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1.
Aquat Toxicol ; 231: 105740, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33440272

RESUMO

Ensuring that oocytes are fertilized by a single sperm during broadcast spawning is crucial for the fertilization success of many marine invertebrates. Although the adverse impacts of ocean acidification (OA) on various marine species have been revealed in recent years, its impact on polyspermy and the underlying mechanisms involved remain largely unknown. Therefore, in the present study, the effect of OA on polyspermy risk was assessed in a broadcast spawning bivalve, Tegillarca granosa. In addition, the impacts of OA on the two polyspermy blocking processes, the fast block (membrane depolarization) and the permanent block (cortical reaction), were investigated. The results show that the exposure of oocytes to two future OA scenarios (pH 7.8 and pH 7.4) leads to significant increases in polyspermy risk, about 1.70 and 2.38 times higher than the control, respectively. The maximum change in the membrane potential during oocyte membrane depolarization markedly decreased to 15.79 % (pH 7.8) and 34.06 % (pH 7.4) of the control value. Moreover, the duration of oocyte membrane depolarization was significantly reduced to approximately 63.38 % (pH 7.8) and 21.91 % (pH 7.4) of the control. In addition, cortical granule exocytosis, as well as microfilament migration, were significantly arrested by OA treatment. Exposure to future OA scenarios also led to significant reductions in the ATP and Ca2+ content of the oocytes, which may explain the hampered polyspermy blocking. Overall, the present study suggests that OA may significantly increase polyspermy risk in T. granosa by inhibiting membrane depolarization and arresting cortical granule exocytosis.


Assuntos
Ácidos/química , Bivalves/fisiologia , Grânulos Citoplasmáticos/metabolismo , Exocitose , Potenciais da Membrana/fisiologia , Oceanos e Mares , Espermatozoides/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Feminino , Fertilização/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Masculino , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Poluentes Químicos da Água/toxicidade
2.
PLoS One ; 15(9): e0238572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32898143

RESUMO

Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multi-kinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Rabdomiossarcoma/tratamento farmacológico , Tiazolidinas/farmacologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia
3.
Int J Nanomedicine ; 15: 5061-5072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764936

RESUMO

Purpose: Zirconia is one of the most promising implant materials due to its favorable physical, mechanical and biological properties. However, until now, we know little about the mechanism of osseointegration on zirconia. The purpose of this study is to evaluate the effect of Syndecan (Sdc) on osteoblastic cell (MC3T3-E1) adhesion and proliferation onto zirconia materials. Materials and Methods: The mirror-polished disks 15 mm in diameter and 1.5 mm in thick of commercial pure titanium (CpTi), 3mol% yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) and nano-zirconia (NanoZr) are used in this study. MC3T3-E1 cells were seeded onto specimen surfaces and subjected to RNA interference (RNAi) for Syndecan-1, Syndecan-2, Syndecan-3, and Syndecan-4. At 48h post-transfection, the cell morphology, actin cytoskeleton, and focal adhesion were observed using scanning electron microscopy or laser scanning confocal fluorescence microscopy. At 24h and 48h post-transfection, cell counting kit-8 (CCK-8) assay was used to investigate cell proliferation. Results: The cell morphology of MC3T3-E1 cells on CpTi, 3Y-TZP, and NanoZr changed into abnormal shape after gene silencing of Syndecan. Among the Syndecan family, Sdc-2 is responsible for NanoZr-specific morphology regulation, via maintenance of cytoskeletal conformation without affecting cellular attachment. According to CCK-8 assay, Sdc-2 affects the osteoblastic cell proliferation onto NanoZr. Conclusion: Within the limitation of this study, we suggest that Syndecan affects osteoblastic cell adhesion on CpTi, 3Y-TZP, and NanoZr. Sdc-2 might be an important heparin-sensitive cell membrane regulator in osteoblastic cell adhesion, specifically on NanoZr, through the organization of actin cytoskeleton and affects osteoblastic cell proliferation.


Assuntos
Osseointegração/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Sindecanas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Adesão Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Microscopia Eletrônica de Varredura , Osseointegração/genética , Propriedades de Superfície , Sindecana-2/genética , Sindecana-2/metabolismo , Sindecanas/genética , Titânio/química , Ítrio/química , Zircônio/química
4.
Proc Natl Acad Sci U S A ; 117(29): 17399-17408, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641513

RESUMO

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.


Assuntos
Fenômenos Fisiológicos Celulares , Forma Celular/fisiologia , Citoesqueleto/fisiologia , Células Vegetais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas , Arabidopsis/metabolismo , Citocalasina D/farmacologia , Microtúbulos/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/ultraestrutura , Protoplastos
5.
Mol Cell Biochem ; 468(1-2): 129-142, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32185674

RESUMO

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Cirrose Hepática/metabolismo , Substâncias Protetoras/farmacologia , Silimarina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Cromatografia Gasosa , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Dimetil Sulfóxido/toxicidade , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Espectrometria de Massas , Triglicerídeos/metabolismo
6.
Med Sci Monit ; 26: e921626, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32034900

RESUMO

BACKGROUND The stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved. MATERIAL AND METHODS A compressive force of 25 g/m² was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin alpha5ß1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin alpha5ß1 inhibitor (Ac-PHSCN-NH 2, ATN-161). RESULTS Mechanical force increased the expression of integrin alpha5ß1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin alpha5ß1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited. CONCLUSIONS The integrin alpha5ß1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin alpha5ß1 pathway.


