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1.
Fish Shellfish Immunol ; 91: 148-158, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31082520

RESUMO

The Rho family GTPase Rac1 acts as a molecular switch for signal transduction to regulate various cellular functions. Here, a Rac1 homolog (LcRac1) was identified in large yellow croaker (Larimichthys crocea), one of the most economically important marine fishes. The LcRac1 protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LcRac1). LcRac1 was ubiquitously expressed in all 12 tissues we examined, with the highest expression in heart and blood and the weakest expression in head-kidney and spleen. Moreover, time course analysis revealed that LcRac1 expression was obviously up-regulated in liver, spleen and head-kidney after immunization with Poly I:C, LPS and Vibrio parahemolyticus. On the other hand, on the basis of protein interaction, it was found that the LcRac1 interacted with Tropomyosin, a crucial protein in the process of phagocytosis. Furthermore, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the LcRac1 gene was silenced by sequence-specific siRNA. Fluorescence microscopy assays revealed FITC-labeled V. parahemolyticus were remarkably decreased after LcRac1 was silenced by sequence-specific siRNA at 24 h. These findings implicate the vital role of LcRac1 in innate immunity in the large yellow croaker.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Fagocitose/genética , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária , Tropomiosina , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia , Proteínas rac1 de Ligação ao GTP/química
2.
Cell Mol Life Sci ; 76(19): 3891-3898, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31053884

RESUMO

Statins are potent lipid-lowering drugs. Large prospective clinical trials have shown the anti-thrombotic effect of statins, e.g., preventing deep vein thrombosis. However, the mechanism underlying the beneficial effect of statins in reducing thrombus formation remains to be established. We, thus, conduct this study to investigate the potential molecular mechanisms. The cultured human hepatoma cells (HepG2) were used as the in vitro model. The human protein C gene promoter was cloned into the luciferase reporter to study the transcriptional regulation of human protein C gene. Wistar rats fed with simvastatin (5 mg/kg day) were used as the in vivo model. We found that simvastatin increased the expression of protein C in hepatocytes (361 ± 64% and 313 ± 59% after 2 h and 6 h of stimulation, respectively, both p < 0.01). In the animal study, the serum protein C levels were increased in the simvastatin-treated group (7 ± 2.2 unit/ml vs 23.4 ± 19.3 unit/ml and 23.4 ± 18.2 unit/ml and 1 and 2 weeks of treatment, respectively, both p < 0.05). Regarding the possible molecular mechanism, we found that the level of hepatocyte nuclear factor 1α (HNF1α) was also increased in both the in vivo and in vitro models. We found that the protein C promoter activity was increased by simvastatin, and this effect was inhibited by HNF1α knockdown and constitutively active Rac1. Therefore, stains may modulate protein C expression through small GTPase Rac 1 and HNF1α.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteína C/genética , Animais , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína C/metabolismo , Ratos Wistar , Sinvastatina/farmacologia , Transcrição Genética/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/genética
3.
Cancer Sci ; 110(6): 1883-1896, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955235

RESUMO

MicroRNAs have been reported to play critical roles in the regulation of non-small-cell cancer (NSCLC) development, but the role of microRNA (miR)-331-3p in NSCLC is still unclear. In this study, the expression levels of miR-331-3p in NSCLC tumor tissues and adjacent normal tissues were examined by quantitative RT-PCR, and the relationship between miR-331-3p expression and patient clinicopathological characteristics was analyzed. The effects of miR-331-3p on epithelial-mesenchymal transition (EMT), migration, and metastasis of NSCLC cells were determined in vitro and vivo. Direct functional targets of miR-331-3p were identified by luciferase reporter assay, western blot assay, immunohistochemical staining, and rescue assay. The downstream pathway regulated by miR-331-3p was identified by immunofluorescence, immunoprecipitation, and Rac1 activity examination. Our results showed that miR-331-3p was significantly downregulated in NSCLC tumor tissues and was correlated with clinicopathological characteristics, and miR-331-3p could be an independent prognostic marker for NSCLC patients. Furthermore, miR-331-3p significantly suppressed EMT, migration and metastasis of NSCLC cells in vitro and in vivo. Both ErbB2 and VAV2 were direct functional targets of miR-331-3p. The activities of Rac1, PAK1, and ß-catenin were regulated by miR-331-3p through ErbB2 and VAV2 targeting. These results indicated that miR-331-3p suppresses EMT, migratory capacity, and metastatic ability by targeting ErbB2 and VAV2 through the Rac1/PAK1/ß-catenin axis in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-vav/genética , Receptor ErbB-2/genética , Transdução de Sinais/genética , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor ErbB-2/metabolismo , beta Catenina/genética , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética
4.
Toxicol Lett ; 310: 61-69, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31018152

