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1.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068233

RESUMO

The blood-brain barrier (BBB) is critical to maintaining central nervous system (CNS) homeostasis. However, the effects of microgravity (MG) on the BBB remain unclear. This study aimed to investigate the influence of simulated MG (SMG) on the BBB and explore its potential mechanism using a proteomic approach. Rats were tail-suspended to simulate MG for 21 days. SMG could disrupt the BBB, including increased oxidative stress levels, proinflammatory cytokine levels, and permeability, damaged BBB ultrastructure, and downregulated tight junctions (TJs) and adherens junctions (AJs) protein expression in the rat brain. A total of 554 differentially expressed proteins (DEPs) induced by SMG were determined based on the label-free quantitative proteomic strategy. The bioinformatics analysis suggested that DEPs were mainly enriched in regulating the cell-cell junction and cell-extracellular matrix biological pathways. The inhibited Ras-related C3 botulinum toxin substrate 1 (Rac1)/Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (Wave2)/actin-related protein 3 (Arp3) pathway and the decreased ratio of filamentous actin (F-actin) to globular actin contributed to BBB dysfunction induced by SMG. In the human brain microvascular endothelial cell (HBMECs), SMG increased the oxidative stress levels and proinflammatory cytokine levels, promoted apoptosis, and arrested the cell cycle phase. Expression of TJs and AJs proteins were downregulated and the distribution of F-actin was altered in SMG-treated HBMECs. The key role of the Rac1/Wave2/Arp3 pathway in BBB dysfunction was confirmed in HBMECs with a specific Rac1 agonist. This study demonstrated that SMG induced BBB dysfunction and revealed that Rac1/Wave2/Arp3 could be a potential signaling pathway responsible for BBB disruption under SMG. These results might shed a novel light on maintaining astronaut CNS homeostasis during space travel.


Assuntos
Proteína 3 Relacionada a Actina/metabolismo , Barreira Hematoencefálica/patologia , Regulação da Expressão Gênica , Proteoma/metabolismo , Simulação de Ausência de Peso/efeitos adversos , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Citoesqueleto de Actina , Animais , Barreira Hematoencefálica/metabolismo , Masculino , Proteoma/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Junções Íntimas
2.
Nat Commun ; 12(1): 3519, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112781

RESUMO

TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.


Assuntos
Infecções Bacterianas/imunologia , Imunidade Inata , Canais Iônicos/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Infecções por Escherichia coli/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
3.
Biomaterials ; 274: 120829, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33933985

RESUMO

Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.


Assuntos
Actinas , Células-Tronco Mesenquimais , Actinas/metabolismo , Colágeno , Humanos , Células-Tronco Mesenquimais/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Biochem Biophys Res Commun ; 555: 74-80, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33813279

RESUMO

The engagement of the receptor for advanced glycation end-products (receptor for AGEs, RAGE) with diverse ligands could elicit chronic vascular inflammation, such as atherosclerosis. Binding of cytoplasmic tail RAGE (ctRAGE) to diaphanous-related formin 1 (Diaph1) is known to yield RAGE intracellular signal transduction and subsequent cellular responses. However, the effectiveness of an inhibitor of the ctRAGE/Diaph1 interaction in attenuating the development of atherosclerosis is unclear. In this study, using macrophages from Ager+/+ and Ager-/- mice, we validated the effects of an inhibitor on AGEs-RAGE-induced foam cell formation. The inhibitor significantly suppressed AGEs-RAGE-evoked Rac1 activity, cell invasion, and uptake of oxidized low-density lipoprotein, as well as AGEs-induced NF-κB activation and upregulation of proinflammatory gene expression. Moreover, expression of Il-10, an anti-inflammatory gene, was restored by this antagonist. These findings suggest that the RAGE-Diaph1 inhibitor could be a potential therapeutic drug against RAGE-related diseases, such as chronic inflammation and atherosclerosis.


