RESUMO
Janus kinase 2 (JAK2) gene mutations are the main drivers for polycythemia vera (PV) and essential thrombocythemia (ET). The mechanisms of single altered gene causing two different diseases are unclear. Additionally, novel treatments specifically targeting mutated JAK2 proteins are needed. In this study, the induced pluripotent stem cells (iPSCs) were virally transduced to express wild-type JAK2 (JAK2WT), JAK2p.V617F (JAK2V617F) or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs which were differentiated into megakaryocytes in the presence vs. absence of doxycycline were compared to ensure that the differences were solely from mutated JAK2 expressions. The JAK2V617-expressing iPSCs yielded significantly higher numbers of megakaryocytes consistent with the ET phenotype, while there was no enhancement by JAK2exon12 expression compatible with the pure erythrocytosis in humans. Capillary Western analyses revealed significantly greater JAK2 phosphorylation in iPSCs carrying JAK2V617F but not in JAK2WT and JAK2exon12 iPSCs. Activation of STAT3, STAT5 and AKT was increased by JAK2V617F, while they were decreased in JAK2exon12 iPSCs. Notably, interferon alpha and/or arsenic trioxide inhibited megakaryocytes proliferation and reduced JAK2, STAT3, STAT5 and AKT phosphorylation in mutant JAK2-expressing iPSCs compared with those without induction. In conclusion, JAK2V617F expression in iPSCs preferentially promoted megakaryocytes with a signaling profile distinctive from JAK2exon12 expression. Treatments with interferon alpha or arsenic trioxide preferentially suppressed the mutated over wild-type JAK2 signaling. This iPSC model is helpful in mechanistic studies and novel therapy screen for myeloproliferative neoplasm.
Assuntos
Células-Tronco Pluripotentes Induzidas , Janus Quinase 2 , Transdução de Sinais , Trombocitemia Essencial , Trióxido de Arsênio/farmacologia , Doxiciclina , Humanos , Interferon-alfa/farmacologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Trombocitemia Essencial/genéticaRESUMO
Activating mutations affecting the JAK-STAT signal transduction is the genetic driver of myeloproliferative neoplasms (MPNs) which comprise polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. The JAK2p.V617F mutation can produce both erythrocytosis in PV and thrombocytosis in ET, while JAK2 exon 12 mutations cause only erythrocytosis. We hypothesized that these two mutations activated different intracellular signals. In this study, the induced pluripotent stem cells (iPSCs) were used to model JAK2-mutated MPNs. Normal iPSCs underwent lentiviral transduction to overexpress JAK2p.V617F or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs were differentiated into erythroid cells. Compared with JAK2V617F-iPSCs, JAK2exon12-iPSCs yielded more total CD71+GlycophorinA+ erythroid cells, displayed more mature morphology and expressed more adult hemoglobin after doxycycline induction. Capillary Western immunoassay revealed significantly higher phospho-STAT1 but lower phospho-STAT3 and lower Phospho-AKT in JAK2exon12-iPSCs compared with those of JAK2V617F-iPSCs in response to erythropoietin. Furthermore, interferon alpha and arsenic trioxide were tested on these modified iPSCs to explore their potentials for MPN therapy. Both agents preferentially inhibited proliferation and promoted apoptosis of the iPSCs expressing mutant JAK2 compared with those without doxycycline induction. In conclusion, the modified iPSC model can be used to investigate the mechanisms and search for new therapy of MPNs.
Assuntos
Janus Quinase 2/genética , Policitemia Vera/genética , Policitemia/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Eritropoese/genética , Éxons , Regulação da Expressão Gênica/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Policitemia/patologia , Policitemia Vera/patologia , Mielofibrose Primária , Fatores de Transcrição STAT/genética , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia , Trombocitose/genética , Trombocitose/patologiaRESUMO
Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 µM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions.
Assuntos
Metaloproteases/metabolismo , Venenos de Serpentes/química , Trimeresurus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Colágeno Tipo IV/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Concentração Inibidora 50 , Metaloproteases/genética , Dados de Sequência Molecular , Pichia/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The incidence of venomous snake bites increases every year in Thailand, especially due to green pit viper. After the bite, there is bleeding due to thrombin-like property of the venom. The mean platelet volume has been reported to be decreased in those who have been bitten by this snake. In this study we investigate the effect of green pit viper venom (Trimeresurus albolabris) on platelet volume (MPV), number and morphology of platelets in vitro. The test was carried out by washing platelets in phosphate buffer at pH 7.2 to remove fibrinogen, then the washed platelets were mixed with green pit viper venom. Platelet morphology was examined by scanning electron microscope (SEM). The morphology of platelets was smaller than normal which ranges from 1.1- 1.2 microm. Green pit viper venom can directly effect platelet morphology, decreasing platelet volume.