Falchi Mario, Varricchio Lilian, Martelli Fabrizio, Marra Manuela, Picconi Orietta, Tafuri Agostino, Girelli Gabriella, Uversky Vladimir N, Migliaccio Anna Rita
National AIDS Center, Istituto Superiore Sanita, Rome, Italy.
Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Exp Hematol. 2017 Jun;50:53-76. doi: 10.1016/j.exphem.2017.02.001. Epub 2017 Feb 21.
Calreticulin (CALR) is a Ca-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 Ca 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, Ca 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, Ca 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 Ca 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 Ca flux inhibitors. These results indicates that Ca-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.
钙网蛋白（CALR）是一种钙结合蛋白，它与结合在其N/P结构域的蛋白质在细胞区室之间穿梭。人类促红细胞生成素受体的激活会诱导钙通量这一认识促使我们研究CALR在人类红细胞生成中的作用。如蛋白质印迹分析所示，从正常来源和JAK2V617F真性红细胞增多症（PV）患者体外生成的成红细胞表达高水平的CALR。然而，钙仅在正常细胞中调节CALR构象。正常成红细胞在其细胞表面主要表达CALR的N端结构域（N-CALR）（流式细胞术显示），在其细胞质中表达C端结构域（C-CALR）（共聚焦显微镜显示），并且随着成熟，这两个表位的表达均降低。在早幼红细胞（proEry）细胞质中，C-CALR与糖皮质激素受体（GR）相关联，后者启动应激反应。在这些细胞中，钙剥夺和核输出抑制增加了GR的核定位，同时减少了C-CALR和C-CALR/GR关联的细胞质检测以及对GR激动剂地塞米松（Dex）的增殖反应。相反，钙和促红细胞生成素增加了C-CALR/GR关联和对Dex的反应性。相比之下，JAK2V617F proErys表达正常细胞表面水平的N-CALR，但几乎检测不到细胞质水平的C-CALR。这些细胞中的GR主要存在于细胞核中，对Dex无反应。鲁索替尼挽救了JAK2V617F proErys中C-CALR的细胞质检测、C-CALR/GR关联和对Dex的反应性，其作用被核输出和钙通量抑制剂拮抗。这些结果表明，钙诱导的CALR构象变化调节正常成红细胞中GR的核输出，并且JAK2V617F在PV中使该功能失调。
