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1.
Mol Immunol ; 112: 206-214, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176200

RESUMO

Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of a zebrafish microRNA, dre-miR-722, in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2). Consistently, polarization of the actin cytoskeleton, cell migration and generation of the reactive oxygen species are significantly inhibited upon induced miR-722 overexpression. Together, zebrafish miR-722 is a suppressor for migration and signaling in human neutrophil like cells.


Assuntos
Quimiotaxia/genética , MicroRNAs/genética , Neutrófilos/fisiologia , Peixe-Zebra/genética , Actinas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Células HEK293 , Células HL-60 , Humanos , Inflamação/genética , Leucemia/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética
2.
Cell Prolif ; 52(4): e12614, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30983072

RESUMO

OBJECTIVES: To reveal the role of circular RNA (circRNA) DOCK1 (circDOCK1) as a potential biomarker and therapeutic target and its competing endogenous RNA mechanism in bladder carcinoma (BC). METHODS: The next-generation sequencing (NGS) technology was introduced to screen the circRNA expression profiles of BC using microarray. qPCR and Western blots assay were employed to measure the gene expression in different groups. Cell counting kit-8, EdU and transwell assays were applied to detect the cell viability, proliferation and migration potential, respectively. Luciferase reporter assay was used to test the binds between hsa-miR-132-3p/Sox5. Xenografted tumour growth of nude mice was performed to test the role of circDOCK1 in vivo. RESULTS: CircDOCK1 was upregulated in BC tissues and cell lines. Repression of circDOCK1 reduced cell viability, inhibited cell proliferation and curbed the cell migration potential of BC cell. CircDOCK1 played its role via regulation of circDOCK1/hsa-miR-132-3p/Sox5 pathway in BC cells. Suppression circDOCK1 inhibited the tumour growth in vivo. CONCLUSION: In this study, we revealed that circDOCK1 affected the progression of BC via modulation of circDOCK1/hsa-miR-132-3p/Sox5 pathway both in vitro and in vivo and providing a potential biomarker and therapeutic targets for BC.


Assuntos
MicroRNAs/genética , Fatores de Transcrição SOXD/genética , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/genética , Proteínas rac de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA/genética , Regulação para Cima/genética , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia
3.
Nat Commun ; 10(1): 684, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737382

RESUMO

Retrograde BMP trans-synaptic signaling is essential for synaptic development. Despite the importance of endocytosis-regulated BMP receptor (BMPR) control of this developmental signaling, the mechanism remains unknown. Here, we provide evidence that Abelson interactor (Abi), a substrate for Abl kinase and component of the SCAR/WAVE complex, links Abl and Rac1 GTPase signaling to BMPR macropinocytosis to restrain BMP-mediated synaptic development. We find that Abi acts downstream of Abl and Rac1, and that BMP ligand Glass bottom boat (Gbb) induces macropinocytosis dependent on Rac1/SCAR signaling, Abl-mediated Abi phosphorylation, and BMPR activation. Macropinocytosis acts as the major internalization route for BMPRs at the synapse in a process driven by Gbb activation and resulting in receptor degradation. Key regulators of macropinocytosis (Rabankyrin and CtBP) control BMPR trafficking to limit BMP trans-synaptic signaling. We conclude that BMP-induced macropinocytosis acts as a BMPR homeostatic mechanism to regulate BMP-mediated synaptic development.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte/genética , Drosophila , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Proteínas rac de Ligação ao GTP/genética
4.
Blood ; 133(18): 1977-1988, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30723080

RESUMO

Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67 phox , activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)-bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.