RESUMO

PM2.5 is the main particulate air pollutant that is capable of inducing airway injury. Previous studies have indicated that Rac1 is involved in cigarette smoke-induced lung inflammation and lipopolysaccharide-mediated pulmonary injury. However, the contribution of Rac1 activity to PM2.5-induced lung inflammation remains largely unclear. Here, we investigated the regulation of Rac1 in PM2.5-induced inflammation in mouse airways and human bronchial epithelial cells (16HBE). The lungs of mice exposed to PM2.5 showed increased IL-1ß expression and an accumulation of inflammatory cells, thereby indicating high Rac1 activity. The exposure of 16HBE cells to PM2.5 resulted in elevated Rac1 levels, as well as an increased release of IL-1ß. Particularly, the selective inhibition of Rac1 ameliorated the IL-1ß release and inflammation in model lungs. Histological assessment showed that treatment with a Rac1 inhibitor, NSC23766, reduced the infiltration of neutrophils and macrophages into the airway lumen. Moreover, the selective inhibition or knockdown of Rac1 decreased IL-1ß release in 16HBE cells induced by PM2.5, which correlated with PM2.5-induced Rac1-regulated AKT signaling. Our data suggest an important role for Rac1 in the pathological alterations associated with PM2.5-mediated lung inflammation. Rac1 may be a promising therapeutic target for the treatment of the inflammatory diseases induced by PM2.5 inhalation.


Assuntos
Aminoquinolinas/farmacologia , Anti-Inflamatórios/farmacologia , Pulmão/efeitos dos fármacos , Neuropeptídeos/antagonistas & inibidores , Material Particulado/toxicidade , Pneumonia/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Exposição por Inalação/efeitos adversos , Interleucina-1beta/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos ICR , Neuropeptídeos/metabolismo , Tamanho da Partícula , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/patologia , Interferência de RNA , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
J Mol Neurosci ; 68(2): 171-180, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30888622

RESUMO

Synaptic cell adhesion molecules, including neurexins and neuroligins, mediate the formation and maintenance of connections between neuronal cells. Although neurexins and neuroligins are known to interact with each other in a calcium-dependent manner and several neuropeptides have been shown to act through G protein-coupled receptors to increase intracellular calcium levels, no studies have examined the role of the neuropeptide oxytocin in association with adhesion molecules. Given that oxytocin receptors are located on presynaptic and postsynaptic membranes and that oxytocin exerts direct effects on neuronal excitability, it could be hypothesized that oxytocin affects the expression of cell surface adhesion molecules. In the present study, we show that incubation in the presence of oxytocin (1 µM, 48 h) exerted cell-specific effects on the levels of neurexin 2α, neurexin 2ß, and neuroligin 3. Oxytocin significantly increased the mRNA expression levels of neurexin 2α, neurexin 2ß, and neuroligin 3 in SH-SY5Y, U-87MG, and primary cerebellar cells. The effect of inhibiting oxytocin receptors on the expression of neurexin 2ß was more dramatic in U-87MG cells than in SH-SY5Y cells. Oxytocin did not exert effects in primary corticohippocampal cells. Additionally, we measured the expression of selected GTPases to determine whether they could mediate the effects of oxytocin. Oxytocin induced a decrease in the mRNA level of Rac1 in U-87MG and primary cerebellar cells and exerted a stimulatory effect on the expression of RhoB at the gene and protein level in SH-SY5Y cells. These results suggest that the regulation of neurexins and neuroligins involves the activation of oxytocin receptors. These effects are likely mediated by the stimulation of RhoB GTPase, at least in certain types of cells.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cerebelo/citologia , Humanos , Hipotálamo/citologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ocitocina/farmacologia , Ratos , Ratos Wistar , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/metabolismo
6.
Cell Oncol (Dordr) ; 42(3): 287-301, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30859392

