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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.
Life Sci ; 256: 117976, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32561397

RESUMO

AIMS: We have previously reported that Centchroman (CC), an oral contraceptive drug, inhibits breast cancer progression and metastasis. In this study, we investigated whether CC inhibits local invasion of tumor cells and/or their metastatic colonization with detailed underlying mechanisms. MAIN METHODS: The effect of CC on the experimental metastasis and spontaneous metastasis was demonstrated by using tail-vein and orthotopic 4T1-syngeneic mouse tumor models, respectively. The anti-angiogenic potential of CC was evaluated using well established in vitro and in vivo models. The role of RAC1/PAK1/ß-catenin signaling axis in the metastasis was investigated and validated using siRNA-mediated knockdown of PAK1 as well as by pharmacological PAK1-inhibitor. KEY FINDINGS: The oral administration of CC significantly suppressed the formation of metastatic lung nodules in the 4T1-syngeneic orthotopic as well as experimental metastatic models. More importantly, CC treatment suppressed the tube formation and migration capacities of human umbilical vein endothelial cells (HUVEC) and inhibited pre-existing vasculature as well as the formation of neovasculature. The suppression of migration and invasion capacities of metastatic breast cancer cells upon CC treatment was associated with the inhibition of small GTPases (Rac1 and Cdc42) concomitant with the downregulation of PAK1 and downstream ß-catenin signaling. In addition, CC upregulated the expression of miR-145, which is known to target PAK1. SIGNIFICANCE: This study warrants the repurposing of CC as a potential therapeutic agent against metastatic breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Centocromano/farmacologia , Antagonistas de Estrogênios/farmacologia , Neuropeptídeos/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Quinases Ativadas por p21/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Centocromano/uso terapêutico , Antagonistas de Estrogênios/uso terapêutico , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neuropeptídeos/metabolismo , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
3.
PLoS One ; 15(4): e0230814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251425

RESUMO

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.


Assuntos
Crescimento Neuronal/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologia , Estatmina/metabolismo , Tubulina (Proteína)/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Acetilação , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia
4.
J Immunol Res ; 2020: 1906204, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190703

RESUMO

Background: Macrophage-derived foam cells play a central role in atherosclerosis, and their ultimate fate includes apoptosis, promotion of vascular inflammation, or migration to other tissues. Nε-Carboxymethyl-lysine (CML), the key active component of advanced glycation end products, induced foam cell formation and apoptosis. Previous studies have shown that the Vav1/Rac1 pathway affects the macrophage cytoskeleton and cell migration, but its role in the pathogenesis of diabetic atherosclerosis is unknown. Methods and Results: In this study, we used anterior tibiofibular vascular samples from diabetic foot amputation patients and accident amputation patients, and histological and cytological tests were performed using a diabetic ApoE-/- mouse model and primary peritoneal macrophages, respectively. The results showed that the atherosclerotic plaques of diabetic foot amputation patients and diabetic ApoE-/- mice were larger than those of the control group. Inhibition of the Vav1/Rac1 pathway reduced vascular plaques and promoted the migration of macrophages to lymph nodes. Transwell and wound healing assays showed that the migratory ability of macrophage-derived foam cells was inhibited by CML. Cytoskeletal staining showed that advanced glycation end products inhibited the formation of lamellipodia in foam cells, and inhibition of the Vav1/Rac1 pathway restored the formation of lamellipodia. Conclusion: CML inhibits the migration of foam cells from blood vessels via the Vav1/Rac1 pathway, and this process affects the formation of lamellipodia.


Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Pé Diabético/metabolismo , Células Espumosas/fisiologia , Lisina/análogos & derivados , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Amputação , Animais , Apolipoproteínas E/genética , Aterosclerose/patologia , Movimento Celular , Células Cultivadas , Pé Diabético/patologia , Humanos , Lisina/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais
5.
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
6.
Jpn J Clin Oncol ; 50(4): 465-472, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32134451

