The effect of JAK2V617F Mutation to the Endothelial Cells and the Expression Profiles of SOCS1-4 Genes in Myeloproliferative Neoplasms

Abstract

Objective: Patients with myeloproliferative neoplasms (MPN) are at an increased risk of developing thrombosis which leads to significant levels of morbidity and mortality. Although this propensity for thrombosis has been attributed to increased blood counts, other factors such as endothelial cell (EC) dysfunction likely play an important role. Previous studies have provided evidence of EC involvement by JAK2V617F which is known to be present in blood cells of 50-95% of patients with MPN. Furthermore, the morphological and functional changes of this mutation, a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) has not been shown in EC. In addition, the role of epigenetic factors including suppressors of cytokine signaling (SOCS) proteins is also unknown for MPN. In this study, the effects of the JAK2V617F mutation on EC and the SOCS 1-4 genes expression and their methylation profiles were investigated. Materials and Methods: The in vitro transduction method of green fluorescent protein (GFP) labeled lentivirus carrying the JAK2 (wild form) or JAK2V617F mutation into human cord vein endothelial cell (HUVEC) was applied. GFP was determined and analyzed with the flow cytometer. Afterwards, the genetically modified EC population was isolated, gating for GFP signal by cell sorter and then DNA and RNA isolations were performed. Gene expressional changes of SOCS1-4 genes were determined in real time RT-PCR and methylation specific PCR was performed with bisulfite modification of DNA. Results: The successful infection of JAK2, JAK2V617F and GFP vectors into EC were achieved. The comparison of the genetically modified EC revealed no morphological differences. A significant increase in the expressions of SOCS1-4 were detected in mutant EH compared to JAK2 and GFP. It was determined that methylation had no effect on suppression of SOCS1-4 expression detected in GFP and JAK2 EC. Conclusion: Future studies will further enlighten the effects of JAK2V617F on EC and its role in thrombotic events.

Keywords

• Myeloroliferative Neoplasm (MPN)
• JAK2V617F
• Endothelial Cells
• Lentivirus
• SOCS

Miyeloproliferatif Neoplazilerde JAK2V617F Mutasyonunun Endotel Hücresine Etkisi ve SOCS1-4 Gen Anlatımlarına Yansıması

Abstract

Amaç: Miyeloproliferatif neoplazi (MPN)’li hastalar morbidite ve mortalitesi yüksek tromboz riski taşırlar. Tromboza olan bu yatkınlık, artmış kan hücre sayısı ile bağdaştırılmış olsa da, endotel hücre (EH) fonksiyon bozukluğu gibi faktörler de önemli rol oynamaktadır. EH’nin miyeloproliferatif bozukluklarla olan bağlantısını göstermek üzere yapılan önceki çalışmalarda EH’nin MPN kan hücrelerinde %50-95 oranında mevcut olan JAK2V617F mutasyonunu taşıdığı gösterilmiş, ancak sitozolik tirozin kinaz olan Janus kinase 2 (JAK2) de meydana gelen bu mutasyonun EH üzerinde göstermiş olduğu morfolojik ve fonksiyonel değişiklik gösterilmemiştir. Ayrıca epigenetik faktörlerden Sitokin Sinyal Süpresör (SOCS) proteinleri‘nin MPN’deki etkisi de tam olarak bilinmemektedir. Bu çalışma ile JAK2V617F mutasyonunun EH'de yapmış olduğu etkileri epigenetik açıdan önemli olan, SOCS 1-4 gen anlatımları ve metilasyon durumları incelenmiştir. Gereç ve Yöntem: In vitro yeşil floresan proteini (GFP) ile işaretlenmiş JAK2 (yabanil formu) veya JAK2V617F mutasyonu taşıyan lentivirusün insan kordon veni endotel hücresi (HUVEC) içine transdüksiyon metodu kullanılmıştır. Genetik olarak modifiye edilmiş EH populasyonunda, akımölçer cihazı ile GFP tespit edilmiş ve analizleri yapılmıştır. Ardından hücre ayırıcı ile seçilmiş olan GFP+ hücrelerde DNA ve RNA izolasyonları gerçekleştirilmiştir. SOCS1-4 genlerinin anlatım değerleri gerçek zamanlı RT-PZR tespit edilmiş ve bisülfit modifikasyona maruz bırakılan DNA yardımıyla metilasyona özgü PZR yapılmıştır. Bulgular: JAK2, JAK2V617F ve GFP taşıyan lentiviral vektörlerle başarılı bir şekilde EH enfeksiyonu gerçekleştirilmiştir. Genetiği modifiye edilmiş EH'ler birbirleri ile kıyaslandığında morfolojik bir farklılık saptanmamıştır. SOCS1-4 genlerinde, mutant EH de JAK2 ve GFP'e göre gen anlatım düzeyinde anlamlı artış tespit edilmiştir. GFP ve JAK2 de tespit edilen SOCS1-4 gen anlatım baskılanmasında bu genlerde oluşması muhtemel bir metilasyon olmadığı tespit edilmiştir. Sonuç: Bu konuda gelecekte yapılacak çalışmalar, JAK2V617F’in EH’ler üzerindeki etkilerini ve trombotik olaylarla bağlantılısını açıklamamıza yardımcı olacaktır.