RESUMO

PURPOSE: Anti-apoptotic and pro-migratory phenotypes are hallmarks of neoplastic diseases, including primary brain malignancies. In this work, we examined whether reprogramming of the apoptotic and migratory machineries through a multi-targeting approach would induce enhanced cell death and enhanced inhibition of the migratory capacity of glioblastoma cells. METHODS: Preclinical testing and molecular analyses of combined inhibition of Bcl-2/Bcl-xL and RAC1 were performed in established, primary cultured and stem-like glioblastoma cell systems. RESULTS: We found that the combined inhibition of Bcl-2/Bcl-xL and RAC1 resulted in synergistic pro-apoptotic and anti-migratory effects in a broad range of different glioblastoma cells. At the molecular level, we found that RAC1 inhibition led to a decreased expression of the deubiquitinase Usp9X, followed by a decreased stability of Mcl-1. We also found that the combined inhibition led to a significantly decreased migratory activity and that tumor formation of glioblastoma cells on chorion allantoic membranes of chicken embryos was markedly impaired following the combined inhibition. CONCLUSIONS: Our data indicate that concomitant inhibition of RAC1 and Bcl-2/Bcl-xL induces pro-apoptotic and anti-migratory glioblastoma phenotypes as well as synergistic anti-neoplastic activities. The clinical efficacy of this inhibitory therapeutic strategy warrants further evaluation.


Assuntos
Compostos de Anilina/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Sulfonamidas/farmacologia , Ubiquitina Tiolesterase/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitina Tiolesterase/genética , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Int Immunopharmacol ; 70: 268-273, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851707

RESUMO

The TRPV2 cation channel has been recently implicated in the regulation of arthritis severity, joint damage, and in the invasive behavior of the fibroblast-like synoviocyte (FLS). However, its mechanism of action was unknown. In this study we characterize the cell signaling events mediating the TRPV2 suppressive activity in FLS invasiveness. Studies with FLS cell lines derived from patients with RA revealed that TRPV2-specific stimulation significantly reduced FLS adhesion to different extracellular matrices that shared binding to αν, ß1 and ß3 integrins. Localization of these integrins to the plasma membrane and numbers of thick and organized actin filaments were diminished by TRPV2 specific stimulation, and cells developed a round and non-polarized morphology. TRPV2 stimulation significantly reduced levels of activated RhoA, Rac1 and cofilin. RhoA activators were able to overcome the TRPV2-induced suppression on both RhoA activation and invasion. These new discoveries suggest that TRPV2 regulates key intracellular processes implicated in cell invasion in arthritis and other processes such as cancer, and has the potential to become a useful target for drug development.


Assuntos
Citoesqueleto de Actina/metabolismo , Artrite Reumatoide/metabolismo , Fibroblastos/fisiologia , Sinoviócitos/fisiologia , Canais de Cátion TRPV/metabolismo , Citoesqueleto de Actina/patologia , Fatores de Despolimerização de Actina/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Forma Celular , Ativação Enzimática , Humanos , Integrinas/metabolismo , Invasividade Neoplásica , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
8.
Cell Biochem Funct ; 37(2): 62-71, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30773655

