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1.
Commun Biol ; 4(1): 850, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239035

RESUMO

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.


Assuntos
Polaridade Celular/fisiologia , Endocitose , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Cadeia A de beta-Cristalina/genética
2.
Biomolecules ; 11(6)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200023

RESUMO

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. MS is characterized by infiltrations of leukocytes such as T and B lymphocytes and macrophages. Macrophages have been identified as major effectors of inflammation and demyelination in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the activation and heterogeneity of macrophages in MS has been poorly investigated. Thus, in this study, we evaluated M1 and M2 macrophages immunophenotype from EAE and control mice by analyzing over 30 surface and intracellular markers through polychromatic flow cytometry, qRT-PCR, and ELISA assay. We showed that M1 macrophages possessed a higher proinflammatory profile in EAE compared to control mice, since they expressed higher levels of activation/co-stimulatory markers (iNOS, CD40, and CD80) and cytokines/chemokines (IL-6, IL-12, CCL2, and CXCL10), whereas M2 lost their M2-like phenotype by showing a decreased expression of their signature markers CD206 and CCL22, as well as a concomitant upregulation of several M1 makers. Furthermore, immunization of M1 and M2 macrophages with MOG35-55 led to a significant hyperactivation of M1 and a concomitant shift of anti-inflammatory M2 to pro-inflammatory M1 macrophages. Overall, we provide evidence for a phenotypic alteration of M1/M2 balance during MS, which can be of crucial importance not only for a better understanding of the immunopathology of this neurodegenerative disease but also to potentially develop new macrophage-centered therapeutic strategies.


Assuntos
Polaridade Celular/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Macrófagos/imunologia , Esclerose Múltipla/imunologia , Plasticidade Neuronal/fisiologia , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Imunofenotipagem/métodos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia
3.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299330

RESUMO

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer's disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-ß peptide (Aß). The morphological evaluation showed that Aß treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aß. Moreover, URB597 reduced both the increase of Rho protein activation in Aß-treated BV-2 cells and the Aß-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.


Assuntos
Amidoidrolases/antagonistas & inibidores , Benzamidas/farmacologia , Carbamatos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amidoidrolases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Citocinas/metabolismo , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Camundongos , Microglia/patologia , Alcamidas Poli-Insaturadas/metabolismo
4.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131730

RESUMO

Noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling has been implicated in endoderm morphogenesis. However, the underlying cellular and molecular mechanisms of this process are unclear. We found that, during convergence and extension (C&E) in zebrafish, gut endodermal cells are polarized mediolaterally, with GFP-Vangl2 enriched at the anterior edges. Endoderm cell polarity is lost and intercalation is impaired in the absence of glypican 4 (gpc4), a heparan-sulfate proteoglycan that promotes Wnt/PCP signaling, suggesting that this signaling is required for endodermal cell polarity. Live imaging revealed that endoderm C&E is accomplished by polarized cell protrusions and junction remodeling, which are impaired in gpc4-deficient endodermal cells. Furthermore, in the absence of gpc4, Cadherin 2 expression on the endodermal cell surface is increased as a result of impaired Rab5c-mediated endocytosis, which partially accounts for the endodermal defects in these mutants. These findings indicate that Gpc4 regulates endodermal planar cell polarity during endoderm C&E by influencing the localization of Cadherin 2. Thus, our study uncovers a new mechanism by which Gpc4 regulates planar cell polarity and reveals the role of Wnt/PCP signaling in endoderm morphogenesis.