Assuntos
Citoesqueleto de Actina/metabolismo , Fibroblastos/citologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Gengiva/citologia , Integrina alfa5beta1/metabolismo , Mecanotransdução Celular , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Adolescente , Células Cultivadas , Criança , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Mecanotransdução Celular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Estresse Mecânico
7.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G504-G517, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31928221

RESUMO

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Cirrose Hepática Experimental/prevenção & controle , Masculino , Camundongos Endogâmicos BALB C , Ácidos Nipecóticos/farmacologia , Proteínas Nucleares/genética , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/genética , Regulação para Cima
8.
Am J Respir Cell Mol Biol ; 62(5): 645-656, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31913659

RESUMO

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Pulmão/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Polimerização , Proteínas Serina-Treonina Quinases/metabolismo , Acetilcolina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Adulto , Biocatálise/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Feminino , Histamina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serotonina/farmacologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
9.
Nanoscale ; 12(6): 3731-3749, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31993609

RESUMO

The actin cytoskeleton is required for the maintenance of the cell shape and viability of bacteria. It remains unknown to which extent nanoparticles (NPs) can orchestrate the mechanical instability by disrupting the cytoskeletal network in bacterial cells. Our work demonstrates that Au-Ag NPs disrupt the bacterial actin cytoskeleton specifically, fluidize the inner membrane and lead to killing of bacterial cells. In this study, we have tried to emphasize on the key parameters important for NP-cell interactions and found that the shape, specific elemental surface localization and enhanced electrostatic interaction developed due to the acquired partial positive charge by silver atoms in the aggregated NPs are some of the major factors contributing towards better NP interactions and subsequent cell death. In vivo studies in bacterial cells showed that the NPs exerted a mild perturbation of the membrane potential. However, its most striking effect was on the actin cytoskeleton MreB resulting in morphological changes in the bacterial cell shape from rods to predominantly spheres. Exposure to NPs resulted in the delocalization of MreB patches from the membrane but not the tubulin homologue FtsZ. Concomitant with the redistribution of MreB localization, a dramatic increase of membrane fluid regions was observed. Our studies reveal for the first time that Au-Ag NPs can mediate bacterial killing and disrupt the actin cytoskeletal functions in bacteria.


Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli , Ouro/farmacologia , Nanopartículas Metálicas/química , Prata/farmacologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Antibacterianos/química , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Ouro/química , Prata/química
10.
Exp Cell Res ; 388(2): 111808, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891685

RESUMO

Recent studies have shown that serum secretory phospholipase A2 group IB (sPLA2-IB) is associated with proteinuric kidney diseases and plays a pivotal role in podocyte injury via its natural receptor. Arachidonic acid (AA), as a major metabolite of sPLA2-IB, regulates the actin bungling remodeling and contributes to the podocyte injury. However, the underlying mechanism of AA in the regulation of podocyte actin remodeling and human podocyte injury is unclear. Here, we reported that AA induced F-actin cytoskeletal ring formation and promoted protein kinase A (PKA), nephrin and c-Abl phosphorylation. Moreover, AA promoted c-Abl translocation from the nucleus to the cytoplasm and increased the recruitment of c-Abl to p-nephrin by the interaction between them. H89 (PKA inhibitor) provided protection against AA-induced F-actin bunching remodeling, down-regulated nephrin phosphorylation, and suppressed the c-Abl translocation and activation. STI571 (c-Abl inhibitor) also improved the AA associated F-actin bunching remodeling. In addition, H89 and STI571 both alleviated apoptosis and adhesion damage of podocyte. These results indicate that an excess of AA treatment is detrimental to the podocyte actin cytoskeleton and promotes podocyte injury due to the activation of PKA-c-Abl signaling.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Podócitos/patologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Células Cultivadas , Humanos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-abl/genética
11.
Sci Adv ; 6(1): eaay2432, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911947

RESUMO

The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Forminas/imunologia , Proteínas de Membrana/genética , Proteína-Tirosina Quinase ZAP-70/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/imunologia , Actinas/antagonistas & inibidores , Actinas/química , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Forminas/genética , Forminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/metabolismo , Células Jurkat/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos
12.
Protoplasma ; 257(1): 89-101, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31342152

RESUMO

The aim of the current study was to examine the effect of different exogenous putrescine concentrations (200, 400, 600, and 800 µM) on the tea pollen performance. It was shown that putrescine has a dose-dependent effect on pollen performance. Results exhibited that pollen germination and tube elongation were induced by 200 and 400 µM putrescine treatment, especially, 400 µM putrescine-enhanced pollen performance. However, pollen performance was inhibited by higher concentrations of putrescine. Putrescine concentrations above 400 µM changed the actin filament distribution in pollen tubes by affecting the distribution of sucrose synthase enzyme. Alterations of the distribution on sucrose synthase enzyme also caused the alterations in the dispersion of cellulose and callose in the cell wall, and morphological alterations such as balloon-shaped and snake-shaped pollen tube tip accompanied them. Moreover, putrescine concentrations above 400 µM caused a decrease of ROS level in apex and led to chromatin condensation of the generative nucleus. In conclusion, exogenous putrescine application can be used as a pollen performance enhancer at low concentrations while the high concentrations cause adverse effects reducing fertilization success.