RESUMO

Osteogenesis is the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. MicroRNAs (miRNAs) are short noncoding RNAs that target specific genes to mediate translational activities. In this study, we investigated how miR-224 regulates the osteoblastic differentiation of human MSCs (hMSCs) as well as the underlying mechanism. The results revealed the upregulation of miR-224 during hMSC differentiation. In vitro experiments showed that the downregulation of miR-224 suppressed the differentiation of hMSCs into osteoblasts. However, upregulation of miR-224 was concomitant with increased expression of relevant genes and augmented activity of alkaline phosphatase. Furthermore, the results indicated that Rac1 acted as the bona fide target of miR-224 and that Rac1 depletion promoted osteogenic differentiation in miR-224-silenced hMSCs. In addition, we found that both JAK/STAT3 and Wnt/ß-catenin pathways were repressed by Rac1 depletion using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and immunofluorescence. Our data indicate a novel molecular mechanism in relation to hMSCs differentiation into osteoblasts, which may facilitate bone anabolism via miR-224. SIGNIFICANCE OF THE STUDY: In this study, we mainly explored the effects of miR-224 on hMSCs differentiation into osteoblasts. We find that induced miR-224 expression in hMSCs is considered closely associated with specific osteogenesis-related genes, alkaline phosphatase activity, and matrix mineralization, indicating that miR-224 may serve as a promising biomarker for osteogenic differentiation. Our data indicate a novel molecular mechanism in relation to hMSCs differentiation into osteoblasts, which may facilitate bone anabolism via miR-224.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular Transformada , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Osteoblastos/citologia , Proteínas rac1 de Ligação ao GTP/genética
9.
PLoS One ; 14(2): e0212219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735546

RESUMO

Translocation of the glucose transporter GLUT4 to the sarcolemma accounts for glucose uptake in skeletal muscle following insulin administration. The protein kinase Akt2 and the small GTPase Rac1 have been implicated as essential regulators of insulin-stimulated GLUT4 translocation. Several lines of evidence suggest that Rac1 is modulated downstream of Akt2, and indeed the guanine nucleotide exchange factor FLJ00068 has been identified as an activator of Rac1. On the other hand, the mechanisms whereby Akt2 and Rac1 are regulated in parallel downstream of phosphoinositide 3-kinase are also proposed. Herein, we aimed to provide additional evidence that support a critical role for Akt2 in insulin regulation of Rac1 in mouse skeletal muscle. Knockdown of Akt2 by RNA interference abolished Rac1 activation following intravenous administration of insulin or ectopic expression of a constitutively activated phosphoinositide 3-kinase mutant. The activation of another small GTPase RalA and GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated form of phosphoinositide 3-kinase, but not Rac1, were also diminished by downregulation of Akt2 expression. Collectively, these results strongly support the notion that Rac1 acts downstream of Akt2 leading to the activation of RalA and GLUT4 translocation to the sarcolemma in skeletal muscle.


Assuntos
Glucose/metabolismo , Insulina/farmacologia , Músculo Esquelético/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Sarcolema/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Masculino , Camundongos , Mutação , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Sarcolema/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(8): 2967-2976, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30728292

RESUMO

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteína Forkhead Box O1/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Ativação Transcricional/genética , Proteínas rac1 de Ligação ao GTP/genética
11.
Scand J Immunol ; 89(5): e12752, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30681176

RESUMO

The accumulation of airway apoptotic cells may be an important factor causing airway hyper-responsiveness (AHR). Whether the apoptotic cells can be promptly removed is related to the occurrence and course of asthma. In recent years, studies have shown that Rac1 is involved in many cellular biological activities including the formation and elimination of apoptotic cells. In this study, based on the analysis of airway local cells and related factors in asthmatic mice, we evaluated the expression of Rac1 in airway epithelial cells or phagocytes and analysed its relationship with the incidence of apoptosis or scavenging of apoptotic cells. Our data showed that the expression level of Rac1 in asthmatic mice decreased significantly, while the expression of IL-33 increased obviously. The airway epithelial cell line was stimulated by curcumin at 50 µmol/L for 24-48 hours; more than 50% of the cells were apoptotic, and of which, about 20% were late apoptosis. Rac1 inhibitor (NSC23766) can enhance the apoptosis effect. In addition, the ability of phagocytosis and migration in the epithelial cells or macrophages was increased following the application of Rac1 inhibitors or specific siRNA in a dose-dependent manner, and the expression level of IL-33 was simultaneously increased after blocking Rac1. It is suggested that the down regulation of Rac1 in asthma may contribute to the apoptosis of airway epithelial cells and affect the clearance of apoptotic cells, which will lead to the aggregation of the apoptotic cells in the respiratory tract and participate in AHR.


