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1.
Nat Commun ; 11(1): 4886, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985500

RESUMO

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.


Assuntos
Calreticulina/genética , Mielofibrose Primária/genética , Trombocitemia Essencial/genética , Animais , Calreticulina/metabolismo , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fenótipo , Mielofibrose Primária/metabolismo , Deleção de Sequência , Trombocitemia Essencial/metabolismo
2.
Life Sci ; 257: 118088, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663573

RESUMO

AIMS: Bone marrow stromal cells (BMSCs) have been reported to interact with multiple myeloma (MM) and exert a vital function of the survival of MM cells. Heme oxygenase-1 (HO-1), a cytoprotective enzyme, has the potential to become a hematological malignancies targeted gene. This study aimed to investigate the role of HO-1 in MM resistance of BMSCs and its possible mechanisms. MAIN METHODS: In this study, the expression of related proteins was detected by RT-qPCR and Western blot. HO-1 expression was regulated by lentivirus transfection. Cell viability and apoptosis were detected by Flow cytometry and CCK-8. Cytokine secretion was assayed by ELISA. The survival and carcinogenic abilities was detected by clone formation assay. KEY FINDINGS: HO-1 expression in the BMSCs of stage III MM patients was substantially increased, compared with that of healthy donors and stage I/II patients. The results of co-culture of BMSCs and MM cells indicated that, the upregulated HO-1 inhibited the apoptosis of co-cultured MM cells, while downregulated HO-1 promoted the chemosensitivity of co-cultured MM cells, moreover, the upregulated HO-1 in BMSCs increased the colony-formation ability of MM cells. This protective capability may be regulated by CXCL12/CXCR4 signaling. High HO-1 expression in BMSCs can promote the phosphorylation of the JAK2/STAT3 pathway, thereby increasing secretion of SDF-1 in BMSCs and activating CXCL12/CXCR4 signaling. In addition, direct contact between BMSCs and MM cells may cause drug resistance. SIGNIFICANCE: These results indicated that the regulation of HO-1 in BMSCs may be a new effective method of MM therapy.


Assuntos
Antineoplásicos/farmacologia , Heme Oxigenase-1/genética , Células-Tronco Mesenquimais/citologia , Mieloma Múltiplo/patologia , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Estudos de Casos e Controles , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Estadiamento de Neoplasias , Fator de Transcrição STAT3/metabolismo
3.
Life Sci ; 257: 118083, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32673665

RESUMO

AIMS: To investigate the preclinical pharmacodynamics and mechanism of JLX001 against myocardial ischemia reperfusion (MI/R) for clinical application. MATERIALS AND METHODS: In vivo, SD rats were given intragastric administration for 5 days, and the MI/R model was established by ligating/releasing the left anterior descending coronary artery. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model was established after the drug was pre-incubated for 24 h in H9C2 cells. The infract size was determined by TTC staining. Left ventricular function of MI/R rats was detected by echocardiography. The level of histopathological score was determined by hematoxylin-eosin (HE) staining. The level of superoxide dismutase (SOD), malondialdehyde (MDA), creatine kinase (CK), lactic dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were determined by relevant kits. The level of apoptosis was measured by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Hoechst staining. The expression of p-Jak2, p-Stat3, Bax, Bcl-2, TNF-α, IL-1ß protein were determined by western blot. KEY FINDINGS: JLX001 can significantly improve left ventricular function, reduce myocardial infract size, histopathological score, the level of MDA, CK, LDH, TNF-α, IL-1ß and the expression of Bax protein, significantly increase the activity of SOD, Bcl-2 protein expression, p-Jak2 protein expression, p-Stat3 protein expression in rat heart tissues and H9C2 cells. These effects can be reversed by AG490 which is a specific inhibitor of Jak2-Stat3 pathway. SIGNIFICANCE: JLX001 can alleviate MI/R injury by inhibiting myocardial apoptosis, inflammation, and oxidative stress via Jak2-Stat3 pathway in vivo and in vitro.


