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1.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
2.
Prostate ; 80(13): 1045-1057, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687658

RESUMO

BACKGROUND: There is a need to develop novel therapies which could be beneficial to patients with prostate cancer (CaP) including those who are predisposed to poor outcome, such as African-Americans. This study investigates the role of ROBO1-pathway in predicting outcome and race-based disparity in patients with CaP. METHODS AND RESULTS: Aided by RNA sequencing-based DECIPHER-testing and immunohistochemical (IHC) analysis of tumors we show that ROBO1 is lost during the progressive stages of CaP, a prevalent feature in African-Americans. We show that the loss of ROBO1 predicts high-risk of recurrence, metastasis and poor outcome of androgen-deprivation therapy in radical prostatectomy-treated patients. These data identified an aggressive ROBO1deficient /DOCK1+ve sub-class of CaP. Combined genetic and IHC data showed that ROBO1 loss is accompanied by DOCK1/Rac1 elevation in grade-III/IV primary-tumors and Mets. We observed that the hypermethylation of ROBO1-promoter contributes to loss of expression that is highly prevalent in African-Americans. Because of limitations in restoring ROBO1 function, we asked if targeting the DOCK1 could be an ideal strategy to inhibit progression or treat ROBO1deficient metastatic-CaP. We tested the pharmacological efficacy of CPYPP, a selective inhibitor of DOCK1 under in vitro and in vivo conditions. Using ROBO1-ve and ROBO1+ve CaP models, we determined the median effective concentration of CPYPP for growth. DOCK1-inhibitor treatment significantly decreased the (a) Rac1-GTP/ß-catenin activity, (b) transmigration of ROBO1deficient cells across endothelial lining, and (c) metastatic spread of ROBO1deficient cells through the vasculature of transgenicfl Zebrafish model. CONCLUSION: We suggest that ROBO1 status forms as predictive biomarker of outcome in high-risk populations such as African-Americans and DOCK1-targeting therapy has a clinical potential for treating metastatic-CaP.


Assuntos
Afro-Americanos/genética , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/etnologia , Neoplasias da Próstata/genética , Receptores Imunológicos/genética , Proteínas rac de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Metilação de DNA , Grupo com Ancestrais do Continente Europeu/genética , Disparidades nos Níveis de Saúde , Humanos , Imuno-Histoquímica , Masculino , Metástase Neoplásica , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Regiões Promotoras Genéticas , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Receptores Imunológicos/biossíntese , Receptores Imunológicos/deficiência , Peixe-Zebra , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
3.
Nat Cell Biol ; 22(4): 498-511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203420

RESUMO

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.


Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Nat Commun ; 11(1): 42, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896748

RESUMO

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/- animal sera and are secreted in macrophage media after DNA damage. The Er1F/- EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.


Assuntos
Dano ao DNA/fisiologia , Exossomos/metabolismo , Macrófagos/citologia , Animais , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Exossomos/patologia , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Masculino , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
Medicine (Baltimore) ; 99(1): e18445, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31895772

RESUMO

BACKGROUNDS: HER-2 positive breast cancer is a subtype of breast cancer with poor clinical outcome. The aim of this study was to identify differentially expressed genes (DEGs) for HER-2 positive breast cancer and elucidate the potential interactions among them. MATERIAL AND METHODS: Three gene expression profiles (GSE29431, GSE45827, and GSE65194) were derived from the Gene Expression Omnibus (GEO) database. GEO2R tool was applied to obtain DEGs between HER-2 positive breast cancer and normal breast tissues. Gene ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (David) online tool. Protein-protein interaction (PPI) network, hub gene identification and module analysis was conducted by Cytoscape software. Online Kaplan-Meier plotter survival analysis tool was also used to investigate the prognostic values of hub genes in HER-2 positive breast cancer patients. RESULTS: A total of 54 upregulated DEGs and 269 downregulated DEGs were identified. Among them, 10 hub genes including CCNB1, RAC1, TOP2A, KIF20A, RRM2, ASPM, NUSAP1, BIRC5, BUB1B, and CEP55 demonstrated by connectivity degree in the PPI network were screened out. In Kaplan-Meier plotter survival analysis, the overexpression of RAC1 and RRM2 were shown to be associated with an unfavorable prognosis in HER-2 positive breast cancer patients. CONCLUSIONS: This present study identified a number of potential target genes and pathways which might impact the oncogenesis and progression of HER-2 positive breast cancer. These findings could provide new insights into the detection of novel diagnostic and therapeutic biomarkers for this disease.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Ribonucleosídeo Difosfato Redutase/genética , Proteínas rac1 de Ligação ao GTP/genética , Estudos de Casos e Controles , Biologia Computacional , Regulação para Baixo , Feminino , Humanos , Receptor ErbB-2 , Transcriptoma/genética , Regulação para Cima
6.
Arterioscler Thromb Vasc Biol ; 40(3): 714-732, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996022