Keywords

• Miyeloproliferatif Neoplazi (MPN)
• JAK2V617F
• Endotel Hücre
• Lentivirüs
• SOCS

References

1. 1. Levine RL, Wadleigh M, Cools J. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.
2. 2. James C, Ugo V, Le Couedic JP. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434(7037):1144-8.
3. 3. Kralovics R, Passamonti F, Buser AS.. A gain-offunction mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90.
4. 4. Tefferi A, Elliott M. Thrombosis in myeloproliferative disorders: prevalence, prognostic factors, and the role of leukocytes and JAK2V617F. Semin Thromb Hemost 2007;33:313-20.
5. 5. Wolanskyj AP, Schwager SM, McClure RF, Larson DR, Tefferi A. Essential thrombocythemia beyond the first decade: life expectancy, longterm complication rates, and prognostic factors. Mayo Clin Proc 2006;81(2):159-66.
6. 6. Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA, Barosi G, Verstovsek S, Birgegard G, Mesa R, Reilly JT, Gisslinger H, Vannucchi AM, Cervantes F, Finazzi G, Hoffman R, Gilliland DG, Bloomfield CD, Vardiman JW. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood 2007;110(4):1092-7.
7. 7. Wehmeier A, Daum I, Jamin H, Schneider W. Incidence and clinical risk factors for bleeding and thrombotic complications in myeloproliferative disorders. A retrospective analysis of 260 patients. Ann Hematol 1991;63:101-6.
8. 8. Craig MK. Propensity for hemorrhage and thrombosis in chronic myeloproliferative disorders. Seminars in hematology 2004;41(2 Suppl3):10-4.

