RESUMO
BACKGROUND: Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function. METHODS: Thymocytes from mice were treated in vitro with Sema3A for 30min. Scanning electron microscopy was performed to assess cell morphology. Atomic force microscopy was performed to further evaluate cell morphology, membrane roughness, and elasticity. Flow cytometry and/or fluorescence microscopy were performed to assess the F-actin cytoskeleton and ROCK2. Cell adhesion to a bovine serum albumin substrate and transwell migration assays were used to assess cell migration. RESULTS: Sema3A induced filopodia formation in thymocytes, increased membrane stiffness and roughness, and caused a cortical distribution of the cytoskeleton without changes in F-actin levels. Sema3A-treated thymocytes showed reduced substrate adhesion and migratory ability, without changes in cell viability. In addition, Sema3A was able to down-regulate ROCK2. CONCLUSIONS: Sema3A promotes cytoskeletal rearrangement, leading to membrane modifications, including increased stiffness and roughness. This effect in turn affects the adhesion and migration of thymocytes, possibly due to a reduction in ROCK2 expression. GENERAL SIGNIFICANCE: Sema3A treatment impairs thymocyte migration due to biomechanical alterations in cell membranes.
Assuntos
Fenômenos Biomecânicos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Semaforina-3A/farmacologia , Timócitos/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Timócitos/metabolismo , Timócitos/ultraestrutura , Quinases Associadas a rho/metabolismoRESUMO
Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+ /Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+ /Vim- cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+ /Krt+ /ΔNp63α+ /Pax-6low /α6 integrin+ phenotype. J. Cell. Physiol. 232: 818-830, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Movimento Celular , Células Epiteliais/citologia , Epitélio Corneano/citologia , Vimentina/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Integrina alfa6/metabolismo , Queratinas/metabolismo , Mitomicina/farmacologia , Plectina/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Coelhos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.
Assuntos
Comunicação Celular/genética , Redes Reguladoras de Genes , Heme Oxigenase-1/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Pseudópodes/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Cristalografia por Raios X , Meios de Cultivo Condicionados/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Ligação Proteica/efeitos dos fármacos , Proteômica , Pseudópodes/efeitos dos fármacos , Análise de Sequência de RNA , Espectrometria de Massas em Tandem , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
UNLABELLED: Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. SIGNIFICANCE STATEMENT: Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss.
Assuntos
Actinas/metabolismo , Axônios/fisiologia , Endocitose/fisiologia , Galectina 1/metabolismo , Peróxido de Hidrogênio/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Galectina 1/genética , Galectina 1/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Ratos , Semaforina-3A/farmacologia , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Having demonstrated that the bradykinin B2 receptor (B2R) is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK) on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L) for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L) incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L) for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8), modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies.
Assuntos
Bradicinina/farmacologia , Movimento Celular/efeitos dos fármacos , Trofoblastos/fisiologia , Bradicinina/análogos & derivados , Bradicinina/antagonistas & inibidores , Antagonistas de Receptor B1 da Bradicinina , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Receptor B1 da Bradicinina/efeitos dos fármacos , Receptor B2 da Bradicinina/fisiologia , Trofoblastos/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability. MATERIAL AND METHODS: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound-healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active ß1 integrin. Rac activation was evaluated by means of a pull-down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann-Whitney U-test. RESULTS: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin-treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of ß1 integrin activation. During cell spreading, simvastatin diminished Rac activation. CONCLUSION: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing.
Assuntos
Anticolesterolemiantes/farmacologia , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Sinvastatina/farmacologia , Actinas/análise , Adolescente , Adulto , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Ativação Enzimática , Feminino , Imunofluorescência , Gengiva/citologia , Humanos , Integrina beta1/análise , Masculino , Pseudópodes/efeitos dos fármacos , Vinculina/análise , Adulto Jovem , Proteínas rac de Ligação ao GTP/análiseRESUMO
We investigated the effects of citric acid (CA) on cultured human osteoblastic (HOB) cells by evaluating cell adhesion, proliferation, and cytotoxicity. (3)H-Thymidine-labeled HOB cells were incubated in culture medium supplemented or not with 4%, 6%, 8%, or 10% CA for 1 minute. After incubation, cell morphology was evaluated by Nomarski interferential light microscopy, cell proliferation was accessed by measurements of (3)H-thymidine associated to the cells, and cell lysis was monitored by measuring the amount of (3)H-thymidine released by cells. We observed that most of the CA-treated cells presented numerous atypical vacuoles, and such cells were also highly polymorphic, exhibiting round-shaped cells. Nonetheless, CA at all concentrations assayed did not yield cytotoxicity as measured by (3)H-containing DNA release, although significant decrease in cell proliferation was observed (P > .05). Furthermore, cells which were treated with CA at the lowest concentration assayed (4%) restored normal proliferation rates 3 days after treatment.