Assuntos
Síndromes de Imunodeficiência/genética , Proteínas rac de Ligação ao GTP/genética , Adolescente , Adulto , Animais , Pré-Escolar , Citoesqueleto/patologia , Feminino , Mutação com Ganho de Função , Humanos , Lactente , Recém-Nascido , Linfopenia/genética , Camundongos , Camundongos Endogâmicos C57BL , Linhagem , Proteínas rac de Ligação ao GTP/imunologia
5.
Fish Shellfish Immunol ; 84: 998-1006, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399403

RESUMO

Rac1 and Rac2, belonging to the small Rho GTPase family, play an important role during the immune responses. In this study, a Rac1 homolog (CsRac1) and a Rac2 homolog (CsRac2) were cloned from the Cynoglossus semilaevis. The full-length of CsRac1 and CsRac2 cDNA was 1219 bp and 1047 bp, respectively. Both CsRac1 and CsRac2 contain a 579 bp open reading frame (ORF) which encoding a 192 amino acids putative protein. The predicted molecular weight of CsRac1 and CsRac2 was 21.41 kDa and 21.35 kDa, and their theoretical pI was 8.50 and 7.91, respectively. Sequence analysis showed that the conserved RHO domain was detected both from amino acid of CsRac1 and CsRac2. Homologous analysis showed that CsRac1 and CsRac2 share high conservation with other counterparts from different species. The CsRac1 and CsRac2 transcript showed wide tissue distribution, in which CsRac1 and CsRac2 exhibit the highest expression level in liver and gill, respectively. The expression level of CsRac1 and CsRac2 fluctuated in the liver and gill tissues at different time points after challenged by Vibrio harveyi. Specifically, CsRac1 and CsRac2 were significantly up-regulated at 48 h and 96 h post injection. Moreover, the knocking down of CsRac1 and CsRac2 in cell line (TSHKC) reduced the expression of CsPAK1, CsIL1-ß and CsTNF-α. The present data suggests that CsRac1 and CsRac2 might play important roles in the innate immunity of half-smooth tongue sole.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia
6.
Genet Med ; 21(4): 1021-1026, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30293988

RESUMO

PURPOSE: RAC3 is an underexamined member of the Rho GTPase gene family that is expressed in the developing brain and linked to key cellular functions. De novo missense variants in the homolog RAC1 were recently associated with developmental disorders. In the RAC subfamily, transforming missense changes at certain shared residues have been observed in human cancers and previously characterized in experimental studies. The purpose of this study was to determine whether constitutional dysregulation of RAC3 is associated with human disease. METHODS: We discovered a RAC3 variant in the index case using genome sequencing, and searched for additional variants using international data-sharing initiatives. Functional effects of the variants were assessed using a multifaceted approach generalizable to most clinical laboratory settings. RESULTS: We rapidly identified five individuals with de novo monoallelic missense variants in RAC3, including one recurrent change. Every participant had severe intellectual disability and brain malformations. In silico protein modeling, and prior in vivo and in situ experiments, supported a transforming effect for each of the three different RAC3 variants. All variants were observed in databases of somatic variation in cancer. CONCLUSIONS: Missense variants in RAC3 cause a novel brain disorder, likely through a mechanism of constitutive protein activation.


Assuntos
Predisposição Genética para Doença , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas rac de Ligação ao GTP/genética , Adulto , Pré-Escolar , GTP Fosfo-Hidrolases/genética , Humanos , Recém-Nascido , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/fisiopatologia , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/diagnóstico por imagem , Transtornos do Neurodesenvolvimento/fisiopatologia , Fenótipo , Sequenciamento Completo do Genoma
7.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30279207