9. 9. Falanga A, Marchetti M, Barbui T, Smith CW. Pathogenesis of thrombosis in essential thrombocythemia and polycythemia vera: the role of neutrophils. Semin Hematol 2005;42(4):239-47.
10. 10. Falanga A, Marchetti M, Evangelista V. Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera. Blood 2000; 96(13):4261-6.
11. 11. Blann A, Caine G, Bareford D.Abnormal vascular, platelet and coagulation markers in primary thrombocythaemia are not reversed by treatments that reduce the platelet count. Platelets 2004;15(7): 447-9.
12. 12. Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, Villamor N, Colomer D, Cervantes F. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status. Haematologica 2006; 91(2):169-75.
13. 13. Robertson B, Urquhart C, Ford I, Townend J, Watson HG, Vickers MA, Greaves M. Platelet and coagulation activation markers in myeloproliferative diseases: relationships with JAK2 V6I7 F status, clonality, and antiphospholipid antibodies. J Thromb Haemost 2007;5(8):1679-85.
14. 14. Bellucci S, Michiels JJ. The role of JAK2 V617F mutation, spontaneous erythropoiesis and megakaryocytopoiesis, hypersensitive platelets, activated leukocytes, and endothelial cells in the etiology of thrombotic manifestations in polycythemia vera and essential thrombocythemia. Semin Thromb Hemost 2006; 32(4 Pt 2):381-98.
15. 15. Duda DG, Fukumura D, Jain RK. Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends Mol Med 2004;10(4):143-5.
16. 16. Sozer S, Fiel MI, Schiano T, Xu M, Mascarenhas J, Hoffman R.The presence of JAK2V617F mutation in the liver endothelial cells of patients with Budd-Chiari syndrome. Blood 2009;113(21): 5246-9.
17. 17. Teofili L, Martini M, Iachininoto MG, Capodimonti S, Nuzzolo ER, Torti L, Cenci T, Larocca LM, Leone G. Endothelial progenitor cells are clonal and exhibit JAK2V617F mutation in a subset of thrombotic patients with Phnegative myeloproliferative neoplasms. Blood 2011;117(9):2700-7.
18. 18. Mascarenhas J, Roper N, Chaurasia P, Hoffman R. Epigenetic abnormalities in myeloproliferative neoplasms: a target for novel therapeutic strategies. Clin Epigenetics 2011;2(2): 197-212.
19. 19. Kazi JU, Kabir NN, Flores-Morales A, Rönnstrand L. SOCS proteins in regulation of receptor tyrosine kinase signaling. Cell Mol Life Sci 2014;71(17): 3297-310.
20. 20. Cooney RN. Suppressors of cytokine signaling (SOCS): inhibitors of the JAK/STAT pathway. Shock 2002;17(2): 83-90.
21. 21. Qin W, Li LL, Lu HN, Huang BB, Xiu B, Bo LJ, Gao QM, Zhang WJ, Fu JF. [Study of hypermethylation of SOCS gene in typical myeloproliferative disease]. Zhonghua Xue Ye Xue Za Zhi 2011;32(11):772-6.
22. 22. Quentmeier H, Geffers R, Jost E, Macleod RA, Nagel S, Röhrs S, Romani J, Scherr M, Zaborski M, Drexler HG. SOCS2: inhibitor of JAK2V617F-mediated signal transduction. Leukemia 2008;22(12):2169-75.
23. 23. Haan S, Wüller S, Kaczor J, Rolvering C, Nöcker T, Behrmann I, Haan C. SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling. Oncogene 2009;28(34):3069-80.
24. 24. Jost E, do ON, Dahl E, Maintz CE, Jousten P, Habets L, Wilop S, Herman JG, Osieka R, Galm O. Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ ABL-negative myeloproliferative disorders. Leukemia 2007;21(3): 505-10.
25. 25. Guo W, Li W, Yuan L, Mei X, Hu W. MicroRNA- 106a-3p Induces Apatinib Resistance and Activates Janus-Activated Kinase 2 (JAK2)/ Signal Transducer and Activator of Transcription 3 (STAT3) by Targeting the SOCS System in Gastric Cancer. Med Sci Monit 2019;25:10122-8.
26. 26. Bullock AN, Rodriguez MC, Debreczeni JE, Songyang Z, Knapp S. Structure of the SOCS4- ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation. Structure 2007;15(11):1493-504.
27. 27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods 2001;25(4):402-8.
