RESUMO
The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.
Assuntos
Interleucina-7 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Interleucina-7/genética , Interleucina-7/metabolismo , Fosfatidilinositol 4,5-Difosfato , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Transdução de Sinais/genéticaRESUMO
Rasa3 is a GTPase activating protein of the GAP1 family which targets R-Ras and Rap1. Although catalytic inactivation or deletion of Rasa3 in mice leads to severe hemorrhages and embryonic lethality, the biological function and cellular location of Rasa3 underlying these defects remains unknown. Here, using a combination of loss of function studies in mouse and zebrafish as well as in vitro cell biology approaches, we identify a key role for Rasa3 in endothelial cells and vascular lumen integrity. Specific ablation of Rasa3 in the mouse endothelium, but not in megakaryocytes and platelets, lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice. Reduced plexus/sprouts formation and vascular lumenization defects were observed when Rasa3 was specifically inactivated in mouse endothelial cells at the postnatal or adult stages. Similar results were obtained in zebrafish after decreasing Rasa3 expression. In vitro, depletion of Rasa3 in cultured endothelial cells increased ß1 integrin activation and cell adhesion to extracellular matrix components, decreased cell migration and blocked tubulogenesis. During migration, these Rasa3-depleted cells exhibited larger and more mature adhesions resulting from a perturbed dynamics of adhesion assembly and disassembly which significantly increased their life time. These defects were due to a hyperactivation of the Rap1 GTPase and blockade of FAK/Src signaling. Finally, Rasa3-depleted cells showed reduced turnover of VE-cadherin-based adhesions resulting in more stable endothelial cell-cell adhesion and decreased endothelial permeability. Altogether, our results indicate that Rasa3 is a critical regulator of Rap1 in endothelial cells which controls adhesions properties and vascular lumen integrity; its specific endothelial cell inactivation results in occluded blood vessels, hemorrhages and early embryonic death in mouse, mimicking thus the Rasa3-/- mouse phenotype.
Assuntos
Permeabilidade Capilar/genética , Adesão Celular/genética , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião de Mamíferos , Embrião não Mamífero , Feminino , Proteínas Ativadoras de GTPase/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Peixe-Zebra , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.
Assuntos
Plaquetas/metabolismo , Proteínas Ativadoras de GTPase/genética , Hematopoese/genética , Proteínas de Ligação a Telômeros/genética , Animais , Plaquetas/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular/genética , Fêmur/metabolismo , Fêmur/patologia , Citometria de Fluxo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Integrinas/genética , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Complexo Shelterina , Transdução de Sinais/genética , Baço/metabolismo , Baço/patologia , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Snail2 is a zinc finger transcription factor involved in driving epithelial to mesenchymal transitions. Snail2 null mice are viable, but display defects in melanogenesis, gametogenesis and hematopoiesis, and are markedly radiosensitive. Here, using mouse genetics, we have studied the contributions of Snail2 to epidermal homeostasis and skin carcinogenesis. Snail2 (-/-) mice presented a defective epidermal terminal differentiation and, unexpectedly, an increase in number, size and malignancy of tumor lesions when subjected to the two-stage mouse skin chemical carcinogenesis protocol, compared with controls. Additionally, tumor lesions from Snail2 (-/-) mice presented a high inflammatory component with an elevated percentage of myeloid precursors in tumor lesions that was further increased in the presence of the anti-inflammatory agent dexamethasone. In vitro studies in Snail2 null keratinocytes showed that loss of Snail2 leads to a decrease in proliferation indicating a non-cell autonomous role for Snail2 in the skin carcinogenic response observed in vivo. Bone marrow (BM) cross-reconstitution assays between Snail2 wild-type and null mice showed that Snail2 absence in the hematopoietic system fully reproduces the tumor behavior of the Snail2 null mice and triggers the accumulation of myeloid precursors in the BM, blood and tumor lesions. These results indicate a new role for Snail2 in preventing myeloid precursors recruitment impairing skin chemical carcinogenesis progression.