RESUMO

OBJECTIVE: Calcification of atherosclerotic plaque is traditionally associated with increased cardiovascular event risk; however, recent studies have found increased calcium density to be associated with more stable disease. 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors or statins reduce cardiovascular events. Invasive clinical studies have found that statins alter both the lipid and calcium composition of plaque but the molecular mechanisms of statin-mediated effects on plaque calcium composition remain unclear. We recently defined a macrophage Rac (Ras-related C3 botulinum toxin substrate)-IL-1ß (interleukin-1 beta) signaling axis to be a key mechanism in promoting atherosclerotic calcification and sought to define the impact of statin therapy on this pathway. Approach and Results: Here, we demonstrate that statin therapy is independently associated with elevated coronary calcification in a high-risk patient population and that statins disrupt the complex between Rac1 and its inhibitor RhoGDI (Rho GDP-dissociation inhibitor), leading to increased active (GTP bound) Rac1 in primary monocytes/macrophages. Rac1 activation is prevented by rescue with the isoprenyl precursor geranylgeranyl diphosphate. Statin-treated macrophages exhibit increased activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), increased IL-1ß mRNA, and increased Rac1-dependent IL-1ß protein secretion in response to inflammasome stimulation. Using an animal model of calcific atherosclerosis, inclusion of statin in the atherogenic diet led to a myeloid Rac1-dependent increase in atherosclerotic calcification, which was associated with increased serum IL-1ß expression, increased plaque Rac1 activation, and increased plaque expression of the osteogenic markers, alkaline phosphatase and RUNX2 (Runt-related transcription factor 2). CONCLUSIONS: Statins are capable of increasing atherosclerotic calcification through disinhibition of a macrophage Rac1-IL-1ß signaling axis.


Assuntos
Aterosclerose/tratamento farmacológico , Atorvastatina/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Macrófagos/efeitos dos fármacos , Neuropeptídeos/metabolismo , Placa Aterosclerótica , Calcificação Vascular/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Idoso , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Camundongos Knockout para ApoE , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Prenilação , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Estudos Retrospectivos , Transdução de Sinais , Calcificação Vascular/genética , Calcificação Vascular/patologia , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
7.
Int J Mol Sci ; 21(2)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936261

RESUMO

The regulation of the differentiation of the bone-forming cells, the osteoblasts, is complex. Many signaling pathways converge on the master regulator of osteoblast differentiation Runx2. The role of molecules that integrate several signaling pathways such as the Rho GTPases need to be better understood. We, therefore, asked at which stage Rac1, one of the Rho GTPase, is needed for osteoblast differentiation and whether it is involved in two pathways, the anabolic response to parathyroid hormone and the stimulatory effect of fibronectin isoforms on integrins. Genetic deletion of Rac1 in preosteoblasts using the osterix promoter diminished osteoblast differentiation in vitro. This effect was however similar to the presence of the promoter by itself. We, therefore, applied a Rac1 inhibitor and confirmed a decrease in differentiation. In vivo, Rac1 deletion using the osterix promoter decreased bone mineral density as well as histomorphometric measures of osteoblast function. In contrast, deleting Rac1 in differentiating osteoblasts using the collagen α1(I) promoter had no effects. We then evaluated whether intermittent parathyroid hormone (PTH) was able to affect bone mineral density in the absence of Rac1 in preosteoblasts. The increase in bone mineral density was similar in control animals and in mice in which Rac1 was deleted using the osterix promoter. Furthermore, stimulation of integrin by integrin isoforms was able to enhance osteoblast differentiation, despite the deletion of Rac1. In summary, Rac1 in preosteoblasts is required for normal osteoblast function and bone density, but it is neither needed for PTH-mediated anabolic effects nor for integrin-mediated enhancement of differentiation.