Assuntos
Asma/imunologia , Fagócitos/imunologia , Hipersensibilidade Respiratória/imunologia , Mucosa Respiratória/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Aminoquinolinas/farmacologia , Animais , Apoptose , Hiper-Reatividade Brônquica , Curcumina/metabolismo , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Mucosa Respiratória/patologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética
12.
Oncogene ; 38(19): 3651-3666, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659265

RESUMO

Oxygen is an indispensable element for cell survival and maintenance. Eukaryotic cells are equipped with a series of signaling pathways that cope with hypoxia. The dioxygenase factor inhibiting HIF (FIH) is an oxygen sensor that regulates the transcriptional activity of hypoxia-inducible factor (HIF) through asparaginyl hydroxylation. Given that HACE1 was detected as an FIH-interacting protein in a previous proteomics study, we tested whether the E3 ubiquitin ligase HACE1 is a substrate for FIH. FIH interacted with HACE1, in cells and in vitro, and was determined to hydroxylate HACE1 at the N191 residue within the ankyrin repeat domain. Hydroxylation disrupted the physical association between HACE1 and its representative target, Rac1. Under hypoxic conditions, HACE1 is less hydroxylated due to the inactivation of FIH, and subsequently functions to ubiquitinate the active form of Rac1, leading to the proteasomal degradation of Rac1. Since Rac1 stimulates cell movement, HACE1 inhibits cell migration and invasion in breast cancer by removing active Rac1. Such an effect of HACE1 is reinforced under hypoxia because HACE1 escapes from FIH-mediated hydroxylation. In clinical datasets, HACE1 downregulation is associated with poor outcomes in patients with breast cancer. Taken together, FIH is likely to act as an oxygen sensor that determines oxygen-dependent cancer progression.


Assuntos
Neoplasias da Mama/patologia , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Repetição de Anquirina , Asparagina/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Hidroxilação , Oxigênio/metabolismo , Hipóxia Tumoral , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/genética
13.
Mol Carcinog ; 58(1): 55-65, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30182377

RESUMO

Liver cancer stem cells (CSCs) were involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). miR-365 was downregulated in hepatocellular carcinoma and inhibited HCC cell proliferation and invasion. However, the role of miR-365 in liver cancer stem cells was unknown. Herein, we observed a remarkable decrease of miR-365 expression in CD133 or EpCAM-positive liver CSCs as well as in CSC-enriched hepatoma spheres. Up-regulated miR-365 suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self-renewal ability of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in hepatoma cells downregulated the RAC1 mRNA and protein expression. RAC1 also could promote the expansion of liver CSCs. The special RAC1 inhibitor EHop-106 or RAC1 overexpression abolished the discrepancy in liver CSC proportion and the self-renewal capacity between miR-365 overexpression hepatoma cells and control cells, which further confirmed that RAC1 was required in miR-365-suppressed liver CSCs expansion. miR-365 was downregulated in liver CSCs and could inhibit HCC cells dedifferentiation and liver CSCs expansion by targeting RAC1 signaling.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genética
14.
J Neuroinflammation ; 15(1): 343, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553270