28. 28. Gharavi NM, Alva JA, Mouillesseaux KP, Lai C, Yeh M, Yeung W, Johnson J, Szeto WL, Hong L, Fishbein M, Wei L, Pfeffer LM, Berliner JA. 2007. Role of the JAK/STAT Pathway in the Regulation of Interleukin-8 Transcription by Oxidized Phospholipids in Vitro and in Atherosclerosis in Vivo. J. Biol. Chem. 282: 31460-8.
29. 29. Huang SP, Wu MS, Shun CT, Wang HP, Lin MT, Kuo ML, Lin JT. 2004. Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma. J Biomed Sci 11: 517-27.
30. 30. Tawfik A, Jin L, Banes-Berceli AKL, Caldwell RB, Ogbi S, Shirley A, Barber D, Catravas JD, Stern DM, Fulton D, Caldwell RW, Marrero MB. Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2. Vascular Pharmacology 2005;43(5):320-6.
31. 31. Sandberg EM, Sayeski PP. Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells. J Biol Chem 2004;279(13):34547-52.
32. 32. Gorina R, Petegnief V, Chamorro A, Planas AM. AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway. J Neurochem 2005;92(3):505-18.
33. 33. Neria F, Caramelo C, Peinado H, Gonzalez- Pacheco FR, Deudero JJ, de Solis AJ, Fernandez- Sanchez R, Penate S, Cano A, Castilla MA. Mechanisms of endothelial cell protection by blockade of the JAK2 pathway. Am J Physiol Cell Physiol 2007; 292(3): C1123-31.
34. 34. Neria F, Castilla MA, Sanchez RF, Gonzalez Pacheco FR, Deudero JJ, Calabia O, Tejedor A, Manzarbeitia F, Ortiz A, Caramelo C. Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity. Kidney Int 2008;75(2):227-34.
35. 35. Santilli F, Romano M, Recchiuti A, Dragani A, Falco A, Lessiani G, Fioritoni F, Lattanzio S, Mattoscio D, De Cristofaro R, Rocca B, Davi G. Circulating endothelial progenitor cells and residual in vivo thromboxane biosynthesis in low-dose aspirin-treated polycythemia vera patients. Blood 2008;112(4):1085-90.
36. 36. Cucuianu A, Stoia M, Farcas A, Dima D, Zdrenghea M, Patiu M, Olinic D, Petrov L. Arterial stenosis and atherothrombotic events in polycythemia vera and essential thrombocythemia. Rom J Intern Med 2006;44(4):397-406.
37. 37. Cosgrove ME, Suman R, Harrison HJ, Jackson GE, Howard MR, Hitchcock IS. Endothelial JAK2V617F Expression Drives Inflammation and Cellular Senescence; New Evidence for the Roles of Endothelial Cells in MPN-Related Clotting Abnormalities? Blood 2016;128(22):3134-3134.
38. 38. Pedroso JAB, Ramos-Lobo AM, Donato J, Jr. SOCS3 as a future target to treat metabolic disorders. Hormones (Athens) 2019;18(2):127-36.
39. 39. Kim WS, Kim MJ, Kim DO, Byun JE, Huy H, Song HY, Park YJ, Kim TD, Yoon SR, Choi EJ, Jung H, Choi I. Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity. Sci Rep 2017;7:46153.
40. 40. Nischal S, Bhattacharyya S, Christopeit M, Yu Y, Zhou L, Bhagat TD, Sohal D, Will B, Mo Y, Suzuki M, Pardanani A, McDevitt M, Maciejewski JP, Melnick AM, Greally JM, Steidl U, Moliterno A, Verma A. Methylome Profiling Reveals Distinct Alterations in Phenotypic and Mutational Subgroups of Myeloproliferative Neoplasms. Cancer Research 2013;73(3):1076-85.
41. 41. Brasier AR. The NF-kappaB regulatory network. Cardiovasc Toxicol 2006;6(2):111-30.
42. 42. Pérez C, Pascual M, Martín-Subero JI, Bellosillo B, Segura V, Delabesse E, Álvarez S, Larrayoz MJ, Rifón J, Cigudosa JC, Besses C, Calasanz MJ, Cross NCP, Prósper F, Agirre X. Aberrant DNA methylation profile of chronic and transformed classic Philadelphia-negative myeloproliferative neoplasms. Haematologica 2013;98(9):1414-20.
43. 43. Izzi B, Noro F, Cludts K, Freson K, Hoylaerts MF. Cell-Specific PEAR1 Methylation Studies Reveal a Locus that Coordinates Expression of Multiple Genes. Int J Mol Sci 2018;19(4):1069.
44. 44. Han S, Tan C, Ding J, Wang J, Ma’ayan A, Gouon-Evans V. Endothelial cells instruct liver specification of embryonic stem cellderived endoderm through endothelial VEGFR2 signaling and endoderm epigenetic modifications. Stem Cell Res 2018;30: 163-70.