Assuntos
Ácido Cítrico/farmacologia , Osteoblastos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Ácido Cítrico/toxicidade , Meios de Cultura , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Interferência , Pseudópodes/efeitos dos fármacos , Compostos Radiofarmacêuticos , Timidina , Fatores de Tempo , Trítio , Vacúolos/efeitos dos fármacosRESUMO
Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 microM), an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 microM, resveratrol at 5 microM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 microM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 microM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominant-negative Rac retain filopodia response to 50 microM resveratrol. Lamellipodia response to 5 microM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 microM) signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.
Assuntos
Actinas/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Citoesqueleto/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios , Neoplasias Hormônio-Dependentes/patologia , Estilbenos/farmacologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/ultraestrutura , Movimento Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Feminino , Genes Dominantes , Humanos , Invasividade Neoplásica , Neoplasias Hormônio-Dependentes/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Resveratrol , Transfecção , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genéticaRESUMO
The involvement of actin filaments from the host cell on the process of invasion of trypomastigote forms of Trypanosma cruzi was analyzed in seven different cell lines. Prior incubation of all cell lines with cytochalasin D, under conditions which interfere with actin filaments, markedly inhibited parasite internalization and increased parasite attachment. Attached parasites were readily ingested following washing of the drug-treated cells. Cytochalasin treatment interfered with the distribution of actin filaments of the host cell as evaluated by visualization of the filaments using confocal laser scanning microscopy of cells incubated in the presence of FITC-phalloidin. Concentration of actin filaments could be observed in most, but not all, parasites in the process of internalization. We also treated LLCMK 2 and macrophage cells with Jasplakinolide, a drug that stabilizes actin filaments, before interaction with the trypomastigote forms. This drug partially inhibits parasite invasion into the cells. Prior incubation of the host cells in the presence of colchicine, which interfere with microtubules, also inhibited parasite internalization into the cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Doença de Chagas/parasitologia , Depsipeptídeos , Interações Hospedeiro-Parasita/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Antineoplásicos/farmacologia , Chlorocebus aethiops , Colchicina/farmacologia , Citocalasina D/farmacologia , Imunofluorescência , Interações Hospedeiro-Parasita/efeitos dos fármacos , Macrófagos/parasitologia , Microscopia Eletrônica de Varredura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Peptídeos Cíclicos/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/ultraestrutura , Células VeroRESUMO
In this study we show that insulin-like growth factor (IGF)-I selectively promotes survival and differentiation of amacrine neurons. In cultures lacking this factor, an initial degeneration pathway, selectively affecting amacrine neurons, led to no lamellipodia development and little axon outgrowth. Cell lysis initially affected 50% of amacrine neurons; those remaining underwent apoptosis leading to the death of approximately 95% of them by day 10. Apoptosis was preceded by a marked increase in c-Jun expression. Addition of IGF-I or high concentrations (over 1 microM) of either insulin or IGF-II to the cultures prevented the degeneration of amacrine neurons, stimulated their neurite outgrowth, increased phospho-Akt expression and decreased c-Jun expression. The high insulin and IGF-II concentrations required to protect amacrine cells suggest that these neurons depend on IGF-I for their survival, IGF-II and insulin probably acting through IGF-I receptors to mimic IGF-I effects. Inhibition of phosphatidylinositol-3 kinase (PI 3-kinase) with wortmannin blocked insulin-mediated survival. Wortmannin addition had similar effects to IGF-I deprivation: it prevented neurite outgrowth, increased c-Jun expression and induced apoptosis. These results suggest that IGF-I is essential for the survival and differentiation of amacrine neurons, and activation of PI 3-kinase is involved in the intracellular signaling pathways mediating these effects.