Assuntos
Densidade Óssea/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Neuropeptídeos/genética , Hormônio Paratireóideo/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Diferenciação Celular/genética , Colágeno Tipo I/genética , Fibronectinas/genética , Humanos , Integrinas/genética , Camundongos , Osteoblastos/metabolismo , Osteogênese/genética , Transdução de Sinais/genética , Fator de Transcrição Sp7/genética , Proteínas rho de Ligação ao GTP/genética
8.
Oral Dis ; 26(2): 302-312, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31793126

RESUMO

OBJECTIVES: This study aimed to explore whether RhoG/Rac1 was involved in migration and invasion of salivary adenoid cystic carcinoma (SACC). MATERIALS AND METHODS: RhoG and Rac1 were evaluated in two SACC cell lines, namely SACC-83 and SACC-LM, with low and high rates of lung metastasis, respectively. Functional changes were evaluated using cell proliferation, transwell, and wound-healing assays, and molecular events were investigated using real-time PCR and Western blot assays. RESULTS: RhoG and Rac1 were highly expressed and more activated in SACC-LM cells than in SACC-83 cells. RhoG overexpression promoted SACC-83 cell migration and invasion through activating Rac1. The knockdown of RhoG or Rac1 partially blocked epiregulin-induced migration and invasion in SACC-83 cells. Epiregulin-induced activation of RhoG/Rac1 in SACC-83 cells was blocked by a Src inhibitor, or an AKT inhibitor or AKT siRNA, or an ERK1/2 inhibitor. Moreover, the epiregulin-induced phosphorylation of AKT and ERK1/2 in SACC-83 cells was blocked by a Src inhibitor, and the epiregulin-induced phosphorylation of ERK1/2 was blocked by an AKT inhibitor or AKT siRNA. Overexpression of activated AKT induced activation of ERK1/2 and RhoG. CONCLUSIONS: RhoG/Rac1 signaling pathway was involved in SACC cell migration and invasion. RhoG/Rac1 at least partially mediated epiregulin/Src/AKT/ERK1/2 signaling to promote SACC cell migration and invasion.


Assuntos
Carcinoma Adenoide Cístico/enzimologia , Carcinoma Adenoide Cístico/patologia , Neoplasias das Glândulas Salivares/enzimologia , Neoplasias das Glândulas Salivares/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Epirregulina/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
9.
J Biol Chem ; 295(5): 1300-1314, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31871052

RESUMO

ß1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the ß2- and ß3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of ß1-chimaerin, ß1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that ß1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, ß1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that ß1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in ß1-chimaerin that is absent in ß1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that ß1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.


Assuntos
Núcleo Celular/metabolismo , Proteínas Quimerinas/genética , Proteínas Quimerinas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Processamento Alternativo , Motivos de Aminoácidos/genética , Animais , Células COS , Ciclo Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Diglicerídeos/metabolismo , Éxons/genética , Inativação Gênica , Humanos , Domínios Proteicos/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Deleção de Sequência , Proteínas rac1 de Ligação ao GTP/genética
10.
Am J Physiol Endocrinol Metab ; 318(3): E330-E342, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31846370

RESUMO

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Proteína Axina/fisiologia , Glucose/metabolismo , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Neuropeptídeos/fisiologia , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Proteína Axina/genética , Linhagem Celular , Estimulação Elétrica , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genética
11.
PLoS One ; 14(12): e0225051, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805065

RESUMO

As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK's function in endothelial cells remains unclear. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK's function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater in Mertk-/- mice. To analyze endothelial MERTK's involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. These results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response.


