RESUMO
Loss of hematopoietic stem cell (HSC) function and increased risk of developing hematopoietic malignancies are severe and concerning complications of anticancer radiotherapy and chemotherapy. We have previously shown that thrombopoietin (TPO), a critical HSC regulator, ensures HSC chromosomal integrity and function in response to γ-irradiation by regulating their DNA-damage response. TPO directly affects the double-strand break (DSB) repair machinery through increased DNA-protein kinase (DNA-PK) phosphorylation and nonhomologous end-joining (NHEJ) repair efficiency and fidelity. This effect is not shared by other HSC growth factors, suggesting that TPO triggers a specific signal in HSCs facilitating DNA-PK activation upon DNA damage. The discovery of these unique signaling pathways will provide a means of enhancing TPO-desirable effects on HSCs and improving the safety of anticancer DNA agents. We show here that TPO specifically triggers Erk and nuclear factor κB (NF-κB) pathways in mouse hematopoietic stem and progenitor cells (HSPCs). Both of these pathways are required for a TPO-mediated increase in DSB repair. They cooperate to induce and activate the early stress-response gene, Iex-1 (ier3), upon DNA damage. Iex-1 forms a complex with pERK and the catalytic subunit of DNA-PK, which is necessary and sufficient to promote TPO-increased DNA-PK activation and NHEJ DSB repair in both mouse and human HSPCs.
Assuntos
Reparo do DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Proteínas Imediatamente Precoces/metabolismo , NF-kappa B/metabolismo , Trombopoetina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Antineoplásicos/química , Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Transdução de Sinais , Células-Tronco/citologiaRESUMO
BACKGROUND: How platelets interact with and influence the tumor microenvironment (TME) remains poorly characterized. METHODS: We compared the presence and participation of platelets in the TME of two tumors characterized by highly different TME, PyMT AT-3 mammary tumors and B16F1 melanoma. RESULTS: We show that whereas firmly adherent platelets continuously line tumor vessels of both AT-3 and B16F1 tumors, abundant extravascular stromal clusters of platelets from thrombopoietin-independent origin were present only in AT-3 mammary tumors. We further show that platelets influence the angiogenic and inflammatory profiles of AT-3 and B16F1 tumors, though with very different outcomes according to tumor type. Whereas thrombocytopenia increased bleeding in both tumor types, it further caused severe endothelial degeneration associated with massive vascular leakage, tumor swelling, and increased infiltration of cytotoxic cells, only in AT-3 tumors. CONCLUSIONS: These results indicate that while platelets are integral components of solid tumors, their localization and origin in the TME, as well as their impact on its shaping, are tumor type-dependent.
Assuntos
Neoplasias Mamárias Animais , Microambiente Tumoral , Animais , HumanosRESUMO
This article highlights the presentations from the 2021 scientific meeting of the Club Hematopoiesis and Oncogenesis. This annual meeting focuses on hematopoiesis and oncogenic mechanisms. Various topics were presented: expansion of hematopoietic stem cells with in vivo and ex vivo strategies, the role of the hematopoietic stem cell niches in aging and leukemic resistance, the crossroad between hematology and immunology, the importance of the metabolism in normal hematopoiesis and hematopoietic defects, solid tumors and oncogenesis, the noncoding genome, inflammation in monocyte differentiation and leukemia, and importantly, the recent advances in myeloid malignancies, lymphoid leukemia and lymphoma.
Assuntos
Leucemia , Linfoma , Humanos , Hematopoese/genética , Células-Tronco Hematopoéticas , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologiaRESUMO
Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2P95H with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFß1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Transtornos Mieloproliferativos , Mielofibrose Primária , Animais , Camundongos , Janus Quinase 2/genética , Janus Quinases/genética , Mutação , Transtornos Mieloproliferativos/genética , Mielofibrose Primária/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição STAT/genéticaRESUMO
The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1(-/-) mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)-dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1(-/-) spleens. The ERK1(-/-) phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.
Assuntos
Anemia/patologia , Células Precursoras Eritroides/fisiologia , Eritropoese/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Baço/metabolismo , Anemia/induzido quimicamente , Animais , Apoptose , Western Blotting , Transplante de Medula Óssea , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/citologia , Eritropoetina/metabolismo , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/toxicidade , Fenil-Hidrazinas/toxicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Baço/citologiaRESUMO
Ionizing radiations (IR) alter hematopoietic stem cell (HSC) function on the long term, but the mechanisms underlying these effects are still poorly understood. We recently showed that IR induces the derepression of L1Md, the mouse young subfamilies of LINE-1/L1 retroelements. L1 contributes to gene regulatory networks. However, how L1Md are derepressed and impact HSC gene expression are not known. Here, we show that IR triggers genome-wide H3K9me3 decrease that occurs mainly at L1Md. Loss of H3K9me3 at intronic L1Md harboring NF-κB binding sites motifs but not at promoters is associated with the repression of HSC-specific genes. This is correlated with reduced NFKB1 repressor expression. TNF-α treatment rescued all these effects and prevented IR-induced HSC loss of function in vivo. This TNF-α/NF-κB/H3K9me3/L1Md axis might be important to maintain HSCs while allowing expression of immune genes during myeloid regeneration or damage-induced bone marrow ablation.