Assuntos
Janus Quinase 2/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tirfostinas/farmacologia
4.
Int Immunopharmacol ; 86: 106749, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32645632

RESUMO

In December 2019, a novel coronavirus pneumonia (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suddenly broke out in China and rapidly spread all over the world. Recently, a cell surface protein, known as angiotensin-converting enzyme II (ACE2), has been identified to be involved in receptor-mediated endocytosis for SARS-CoV-2 entry to the cells. Many studies have reported the clinical characteristics of COVID-19: sudden deterioration of disease around 1-2 weeks after onset; much lower level of lymphocytes, especially natural killer (NK) cells in peripheral blood; extremely high pro-inflammatory cytokines and C reactive protein (CRP). About 15.7% of patients develop severe pneumonia, and cytokine storm is an important factor leading to rapid disease progression. Currently, there are no specific drugs for COVID-19 and the cytokine storm it causes. Baricitinib intracellularly inhibits the proinflammatory signal of several cytokines by suppressing Janus kinase (JAK) JAK1/JAK2. It has been demonstrated clinical benefits for the patients with rheumatoid arthritis (RA), active systemic lupus erythematosus and atopic dermatitis with good efficacy and safety records. Baricitinib is expected to interrupt the passage and intracellular assembly of SARS-CoV-2 into the target cells mediated by ACE2 receptor, and treat cytokine storm caused by COVID-19. Several clinical trials are currently investigating the drug, and one of which has been completed with encouraging results. In this paper, we will elaborate the role of cytokine storm mediated by JAK-STAT pathway in severe COVID-19, the possible mechanisms of baricitinib on reducing the viral entry into the target cells and cytokine storm, the key points of pharmaceutical care based on the latest research reports, clinical trials progress and drug instruction from the US FDA, so as to provide reference for the treatment of severe COVID-19.


Assuntos
Azetidinas/uso terapêutico , Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Azetidinas/farmacologia , Betacoronavirus/metabolismo , Ensaios Clínicos como Assunto , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/diagnóstico , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/virologia , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Resultado do Tratamento , Montagem de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
5.
Mol Cell ; 78(6): 1207-1223.e8, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32504554

RESUMO

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.


Assuntos
Antígeno B7-H1/genética , Interferon gama/metabolismo , RNA Longo não Codificante/genética , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Interferon gama/genética , Interferons/genética , Interferons/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Proteína 2 Ligante de Morte Celular Programada 1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos
6.
Proc Natl Acad Sci U S A ; 117(26): 15047-15054, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32532922

RESUMO

Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2 By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.


Assuntos
Neoplasias da Mama/enzimologia , Proteínas Quinases Associadas com Morte Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Tamoxifeno/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Feminino , Humanos , Janus Quinase 2/genética , Camundongos , Camundongos SCID , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fator de Transcrição STAT3/genética
7.
PLoS One ; 15(6): e0232801, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479500

RESUMO

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis are hematopoietic stem cell disorders and share driver mutations that either directly activate the thrombopoietin receptor, MPL, or activate it indirectly through gain-of-function mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, MPL surface expression in hematopoietic stem cells is also reduced in the myeloproliferative neoplasms due to abnormal post-translational glycosylation and premature destruction of JAK2, suggesting that the myeloproliferative neoplasms are disorders of MPL processing since MPL is the only hematopoietic growth factor receptor in hematopoietic stem cells. To examine this possibility, we genetically manipulated MPL expression and maturation in a JAK2V617F transgenic mouse model of polycythemia vera. Elimination of MPL expression completely abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could only be partially restored by expression of one MPL allele. Most importantly, elimination of thrombopoietin gene expression abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could be completely restored by expression of a single thrombopoietin allele. These data indicate that polycythemia vera is in part a thrombopoietin-dependent disorder and that targeting the MPL-thrombopoietin axis could be an effective, nonmyelotoxic therapeutic strategy in this disorder.


Assuntos
Janus Quinase 2/genética , Policitemia Vera/genética , Policitemia Vera/metabolismo , Trombopoetina/genética , Trombopoetina/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Transtornos Mieloproliferativos/genética , Fenótipo , Policitemia Vera/patologia , Mielofibrose Primária/genética , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética
8.
PLoS Genet ; 16(6): e1008715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559233

RESUMO

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.


Assuntos
Proteínas de Drosophila/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas ras/metabolismo , Animais , Células Cultivadas , Sequência Conservada , Drosophila , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Janus Quinase 2/metabolismo , Fosforilação , Tirosina/química , Tirosina/genética , Ubiquitinação , Proteínas ras/química , Proteínas ras/genética , Quinases da Família src/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(24): 13670-13679, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32471953

RESUMO

Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Inibidores Enzimáticos/administração & dosagem , Histona Desmetilases/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Fator Estimulador de Colônias/genética , Fatores de Transcrição STAT/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fator Estimulador de Colônias/metabolismo , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Transdução de Sinais/efeitos dos fármacos
10.
Mol Biol (Mosk) ; 54(2): 293-299, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392199

RESUMO

Novel treatments for various types of malignant diseases are warranted. In this study, we evaluated JAK2 inhibitors (Janus kinase 2) for suppressing the growth of malignant neuroblastoma and glioblastoma cells as well as breast and non-small cell lung cancers. Neuroblastoma and glioblastoma cells are the most sensitive to the JAK2 inhibitor AG490. A study of the relative expression of receptors that can activate JAK2 suggests that cell line sensitivity to AG490 may be mediated by IL6-R, IL11-R and/or CSF1-R. AG490 enhances the effect of doxorubicin on neuroblastoma cells. Our findings suggest the possible relevance of JAK2 inhibitors for neuroblastoma therapy, especially in combination with doxorubicin.