Assuntos
Caderinas/metabolismo , Polaridade Celular/fisiologia , Endoderma/metabolismo , Glipicanas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas de Heparan Sulfato/metabolismo , Morfogênese , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP
5.
Biochem Biophys Res Commun ; 561: 172-179, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34023783

RESUMO

Loss of polarity protein Par3 promotes breast cancer tumorigenesis and metastasis. The underlying molecular mechanisms of Par3 down-regulation and related prognostic significance in breast cancer remain unclear. Here, we discovered that Par3 down-regulation was associated with shorter relapse-free survival in Luminal A subtype of breast cancer. Par3 knockdown promoted breast cancer cells migration and invasion. Importantly, we identified that transcription factor Sp1 bound to PARD3 promoter region and induced Par3 expression. Breast cancer patients with low Sp1 showed significantly worse RFS and low expression level of Par3. Par3 over-expression partially reversed Sp1 knockdown induced migration and invasion. Together, decreased Sp1 level mediates Par3 down-regulation, which correlated with poor prognosis of ER + breast cancer patients, via reduced binding with PARD3 promoter.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Recidiva Local de Neoplasia/patologia , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Feminino , Humanos , Gradação de Tumores , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Taxa de Sobrevida
6.
PLoS Biol ; 19(5): e3001250, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33983920

RESUMO

The repeated evolution of multicellularity led to a wide diversity of organisms, many of which are sessile, including land plants, many fungi, and colonial animals. Sessile organisms adhere to a surface for most of their lives, where they grow and compete for space. Despite the prevalence of surface-associated multicellularity, little is known about its evolutionary origin. Here, we introduce a novel theoretical approach, based on spatial lineage tracking of cells, to study this origin. We show that multicellularity can rapidly evolve from two widespread cellular properties: cell adhesion and the regulatory control of adhesion. By evolving adhesion, cells attach to a surface, where they spontaneously give rise to primitive cell collectives that differ in size, life span, and mode of propagation. Selection in favor of large collectives increases the fraction of adhesive cells until a surface becomes fully occupied. Through kin recognition, collectives then evolve a central-peripheral polarity in cell adhesion that supports a division of labor between cells and profoundly impacts growth. Despite this spatial organization, nascent collectives remain cryptic, lack well-defined boundaries, and would require experimental lineage tracking technologies for their identification. Our results suggest that cryptic multicellularity could readily evolve and originate well before multicellular individuals become morphologically evident.


Assuntos
Aderência Bacteriana/fisiologia , Adesão Celular/fisiologia , Animais , Bactérias/metabolismo , Evolução Biológica , Comunicação Celular/fisiologia , Polaridade Celular/fisiologia , Evolução Molecular , Fungos/metabolismo , Humanos
7.
Life Sci ; 277: 119567, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33965378

RESUMO

AIM: This study aimed to evaluate the effects of Asiatic acid (AA), a naturally occurring compound of pentacyclic triterpenoid, on the pathological processes of diabetic retinopathy (DR). METHODS: SD rats were induced to develop early DR by intraperitoneal injection of STZ (60 mg/kg). Four weeks after injection, the diabetic rats were orally administrated with 37.5 mg/kg or 75 mg/kg AA every day for four weeks. The integrity of blood-retinal barrier (BRB) was measured by Evans blue staining. The polarization of microglia was determined by real-time PCR, western blot, and ELISA assays. The inner BRB (iBRB) or outer BRB (oBRB) breakdown was induced in human retinal endothelial cells or APRE19 cells through co-culture with high glucose and LPS-stimulated microglia BV2 cells. The damage to the iBRB and oBRB was measured using transendothelial/transepithelial electrical resistance (TEER/TER) and FITC-conjugated dextran cell permeability assays. KEY FINDINGS: Results demonstrated that AA alleviated BRB breakdown, as evidenced by decreased protein expression of occludin, claudin-5, and ZO-1. Furthermore, AA treatment suppressed inflammation and M1 polarization, while it increased M2 polarization in the retina of DR rats. In vitro, the iBRB or oBRB breakdown was alleviated by AA. LPS-induced M1-polarization of BV2 cells under high glucose condition was also repressed through AA administration. Finally, we demonstrated that AA weakened the TLR4/MyD88/NF-κB p65 signaling pathway both in vivo and in vitro. SIGNIFICANCE: AA ameliorated early DR by regulating microglia polarization via the TLR4/MyD88/NF-κB p65 pathway. These data indicate that AA is a potential candidate for DR treatment.