Assuntos
Células Endoteliais/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , c-Mer Tirosina Quinase/metabolismo , Junções Aderentes/metabolismo , Animais , Permeabilidade Capilar/fisiologia , Criança , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , c-Mer Tirosina Quinase/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
12.
Med Sci Monit ; 25: 9227-9236, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31796725

RESUMO

BACKGROUND The purpose of this study was to investigate whether Orai1 plays a role in the metastasis of osteosarcoma. MATERIAL AND METHODS The expression of Orai1 was silenced by small interfering RNAs against Orai1 (Orai1 siRNA) in osteosarcoma MG-63 cells. Various experiments were carried out to detect the changes in migration, invasion, and adhesion ability of these osteosarcoma cells. Furthermore, the activity of Rac1, Wave2, and Ras was detected using Western blot analysis. Moreover, the Rac1 and Ras inhibitors were used to confirm whether the Ras-Rac1-WAVE2 signaling pathway was involved in osteosarcoma metastasis promoted by Orai1. RESULTS We found that the migration, invasion, and adhesion ability of MG-63 cells were significantly reduced after silencing Orai1 expression (p<0.05). Moreover, the activity of the Rac1-WAVE2 signaling pathway was significantly inhibited after silencing of Orai1 expression (p<0.05). After the Rac1 inhibitor was added, Orai1 siRNA could not further inhibit migration, invasion, and adhesion of the osteosarcoma cells. Further experiments showed that Ras activity was significantly inhibited after silencing Orai1 expression (p<0.05). Moreover, Orai1 siRNA did not further inhibit the activity of the Rac1-WAVE2 signaling pathway nor did it further inhibit the migration, invasion, and adhesion ability of osteosarcoma cells following the addition of Ras inhibitors. CONCLUSIONS Orai1 activates the Ras-Rac1-WAVE2 signaling pathway to promote metastasis of osteosarcoma. Abnormal expression or function of Orai1 may be an important cause of osteosarcoma metastasis.


Assuntos
Neoplasias Ósseas/metabolismo , Proteína ORAI1/metabolismo , Osteossarcoma/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteína ORAI1/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ras/genética
13.
Blood ; 134(25): 2318-2329, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31697813

RESUMO

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1-interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbß3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.


Assuntos
Plaquetas/metabolismo , Pseudópodes/metabolismo , Trombose/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Plaquetas/patologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Selectina-P/genética , Selectina-P/metabolismo , Pseudópodes/genética , Trombose/genética , Trombose/patologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
Int J Mol Sci ; 20(22)2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703278

RESUMO

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid-specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.


Assuntos
Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Teste de Complementação Genética , Humanos , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética
15.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683681

RESUMO

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.


Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Camundongos , Microscopia de Fluorescência , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
16.
Nat Commun ; 10(1): 3975, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484924

RESUMO

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.


Assuntos
Imunidade Inata/genética , Prenilação de Proteína , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Citocinas/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Células RAW 264.7 , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
17.
Oncol Rep ; 42(5): 2039-2048, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31485624

RESUMO

The clinical treatment of renal cell carcinoma (RCC) remains a major challenge. A number of novel agents and therapeutic strategies are currently undergoing active investigation. In the present study, we investigated the potential of GNF­5837, a Trk inhibitor, in the treatment of RCC. Tropomyosin­related kinases (Trk), a family of neurotrophin receptors, are vital for neural development and have also been identified as prognostic markers in malignancies of diverse origins. In the present study, we demonstrated that GNF­5837, an inhibitor of TrkA and TrkB, suppressed the cell viability of renal carcinoma 786O and Caki­2 cells in a concentration­dependent manner. GNF­5837 treatment led to decreased activities of TrkA and TrkB signaling, accompanied by reduced phosphorylation levels of AKT and extracellular signal­regulated kinase (ERK) kinases, which was detected by western blot assay. GNF­5837 induced G0/G1­phase arrest and apoptosis. Consistently, GNF­5837 affected the expression of p21, c­Myc, and survivin proteins. Meanwhile, a wound healing assay showed that GNF­5837 inhibited the migration ability of RCC cells by impairing Rac1 activity. GNF­5837 also enhanced the cytotoxic effects of sunitinib via inhibition of ERK kinase. Taken together, these results identify the pharmacological potential of targeting Trk signaling as a therapeutic strategy for RCC.


Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/genética
18.
Oncogene ; 38(50): 7447-7456, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31427738

RESUMO

The Rho GTPases RhoA, Rac1, and Cdc42 are important regulators of cytoskeletal dynamics. Although many in vitro and in vivo data indicate tumor-promoting effects of activated Rho GTPases, also tumor suppressive functions have been described, suggesting either highly cell-type-specific functions for Rho GTPases in cancer or insufficient cancer models. The availability of a large number of cancer genome-sequencing data by The Cancer Genome Atlas (TCGA) allows for the investigation of Rho GTPase function in human cancers in silico. This information should be used to improve our in vitro and in vivo cancer models, which are essential for a molecular understanding of Rho GTPase function in malignant tumors and for the potential development of cancer drugs targeting Rho GTPase signaling.


Assuntos
Genes Supressores de Tumor , Neoplasias/genética , Oncogenes/genética , Proteínas rho de Ligação ao GTP/genética , Antineoplásicos/uso terapêutico , Citoesqueleto/genética , Genoma Humano/genética , Humanos , Neoplasias/tratamento farmacológico , Transdução de Sinais/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética
19.
Cell Mol Biol Lett ; 24: 55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31462898

RESUMO

Background: Hypoxia, a major condition associated with the tumor microenvironment, stimulates the migration of cancer cells. SOX2 is a powerful transcription factor that shows higher expression in several cancers, however, its role in hypoxia-induced breast cancer cell migration remains largely elusive. Methods: The human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were cultured under hypoxic conditions. The cell migration rate was determined using the wound-healing and transwell assays. The protein levels of SOX2, NEDD9 and HIF-1α were evaluated via western blotting analysis. The NEDD9 mRNA levels were evaluated using qPCR. The activation of Rac1 was detected with the pulldown assay. The binding of SOX2 to the NEDD9 promoter was checked using the luciferase reporter assay. We also transfected breast cancer cells with specific siRNA for SOX2, NEDD9 or the Rac1 inactive mutant (T17 N) to investigate the role of SOX2, NEDD9 and Rac1 in the response to hypoxia. Results: Hypoxia markedly increased SOX2 protein levels in a time-dependent manner. SiRNA-mediated disruption of SOX2 inhibited cell migration under hypoxic conditions. Hypoxia also significantly augmented the NEDD9 mRNA and protein levels. Interestingly, SOX2 is a positive transcriptional regulator of NEDD9. Knockdown of SOX2 inhibited hypoxia-induced NEDD9 mRNA and protein expressions. Furthermore, hypoxia-induced upregulation of Rac1 activity and HIF-1α expression was attenuated by SOX2 or NEDD9 silencing, and Rac1-T17 N abolished HIF-1α expression as well as cell migration in cells subjected to hypoxia. Conclusions: Our results highlight the essential role of SOX2 in breast cancer cell motility. The upregulation of SOX2 under hypoxic conditions may facilitate NEDD9 transcription and expression, and subsequent activation of Rac1 and HIF-1α expression. This could accelerate breast cancer cell migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fatores de Transcrição SOXB1/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Hipóxia Tumoral , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
Sci Adv ; 5(8): eaax1595, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31457101

RESUMO

Ras-related C3 botulinum toxin substrate 1 (Rac1) functions as a molecular switch by cycling between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. An oncogenic mutant of Rac1, an N92I mutant, strongly promotes cell proliferation and subsequent oncogenic activities by facilitating the intrinsic GDP dissociation in the inactive GDP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the N92I mutant. We found that the static structure of the GDP binding site is not markedly perturbed by the mutation, but the overall conformational stability decreases in the N92I mutant, which then facilitates GDP dissociation by lowering the activation energy for the dissociation reaction. On the basis of these results, we proposed the activation mechanism of the N92I mutant, in which the decreased conformational stability plays important roles in its activation process.


Assuntos
Proteínas rac1 de Ligação ao GTP/metabolismo , Sítios de Ligação , Guanosina Difosfato/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura , Proteínas rac1 de Ligação ao GTP/genética
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