Assuntos
Células Espumosas/metabolismo , Macrófagos Peritoneais/patologia , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Expressão Gênica , Inflamação/genética , Inflamação/patologia , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Neuropeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
Chem Pharm Bull (Tokyo) ; 69(4): 325-332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790078

RESUMO

Chemoresistance is one of the main factors of treatment failure of cervical cancer (CC). Here, we intended to discover the role and mechanism of miR-509-5p in the paclitaxel chemoresistance of CC cells. RT-PCR was conducted to verify miR-509-3p expression. HCC94 and C-33A paclitaxel-resistant CC cell models were constructed. Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were performed to verify the viability and apoptosis of HCC94 and C-33A cells after upregulating miR-509-3p. Besides, the downstream target of miR-509-3p was analyzed by bioinformatics, and the targeted relationship between miR-509-3p and RAC1 was identified by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Further, the expression of apoptotic proteins (Bcl2, Bax, and Caspase3) and the RAC1/PAK1/LIMK1/Cofilin pathway was monitored by Western blot. The result showed that upregulating miR-509-3p markedly inhibited the viability and promoted the apoptosis of CC cells. On the other hand, miR-509-3p was distinctly downregulated in paclitaxel-resistant HCC94 and C-33A cells (vs. normal cells). The transfection of miR-509-3p mimics notably increased their sensitivity to paclitaxel. Meanwhile, RAC1 was found as the potential target of miR-509-3p in bioinformatics analysis. Moreover, the RAC1/p21 (RAC1) activated kinase 1 (PAK1)/LIM kinase 1 (LIMK1)/Cofilin pathway was significantly activated in paclitaxel-resistant HCC94 and C-33A cells, while miR-509-3p overexpression significantly inactivated this pathway. Additionally, downregulation of RAC1 also partly reversed the paclitaxel-resistance of CC cells and inhibited PAK1/LIMK1/Cofilin. All in all, miR-509-3p enhances the apoptosis and chemosensitivity of CC cells by regulating the RAC1/PAK1/LIMK1/Cofilin pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/genética , Paclitaxel/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cofilina 1/genética , Cofilina 1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases Lim/genética , Quinases Lim/metabolismo , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
6.
J Phys Chem B ; 125(15): 3790-3802, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33848152

RESUMO

Rac1 is a small GTPase that plays key roles in actin reorganization, cell motility, and cell survival/growth as well as in various cancer types and neurodegenerative diseases. Similar to other Ras superfamily GTPases, Rac1 switches between active GTP-bound and inactive GDP-bound states. Switch I and II regions open and close during GDP/GTP exchange. P29S and A159V (paralogous to K-RasA146) mutations are the two most common somatic mutations of Rac1. Rac1P29S is a known hotspot for melanoma, whereas Rac1A159V most commonly occurs in head and neck cancer. To investigate how these substitutions induce the Rac1 dynamics, we used atomistic molecular dynamics simulations on the wild-type Rac1 and two mutant systems (P29S and A159V) in the GTP bound state, and on the wild-type Rac1 and P29S mutated system in the GDP bound state. Here, we show that P29S and A159V mutations activate Rac1 with different mechanisms. In Rac1P29S-GTP, the substitution increases the flexibility of Switch I based on RMSF and dihedral angle calculations and leads to an open conformation. We propose that the open Switch I conformation is one of the underlying reasons for rapid GDP/GTP exchange of Rac1P29S. On the other hand, in Rac1A159V-GTP, some of the contacts of the guanosine ring of GTP with Rac1 are temporarily lost, enabling the guanosine ring to move toward Switch I and subsequently close the switch. Rac1A159V-GTP adopts a Ras state 2 like conformation, where both switch regions are in closed conformation and Thr35 forms a hydrogen bond with the nucleotide.


Assuntos
Melanoma , Proteínas rac1 de Ligação ao GTP , Guanosina Trifosfato , Humanos , Conformação Molecular , Mutação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras
7.
Nat Commun ; 12(1): 2335, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879799

RESUMO

Current therapeutic options for treating colorectal cancer have little clinical efficacy and acquired resistance during treatment is common, even following patient stratification. Understanding the mechanisms that promote therapy resistance may lead to the development of novel therapeutic options that complement existing treatments and improve patient outcome. Here, we identify RAC1B as an important mediator of colorectal tumourigenesis and a potential target for enhancing the efficacy of EGFR inhibitor treatment. We find that high RAC1B expression in human colorectal cancer is associated with aggressive disease and poor prognosis and deletion of Rac1b in a mouse colorectal cancer model reduces tumourigenesis. We demonstrate that RAC1B interacts with, and is required for efficient activation of the EGFR signalling pathway. Moreover, RAC1B inhibition sensitises cetuximab resistant human tumour organoids to the effects of EGFR inhibition, outlining a potential therapeutic target for improving the clinical efficacy of EGFR inhibitors in colorectal cancer.