Assuntos
Doxorrubicina/farmacologia , Janus Quinase 2/antagonistas & inibidores , Neuroblastoma/patologia , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Janus Quinase 2/metabolismo , Fosforilação , Transdução de Sinais , Tirfostinas/farmacologia
11.
Nucleic Acids Res ; 48(9): 4780-4796, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232334

RESUMO

Previously, we have shown that human sperm Prohibitin (PHB) expression is significantly negatively correlated with mitochondrial ROS levels but positively correlated with mitochondrial membrane potential and motility. However, the possible role of PHB in mammalian spermatogenesis has not been investigated. Here we document the presence of PHB in spermatocytes and its functional roles in meiosis by generating the first male germ cell-specific Phb-cKO mouse. Loss of PHB in spermatocytes resulted in complete male infertility, associated with not only meiotic pachytene arrest with accompanying apoptosis, but also apoptosis resulting from mitochondrial morphology and function impairment. Our mechanistic studies show that PHB in spermatocytes regulates the expression of STAG3, a key component of the meiotic cohesin complex, via a non-canonical JAK/STAT pathway, and consequently promotes meiotic DSB repair and homologous recombination. Furthermore, the PHB/JAK2 axis was found as a novel mechanism in the maintenance of stabilization of meiotic STAG3 cohesin complex and the modulation of heterochromatin formation in spermatocytes during meiosis. The observed JAK2-mediated epigenetic changes in histone modifications, reflected in a reduction of histone 3 tyrosine 41 phosphorylation (H3Y41ph) and a retention of H3K9me3 at the Stag3 locus, could be responsible for Stag3 dysregulation in spermatocytes with the loss of PHB.


Assuntos
Código das Histonas , Meiose/genética , Proteínas Repressoras/fisiologia , Espermatócitos/metabolismo , Espermatogênese/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Pareamento Cromossômico , Epigenoma , Histonas/metabolismo , Recombinação Homóloga , Infertilidade/genética , Janus Quinase 2/metabolismo , Janus Quinases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Estágio Paquíteno , Fosforilação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Espermatócitos/enzimologia , Espermatócitos/ultraestrutura , Testículo/metabolismo
12.
J Med Chem ; 63(10): 5324-5340, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32329617

RESUMO

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.


Assuntos
Amitrol (Herbicida)/química , Amitrol (Herbicida)/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Animais , Células HEK293 , Humanos , Ligantes , Ligação Proteica/fisiologia , Células Sf9 , Difração de Raios X/métodos
13.
Proc Natl Acad Sci U S A ; 117(11): 6067-6074, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123064

RESUMO

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.


Assuntos
Células Epiteliais/patologia , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Transdução de Sinais/imunologia , Uveíte/patologia , Animais , Linhagem Celular , Corpo Ciliar/citologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Janus Quinase 2/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/imunologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/imunologia , Fator de Transcrição STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Sermorelina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Uveíte/tratamento farmacológico , Uveíte/imunologia
14.
Pediatr Blood Cancer ; 67(5): e28232, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32134181

RESUMO

OBJECTIVES: Pediatric myelofibrosis is a rare entity with the largest reported series of 19 cases. We describe here the clinicopathological spectrum and outcomes of 15 cases of pediatric myelofibrosis. METHODS: Case files of myelofibrosis of patients less than 18 years were retrieved from January 2016 to January 2019, and patients with idiopathic myelofibrosis after exhaustive work-up were studied. Their clinicopathological profiles were studied and then followed up for resolution and malignant transformation. RESULTS: Of the 15 cases of idiopathic myelofibrosis, transfusion-dependent anemia (14/15) was most common presentation. Only one patient showed leukoerythroblastosis with dacryocytes. Myeloid hyperplasia was seen in 13 of 15 patients and megakaryocytic hyperplasia in 10 patients. Dysmegakaryopoiesis was seen in 8 of 15 patients, and only three had small loose megakaryocytic clustering. None showed hyperchromatic megakaryocytes, intrasinusoidal hematopoiesis, or osteosclerosis. One patient with trisomy 8 tested positive for JAK2V617F. Bone marrow biopsy was hypercellular in 13, and 8 had world health organization (WHO) MF-3 fibrosis. None of the patients developed malignancy, one had spontaneous resolution, and one patient required allogenic stem cell transplant. CONCLUSIONS: Pediatric myelofibrosis is a distinct entity from primary myelofibrosis in adults and merits mention in the WHO manual as a distinct entity.