RESUMO

The aim of the present study was to explore the expression profile and the potential regulatory mechanism of two long non-coding RNAs (lncRNAs) (RP13-650J16.1 and TCONS_00023979) in prostate cancer (PCa). Expression profile of lncRNAs in PCa and paracancerous tissues were investgated by the high-throughput gene chip technology. Specific siRNA of RP13-650J16.1 or TCONS_00023979 was transfected into DU145 cells. Then, the relative expression of RP13-650J16.1, receptor-associated coactivator 3 (RAC3), promyelocytic leukemia (PML), and TCONS_00023979 was detected by quantitative real-time PCR and Western blotting. MTT assay was used to detect the proliferation of DU145 cells. The migration ability of DU145 cells was measured by Transwell chambers. Single cell proliferation and clonogenic ability were detected by plate clone formation assay. RP13-650J16.1 and RAC3 expression was up-regulated, and TCONS_00023979 and PML expression was down-regulated in PCa tissues. Silencing RP13-650J16.1 could decrease RAC3 expression, and knockout of TCONS_00023979 also reduced PML expression. Moreover, the ability of proliferation, migration, and colony formation of DU145 cells was decreased after transfected with si-RP13-650J16.1, while these abilities were increased after transfected with si-TCONS_00023979. Collectively, our findings demonstrated that RP13-650J16.1 might be an oncogene and TCONS_00023979 might be an antioncogene in PCa.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Proteínas rac de Ligação ao GTP/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Próstata/metabolismo , Próstata/patologia , Próstata/cirurgia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Microambiente Tumoral , Proteínas rac de Ligação ao GTP/metabolismo
8.
PLoS Genet ; 14(9): e1007670, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30265669

RESUMO

Rac GTPases act as master switches to coordinate multiple interweaved signaling pathways. A major function for Rac GTPases is to control neurite development by influencing downstream effector molecules and pathways. In Caenorhabditis elegans, the Rac proteins CED-10, RAC-2 and MIG-2 act in parallel to control axon outgrowth and guidance. Here, we have identified a single glycine residue in the CED-10/Rac1 Switch 1 region that confers a non-redundant function in axon outgrowth but not guidance. Mutation of this glycine to glutamic acid (G30E) reduces GTP binding and inhibits axon outgrowth but does not affect other canonical CED-10 functions. This demonstrates previously unappreciated domain-specific functions within the CED-10 protein. Further, we reveal that when CED-10 function is diminished, the adaptor protein NAB-1 (Neurabin) and its interacting partner SYD-1 (Rho-GAP-like protein) can act as inhibitors of axon outgrowth. Together, we reveal that specific domains and residues within Rac GTPases can confer context-dependent functions during animal development.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Crescimento Neuronal/genética , Domínios Proteicos/fisiologia , Proteínas rac de Ligação ao GTP/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Feminino , Ácido Glutâmico/genética , Glicina/genética , Masculino , Mutagênese , Domínios Proteicos/genética , Proteínas rac de Ligação ao GTP/metabolismo
9.
J Biol Chem ; 293(40): 15397-15418, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30108175

RESUMO

The MET proto-oncogene-encoded receptor tyrosine kinase (MET) and AXL receptor tyrosine kinase (AXL) are independently operating receptor tyrosine kinases (RTKs) that are functionally associated with aggressive and invasive cancer cell growth. However, how MET and AXL regulate the migratory properties of cancer cells remains largely unclear. We report here that the addition of hepatocyte growth factor (HGF), the natural ligand of MET, to serum-starved human glioblastoma cells induces the rapid activation of both MET and AXL and formation of highly polarized MET-AXL clusters on the plasma membrane. HGF also promoted the formation of the MET and AXL protein complexes and phosphorylation of AXL, independent of AXL's ligand, growth arrest-specific 6 (GAS6). The HGF-induced MET-AXL complex stimulated rapid and dynamic cytoskeleton reorganization by activating the small GTPase RAC1, a process requiring both MET and AXL kinase activities. We further found that HGF also promotes the recruitment of ELMO2 and DOCK180, a bipartite guanine nucleotide exchange factor for RAC1, to the MET-AXL complex and thereby stimulates the RAC1-dependent cytoskeleton reorganization. We also demonstrated that the MET-AXL-ELMO2-DOCK180 complex is critical for HGF-induced cell migration and invasion in glioblastoma or other cancer cells. Our findings uncover a critical HGF-dependent signaling pathway that involves the assembly of a large protein complex consisting of MET, AXL, ELMO2, and DOCK180 on the plasma membrane, leading to RAC1-dependent cell migration and invasion in various cancer cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Invasividade Neoplásica , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo
10.
Methods Mol Biol ; 1821: 235-246, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30062416