Assuntos
Retinopatia Diabética/metabolismo , Triterpenos Pentacíclicos/farmacologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Polaridade Celular/fisiologia , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/tratamento farmacológico , Inflamação/patologia , Masculino , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Triterpenos Pentacíclicos/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo
8.
Exp Cell Res ; 404(1): 112630, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33971195

RESUMO

The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development-related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.


Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/citologia , Túbulos Renais Proximais/citologia , Túbulos Renais/metabolismo , Transporte Biológico , Linhagem Celular , Matriz Extracelular/metabolismo , Humanos , Rim/metabolismo , Sódio/metabolismo
9.
Cancer Sci ; 112(7): 2625-2641, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33931921

RESUMO

Bladder cancer (BLCA) remains the leading cause of cancer-related mortality among genitourinary malignancies worldwide. BLCA metastasis represents the primary reason for its poor prognosis. In this study, we report that decreased expression of partitioning defective 3 (Par3), a polarity protein (encoded by PARD3), is associated with tumor aggressive phenotypes and poor prognosis in BLCA patients. Consistently, ablation of Par3 promotes the metastasis and invasion of BLCA cells in vitro and in vivo. Further studies reveal that zinc finger protein Snail represses the expression of Par3 by binding to E2-box (CAGGTG) of PARD3 promoter-proximal. Inhibition of GSK-3ß promotes the expression and nuclear localization of Snail and then reduces the expression of Par3, resulting in the metastasis and invasion of BLCA cells. Moreover, we detected the interaction between Par3 (936-1356 aa) and ZO-1 (1372-1748 aa), which is involved in the maintenance of tight junction. Together, our results demonstrate that the GSK-3ß/Snail/Par3/ZO-1 axis regulates BLCA metastasis, and Snail is a major regulator for Par3 protein expression in BLCA.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Polaridade Celular/fisiologia , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Fosforilação , Prognóstico , Distribuição Aleatória , Fatores de Transcrição da Família Snail/genética , Junções Íntimas/fisiologia , Proteína da Zônula de Oclusão-1/metabolismo
10.
Ann Clin Lab Sci ; 51(2): 220-230, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33941562

RESUMO

OBJECTIVE: Progranulin (PGRN) has been confirmed to exhibit anti-inflammatory activity. Nevertheless, the mechanisms of PGRN in acute lung injury (ALI) remain uncertain. The aim of this study was to explore the role of PGRN in a lipopolysaccharide (LPS)-induced ALI model and in primary bone marrow-derived macrophages, as well as to investigate the underlying mechanism of PGRN. METHODS: Mice were treated with recombinant PGRN protein to detect the effect of PGRN on mouse ALI. Bronchial alveolar lavage fluid (BALF) was analyzed to quantify the inflammatory cytokines, and the lung wet-to-dry ratio was calculated to assess the degree of pulmonary edema. Histological staining was completed on the lung tissues. CCK-8 assay was used to measure cell viability. Western blotting and quantitative polymerase chain reaction were performed to study the transcriptomic profiles during the MAPK pathway. RESULTS: Recombinant human PGRN significantly suppressed cellular inflammatory response, increased lung permeability, and reduced the expression of inflammatory proteins in the BALF and serum, which further improved survival time. Also, PGRN inhibited the LPS-induced M1 marker gene expression and enhanced the M2 marker gene expression in vivo and in vitro. Further analysis revealed that PGRN suppresses the activity of the MAPK pathway in LPS-treated macrophages in a dose-dependent manner. CONCLUSION: PGRN exhibited anti-inflammatory activity and regulated macrophage polarization by suppressing the phosphorylation of the MAPK pathway.


