems, Minneapolis, MN, USA), Dex (10−6 mol/L) and estradiol (10−6 mol/L) (both from Sigma-Aldrich, St Louis, MO, USA) as described previously [26Migliaccio G. Di Pietro R. di Giacomo V. et al.In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients.Blood Cells Mol Dis. 2002; 28: 169-180Crossref PubMed Scopus (115) Google Scholar]. At day 10, cells were analyzed by flow cytometry with CD36 and CD235a to assess maturation [27Varricchio L. Masselli E. Alfani E. et al.The dominant negative beta isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.Blood. 2011; 118: 425-436Crossref PubMed Scopus (32) Google Scholar, 29Falchi M. Varricchio L. Martelli F. et al.Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.Haematologica. 2015; 100: 178-187Crossref PubMed Scopus (31) Google Scholar] and subjected to further analyses. Day 10 Erys were first GF deprived (GFD) for 4 hours in Iscove's modified Dulbecco's medium (#21980, Life Technologies, Carlsbad, California, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) that had been charcoal treated to remove glucocorticoids (F6765, Sigma-Aldrich) and then exposed for 0, 15 minutes, and 4 hours to GFs (SCF, IL-3, or EPO) with or without Dex (10−6 mol/L) or to Dex alone. In selected experiments, cells were exposed to GFs and Dex in combination with the pan-GR inhibitor RU486 (5 μmol/L, Danco Laboratories) [30Bourgeois S. Pfahl M. Baulieu E.E. DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486.EMBO J. 1984; 3: 751-755Crossref PubMed Scopus (116) Google Scholar]. Protein half-life determinations were performed on day 10 Erys exposed to the protein synthesis inhibitor cycloheximide (50 μg/mL, #C4859, Sigma-Aldrich) [31Siegel M.R. Sisler H.D. Inhibition of protein synthesis in vitro by cycloheximide.Nature. 1963; 200: 675-676Crossref PubMed Scopus (76) Google Scholar] for 0, 4, 8, 12, 16, and 24 hours. Day 10 Erys were pretreated in HEMA conditions for 4 hours with the selective intracellular Ca2+ chelator BAPTA [32Dieter P. Fitzke E. Duyster J. BAPTA induces a decrease of intracellular free calcium and a translocation and inactivation of protein kinase C in macrophages.Biol Chem Hoppe Seyler. 1993; 374: 171-174Crossref PubMed Scopus (39) Google Scholar] (10 μmol/L, #120503, Abcam, Cambridge, MA, USA) in combination with either a compound that raises intracellular Ca2+ levels (ionomycin [33Morgan A.J. Jacob R. Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane.Biochem J. 1994; 300: 665-672Crossref PubMed Scopus (240) Google Scholar], Io, 100 nmol/L, #sc30085, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or an inhibitor of Ca2+ in the ER (thapsigargin [34Lytton J. Westlin M. Hanley M.R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.J Biol Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar], TG, 100 nmol/L, #586005, Calbiochem, San Diego, CA, USA) as described previously [16Holaska J.M. Black B.E. Rastinejad F. Paschal B.M. Ca2+-dependent nuclear export mediated by calreticulin.Mol Cell Biol. 2002; 22: 6286-6297Crossref PubMed Scopus (91) Google Scholar]. Day 10 Erys were cultured for 30–60 minutes in HEMA conditions with or without the inhibitor of nuclear export leptomycin (3 μg/mL, #L2913, Sigma-Aldrich) [35Kudo N. Wolff B. Sekimoto T. et al.Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1.Exp Cell Res. 1998; 242: 540-547Crossref PubMed Scopus (700) Google Scholar]. Day 10 Erys were cultured for 2–24 hours in HEMA culture with and without the pan-JAK inhibitor ruxolitinib (10 μmol/L, #S1378, Sellechem, Houston, TX, USA) [36Zhou T. Georgeon S. Moser R. Moore D.J. Caflisch A. Hantschel O. Specificity and mechanism-of-action of the JAK2 tyrosine kinase inhibitors ruxolitinib and SAR302503 (TG101348).Leukemia. 2014; 28: 404-407Crossref PubMed Scopus (80) Google Scholar]. Cells were washed twice in CaCl2-and MgCl2-free phosphate-buffered saline (PBS, #70011-044, Invitrogen, Carlsbad, CA, USA) and lysed in lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4; 1% NP40; 0.25% sodium deoxycholate; 150 mmol/L NaCl; 1 mmol/L of the Ca2+ chelator EDTA; 1 mmol/L PMSF; 1μ/mL each aprotinin, leupeptin, and pepstatin; 1 mmol/L Na3VO4; 1 mmol/L NaF (all from Sigma-Aldrich) as described previously [27Varricchio L. Masselli E. Alfani E. et al.The dominant negative beta isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.Blood. 2011; 118: 425-436Crossref PubMed Scopus (32) Google Scholar]. This procedure reduces the Ca2+ content of the medium in which the proteins are dissolved. To determine the effects of Ca2+ on the conformation of CALR in solution, in selected experiments, cells were washed twice in PBS containing 0.9 or 10 mmol/L CaCl2 and then lysed in buffer prepared as above but without EDTA. Protein extracts (30 μg/lane) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing β-mercaptoethanol (5% v/v), were blotted on nitrocellulose membranes as described previously [27Varricchio L. Masselli E. Alfani E. et al.The dominant negative beta isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.Blood. 