Assuntos
Inflamação/patologia , Queratinócitos/patologia , Células Progenitoras Mieloides/patologia , Neoplasias Experimentais/patologia , Neoplasias Cutâneas/patologia , Fatores de Transcrição/fisiologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Apoptose , Western Blotting , Carcinógenos/toxicidade , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Imunofluorescência , Hematopoese , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Inflamação/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Progenitoras Mieloides/efeitos dos fármacos , Células Progenitoras Mieloides/metabolismo , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição da Família SnailRESUMO
Snail1 (Snail) and Snail2 (Slug) are transcription factors that share a similar DNA binding structure of four and five C2H2 zinc finger motifs (ZF), respectively. Both factors bind specifically to a subset of E-box motifs (E2-box: CAGGTG/CACCTG) in target promoters like the E-cadherin promoter and are key mediators of epithelial-to-mesenchymal transition (EMT). However, there are differences in the biological actions, in binding affinities to E-cadherin promoter, and in the target genes of Snail1 and Snail2, although the molecular bases are presently unknown. In particular, the role of each Snail1 and Snail2 ZF in the binding to E-boxes and in EMT induction has not been previously explored. We have approached this question by modeling Snail1 and Snail2 protein-DNA interactions and through mutational and functional assays of different ZFs. Results show that Snail1 efficient repression and binding to human and mouse E-cadherin promoter as well as EMT-inducing ability require intact ZF1 and ZF2, while for Snail2, either ZF3 or ZF4 is essential for those functions. Furthermore, the differential distribution of E2-boxes in mouse and human E-cadherin promoters also contributes to the differential Snail factor activity. These data indicate a non-equivalent role of Snail1 and Snail2 ZFs in gene repression, contributing to the elucidation of the molecular differences between these important EMT regulators.
Assuntos
Caderinas/genética , Transição Epitelial-Mesenquimal/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição da Família Snail , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Dedos de Zinco/genéticaRESUMO
The enzyme spermine oxidase (SMOX) is involved in polyamine catabolism and converts spermine to spermidine. The enzymatic reaction generates reactive hydrogen peroxide and aldehydes as by-products that can damage DNA and other biomolecules. Increased expression of SMOX is frequently found in lung, prostate, colon, stomach and liver cancer models, and the enzyme also appears to play a role in neuronal dysfunction and vascular retinopathy. Because of growing evidence that links SMOX activity with DNA damage, inflammation, and carcinogenesis, the enzyme has come into view as a potential drug target. A major challenge in cancer research is the lack of characterization of antibodies used for identification of target proteins. To overcome this limitation, we generated a panel of high-affinity rabbit monoclonal antibodies against various SMOX epitopes and selected antibodies for use in immunoblotting, SMOX quantification assays, immunofluorescence microscopy and immunohistochemistry. Immunohistochemistry analysis with the antibody SMAB10 in normal and transformed tissues confirms that SMOX is upregulated in several different cancers. Together, the panel of antibodies generated herein adds to the toolbox of high-quality reagents to study SMOX biology and to facilitate SMOX drug development.
Assuntos
Antineoplásicos Imunológicos , Neoplasias , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Anticorpos Monoclonais , Humanos , Imuno-Histoquímica , Masculino , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermina/metabolismo , Poliamina OxidaseRESUMO
Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.
Assuntos
Condrócitos/metabolismo , MAP Quinase Quinase Quinases/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Osteocalcina/genética , Osteocondrodisplasias/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Aminoacetonitrila/análogos & derivados , Aminoacetonitrila/farmacologia , Animais , Calcificação Fisiológica/genética , Diferenciação Celular , Condrócitos/patologia , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteocalcina/antagonistas & inibidores , Osteocalcina/metabolismo , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Osteogênese/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tiofenos/farmacologiaRESUMO
We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.