RESUMO

BACKGROUND: Over-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration. Reversion of over-activated to neuroprotective microglia phenotype could regenerate a healthy CNS-supporting microglia environment. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of microglia to a ramified, neuroprotective phenotype. METHODS: We exploited the anti-inflammatory and neuroprotective properties of conditioned medium of adipose-derived mesenchymal stem cells (CM) as a tool to generate the neuroprotective phenotype of microglia in vitro, and we set up a microscopy-based siRNA screen to identify its hits by cell morphology. RESULTS: We initially assayed an array of 157 siRNAs against genes that codify proteins and factors of cytoskeleton and activation/inflammatory pathways in microglia. From them, 45 siRNAs significantly inhibited the CM-induced transition from a neurotoxic to a neuroprotective phenotype of microglia, and 50 siRNAs had the opposite effect. As a proof-of-concept, ten of these targets were validated with individual siRNAs and by downregulation of protein expression. This validation step resulted essential, because three of the potential targets were false positives. The seven validated targets were assayed in a functional screen that revealed that the atypical RhoGTPase RhoE/Rnd3 is necessary for BDNF expression and plays an essential role in controlling microglial migration. CONCLUSIONS: Besides the identification of RhoE/Rnd3 as a novel inducer of a potential neuroprotective phenotype in microglia, we propose a list of potential targets to be further confirmed with selective activators or inhibitors.


Assuntos
Citocinas/metabolismo , Microglia/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Movimento Celular/fisiologia , Forma Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Feminino , Regulação da Expressão Gênica/genética , Células-Tronco Mesenquimais/química , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética
15.
J Agric Food Chem ; 66(44): 11767-11774, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30269504

RESUMO

Nano titanium dioxide (Nano-TiO2) has been applied in food packaging systems and food additives, but it may cause potential neurotoxicity for human and animals. In our study, the effects of nano-TiO2 exposure during pregnancy/lactation on the development of the central nervous system in offspring mice were examined and its molecular mechanism involving Rho family was investigated. Our findings showed that pregnancy/lactation exposure to nano-TiO2 resulted in thinning of cerebral and cerebellar cortex, decrease in number of neurons per unit area of cerebrum, edema and nuclear condensation, dysplasia of neurites in hippocampal pyramidal cells, thinning in pyramidal cell layer in hippocampus, and decrease in learning and memory of offspring mice. Furthermore, expressions of Rac1 and Cdc42 involved in axon and dendritic development were decreased, whereas RhoA expression and ratio of RhoA/Rac1 were increased in offspring brain. It implies that exposure to nano-TiO2 during pregnancy/lactation could result in brain retardation and cognitive impairment in offspring mice, which was closely related to alterations in the expression of Rho protein family. Therefore, application of nano-TiO2 in daily life should be performed with caution.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/crescimento & desenvolvimento , Exposição Materna/efeitos adversos , Nanopartículas Metálicas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Titânio/toxicidade , Animais , Sistema Nervoso Central/metabolismo , Feminino , Humanos , Masculino , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
PLoS Genet ; 14(5): e1007370, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29734338

RESUMO

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.


Assuntos
Actinas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Movimento Celular , Cercopithecus aethiops , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/metabolismo , Mutação , Ligação Proteica , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ras/genética
17.
Biomed Pharmacother ; 99: 622-628, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29710460

RESUMO

According to many studies, miRNAs are involved in the control of bone cell differentiation and function. Hence, an understanding of the pathways regulated by miRNAs involved in skeletal function is essential for the development of miRNA-based therapeutic strategies for bone diseases. In this study we evaluated the role of miR-25 in osteoblast differentiation by examining the expressions of key osteoblast differentiation markers like Runx2 and Ocn and also evaluated the effects of miR-25-Rac1 axis on PI3K/AKT and JNK pathways. MC3T3-E1 osteoprogenitors were treated with osteogenic differentiation media which was refreshed every 48?h after the initial differentiation treatment and were then quantified for total miRNA content. The viability, migration as well as Runx2 and Ocn expressions in cells transfected with miR-25 mimic, miR-25 inhibitor, and si-Rac1 were evaluated by CCK-8 assay, wound migration assay, qRT-PCR and Western blot. Finally, the effects of miR-25-Rac1 axis on PI3K/AKT and JNK pathways were studied. MiR-25 was found to significantly enhance cell viability and migration and up-regulate the expressions of Runx2 and Ocn. MiR-25 was also found to enhance the expression levels of Rac1, which contributed to the effects of miR-25 on osteoblastic cell lines. MiR-25 activated PI3K/AKT and JNK pathways possibly by up-regulation of Rac1. Enhanced expression of miR-25 led to the promotion of cell viability and migration, as well as up-regulation of Runx2 and Ocn markers by enhancing Rac1 expression.


