Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion
Abstract Background Somatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential...
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| Format: | Article |
| Language: | English |
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BMC
2016-05-01
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| Series: | Journal of Hematology & Oncology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13045-016-0275-0 |
| _version_ | 1819262631005913088 |
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| author | Lijuan Han Claudia Schubert Johanna Köhler Mirle Schemionek Susanne Isfort Tim H. Brümmendorf Steffen Koschmieder Nicolas Chatain |
| author_facet | Lijuan Han Claudia Schubert Johanna Köhler Mirle Schemionek Susanne Isfort Tim H. Brümmendorf Steffen Koschmieder Nicolas Chatain |
| author_sort | Lijuan Han |
| collection | DOAJ |
| description | Abstract Background Somatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation. |
| first_indexed | 2024-12-23T20:00:45Z |
| format | Article |
| id | doaj.art-05519d64505b4c6ebd53b93217bcc276 |
| institution | Directory Open Access Journal |
| issn | 1756-8722 |
| language | English |
| last_indexed | 2024-12-23T20:00:45Z |
| publishDate | 2016-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Hematology & Oncology |
| spelling | doaj.art-05519d64505b4c6ebd53b93217bcc2762022-12-21T17:33:05ZengBMCJournal of Hematology & Oncology1756-87222016-05-019111410.1186/s13045-016-0275-0Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretionLijuan Han0Claudia Schubert1Johanna Köhler2Mirle Schemionek3Susanne Isfort4Tim H. Brümmendorf5Steffen Koschmieder6Nicolas Chatain7Department 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 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 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 UniversityAbstract Background Somatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.http://link.springer.com/article/10.1186/s13045-016-0275-0CalreticulinFrameshift mutantsdel52Myeloproliferative neoplasmsMPNMegakaryopoiesis |
| spellingShingle | Lijuan Han Claudia Schubert Johanna Köhler Mirle Schemionek Susanne Isfort Tim H. Brümmendorf Steffen Koschmieder Nicolas Chatain Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion Journal of Hematology & Oncology Calreticulin Frameshift mutants del52 Myeloproliferative neoplasms MPN Megakaryopoiesis |
| title | Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion |
| title_full | Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion |
| title_fullStr | Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion |
| title_full_unstemmed | Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion |
| title_short | Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion |
| title_sort | calreticulin mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and golgi mediated secretion |
| topic | Calreticulin Frameshift mutants del52 Myeloproliferative neoplasms MPN Megakaryopoiesis |
| url | http://link.springer.com/article/10.1186/s13045-016-0275-0 |
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