Assuntos
Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Carcinogênese , Linhagem Celular Tumoral , Cetuximab/farmacologia , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transdução de Sinais , Regulação para Cima , Via de Sinalização Wnt , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética
8.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800361

RESUMO

Dragon's Blood is a red resin from Dracaena cochinchinensis (Lour.) S.C. Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has shown protective effects on intestinal disorders. Microgravity could alter intestinal homeostasis. However, the potential herbal drugs for preventing intestine epithelial barrier (IEB) dysfunction under microgravity are not available. This study aimed to investigate the effects of Dragon's Blood (DB) on microgravity-induced IEB injury and explore its underlying mechanism. A rat tail-suspension model was used to simulate microgravity (SMG). Histomorphology, ultrastructure, permeability, and expression of junction proteins in jejunum, ileum, and colon of SMG rats were determined. Proteomic analysis was used to identify differentially expressed proteins (DEPs) in rat ileum mucosa altered by DB. The potential mechanism of DB to protect IEB dysfunction was validated by western blotting. The effects of several components in DB were evaluated in SMG-treated Caco-2 cells. DB protected against IEB disruption by repairing microvilli and crypts, inhibiting inflammatory factors, lowering the permeability and upregulating the expression of tight and adherens junction proteins in the ileum of SMG rats. Proteomic analysis showed that DB regulated 1080 DEPs in rat ileum mucosa. DEPs were significantly annotated in cell-cell adhesion, focal adhesion, and cytoskeleton regulation. DB increased the expression of Rac1-WAVE2-Arp2/3 pathway proteins and F-actin to G-actin ratio, which promoted the formation of focal adhesions. Loureirin C in DB showed a protective effect on epithelial barrier injury in SMG-treated Caco-2 cells. DB could protect against IEB dysfunction induced by SMG, and its mechanism is associated with the formation of focal adhesions mediated by the Rac1-WAVE2-Arp2/3 pathway, which benefits intestinal epithelial cell migration and barrier repair.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Mucosa Intestinal/metabolismo , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Simulação de Ausência de Peso/efeitos adversos , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Células CACO-2 , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mucosa Intestinal/patologia , Masculino , Ratos , Ratos Sprague-Dawley
9.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799511

RESUMO

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.


Assuntos
Receptores de Lipopolissacarídeos/genética , Lipoproteínas HDL/farmacologia , Neutrófilos/efeitos dos fármacos , Insuficiência Renal Crônica/genética , Uremia/genética , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Lipoproteínas HDL/isolamento & purificação , Masculino , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Neutrófilos/patologia , Cultura Primária de Células , Diálise Renal , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/terapia , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Uremia/metabolismo , Uremia/fisiopatologia , Uremia/terapia , beta-Ciclodextrinas/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
10.
Cell Prolif ; 54(5): e13023, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33759281

RESUMO

BACKGROUND: Vascular smooth muscle cells (VSMC) switch to macrophage-like cells after cholesterol loading, and this change may play an important role in atherogenesis. Muscleblind-like splicing regulator 1 (MBNL1) is a well-known splicing factor that has been implicated in many cellular processes. However, the role of MBNL1 in VSMC macrophage-like transdifferentiation is largely unknown. In this study, we aim to characterize the role of MBNL1-induced gene splicing during atherogenesis. METHODS: The expression of MBNL1 and Abelson interactor 1 (Abi1) splice variants (Abi1-e10 and Abi1-Δe10) was compared between artery tissues from healthy donors and atherosclerosis patients. Regulatory mechanisms of MBNL1-induced Abi1 gene splicing were studied, and the signal pathways mediated by Abi1 splice variants were investigated in VSMC. RESULTS: Loss of MBNL1 was found in the macrophage-like VSMC (VSMC-M) in artery wall from atherosclerosis patients. In vitro and in vivo evidence confirmed that Abi1 is one of the MBNL1 target genes. Loss of MBNL1 significantly induces the Abi1-Δe10 isoform expression. Compared to the known actin organization activities of the Abi1 gene, we discovered a novel action of Abi1-Δe10, whereby Abi1-Δe10 activates Rac1 independent of upstream stimulation and triggers the Rac1-NOX1-ROS pathway, which results in increased expression of transcription factor Kruppel-like factor 4 (KLF4). While Abi1-Δe10 inhibits contractile VSMC biomarkers expression and cell contraction, it stimulates VSMC proliferation, migration and macrophage-like transdifferentiation. CONCLUSION: Loss-of-function of MBNL1 activates VSMC-M transdifferentiation to promote atherogenesis through regulating Abi1 RNA splicing.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Músculo Liso Vascular/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Desdiferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Músculo Liso Vascular/citologia , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Fenótipo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo
11.
Nucleic Acids Res ; 49(6): 3322-3337, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33704464

RESUMO

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.