Assuntos
Células-Tronco Hematopoéticas , Histonas , Elementos Nucleotídeos Longos e Dispersos , NF-kappa B , Fator de Necrose Tumoral alfa , Animais , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Camundongos , NF-kappa B/metabolismo , Radiação Ionizante , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Recém-Nascido , Fatores Inibidores da Migração de Macrófagos/genética , CamundongosRESUMO
Growth Factor Independent-1B (Gfi-1B) is a transcriptional repressor that plays critical roles in the control of erythropoiesis and megakaryopoiesis. Gfi-1B expression was described to be repressed by an autoregulatory feedback control loop. Here, we show that Gfi-1 transcription is positively regulated early after induction of erythroid differentiation and remains highly active to late erythroblasts. Using chromatin immunoprecipitation assays in CD34+ cells from human cord blood, we found that Gfi-1 and GATA-2 in immature progenitors and then Gfi-1B and GATA-1 in erythroblasts are bound to the Gfi-1B promoter as well as to the promoter of c-myc, a known Gfi-1B target gene. Surprisingly, this Gfi-1/GATA-2-Gfi-1B/GATA-1 switch observed at erythroblast stages is associated to an increase in the Gfi-1B transcription whereas it triggers repression of c-myc transcription. Accordingly, analysis of chromatin modification patterns shows that HDAC, CoREST, and LSD1 are recruited to the c-myc promoter leading to appearance of repressive chromatin marks. In contrast, the Gfi-1B promoter remains associated with a transcriptionally active chromatin configuration as highlighted by an increase in histone H3 acetylation and concomitant release of the LSD1 and CoREST corepressors. The repressive function of Gfi-1B therefore depends on the nature of the proteins recruited to the target gene promoters and on chromatin modifications. We conclude that Gfi-1B behaves as a lineage-affiliated gene with an open chromatin configuration in multipotent progenitors and sustained activation as cells progress throughout erythroid differentiation.
Assuntos
Cromatina/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Western Blotting , Diferenciação Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas Correpressoras , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Vetores Genéticos , Histona Desmetilases/metabolismo , Humanos , Lentivirus , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , TransfecçãoRESUMO
Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45(-)/CD71(+)/GPA(-)cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents.
Assuntos
Eritroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Hematínicos/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Síndromes Mielodisplásicas/enzimologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eritroblastos/patologia , Eritropoese/efeitos dos fármacos , Eritropoetina/sangue , Eritropoetina/farmacologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologiaRESUMO
Maintenance of genomic integrity is crucial for the preservation of hematopoietic stem cell (HSC) potential. Retrotransposons, spreading in the genome through an RNA intermediate, have been associated with loss of self-renewal, aging, and DNA damage. However, their role in HSCs has not been addressed. Here, we show that mouse HSCs express various retroelements (REs), including long interspersed element-1 (L1) recent family members that further increase upon irradiation. Using mice expressing an engineered human L1 retrotransposition reporter cassette and reverse transcription inhibitors, we demonstrate that L1 retransposition occurs in vivo and is involved in irradiation-induced persistent γH2AX foci and HSC loss of function. Thus, RE represents an important intrinsic HSC threat. Furthermore, we show that RE activity is restrained by thrombopoietin, a critical HSC maintenance factor, through its ability to promote a potent interferon-like, antiviral gene response in HSCs. This uncovers a novel mechanism allowing HSCs to minimize irradiation-induced injury and reinforces the links between DNA damage, REs, and antiviral immunity.