Assuntos
Pneumonia/patologia , Progranulinas/farmacologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Polaridade Celular/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Progranulinas/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
J Neuroimmunol ; 355: 577567, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33887539

RESUMO

There is growing evidence that fine particulate matter (PM2.5) is a considerable risk factor for neurodegenerative diseases. Scorpion venom heat-resistant synthetic peptide (SVHRSP) plays a neuroprotective effect by promoting neurogenesis and neuron axon growth. In this study, SVHRSP inhibited the level of TLR4, autophagy and PM2.5-induced microglia M1 polarization, thereby promoting Phosphorylation of PI3K and AKT, inhibiting the expression of NF-κB. Moreover, SVHRSP suppressed the cytotoxic factors and increased the cytoprotective factor. This research demonstrates that SVHRSP relieves PM2.5-induced microglial polarization via TLR4-mediated autophagy activating PI3K/AKT/NF-κB signaling pathway, which provides new insights for the treatment of PM2.5-induced neurodegenerative diseases.


Assuntos
Microglia/metabolismo , NF-kappa B/metabolismo , Material Particulado/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Venenos de Escorpião/farmacologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Camundongos , Microglia/efeitos dos fármacos
12.
PLoS Biol ; 19(4): e3001183, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33891588

RESUMO

The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. Here, we report that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte.


Assuntos
Proteínas de Drosophila , Proteínas do Ovo/metabolismo , Oócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas do Ovo/fisiologia , Endocitose/fisiologia , Feminino , Oogênese/fisiologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/fisiologia , Vitelogênese/fisiologia , Vitelogeninas/fisiologia
13.
Aging (Albany NY) ; 13(7): 10415-10430, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33752173

RESUMO

Exosome-mediated intercellular communication is considered to be an effective mode for malignant cells to transform biological behaviors in stromal cells. However, the mechanisms by which exosomes modulate macrophages within tumor microenvironment remain largely unclear. In this study, we found that both adriamycin-resistant breast cancer (BCa) cells and the corresponding exosomes (A/exo) were capable of inducing macrophages M2 polarization, which promoted the mobility, proliferation, migration and invasion of BCa cells. Since exosomes deliver microRNAs to affect cellular functions in recipient cells, we confirmed that miR-222 was significantly enriched in A/exo and could be successfully transferred to macrophages. Increased miR-222 level was also detected in exosomes derived from plasma and tissues of chemoresistant patients. Moreover, exosomal miR-222 from A/exo polarized M2 macrophages by targeting PTEN and activating Akt signaling pathway, which promoted BCa cells progression in a feed back loop. Co-culture of adriamycin-resistant BCa cells with macrophages in which miR-222 was upregulated or treated with A/exo facilitated tumor growth in vivo. Collectively, our data demonstrated that chemoresistant BCa cells could remodel macrophages within tumor microenvironment by secreting exosomal miR-222, which directly targeted PTEN and caused Akt cascade activation and macrophages M2 polarization. Our findings may provide a foundation for a promising strategy of BCa treatment by targeting exosomes or exosomal miR-222.


Assuntos
Polaridade Celular/fisiologia , Exossomos/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Progressão da Doença , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Humanos , Células MCF-7 , Microambiente Tumoral
14.
FASEB J ; 35(4): e21508, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710706

RESUMO

Migrating tumor cells are characterized by a sustained front-rear asymmetry, with a front enriched in filamentous actin, which is induced by Rho small GTPase Rac. Regulation of Rac activity by its regulators should be required for effective motility. Here, we show that FilGAP, a GTPase-activating protein (GAP) for Rac, controls front-rear polarity and contributes to maintain effective tumor cell migration through the extracellular matrix (ECM). Overexpression of FilGAP in breast cancer cells induced polarized morphology and led to increased migration speed in collagen matrices, while depletion of FilGAP impaired the cell polarity and migration. FilGAP localizes to the cell front through its pleckstrin-homology (PH) domain in a phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent manner and appears to inactivate Rac at its site. We found that the affinity of PH domain to PIP3 is critically involved in the maintenance of cell polarity. Moreover, small GTPase ADP-ribosylation factor 6 (Arf6), which binds to the FilGAP PH domain, also regulates FilGAP-mediated cell polarity and migration of breast cancer cells. We propose that FilGAP regulates front-rear polarity through its PIP3 and Arf6 binding in tumor cell migration through the ECM.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Quinases Associadas a rho/metabolismo
15.
PLoS One ; 16(3): e0248293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33735291

