Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry
Cell Communication and Signaling, vol. 22, pp. 186
Abstract
Abstract Background Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. Methods Intracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. Results Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. Conclusions This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.
Authors 11
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Vikas Bhuria corresponding
Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Center for Health and Medical Prevention - CHaMP, Otto-von-Guericke University, Magdeburg, Germany. vikas.bhuria@med.ovgu.de
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany. vikas.bhuria@med.ovgu.de
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany. vikas.bhuria@med.ovgu.de
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Department of Hematology and Oncology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany
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Affiliation as printed
Center of Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen, Germany
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany
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Affiliation as printed
Center of Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen, Germany
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany
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Affiliation as printed
Center of Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen, Germany
Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Department of Hematology and Oncology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Center for Health and Medical Prevention - CHaMP, Otto-von-Guericke University, Magdeburg, Germany
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
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Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Center for Health and Medical Prevention - CHaMP, Otto-von-Guericke University, Magdeburg, Germany
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
Institute of Medical Microbiology and Hospital Hygiene, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
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Thomas Fischer corresponding
Otto-von-Guericke-Universität Magdeburg
Affiliation as printed
Center for Health and Medical Prevention - CHaMP, Otto-von-Guericke University, Magdeburg, Germany. thomas.fischer@med.ovgu.de
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany. thomas.fischer@med.ovgu.de
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany. thomas.fischer@med.ovgu.de
Health-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke University, Magdeburg, Germany
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