Assuntos
Transformação Celular Neoplásica , Janus Quinase 2 , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Trombopoese , Adolescente , Adulto , Substituição de Aminoácidos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteosclerose/genética , Osteosclerose/metabolismo , Osteosclerose/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Estudos Retrospectivos
15.
Arch Biochem Biophys ; 685: 108330, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32156533

RESUMO

Switching microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype represents a novel therapeutic strategy for diabetic neuropathic pain (DNP). This study aims to determine the role and mechanism of interleukin (IL)-35 in regulating microglial M1/M2 polarization in DNP. A rat model of DNP was induced by a single streptozocin injection and recombinant IL-35 (rIL-35) was then intrathecally administered to the rats for 14 days. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to assess the therapeutic effect of IL-35. Highly aggressive proliferating immortalized (HAPI), a rat microglia cell line, was treated with lipopolysaccharide (LPS) for M1 polarization or IL-4 for M2 polarization. The M1 markers (CD68, iNOS, TNF-α, IL-6) and M2 markers (CD206, Arg-1, IL-10) were examined. rIL-35 administration in DNP model rats elevated MWT and TWL, induced microglial polarization toward the M2 phenotype, suppressed JNK signaling and activated JAK2/STAT6 signaling. In vitro assay confirmed that rIL-35 induced microglial M2 polarization in HAPI cells through inhibiting JNK signaling and activating JAK2/STAT6 signaling. Collectively, the mechanism underlying therapeutic effect of IL-35 on DNP may relate to its promotion of microglial M2 polarization by regulating JNK signaling and JAK2/STAT6 signaling.


Assuntos
Neuropatias Diabéticas/metabolismo , Subunidade p35 da Interleucina-12/metabolismo , Microglia/metabolismo , Neuralgia/metabolismo , Animais , Linhagem Celular , Neuropatias Diabéticas/induzido quimicamente , Neuropatias Diabéticas/complicações , Janus Quinase 2/metabolismo , MAP Quinase Quinase 4/metabolismo , Masculino , Neuralgia/induzido quimicamente , Neuralgia/complicações , Ratos Sprague-Dawley , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/fisiologia , Estreptozocina
16.
Clin Sci (Lond) ; 134(7): 807-825, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32219336

RESUMO

It has been generally believed that cancer-associated fibroblasts (CAFs) have the ability to increase the process of tumor angiogenesis. However, the potential mechanisms by which cancer-derived exosomes in lung cancer (LC) remains to be investigated. LC-derived exosomes were administrated to NIH/3T3 cells. A variety of experiments were conducted to investigate the proangiogenic factors of CAFs, including Western blot, RT-PCR, colony formation assay, tube formation assay, Matrigel plug assay et al. In addition, the impact of JAK2/STAT3 signaling pathway were also explored. The role of hsa-miR-210 was identified with microarray profiling and validated in vitro and in vivo assays. The target of miR-210 was screened by RNA pull down, RNA-sequencing and then verified. It was shown that LC-derived exosomes could induce cell reprogramming, thus promoting the fibroblasts transferring into CAFs. In addition, the exosomes with overexpressed miR-210 could increase the level of angiogenesis and vice versa, which suggested the miR-210 secreted by the LC-derived exosomes may initiate the CAF proangiogenic switch. According to our analysis, the miR-210 had the ability of elevating the expression of some proangiogenic factors such as MMP9, FGF2 and vascular endothelial growth factor (VEGF) a (VEGFa) by activating the JAK2/STAT3 signaling pathway, ten-eleven translocation 2 (TET2) was identified as the target of miR-210 in CAFs which has been involved in proangiogenic switch. miR-210 was overexpressed in serum exosomes of untreated non-small cell LC (NSCLC) patients. We concluded that the promotion effect of exosomal miR-210 on proangiogenic switch of CAFs may be explained by the modulation of JAK2/STAT3 signaling pathway and TET2 in recipient fibroblasts.