Assuntos
Proliferação de Células/genética , MicroRNAs/genética , Neuropeptídeos/genética , Osteoblastos/citologia , Proteínas rac1 de Ligação ao GTP/genética , Animais , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Osteogênese/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
18.
J Immunol Res ; 2018: 4564328, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29651441

RESUMO

Background: In our previous study, mouse double minute 2 homolog (MDM2), insulin-like growth factor 1 (IGF1), signal transducer and activator of transcription 1 (STAT1), and Rac family small GTPase 1 (RAC1) were correlated with the recurrence of giant cell tumor of bone (GCT). The aim of this study is to use a large cohort study to confirm the involvement of these four genes in GCT recurrence. Methods: The expression of these four genes was detected and compared between GCT patients with or without recurrence. The correlation between the expression of these four genes and clinical characteristics was evaluated. Protein-protein interaction (PPI) network was constructed for functional enrichment analysis. Results: It showed that the expression levels of MDM2, IGF1, STAT1, and RAC1 in GCT patients with recurrence were significantly higher than those in GCT patients without recurrence (P < 0.05). Multivariate logistic regression analysis suggested that several clinical characteristics may influence prognosis. A PPI network was constructed using the four genes as hub genes. Functional enrichment analysis showed that this network involves many important biological progress mediated by these four genes, including immune response. Conclusion: MDM2, IGF1, STAT1, and RAC1 are associated with GCT recurrence, which might serve as biomarkers for GCT recurrence.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Tumor de Células Gigantes do Osso/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fator de Transcrição STAT1/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/metabolismo , Estudos de Coortes , Feminino , Tumor de Células Gigantes do Osso/diagnóstico , Tumor de Células Gigantes do Osso/metabolismo , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/genética , Fator de Transcrição STAT1/genética , Adulto Jovem , Proteínas rac1 de Ligação ao GTP/genética
19.
Nat Commun ; 9(1): 477, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396460

RESUMO

Major depressive disorder is associated with abnormalities in the brain and the immune system. Chronic stress in animals showed that epigenetic and inflammatory mechanisms play important roles in mediating resilience and susceptibility to depression. Here, through a high-throughput screening, we identify two phytochemicals, dihydrocaffeic acid (DHCA) and malvidin-3'-O-glucoside (Mal-gluc) that are effective in promoting resilience against stress by modulating brain synaptic plasticity and peripheral inflammation. DHCA/Mal-gluc also significantly reduces depression-like phenotypes in a mouse model of increased systemic inflammation induced by transplantation of hematopoietic progenitor cells from stress-susceptible mice. DHCA reduces pro-inflammatory interleukin 6 (IL-6) generations by inhibiting DNA methylation at the CpG-rich IL-6 sequences introns 1 and 3, while Mal-gluc modulates synaptic plasticity by increasing histone acetylation of the regulatory sequences of the Rac1 gene. Peripheral inflammation and synaptic maladaptation are in line with newly hypothesized clinical intervention targets for depression that are not addressed by currently available antidepressants.


Assuntos
Antocianinas/farmacologia , Ácidos Cafeicos/farmacologia , Epigênese Genética , Glucosídeos/farmacologia , Inflamação/genética , Plasticidade Neuronal/genética , Estresse Psicológico/genética , Animais , Antocianinas/administração & dosagem , Ácidos Cafeicos/administração & dosagem , Ilhas de CpG/efeitos dos fármacos , Depressão/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Glucosídeos/administração & dosagem , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Antígenos Comuns de Leucócito/genética , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Polifenóis/farmacologia , Comportamento Social , Estresse Psicológico/tratamento farmacológico , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
Biochim Biophys Acta Mol Cell Res ; 1865(5): 794-802, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29458077

RESUMO

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.


Assuntos
Caderinas/genética , Caveolina 1/genética , Fator de Transcrição STAT3/genética , Proteínas rac1 de Ligação ao GTP/genética , Células A549 , Animais , Apoptose/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
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