2011; 118: 425-436Crossref PubMed Scopus (32) Google Scholar] and probed with antibodies against N-CALR (#12238, Cell Signaling Technology, Boston, MA, USA), C-CALR (sc-6467, Santa Cruz Biotechnology), GRα (sc-33780), GRαSer211 (#4161s, Cell Signaling Technology), GRβ (#3581, Abcam), STAT5 (sc-835, Santa Cruz Biotechnology), pSTAT5-Y694 (#9351, Cell Signaling Technology), p38 (Cell Signaling Technology), p-Pp38 (#T180/Y182, Cell Signaling Technology), GAPDH (CB1001, Calbiochem), HSP90 (#SMC149A, Stress Marq, Cadboro Bay, CA, USA), or Lamin B1 (#Ab16048, Abcam). The N-CALR and C-CALR antibodies used in this study were raised against synthetic peptides mapping, respectively, residues near the N-terminal or the C-terminal domain not disclosed by the manufacturers and were characterized in previous studies [37Schardt J.A. Eyholzer M. Timchenko N.A. Mueller B.U. Pabst T. Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia.J Cell Mol Med. 2010; 14: 1509-1519Crossref PubMed Scopus (29) Google Scholar, 38Nauseef W.M. McCormick S.J. Clark R.A. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase.J Biol Chem. 1995; 270: 4741-4747Crossref PubMed Scopus (229) Google Scholar]. Membranes were then incubated with appropriate horseradish peroxidase-coupled secondary antibodies (Calbiochem) and immune complexes detected with an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). Nuclear and cytoplasmic fractions were prepared using the kit NE-PER (#78833, Thermo Scientific-Pierce Biotechnology, Rockford, IL). The intensity of each band was quantified with the IS Image Studio Lite version 4.0 program for Windows (Microsoft Software, Inc., North Hampton, MA) and expressed as relative content after normalization with respect to the respective loading control (GAPDH). For protein half-life determinations, the content of each protein was expression as the fold change with respect to the content at time 0. Cells were incubated with CD36 and CD235a (R&D Systems) for maturation assessment [26Migliaccio G. Di Pietro R. di Giacomo V. et al.In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients.Blood Cells Mol Dis. 2002; 28: 169-180Crossref PubMed Scopus (115) Google Scholar, 29Falchi M. Varricchio L. Martelli F. et al.Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.Haematologica. 2015; 100: 178-187Crossref PubMed Scopus (31) Google Scholar], in combination with either anti-N-CALR or C-CALR antibodies or irrelevant IgG. Fluorescent signals were measured with the FACS Aria (Becton Dickinson, Franklin Lakes, NJ, USA), analyzed with FlowJo software (TreeStar, Inc., Ashland, OR, USA), and expressed as mean fluorescence intensity. Dead cells were excluded by Sytox Blue staining (0.002 mmol/L, Molecular Probes, Eugene, OR, USA). Cell maturation states were confirmed by visual examination of cytocentrifuged smears (Cytospin3, Shandon, Astmoor, UK). Cultured cells were fixed with paraformaldehyde, left to attach to polylysine-coated glass slides, permeabilized with 0.1% Triton X-100 (Bio-Rad Laboratories, Richmond, CA, USA) incubated with blocking buffer (bovine serum albumin in PBS 2.5%) for 1 hour, and incubated overnight at 4°C with N-CALR, C-CALR or GR antibodies. After two washes in PBS (Sigma-Aldrich), cells were incubated with Alexa Fluor-conjugated secondary antibodies for 30 minutes at room temperature in the dark, washed twice with PBS, and mounted with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) to visualize nuclei. Evaluations were performed with the FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with a 60× oil-immersion objective. Standard threshold levels were defined in red (emission at 546 nm, N-CALR and C-CALR), green (emission at 488 nm, GR), and blue (emission at 408 nm, DAPI) mode for colored images and in grey mode for single channels, as described previously [39Paddock S.W. Eliceiri K.W. Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.Methods Mol Biol. 2014; 1075: 9-47Crossref PubMed Scopus (46) Google Scholar]. Individual and merged signals were quantified with the freeware software ImageJ (version 1.29, NIH, Bethesda, MD). Magnification bias was minimized by expressing the results as percentages of area covered by single (red or green) or merged (yellow = red + green) signals/total area analyzed. In selected experiments, cells were fixed with ice-cold methanol 100% for 20 minutes and then processed for immunostaining. Er
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