RESUMO

B-cell migration and adhesion are critical to form a germinal center response, the site for B-cell production of high-affinity antibodies. Here, we describe two assays that can be used to examine B-cell cytoskeletal responses needed during the germinal center response: B-cell spreading and homotypic adhesion. Spreading of B cells is dependent on Cdc42, while Rac1 and Rac2 are necessary for homotypic adhesion. These in vitro assays can be used to examine functional responses of B cells mediated by the cell cytoskeleton, for example when comparing B cells from different gene knockout animals.


Assuntos
Linfócitos B/enzimologia , Citoesqueleto/enzimologia , Centro Germinativo/enzimologia , Neuropeptídeos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linfócitos B/citologia , Adesão Celular/fisiologia , Citoesqueleto/genética , Centro Germinativo/citologia , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética
11.
Methods Mol Biol ; 1821: 371-392, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30062425

RESUMO

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.


Assuntos
Quimiotaxia/fisiologia , Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Fatores Quimiotáticos/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/citologia , Dictyostelium/genética , Endocitose/fisiologia , Proteínas de Protozoários/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética
12.
Neural Dev ; 13(1): 17, 2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089513

RESUMO

BACKGROUND: In the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood. METHODS: Through a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes. RESULTS: We show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development. CONCLUSIONS: Together, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Sistema da Linha Lateral/citologia , Mutação/genética , Células de Schwann/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética , Fatores Etários , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Sistema da Linha Lateral/embriologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Microscopia Eletrônica de Transmissão , Proteína Básica da Mielina/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/embriologia , RNA Mensageiro/metabolismo , Células de Schwann/ultraestrutura , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
13.
Sci Rep ; 8(1): 11138, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042445

RESUMO

Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.


Assuntos
Citoesqueleto de Actina/química , Actinas/química , Transtorno do Espectro Autista/genética , Neurônios/química , Citoesqueleto de Actina/genética , Actinas/genética , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Regulação da Expressão Gênica/genética , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Esfoliação de Dente , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética
14.
J Pharmacol Exp Ther ; 367(1): 9-19, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30021868

RESUMO

Histamine induces chemotaxis of mast cells through the H4 receptor. However, little is known about the precise intracellular signaling pathway that mediates this process. In this study, we identified small GTPases Rac1 and Rac2 as intracellular binding partners of the H4 receptor and characterized their roles in H4 receptor signaling. We showed that histamine induced Rac GTPase activation via the H4 receptor. A Rac inhibitor NSC23766 attenuated chemotaxis of mast cells toward histamine, as well as histamine-induced calcium mobilization and extracellular signal-regulated kinase (ERK) activation. Histamine-induced migration of mast cells was also sensitive to PD98059, an inhibitor of the mitogen-activated protein kinase kinase, indicating that the Rac-ERK pathway was involved in chemotaxis through the H4 receptor. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by LY294002 suppressed the histamine-induced chemotaxis and activation of Rac GTPases, suggesting that PI3K regulates chemotaxis upstream of Rac activation. Specific knockdown of Rac1 and Rac2 by short-hairpin RNA revealed that both Rac GTPases are necessary for histamine-induced migration. Downregulation of Rac1 and Rac2 led to attenuated response in calcium mobilization and ERK activation, respectively. These observations suggested that Rac1 and Rac2 have distinct and essential roles in intracellular signaling downstream of H4 receptor-PI3K in histamine-induced chemotaxis of mast cells.


