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.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function

“…In mice, CALR exerts nuclear export function of the nuclear receptors, such as the glucocorticoid receptor (GR) which involves migration to the nucleus (Burns et al, 1994 ; Holaska et al, 2001 ). Although, CALR regulates the nuclear export of GR also in human cells, in human cells this activity does not require nuclear localization of CALR but is exerted in regions of the ER located at the nuclear border (Falchi et al, 2017 ), confirming previous studies which had already indicated that nuclear localization of CALR may be an artifact.…”

“…In addition, the aforementioned biophysical studies on the urea-induced unfolding of CALR and its isolated domains suggest that also the sensitivity of CALR to urea may be limited by the amount of Ca 2+ bound to the protein, possibly as a reflection of the concentration of this ion in the cells being analyzed. The fact that CALR partially retain its 3D-structure in SDS-PAGE explains why CALR migrates in SDS-PAGE with an apparent molecular weight greater (55–60 KDa) than the theoretical 46 KDa expected on the basis of its AA sequence (Michalak et al, 1992 ; Klampfl et al, 2013 ; Nangalia et al, 2013 ; Falchi et al, 2017 ). Although, it is still unclear whether CALR undergoes post-translational modifications, all the aforementioned post-translational modifications have the potential to affect SDS binding at least to some of its domains allowing CALR to retain some globular structure.…”

“…Of note, some of the currently available commercial antibodies were raised against AA sequences in the N-CALR and C-CALR domains whose structure is predicted by computational analyses to be in a metastable equilibrium sensitive to environmental cues (Figure 1 ). For this property, these antibodies may be exploited for epitope mapping studies to identify how the conformation of the protein changes in vivo in response to micro-environmental cues as pioneered by the study of Falchi et al ( 2017 ). The resolution of in vivo epitope mapping studies is however still low and need to be increased by using panels of antibodies against a wider range of domains of intrinsically disordered and ordered, as internal control, regions of CALR (Figure 1A ).…”

Increased von Willebrand factor levels in polycythemia vera and phenotypic differences with essential thrombocythemia

“…A similar association was reported by Mital et al in 142 patients with PV in whom those with VWF:Ag and VWF:Act < 60% and < 50%, respectively, had significantly higher erythrocyte counts. Moreover, PV erythroblasts were found to have increased calreticulin expression on plasma membrane, and calreticulin seems to be involved in the degradation of the circulating VWF/FVIII complex . We also observed a significant association of VWF levels with male sex, not observed in healthy subjects .…”

Calreticulin Affects Hematopoietic Stem/Progenitor Cell Fate by Impacting Erythroid and Megakaryocytic Differentiation

“…So far, the role of CALR mutations in the development of MPN has been only partially elucidated, and no data are available on the physiological function played by CALR during normal hematopoiesis. The only report available is focused on the study of CALR during stress erythropoiesis [12]: the authors demonstrated that in normal proerythroblasts, the CALR C-terminal domain undergoes conformational changes in response to Ca 2+ that activate the nuclear export of the glucocorticoid receptor (GR), resetting the stress response and allowing the cells to undergo terminal differentiation.…”