RESUMO

The distribution of signaling molecules following mechanical or chemical stimulation of a cell defines cell polarization, with regions of high active Cdc42 at the front and low active Cdc42 at the rear. As reaction-diffusion phenomena between signaling molecules, such as Rho GTPases, define the gradient dynamics, we hypothesize that the cell shape influences the maintenance of the "front-to-back" cell polarization patterns. We investigated the influence of cell shape on the Cdc42 patterns using an established computational polarization model. Our simulation results showed that not only cell shape but also Cdc42 and Rho-related (in)activation parameter values affected the distribution of active Cdc42. Despite an initial Cdc42 gradient, the in silico results showed that the maximal Cdc42 concentration shifts in the opposite direction, a phenomenon we propose to call "reverse polarization". Additional in silico analyses indicated that "reverse polarization" only occurred in a particular parameter value space that resulted in a balance between inactivation and activation of Rho GTPases. Future work should focus on a mathematical description of the underpinnings of reverse polarization, in combination with experimental validation using, for example, dedicated FRET-probes to spatiotemporally track Rho GTPase patterns in migrating cells. In summary, the findings of this study enhance our understanding of the role of cell shape in intracellular signaling.


Assuntos
Polaridade Celular/fisiologia , Forma Celular/fisiologia , Modelos Biológicos , Proteína cdc42 de Ligação ao GTP/metabolismo , Simulação por Computador , Difusão , Transdução de Sinais/fisiologia
16.
Front Immunol ; 12: 637335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767704

RESUMO

Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI), increases morbidity and mortality, and is a significant risk factor for chronic kidney disease (CKD). Macrophage infiltration is a common feature after renal IRI, and infiltrating macrophages can be polarized into the following two distinct types: M1 macrophages, i.e., classically activated macrophages, which can not only inhibit infection but also accelerate renal injury, and M2 macrophages, i.e., alternatively activated macrophages, which have a repair phenotype that can promote wound healing and subsequent fibrosis. The role of TSC1, which is a negative regulator of mTOR signaling that regulates macrophage polarization in inflammation-linked diseases, has been well documented, but whether TSC1 contributes to macrophage polarization in the process of IRI is still unknown. Here, by using a mouse model of renal ischemia-reperfusion, we found that myeloid cell-specific TSC1 knockout mice (termed Lyz-TSC1 cKO mice) had higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production than wild-type (WT) mice during the early phase after renal ischemia-reperfusion. Furthermore, the Lyz-TSC1 cKO mice showed attenuated renal fibrosis during the repair phase of IRI with decreased levels of M2 markers on macrophages in the operated kidneys, which was further confirmed in a cell model of hypoxia-reoxygenation (H/R) in vitro. Mechanistically, by using RNA sequencing of sorted renal macrophages, we found that the expression of most M1-related genes was upregulated in the Lyz-TSC1 cKO group (Supplemental Table 1) during the early phase. However, C/EBPß and CD206 expression was decreased during the repair phase compared to in the WT group. Overall, our findings demonstrate that the expression of TSC1 in macrophages contributes to the whole process of IRI but serves as an inflammation suppressor during the early phase and a fibrosis promoter during the repair phase.