RESUMO

BACKGROUND: As a member of the Rho small guanosine triphosphatase family, ras-related C3 botulinum toxin substrate 1 (RAC1) interacts with various specific effectors, and p21-activated kinase 1 (PAK1), which has a role in both carcinogenesis and cellular invasion, binds to RAC1, after which activated PAK1 regulates cellular functions. There have been few reports about the simultaneous analysis of RAC1 and its downstream effector PAK1 in upper urinary tract urothelial carcinoma (UTUC). We assessed the expressions of both RAC1 and PAK1 and evaluated their association with clinicopathological parameters. METHODS: Immunohistochemical studies of RAC1 or PAK1 were performed with specimens from 104 patients with N0M0 UTUC and cancer-free surgical margins. Correlation of the positive expression of RAC1 or PAK1 or both with clinicopathological parameters was evaluated. RESULTS: A hazard model showed that the presence of mixed histologic features and moderate or strong positive expression of both RAC1 and PAK1 were independent factors for shortened disease-specific survival time (Ps = 0.041 and 0.016, respectively), and another hazard model revealed that only moderate or strong positive expression of both RAC1 and PAK1 was an independent factor for shortened recurrence-free survival time in the multivariate analysis (P = 0.036). Neither moderate or strong positive expression of RAC1 alone nor moderate or strong positive expression of PAK1 alone was an independent factor for a worse rate of disease-specific or recurrence-free survival in multivariate analysis. CONCLUSIONS: Patients with N0M0 UTUC, cancer-free surgical margins and moderate or strong positive expression of both RAC1 and PAK1 should be carefully monitored after surgery.


Assuntos
Carcinoma de Células de Transição/cirurgia , Margens de Excisão , Sistema Urinário/patologia , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/cirurgia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Neoplasias Urológicas/patologia
7.
Nat Commun ; 11(1): 1456, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193458

RESUMO

Resistance development to one chemotherapeutic reagent leads frequently to acquired tolerance to other compounds, limiting the therapeutic options for cancer treatment. Herein, we find that overexpression of Rac1 is associated with multi-drug resistance to the neoadjuvant chemotherapy (NAC). Mechanistically, Rac1 activates aldolase A and ERK signaling which up-regulates glycolysis and especially the non-oxidative pentose phosphate pathway (PPP). This leads to increased nucleotides metabolism which protects breast cancer cells from chemotherapeutic-induced DNA damage. To translate this finding, we develop endosomal pH-responsive nanoparticles (NPs) which deliver Rac1-targeting siRNA together with cisplatin and effectively reverses NAC-chemoresistance in PDXs from NAC-resistant breast cancer patients. Altogether, our findings demonstrate that targeting Rac1 is a potential strategy to overcome acquired chemoresistance in breast cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos , Via de Pentose Fosfato , Neoplasias de Mama Triplo Negativas/terapia , Proteínas rac1 de Ligação ao GTP/metabolismo , Adulto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia com Agulha de Grande Calibre , Mama/patologia , Mama/cirurgia , Linhagem Celular Tumoral , Quimioterapia Adjuvante/métodos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Conjuntos de Dados como Assunto , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos , Feminino , Seguimentos , Frutose-Bifosfato Aldolase/metabolismo , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Sistema de Sinalização das MAP Quinases , Mastectomia , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Nucleotídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Am J Hum Genet ; 106(3): 338-355, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109419

RESUMO

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Estudos de Coortes , Feminino , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Masculino , Fenótipo , Proteínas Serina-Treonina Quinases/química , Homologia de Sequência de Aminoácidos
9.
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
10.
BMC Cancer ; 20(1): 32, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931758

RESUMO

BACKGROUND: Vasculogenic mimicry (VM), defined as a capability of aggressive tumor Cells to mimic embryonic vasculogenic networks, caused poor prognosis in hepatocellular carcinoma (HCC). Rho kinases (ROCK), p21-activated kinase (PAK), hypoxia or epithelial-mesenchymal transition (EMT) contributed to the VM potential. However, the details underlying these biological behaviors have not been completely elucidated. METHODS: Kaplan-Meier analysis was conducted to predict relationship with hypoxia Inducible factor (HIF-1α), EMT related markers: Vimentin and patient prognosis. CD34/periodic acid-Schiff (PAS) double staining was examined to differentiate VM-positive (VM+) and VM-negative (VM-) samples. Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on RhoA/ROCK, Rac1/PAK and EMT were evaluated by real time-qPCR and western blot. HIF-1α small interfering RNA (siRNA), overexpressed or short hairpin RNA (shRNA) of ROCK and kinase inhibitors were used to explore the effect of HIF-1α, RhoA/ROCK, Rac1/PAK and Vimentin on VM. RESULTS: HIF-1α or Vimentin was upregulated in VM+ HCC tissues, compared to non-cancerous tissues (P < 0.01), and patients with high expression of HIF-1α or Vimentin had worse prognosis (P < 0.001). We showed hypoxia induced RhoA/ROCK and Rac1/PAK signaling transduction, and EMT could be repressed by HIF-1α siRNA. Notably, RhoA/ROCK or Rac1/PAK stabilized HIF-1α in hypoxia, whereas HIF-1α did not significantly altered RhoA/ROCK or Rac1/PAK signaling in hypoxia. Moreover, we found distinct roles of ROCK1, ROCK2 and PAK in regulating Vimentin phosphorylation. CONCLUSIONS: RhoA/ROCK and Rac/PAK signaling played crucial roles in hypoxia-induced VM via Ser72 and Ser56 Vimentin phosphorylation in HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neovascularização Patológica/metabolismo , Adulto , Idoso , Biomarcadores , Carcinoma Hepatocelular/mortalidade , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Transdução de Sinais , Vimentina/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
11.
Prostate ; 80(5): 412-423, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31995655