Assuntos
Fibroblastos Associados a Câncer/enzimologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Endoteliais/enzimologia , Exossomos/metabolismo , Janus Quinase 2/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Fator de Transcrição STAT3/metabolismo , Células A549 , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/patologia , Exossomos/genética , Exossomos/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Células NIH 3T3 , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
18.
Mol Carcinog ; 59(5): 503-511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32133692

RESUMO

Lung adenocarcinoma (LUAD), as a form of non-small cell lung cancer (NSCLC), is the most frequently diagnosed lung cancer worldwide. To date, a few biomarkers have been reported to provide valuable information in guiding LUAD treatment. The aim of our study was to explore the functional role of pyrroline-5-carboxylate reductase 1 (PYCR1) in LUAD. Based on Oncomine database, we found that PYCR1 was highly expressed in LUAD tissues. We also confirmed an abnormal increase of PYCR1 expression in LUAD cell lines and patients' tissues. Through Kaplan-Meier plotter database, we further studied the prognostic values of PYCR1. The outcomes indicated that overexpressed PYCR1 associated with poor prognosis among LUAD patients. To further study the function of PYCR1 in LUAD, cell counting kit-8, colony-forming, scratch wound healing, and Transwell assays were conducted. The results suggested that knockdown of PYCR1 curbed cell proliferation, migration, and invasion in LUAD cell lines. Subsequently, we identified 50 top genes positively and negatively correlated with PYCR1 in LUAD, and conducted biological pathway enrichment analysis of these genes. Among those enriched pathways, we selected JAK/STAT signaling pathway for further analysis. The results of Western blot assays revealed that PYCR1 knockdown significantly increased the expression of Bcl-2 and c-Myc, and the phosphorylation level of JAK2 and STAT3. Taken together, this study unearthed that PYCR1 knockdown could inhibit tumor growth and affect the JAK/STAT signaling pathway in LUAD. This study may contribute to a better understanding of PYCR1 in LUAD and provide a potential biomarker for cancer prognosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Janus Quinase 2/metabolismo , Pirrolina Carboxilato Redutases/metabolismo , Fator de Transcrição STAT3/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/secundário , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Pirrolina Carboxilato Redutases/genética , Fator de Transcrição STAT3/genética , Taxa de Sobrevida , Células Tumorais Cultivadas
19.
Toxicology ; 433-434: 152404, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32044397

RESUMO

As an alternative to bisphenol A (BPA), bisphenol F (BPF) has been increasingly used in manufacturing various consumer products. Exposured to BPF may lead to imbalanced immune homeostasis, yet the underlying mechanisms have not been fully elucidated. The present study was aimed to investigate the effects of BPF on macrophages and the underlying mechanism in regard to its association with estrogen receptor (ER), janus kinase 2/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (JAK2/STAT3/SOCS3) pathway. In this study, after treatment of RAW264.7 macrophages with BPF (0, 5, 10, 20 µM), the macrophage M1 polarization was promoted, and the gene expression of M1 functional markers and pro-inflammatory cytokines was upregulated, which suggested the involvement of a vicious circle associated with chronic inflammation. Moreover, BPF facilitated SOCS3 expression in the cells in a dose-dependent manner, via activation of the JAK2/STAT3 signaling pathway, which may promote the transcription of many pro-inflammatory factors. Additionally, the above effects of BPF were blocked by either JAK2/STAT3 inhibitor AG490 (10 µM) or ER antagonist ICI 182,780 (10 µM). Taken together, the results of this study indicate that BPF promotes macrophage polarization toward pro-inflammatory M1 subtype, through activation of the ER-JAK2/STAT3/SOCS3 signaling pathway. Our finding may provide a new insight into the link between bisphenol exposure and immune dysfunction.


Assuntos
Compostos Benzidrílicos/toxicidade , Inflamação/induzido quimicamente , Macrófagos/efeitos dos fármacos , Fenóis/toxicidade , Receptores Estrogênicos/metabolismo , Animais , Compostos Benzidrílicos/administração & dosagem , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Fulvestranto/farmacologia , Inflamação/patologia , Janus Quinase 2/metabolismo , Macrófagos/patologia , Camundongos , Fenóis/administração & dosagem , Células RAW 264.7 , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Tirfostinas/farmacologia
20.
Phytomedicine ; 68: 153172, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32004989

RESUMO

BACKGROUND: Aberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties. PURPOSE: In this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells. METHODS: In vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells. RESULTS: Convallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model. CONCLUSION: The result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Estrofantinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinase 2/metabolismo , Masculino , Camundongos Nus , Simulação de Acoplamento Molecular , Neovascularização Patológica/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estrofantinas/química , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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