The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera

• Published in: Experimental hematology Vol. 50; pp. 53 - 76
• Main Authors: Falchi, Mario, Varricchio, Lilian, Martelli, Fabrizio, Marra, Manuela, Picconi, Orietta, Tafuri, Agostino, Girelli, Gabriella, Uversky, Vladimir N., Migliaccio, Anna Rita
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 01.06.2017
• Subjects: Amino Acid Substitution; Calcium - metabolism; Calreticulin - chemistry; Calreticulin - genetics; Calreticulin - metabolism; Cell Differentiation - genetics; Cell Line; Codon; Erythroblasts - metabolism; Erythroid Cells - cytology; Erythroid Cells - metabolism; Erythropoiesis - genetics; Humans; Immunophenotyping; Janus Kinase 2 - genetics; Janus Kinase 2 - metabolism; Mutation; Polycythemia Vera - genetics; Polycythemia Vera - metabolism; Protein Transport; Receptors, Glucocorticoid - metabolism; Signal Transduction
• ISSN: 0301-472X; 1873-2399
• DOI: 10.1016/j.exphem.2017.02.001

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. Model for the role exerted by CALR in the modulation of the stress response in normal erythroid cells and how impairment of CALR function may contribute to erythrocytosis in JAK2+-PV. Under steady-state conditions, EPO/EPO-R interactions control erythropoiesis by activating JAK2, which induces proErys to mature. Under stress, GRα activation “switches” JAK2 signaling to a proliferation mode, rapidly increasing the number of erythroblasts available to differentiate. However, for this increase of erythroblast numbers to be effective in recovering from anemia, an unidentified mechanism must switch the JAK2 signaling in these cells back to the maturation mode. The data presented here suggest that this switch is CALR. In fact, Ca2+-induced conformation changes of this protein, which may be induced in vivo by EPO-R activation, promoted nuclear export of GRα, resetting the stress responsiveness of normal erythroid cells. In contrast, in JAK2+-PV, hyperactivation of JAK2, possibly by increasing intracellular Ca2+ levels even further, impairs the nuclear export functions of CALR, retaining GRα in the nucleus and erythroblasts in proliferation and contributing to erythrocytosis. Ruxolitinib, by suppressing JAK2V717F activity, restores the GRα nuclear export activity of CALR, increasing the Dex responsiveness of these cells and reducing erythrocytosis. The cellular diagram of erythroid maturation is modified from Palis, 2014. [Display omitted] •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.