RESUMO

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Aminoquinolinas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Células PC-3 , Pirimidinas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Sirolimo/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo
12.
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
13.
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
14.
Cell Mol Life Sci ; 77(1): 161-177, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31161284

RESUMO

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bainha de Mielina/metabolismo , Neurogênese , Neurônios/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Axônios/metabolismo , Axônios/ultraestrutura , Movimento Celular , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/ultraestrutura , Células de Schwann/citologia , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
15.
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
16.
Platelets ; 31(1): 112-119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30880553

RESUMO

Platelets play important roles in blood clotting, hemostasis and wound repair, while more and more research show that platelets also have significant contributions in the process of inflammation. Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease. Platelet microparticles, which are membrane vesicles shed by activated platelets, are reported to amplify inflammation in Rheumatoid arthritis. Here we show that either platelet-specific deletion of Rac1 (Rac1-/-) or Rac1-specific inhibitor NSC23766 dramatically inhibit platelet-derived microparticles formation. As we all know, collagen-induced arthritis (CIA) mouse model is the most common autoimmune model of rheumatoid arthritis. Interestingly, NSC23766 alleviated the process of collagen-induced arthritis of DBA mice in vivo, including the reduced hind paw thickness and ankle stiffness, the reduction of arthritic scores and incidence of arthritis. Our work also found that NSC23766-treated CIA mouse spleen is less swollen and contains less enlarged white pulp than PBS control. The histological analysis shows that NSC23766-treated but not solvent control improve the cartilage erosion symptom in the joint of CIA mouse. Interestingly, platelet microparticles in the peripheral blood of NSC23766-treated CIA mice were decreased significantly compared with PBS-treated CIA mice. In conclusion, our work demonstrated that Rac1 inhibition alleviates collagen-induced arthritis through the decrease of platelet microparticles' release. In short, Rac1 aggravate the rheumatoid arthritis deterioration through the regulation of platelet microparticles formation.


Assuntos
Artrite Reumatoide/metabolismo , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Neuropeptídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Artrite Experimental , Artrite Reumatoide/etiologia , Artrite Reumatoide/patologia , Biomarcadores , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Ativação Plaquetária
17.
Nat Commun ; 10(1): 5686, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831727

RESUMO

Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.


Assuntos
Cílios/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endossomos/metabolismo , Glucose/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Receptores da Família Eph/metabolismo , Idoso , Animais , Glicemia , Teste de Tolerância a Glucose , Fatores de Troca do Nucleotídeo Guanina , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Fosforilação , Receptor EphA3/genética , Receptor EphA3/metabolismo , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
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
19.
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
20.
Asian Pac J Cancer Prev ; 20(12): 3539-3545, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870092

RESUMO

BACKGROUND: Metformin, an antidiabetic drug, has been previously reported to have anti-cancer activities. However, its role in the control of cancer cell migration remains elusive. METHODS: To examine the possible effect of metformin on migration of cervical cancer cells. The related mechanisms were further determined by immunocytochemistry and Western's blotting assay. RESULTS: The results showed that metformin treatment substantially inhibited the migration ability of cervical cancer cells. Consistently, the filopodia and lamellipodia formation were depleted after exposure to metformin. The suppression of migration mediated through the regulatory proteins such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), Rac1 and RhoA after metformin treatment. CONCLUSION: Metformin displays antimigration effects in cervical cancer cells by inhibiting filopodia and lamellipodia formation through the suppression of FAK, Akt and its downstream Rac1 and RhoA protein. We propose that metformin could be a novel potential candidate as an antimetastatic cancer drug in the cervical cancer management.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/antagonistas & inibidores , Metformina/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Metástase Neoplásica/tratamento farmacológico , Pseudópodes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
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