Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry
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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-024-01530-z |
| _version_ | 1797247163388919808 |
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| author | Vikas Bhuria Tobias Franz Conny Baldauf Martin Böttcher Nicolas Chatain Steffen Koschmieder Tim H. Brümmendorf Dimitrios Mougiakakos Burkhart Schraven Sascha Kahlfuß Thomas Fischer |
| author_facet | Vikas Bhuria Tobias Franz Conny Baldauf Martin Böttcher Nicolas Chatain Steffen Koschmieder Tim H. Brümmendorf Dimitrios Mougiakakos Burkhart Schraven Sascha Kahlfuß Thomas Fischer |
| author_sort | Vikas Bhuria |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T19:54:19Z |
| format | Article |
| id | doaj.art-db493b9498e045b9a9db0c8fc03508d6 |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-04-24T19:54:19Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-db493b9498e045b9a9db0c8fc03508d62024-03-24T12:27:52ZengBMCCell Communication and Signaling1478-811X2024-03-0122112210.1186/s12964-024-01530-zActivating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entryVikas Bhuria0Tobias Franz1Conny Baldauf2Martin Böttcher3Nicolas Chatain4Steffen Koschmieder5Tim H. Brümmendorf6Dimitrios Mougiakakos7Burkhart Schraven8Sascha Kahlfuß9Thomas Fischer10Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityInstitute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityInstitute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityHealth-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke UniversityDepartment of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen UniversityDepartment of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen UniversityDepartment of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen UniversityHealth-Campus Immunology, Infectiology, and Inflammation (GC-I3), Medical Center, Otto-von-Guericke UniversityInstitute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityInstitute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityInstitute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke UniversityAbstract 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.https://doi.org/10.1186/s12964-024-01530-zCalcium fluxEPO signalingTPO signalingMPNsJAK2-V617FCALR mutation |
| spellingShingle | Vikas Bhuria Tobias Franz Conny Baldauf Martin Böttcher Nicolas Chatain Steffen Koschmieder Tim H. Brümmendorf Dimitrios Mougiakakos Burkhart Schraven Sascha Kahlfuß Thomas Fischer Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry Cell Communication and Signaling Calcium flux EPO signaling TPO signaling MPNs JAK2-V617F CALR mutation |
| title | Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry |
| title_full | Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry |
| title_fullStr | Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry |
| title_full_unstemmed | Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry |
| title_short | Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry |
| title_sort | activating mutations in jak2 and calr differentially affect intracellular calcium flux in store operated calcium entry |
| topic | Calcium flux EPO signaling TPO signaling MPNs JAK2-V617F CALR mutation |
| url | https://doi.org/10.1186/s12964-024-01530-z |
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