Assuntos
Cromatina/metabolismo , Replicação do DNA , Proteínas Ativadoras de GTPase/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteína de Replicação A/metabolismo , Animais , Linhagem Celular Tumoral , Reparo do DNA , Feminino , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fosforilação , Proteólise , Transdução de Sinais , Estresse Fisiológico/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
12.
Biomolecules ; 11(2)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546351

RESUMO

Accumulating evidence has suggested a role of the small GTPase Ras homolog gene family member A (RhoA) in DNA damage response (DDR) in addition to its traditional function of regulating cell morphology. In DDR, 2 key components of DNA repair, ataxia telangiectasia-mutated (ATM) and flap structure-specific endonuclease 1 (FEN1), along with intracellular reactive oxygen species (ROS) have been shown to regulate RhoA activation. In addition, Rho-specific guanine exchange factors (GEFs), neuroepithelial transforming gene 1 (Net1) and epithelial cell transforming sequence 2 (Ect2), have specific functions in DDR, and they also participate in Ras-related C3 botulinum toxin substrate 1 (Rac1)/RhoA interaction, a process which is largely unappreciated yet possibly of significance in DDR. Downstream of RhoA, current evidence has highlighted its role in mediating cell cycle arrest, which is an important step in DNA repair. Unraveling the mechanism by which RhoA modulates DDR may provide more insight into DDR itself and may aid in the future development of cancer therapies.


Assuntos
Dano ao DNA , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular , Sobrevivência Celular , Reparo do DNA , Endonucleases Flap/metabolismo , Humanos , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo
13.
Thorax ; 76(4): 326-334, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542087

RESUMO

BACKGROUND: Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. METHODS AND RESULTS: Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. CONCLUSION: This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipersensibilidade Respiratória , Proteínas rac1 de Ligação ao GTP/metabolismo , Corticosteroides/farmacologia , Aminoquinolinas/administração & dosagem , Aminoquinolinas/farmacologia , Animais , Biópsia , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Proliferação de Células , Modelos Animais de Doenças , Eosinófilos/metabolismo , Células Caliciformes/metabolismo , Humanos , Camundongos , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
14.
BMC Cancer ; 21(1): 155, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579235

RESUMO

BACKGROUND: p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs. METHODS: Fluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α and ERK3 in normal and NMSC specimens. Dunnett's test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay. RESULTS: The expression level of ∆Np63α is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells. CONCLUSIONS: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.


Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Proteína Quinase 6 Ativada por Mitógeno/genética , Fosforilação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas rac1 de Ligação ao GTP/genética
15.
PLoS Genet ; 17(2): e1009308, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33539343

RESUMO

Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.


Assuntos
Aminoquinolinas/farmacologia , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/metabolismo , Pirimidinas/farmacologia , Motilidade Espermática/genética , Motilidade Espermática/fisiologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Região do Complexo-t do Genoma/genética , Animais , Bovinos , Genótipo , Haplótipos , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Fenótipo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Proteínas rac1 de Ligação ao GTP/genética
16.
Nature ; 590(7847): 618-623, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568811

RESUMO

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Células Epiteliais/citologia , Fagócitos/citologia , Fagocitose , Peixe-Zebra/embriologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Apoptose , Movimento Celular , Forma Celular , Extensões da Superfície Celular , Imunidade Inata , Camundongos , Fosfatidilserinas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
17.
Mol Med Rep ; 23(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33398380