Assuntos
Quimiotaxia , Mastócitos/citologia , Receptores Histamínicos H4/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Histamina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/deficiência , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética
15.
J Biol Chem ; 293(28): 11143-11153, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29853638

RESUMO

Inflammation is a major driver of tumor progression and metastasis, although the mechanisms by which proinflammatory cytokines drive metastatic invasion are unknown. Interleukin-6 (IL-6) is a potent proinflammatory cytokine that is elevated in individuals with pancreatic cancer (PDAC), is required for PDAC progression in mice, and increases tumor cell invasion in vitro Here, we provide insights into the mechanisms by which IL-6 activates tumor cell invasion. We found that IL-6 stimulation rapidly and robustly activates the small GTPase cell division cycle 42 (CDC42) in human PDAC cells and promotes the formation of premigratory filopodia. The CDC42 activation was required for IL-6-induced invasion as blocking CDC42 activity rendered the cells insensitive to IL-6's proinvasive effects. Loss of Janus kinase 2 (JAK2) or signal transducer and activator of transcription 3 (STAT3) prevented IL-6-mediated CDC42 activation, indicating that IL-6 activates CDC42 through both JAK2 and STAT3. However, the rapid activation of CDC42 suggested that this activation may be distinct from canonical STAT3-mediated transcriptional activation. Importantly, we observed an interaction between STAT3 and IQ motif-containing GTPase-activating protein 1 (IQGAP1), a scaffolding platform that binds CDC42. STAT3 colocalized with CDC42 and IQGAP1 at the plasma membrane, suggesting cross-talk between IL-6-mediated STAT3 signaling and CDC42 activation. These results suggest that IL-6 promotes metastatic invasion, at least partially, through CDC42 and that, along with its pleiotropic effects on tumor growth and progression, IL-6 signaling also activates proinvasive GTPase signaling, priming tumor cells for metastatic invasion.


Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , Interleucina-6/farmacologia , Neoplasias Pancreáticas/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proliferação de Células , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Tumorais Cultivadas , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
16.
Am J Respir Crit Care Med ; 198(10): 1288-1301, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29897791

RESUMO

RATIONALE: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood. OBJECTIVES: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages. METHODS: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2-/- mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae. MEASUREMENTS AND MAIN RESULTS: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice. CONCLUSIONS: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.


Assuntos
Fumar Cigarros/efeitos adversos , Fumar Cigarros/imunologia , Pneumonia/etiologia , Pneumonia/imunologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata/imunologia , Pulmão/imunologia , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/imunologia , Índice de Gravidade de Doença
17.
Int Urol Nephrol ; 50(9): 1667-1677, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29808448

RESUMO

PURPOSE: Chronic kidney disease of unknown etiology (CKDu), having epidemic characteristics, is being diagnosed increasingly in certain tropical regions of the world, mainly Latin America and Sri Lanka. They have been observed primarily in farming communities and current hypotheses point toward many environmental and occupational triggers. CKDu does not have common etiologies of chronic kidney disease (CKD) such as hypertension, diabetes, or autoimmune disease. We aimed to understand the molecular processes underlying CKDu in Sri Lanka using transcriptome analysis. METHODS: RNA extracted from whole blood was reverse transcribed and used for microarray analysis using the Human HT-12 v.4 array (Illumina). Pathway analysis was carried out using ingenuity pathway analysis (IPA-Qiagen). Microarray results were validated using real-time PCR of five selected genes. RESULTS: Pathways related to innate immune response, including interferon signaling, inflammasome signaling and TREM1 signaling had the most significant positive activation z scores, where as EIF2 signaling and mTOR signaling had the most significant negative activation z scores. Pathways previously linked to fluoride toxicity; G-protein activation, Cdc42 signaling, Rac signaling and RhoA signaling were activated in CKDu patients. The most significantly activated biological functions were cell death, cell movement and antimicrobial response. Significant toxicological functions were mitochondrial dysfunction, oxidative stress and apoptosis. CONCLUSIONS: Based on the molecular pathway analysis in CKDu patients and review of literature, viral infections and fluoride toxicity appear to be contributing to the molecular mechanisms underlying CKDu.