Assuntos
Antivirais/farmacologia , Citoproteção/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Retroelementos/genética , Trombopoetina/farmacologia , Animais , Citoproteção/efeitos da radiação , Dano ao DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Interferons/genética , Interferons/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radiação Ionizante , Fatores de Transcrição STAT/metabolismoRESUMO
During a search to identify resveratrol (3,5,4'-trihydroxy-trans-stilbene, RV) target genes in the human erythroleukemic K562 cell line, we show here that the tensin gene and protein levels are remarkably induced by this dietary polyphenol. Tensin, a cell-matrix adhesion protein binding the integrins and cytoskeletal actin filaments also interacts with PI3-kinase and JNK signaling pathways. Tensin induction by RV is associated with increased K562 cell adhesion to fibronectin, cell spreading and actin polymerization. The same responses were observed in the tensin-deficient MCF7 human breast cancer cell line. In K562 and MCF7 cells treated by RV, tensin was found in punctate and intracytoplasmic areas. In MCF7 epithelial cells, induction of tensin is not exclusively associated with plasma membrane-bound vinculin, suggesting a dual localization of tensin in both focal and fibrillar adhesions. Pharmacological blockade of PI3-kinase and Rho GTPases/Rho-kinase resulted in selective depletion of focal adhesions, disorganization of tensin localization and disruption of stress fibers. RV increased cell motility and attachment to fibronectin in MCF7 cells submitted to mechanical laminar flow stress, and abrogated estrogen-induced MCF7 cancer cell invasion. Our data support the conclusion that induction of tensin by RV contributes to the chemopreventive and anti-invasive activity of this natural dietary compound in tensin-negative and -deficient leukemic cells or epithelioid cancers.
Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/biossíntese , Neoplasias/prevenção & controle , Estilbenos/farmacologia , Actinas/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cicloeximida/farmacologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Immunoblotting , Células K562 , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tensinas , Fatores de TempoRESUMO
The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.
Assuntos
Envelhecimento/imunologia , Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Linfopoese/imunologia , Metiltransferases/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Animais , Antagomirs/genética , Antagomirs/metabolismo , Linfócitos B/imunologia , Sequência de Bases , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Heterocromatina/química , Heterocromatina/metabolismo , Humanos , Linfopoese/genética , Masculino , Metiltransferases/imunologia , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/imunologia , Cultura Primária de Células , Proteínas Repressoras/imunologia , Transdução de SinaisRESUMO
Inhibition of apoptosis or abnormal cell survival can result in tumorigenesis by facilitating the insurgence of various mutations. Immediate-early response gene X-1 (IEX-1), protects T cells from apoptosis induced by the ligation of Fas or the T-cell receptor (TCR)/CD3 complex in Emu-IEX-1 mice that direct the gene expression in both T and B cell lineages under the control of the Emu enhancer. Consistent with a biased effect of IEX-1 towards T cells, Emu-IEX-1 mice selectively developed T-cell lymphomas in the spleen, when they aged, which may be associated with increased levels of IEX-1 phosphorylation in T cells compared to B cells. The lymphomas were single positive (CD4+CD8-, CD4-CD8+), double positive (CD4+CD8+), or double negative (CD4-CD8-) T cells. They resulted from aberrantly clonal expansions of T cells expressing a specific TCR, as suggested by the TCR repertoire analysis using a panel of monoclonal antibodies recognizing TCR Vbeta chain, as well as by TCR beta gene rearrangements. The study provides, for the first time, unambiguous evidence of the oncogenic potential of IEX-1 in a cell-specific manner. The animal model may help our understanding of peripheral T-cell lymphoma development.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Genes Precoces/fisiologia , Linfoma de Células T/genética , Subpopulações de Linfócitos T/imunologia , Linfócitos T/metabolismo , Envelhecimento , Animais , Anticorpos Monoclonais/metabolismo , Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Complexo CD3/metabolismo , Antígenos CD4/genética , Antígenos CD8/genética , Expressão Gênica , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Baço/imunologia , Baço/patologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologiaRESUMO
Activating mutations in the KRAS oncogene are prevalent in pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that pancreatic intraepithelial neoplasia (PanIN) formation, which precedes malignant transformation, associates with the expression of immediate early response 3 (Ier3) as part of a prooncogenic transcriptional pathway. Here, we evaluated the role of IER3 in PanIN formation and PDAC development. In human pancreatic cancer cells, IER3 expression efficiently sustained ERK1/2 phosphorylation by inhibiting phosphatase PP2A activity. Moreover, IER3 enhanced KrasG12D-dependent oncogenesis in the pancreas, as both PanIN and PDAC development were delayed in IER3-deficient KrasG12D mice. IER3 expression was discrete in healthy acinar cells, becoming highly prominent in peritumoral acini, and particularly high in acinar ductal metaplasia (ADM) and PanIN lesions, where IER3 colocalized with phosphorylated ERK1/2. However, IER3 was absent in undifferentiated PDAC, which suggests that the IER3-dependent pathway is an early event in pancreatic tumorigenesis. IER3 expression was induced by both mild and severe pancreatitis, which promoted PanIN formation and progression to PDAC in KrasG12D mice. In IER3-deficient mice, pancreatitis abolished KrasG12D-induced proliferation, which suggests that pancreatitis enhances the oncogenic effect of KRAS through induction of IER3 expression. Together, our data indicate that IER3 supports KRASG12D-associated oncogenesis in the pancreas by sustaining ERK1/2 phosphorylation via phosphatase PP2A inhibition.