Assuntos
Injúria Renal Aguda/patologia , Macrófagos/fisiologia , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/patologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Polaridade Celular/fisiologia , Creatinina/sangue , Citocinas/metabolismo , Fibrose/patologia , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 1 do Complexo Esclerose Tuberosa/genética
17.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33656555

RESUMO

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Fungos/metabolismo , Fungos/fisiologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/fisiologia
18.
Exp Neurol ; 339: 113645, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33600815

RESUMO

Microglia are rapidly activated after acute ischemic stroke, and the polarization of microglial is associated with the prognosis of acute ischemic stroke. Lipoxin A4 (LXA4), an anti-inflammatory agent, has a protective effect against ischemic stroke. However, the role of LXA4 on the polarization of microglial after acute ischemic stroke remains undetermined. We hypothesized that LXA4 may exert the neuroprotective effect though regulating the polarization of microglial. In this study, clinical features of acute ischemic stroke were simulated using a rat model of model of middle cerebral artery occlusion (MCAO) in vivo and the BV2 microglia oxygen-glucose deprivation/reoxygenation model (OGD/R) in vitro. The protective effects of LXA4 on cerebral ischemia-reperfusion injury were determined using TTC staining, HE staining, and TUNEL staining. The expression of targeted genes was assayed using quantitative real-time PCR (qRT-PCR), immunofluorescence, and western blot to investigated the regulation of LXA4 on microglia polarization after acute ischemic stroke. We found that LXA4 exerted protective effects on focal cerebral ischemia-reperfusion injury and reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α. Furthermore, LXA4 inhibited the expression of Notch-1, Hes1, iNOS and CD32 all of which are associated with the differentiation into M1 microglia. By contrast, LXA4 upregulated the expression of Hes5, Arg-1 and CD206 all of which are associated with M2 phenotype in microglia. In addition, blocking the Notch signaling pathway with the inhibitor DAPT significantly mitigated the effect of LXA4 on microglia differentiation. These data suggest that LXA4 may regulate the polarization of microglia after cerebral ischemia-reperfusion injury through the Notch signaling pathway.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Polaridade Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Receptor Notch1/antagonistas & inibidores , Receptores de Lipoxinas/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Isquemia Encefálica/metabolismo , Linhagem Celular , Polaridade Celular/fisiologia , Masculino , Microglia/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch1/biossíntese , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
19.
Mol Biol Cell ; 32(7): 590-604, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33566676

RESUMO

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.


Assuntos
Polaridade Celular/fisiologia , Adesões Focais/metabolismo , Cinesina/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Centrômero/metabolismo , Humanos , Cinesina/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo
20.
Mol Biol Cell ; 32(8): 690-702, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33596087

RESUMO

Par1b/MARK2 is a Ser/Thr kinase with pleiotropic effects that participates in the generation of apico-basal polarity in Caenorhabditis elegans. It is phosphorylated by atypical PKC(ι/λ) in Thr595 and inhibited. Because previous work showed a decrease in atypical protein kinase C (aPKC) activity under proinflammatory conditions, we analyzed the hypothesis that the resulting decrease in Thr595-MARK2 with increased kinase activity may also participate in innate immunity. We confirmed that pT595-MARK2 was decreased under inflammatory stimulation. The increase in MARK2 activity was verified by Par3 delocalization and rescue with a specific inhibitor. MARK2 overexpression significantly enhanced the transcriptional activity of NF-kB for a subset of transcripts. It also resulted in phosphorylation of a single band (∼Mr 80,000) coimmunoprecipitating with RelA, identified as Med17. In vitro phosphorylation showed direct phosphorylation of Med17 in Ser152 by recombinant MARK2. Expression of S152D-Med17 mimicked the effect of MARK2 activation on downstream transcriptional regulation, which was antagonized by S152A-Med17. The decrease in pThr595 phosphorylation was validated in aPKC-deficient mouse jejunal mucosae. The transcriptional effects were confirmed in transcriptome analysis and transcript enrichment determinations in cells expressing S152D-Med17. We conclude that theMARK2-Med17 axis represents a novel form of cross-talk between polarity signaling and transcriptional regulation including, but not restricted to, innate immunity responses.


Assuntos
Polaridade Celular/fisiologia , Complexo Mediador/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/fisiologia , Complexo Mediador/fisiologia , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais
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