Assuntos
Fluoretos/efeitos adversos , Imunidade Inata/genética , Insuficiência Renal Crônica/etiologia , Transdução de Sinais/genética , Viroses/complicações , Adulto , Fator de Iniciação 2 em Eucariotos/genética , Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Humanos , Inflamassomos/genética , Interferons/genética , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA/análise , Sri Lanka , Serina-Treonina Quinases TOR/genética , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética
18.
Mol Med Rep ; 18(1): 333-341, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29749511

RESUMO

Brain ischemia leads to energy depletion, mitochondrial dysfunction and neuronal cell death. The present study was designed to identify key genes and pathways associated with brain ischemia. The gene expression profile GSE52001, including 3 normal brain samples and 3 cerebral ischemia samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified using the limma package. Then functional and pathway enrichment analyses were performed by the MATHT tool. Protein­protein interaction (PPI) network, module selection and microRNA (miRNA)­target gene network were constructed utilizing Cytoscape software. A total of 488 DEGs were identified (including 281 upregulated and 207 downregulated genes). In the PPI network, Rac family small GTPase 2 (RAC2) had higher degrees. RAC2 was significantly enriched in the FcγR­mediated phagocytosis pathway. miR­29A/B/C had a higher degree in the miRNA­target gene network. Insulin like growth factor 1 (Igf1) was identified as the target gene for miR­29A/B/C. RAC2 may function in brain ischemia through mediating the FcγR­mediated phagocytosis pathway. Meanwhile, miR­29A/B/C and their targets gene Igf1 may serve important roles in the development and progression of brain ischemia.


Assuntos
Isquemia Encefálica/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , MicroRNAs/biossíntese , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Proteínas rac de Ligação ao GTP/biossíntese , Proteínas rac de Ligação ao GTP/genética
19.
Sci Signal ; 11(528)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717062

RESUMO

The role of vascular endothelial growth factor (VEGF) signaling in cancer is not only well known in the context of angiogenesis but also important in the functional regulation of tumor cells. Autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears to be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression, and drug resistance. Therefore, understanding the mechanisms involved in VEGF-mediated support of CSCs is critical to successfully treating cancer patients. The expression of the Hippo effector TAZ is associated with breast CSCs and confers stem cell-like properties. We found that VEGF-NRP2 signaling contributed to the activation of TAZ in various breast cancer cells, which mediated a positive feedback loop that promoted mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, thus leading to TAZ activity. In a complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) ß2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, the TAZ-mediated repression of ß2-chimaerin resulted in sustained Rac1 activity in CSCs. Depletion of ß2-chimaerin in non-CSCs increased Rac1 activity, TAZ abundance, and mammosphere formation. Analysis of a breast cancer patient database revealed an inverse correlation between ß2-chimaerin and TAZ expression in tumors. Our findings highlight an unexpected role for ß2-chimaerin in a feed-forward loop of TAZ activation and the acquisition of CSC properties.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas de Neoplasias/genética , Neuropilina-2/genética , Transdução de Sinais/genética , Fatores de Transcrição , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas rac de Ligação ao GTP/genética
20.
Proc Natl Acad Sci U S A ; 115(16): E3731-E3740, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29610331

RESUMO

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.


Assuntos
Actomiosina/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Rombencéfalo/embriologia , Sarcoglicanas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Proteínas rac de Ligação ao GTP/fisiologia , Animais , Padronização Corporal/genética , Sistemas CRISPR-Cas , Movimento Celular , Characidae/genética , Characidae/fisiologia , Cromatina/genética , Cromatina/ultraestrutura , Evolução Molecular , Peixes/classificação , Peixes/genética , Morfogênese , Mutagênese Sítio-Dirigida , Neurogênese , Filogenia , Sarcoglicanas/genética , Especificidade da Espécie , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética
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