Assuntos
Carcinoma in Situ/genética , Carcinoma Ductal Pancreático/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Nus , Camundongos Transgênicos , Mutação de Sentido Incorreto , Transplante de Neoplasias , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Development of novel therapies is critical for T-cell acute leukaemia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleukin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xenografted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.
Assuntos
Interleucina-18/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Células Estromais/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Interleucina-18/sangue , Interleucina-18/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
DNA double-strand breaks (DSBs) represent a serious threat for hematopoietic stem cells (HSCs). How cytokines and environmental signals integrate the DNA damage response and contribute to HSC-intrinsic DNA repair processes remains unknown. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal and expansion. Here, we uncover an unknown function for TPO-Mpl in the regulation of DNA damage response. We show that DNA repair following γ-irradiation (γ-IR) or the action of topoisomerase-II inhibitors is defective in Mpl(-/-) and in wild-type mouse or human hematopoietic stem and progenitor cells treated in the absence of TPO. TPO stimulates DNA repair in vitro and in vivo by increasing DNA-PK-dependent nonhomologous end-joining efficiency. This ensures HSC chromosomal integrity and limits their long-term injury in response to IR. This shows that niche factors can modulate the HSC DSB repair machinery and opens new avenues for administration of TPO agonists for minimizing radiotherapy-induced HSC injury and mutagenesis.
Assuntos
Dano ao DNA/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Mutagênese/fisiologia , Células-Tronco/metabolismo , Trombopoetina/metabolismo , Animais , Ciclo Celular , Ensaio Cometa , Dano ao DNA/genética , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese/genética , Trombopoetina/genéticaRESUMO
The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nicho de Células-Tronco , Animais , Densidade Óssea , Medula Óssea/patologia , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Compartimento Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Microambiente Celular , Deleção de Genes , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Monócitos , Osteoblastos/enzimologia , Osteoblastos/patologia , Osteoclastos/enzimologia , Osteoclastos/patologia , OsteogêneseRESUMO
The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr(308) and Ser(473) residues. PP2A-B56beta and gamma subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK.IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Modificação Traducional de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Células NIH 3T3 , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNARESUMO
The protein phosphatase 2A (PP2A) acts on several kinases in the extracellular signal-regulated kinase (ERK) signaling pathway but whether a specific holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown. By using both RNA interference and overexpression of PP2A B regulatory subunits, we show that B56, but not B, family members of PP2A increase ERK dephosphorylation, without affecting its activation by MEK. Induction of the early gene product and ERK substrate IEX-1 (ier3) by growth factors leads to opposite effects and reverses B56-PP2A-mediated ERK dephosphorylation. IEX-1 binds to B56 subunits and pERK independently, enhances B56 phosphorylation by ERK at a conserved Ser/Pro site in this complex and triggers dissociation from the catalytic subunit. This is the first demonstration of the involvement of B56-containing PP2A in ERK dephosphorylation and of a B56-specific cellular protein inhibitor regulating its activity in an ERK-dependent fashion. In addition, our results raise a new paradigm in ERK signaling in which ERK associated to a substrate can transphosphorylate nearby proteins.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Células COS , Chlorocebus aethiops , Substâncias de Crescimento/metabolismo , Células HeLa , Humanos , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Proteína Fosfatase 2 , Processamento de Proteína Pós-Traducional/fisiologia , Subunidades Proteicas/metabolismoRESUMO
The extracellular signal-regulated kinases (ERKs) are required for thrombopoietin (TPO) functions on hematopoietic cells, but the ERKs targets involved remain unknown. Here we show that the regulation of the immediate early gene X-1 (IEX-1), identified as an ERK substrate in response to TPO, was mediated by an ERK-dependent phosphorylation of AML1. The addition of TPO to UT7-Mpl cells and primary megakaryocytes induced gene expression of IEX-1. Neither erythropoietin (EPO) nor granulocyte macrophage-colony stimulating factor (GM-CSF) was able to activate IEX-1 gene expression in UT7-Mpl cells. The induced expression was mediated by a transcriptional activation of the IEX-1 promoter and required an AML1-binding site located at -1068. The direct involvement of AML1 in the regulation of IEX-1 gene expression was shown by both the use of AML1 mutants and by shRNA experiments targeting endogenous AML1. Finally, the ability of TPO to induce the IEX-1 gene expression was inhibited by U0126, a specific inhibitor of the ERKs activator MEK and AML1 transcriptional activity was shown to be modulated by TPO through ERK-dependent phosphorylation. Taken together, these data suggest that AML1 plays a role in modulating the IEX-1 expression and that the ERK-dependent AML1 phosphorylation regulates the TPO-mediated activation of IEX-1.