Assuntos
Cálcio/metabolismo , Calmodulina/farmacologia , Proteínas/genética , Proteínas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Membrana Celular/fisiologia , Cromatografia de Afinidade , Receptores ErbB/fisiologia , Feminino , Transferência Ressonante de Energia de Fluorescência , Proteína Adaptadora GRB7 , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de SinaisRESUMO
Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) is the last identified member of the inositol 1,4,5-trisphosphate 3-kinases family which phosphorylates inositol 1,4,5-trisphosphate into inositol 1,3,4,5-tetrakisphosphate. Although expression and function of the two other family members ITPKA and ITPKB are rather well characterized, similar information is lacking for ITPKC. Here, we first defined the expression of Itpkc mRNA and protein in mouse tissues and cells using in situ hybridization and new antibodies. Surprisingly, we found that cells positive for ITPKC in the studied tissues express either a multicilium (tracheal and bronchial epithelia, brain ependymal cells), microvilli forming a brush border (small and large intestine, and kidney proximal tubule cells) or a flagellum (spermatozoa), suggesting a role for ITPKC either in the development or the function of these specialized cellular structures. Given this surprising expression, we then analyzed ITPKC function in multiciliated tracheal epithelial cells and sperm cells using our Itpkc knock-out mouse model. Unfortunately, no significant difference was observed between control and mutant mice for any of the parameters tested, leaving the exact in vivo function of this third Ins(1,4,5)P3 3-kinase still open.
Assuntos
Cílios/enzimologia , Células Epiteliais/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Mucosa Respiratória/enzimologia , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Cílios/ultraestrutura , Células Epiteliais/citologia , Expressão Gênica , Hibridização In Situ , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Intestino Grosso/enzimologia , Intestino Delgado/enzimologia , Túbulos Renais Proximais/enzimologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Mucosa Respiratória/citologia , Espermatozoides/enzimologiaRESUMO
RASA3 (or GTPase Activating Protein III, R-Ras GTPase-activating protein, GAP1(IP4BP)) is a GTPase activating protein of the GAP1 subfamily which targets Ras and Rap1. RASA3 was originally purified from pig platelet membranes through its intrinsic ability to bind inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4) with high affinity, hence its first name GAP1(IP4BP) (for GAP1 subfamily member which binds I(1,3,4,5)P4). RASA3 was thus the first I(1,3,4,5)P4 receptor identified and cloned. The in vitro and in vivo functions of RASA3 remained somewhat elusive for a long time. However, recently, using genetically-modified mice and cells derived from these mice, the function of RASA3 during megakaryopoiesis, megakaryocyte adhesion and migration as well as integrin signaling has been reported. The goal of this review is thus to summarize and comment recent and less recent data in the literature on RASA3, in particular on the in vivo function of this specific GAP1 subfamily member.
Assuntos
Movimento Celular/fisiologia , Proteínas Ativadoras de GTPase , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais/fisiologia , Trombopoese/fisiologia , Animais , Adesão Celular/fisiologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fosfatos de Inositol , Camundongos , Camundongos Transgênicos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Snail1 and Snail2, two highly related members of the Snail superfamily, are direct transcriptional repressors of E-cadherin and EMT inducers. Previous comparative gene profiling analyses have revealed important differences in the gene expression pattern regulated by Snail1 and Snail2, indicating functional differences between both factors. The molecular mechanism of Snail1-mediated repression has been elucidated to some extent, but very little is presently known on the repression mediated by Snail2. In the present work, we report on the characterization of Snail2 repression of E-cadherin and its regulation by phosphorylation. Both the N-terminal SNAG and the central SLUG domains of Snail2 are required for efficient repression of the E-cadherin promoter. The co-repressor NCoR interacts with Snail2 through the SNAG domain, while CtBP1 is recruited through the SLUG domain. Interestingly, the SNAG domain is absolutely required for EMT induction while the SLUG domain plays a negative modulation of Snail2 mediated EMT. Additionally, we identify here novel in vivo phosphorylation sites at serine 4 and serine 88 of Snail2 and demonstrate the functional implication of serine 4 in the regulation of Snail2-mediated repressor activity of E-cadherin and in Snail2 induction of EMT.