RESUMO

MicroRNAs (miRNAs/miRs) are a class of non­coding RNAs that serve crucial roles in liver cancer and other liver injury diseases. However, the expression profile and mechanisms underlying miRNAs in liver fibrosis are not completely understood. The present study identified the novel miR­375/Rac family small GTPase 1 (RAC1) regulatory axis in liver fibrosis. Reverse transcription­quantitative PCR was performed to detect miR­375 expression levels. MTT, flow cytometry and western blotting were performed to explore the in vitro roles of miR­375. The dual­luciferase reporter gene assay was performed to determine the potential mechanism underlying miR­375 in liver fibrosis. miR­375 expression was significantly downregulated in liver fibrosis tissues and cells compared with healthy control tissues and hepatocytes, respectively. Compared with the pre­negative control group, miR­375 overexpression inhibited mouse hepatic stellate cell (HSC) viability and epithelial­mesenchymal transition, and alleviated liver fibrosis. The dual­luciferase reporter assay results demonstrated that miR­375 bound to RAC1. Moreover, the results indicated that miR­375 regulated the hedgehog signaling pathway via RAC1 to restrain HSC viability and EMT, thus exerting its anti­liver fibrosis function. The present study identified the miR­375/RAC1 axis as a novel regulatory axis associated with the development of liver fibrosis.


Assuntos
Transição Epitelial-Mesenquimal , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Idoso , Animais , Sobrevivência Celular , Feminino , Proteínas Hedgehog/genética , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas rac1 de Ligação ao GTP/genética
18.
Biophys J ; 120(5): 866-876, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33515600

RESUMO

Rac1 is a small member of the Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, although we focus on Rac1-PAK1 interaction by atomic-force-microscopy-based single-molecule force spectroscopy experiments, we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian network model analysis and molecular dynamics simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion, suggesting the mechanical changes as potential markers of oncogenicity. Indeed, the dissociation of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1, as revealed by single-molecule force spectroscopy experiments. Q61L and Y72C mutations change the mechanics of the Rac1-PAK1 complex by increasing the elasticity of the protein and slowing down the transition to the unbound state. On the other hand, Rac1's intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on switches I and II with Q61L, Y72C, oncogenic P29S and Q61R, and negative T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around the p-loop and switch I decreases in all mutants, mostly in Q61L, whereas some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream and downstream partners including PAK1.


Assuntos
Quinases Ativadas por p21 , Proteínas rac1 de Ligação ao GTP , Guanosina Trifosfato , Mutação , Transdução de Sinais , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
Nat Commun ; 12(1): 56, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397922

RESUMO

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Intestinos/patologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Carcinogênese/genética , Homeostase , Intestinos/ultraestrutura , Camundongos Knockout , Mutação/genética , Especificidade de Órgãos , Fenótipo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Regulação para Cima , Via de Sinalização Wnt
20.
FASEB J ; 35(2): e21235, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33417283

RESUMO

Despite significant evidence that Rac1 is localized to the nucleus, little is known regarding the function and biological significance of nuclear Rac1. Here, we showed that in response to EGF Rac1 was translocated to nuclear speckles and co-localized with the nuclear speckle marker Serine/arginine-rich splicing factor 2 (SRSF2) in Cos-7 cells. We also showed that the nuclear speckle localization of Rac1 was dependent on its T108 phosphorylation and facilitated by Rac1 polybasic region (PBR) that contains a nuclear localization signal and Rac1 GTPase activity. To gain insight into the function of Rac1 in nuclear speckles, we searched for Rac1 binding proteins in the nucleus. We isolated nuclear fraction of HEK 293 cells and incubated with GST-Rac1 and the phosphomimetic GST-Rac1T108E. We identified 463 proteins that were associated with GST-Rac1T108E, but not with GST-Rac1 by LC-MS/MS. Three notable groups of these proteins are: the heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and SRSFs, all of which are involved in pre-mRNA splicing and associated with nuclear speckles. We further showed by co-immunoprecipitation that Rac1 interacts with SRSF2, hnRNPA1, and U2A' in response to EGF. The interaction is dependent on T108 phosphorylation and facilitated by Rac1 PBR and GTPase activity. We showed that hnRNPA1 translocated in and out of nucleus in response to EGF in a similar pattern to Rac1. Rac1 only partially colocalized with U2A' that localizes to the actual splicing sites adjacent to nuclear speckle. Finally, we showed that Rac1 regulated EGF-induced pre-mRNA splicing and this is mediated by T108 phosphorylation. We conclude that in response to EGF, T108 phosphorylated Rac1 is targeted to nuclear speckles, interacts with multiple groups of proteins involved in pre-mRNA splicing, and regulates EGF-induced pre-mRNA splicing.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Proteínas rac1 de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ligação Proteica , Splicing de RNA , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética
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