Assuntos
Actinas/metabolismo , Camellia sinensis/citologia , Camellia sinensis/crescimento & desenvolvimento , Parede Celular/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Putrescina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Camellia sinensis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Parede Celular/efeitos dos fármacos , Tubo Polínico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
13.
Biochem Biophys Res Commun ; 522(2): 335-341, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31761327

RESUMO

Chronic wounds, such as pressure ulcers, are a common complication of impaired peripheral circulation, such as in advanced diabetes. Factors secreted by mesenchymal stromal cells (MSCs) have been shown to enhance wound healing in vitro and in vivo. However, there is little understanding of the impact of the chronic wound environment, namely the limited supply of nutrients and oxygen, on the ability of wound cells to respond to MSCs. In this study, we first established the effects of hypoxia (1% O2) and low serum (1% serum) concentration on the proliferation and migration of keratinocytes. We found that hypoxia and low serum significantly slowed down these processes. Next, we found that supplementation with human MSC-concentrated conditioned media (hMSC-CM) enhanced both cell migration and proliferation in the presence of hypoxia and low serum. Furthermore, low serum and hypoxia decreased cell spreading and F-actin expression, which was reversed in the presence of hMSC-CM. Several wound healing mediators were identified in hMSC-CM, including IL-5, IL-6, IL-8, IL-9, IP-10, MCP-1, FGF-2, and VEGF. This study suggests that the concentrated secretome of human MSCs can reverse the inhibitory effect of hypoxia and low serum on keratinocyte proliferation and migration. This phenomenon may contribute to the beneficial effects of hMSC-CM on wound healing in vivo.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/metabolismo , Soro/metabolismo , Cicatrização/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia
14.
Nanoscale ; 12(1): 115-129, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31773115

RESUMO

Real-time imaging of single virus particles allows the visualization of subtle dynamic events of virus-host interaction. During the human immunodeficiency virus (HIV) infection of resting CD4 T lymphocytes, overcoming cortical actin restriction is an essential step, but the dynamic process and mechanism remain to be characterized. Herein, by using quantum dot (QD) encapsulated fluorescent viral particles and single-virus tracking, we explored detailed scenarios of HIV dynamic entry and crossing the cortical actin barrier. The fine-scale temporal and spatial processes of single HIV virion interaction with the cortical actin were studied in depth during virus entry via plasma membrane fusion. Individual HIV virions modulate the subtle rearrangement of the cortical actin barrier to open a door to facilitate viral entry. The actin-binding protein, α-actinin, was found to be critical for actin dynamics during HIV entry. An α-actinin-derived peptide, actin-binding site 1 peptide (ABS1p), was developed to block HIV infection. Our findings reveal an α-actinin-mediated dynamic cortical actin rearrangement for HIV entry, and identify an antiviral target as well as a corresponding peptide inhibitor based on HIV interaction with the actin cytoskeleton.


Assuntos
Actinas/metabolismo , HIV-1/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Corantes Fluorescentes/química , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/química , Humanos , Microscopia de Fluorescência , Peptídeos/antagonistas & inibidores , Peptídeos/síntese química , Peptídeos/metabolismo , Pontos Quânticos/química , Imagem com Lapso de Tempo , Vírion/química , Vírion/fisiologia , Internalização do Vírus/efeitos dos fármacos
15.
Am J Physiol Lung Cell Mol Physiol ; 318(2): L276-L286, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774302

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterized by a profound remodeling of the collagen in the extracellular matrix (ECM), where the fibers become both denser and more highly aligned. However, it is unknown how this reconfiguration of the collagen matrix affects disease progression. Here, we investigate the role of specific alterations in collagen fiber organization on cell migration dynamics by using biomimetic image-based collagen scaffolds representing normal and fibrotic lung, where the designs are derived directly from high-resolution second harmonic generation microscopy images. The scaffolds are fabricated by multiphoton-excited (MPE) polymerization, where the process is akin to three-dimensional printing, except that it is performed at much greater resolution (∼0.5 microns) and with collagen and collagen analogs. These scaffolds were seeded with early passaged primary human normal and IPF fibroblasts to enable the decoupling of the effect of cell-intrinsic characteristics (normal vs. IPF) versus ECM structure (normal vs. IPF) on migration dynamics. We found that the highly aligned IPF collagen structure promoted enhanced cell elongation and F-actin alignment along with increased cell migration speed and straightness relative to the normal tissues. Collectively, the data are consistent with an enhanced contact guidance mechanism on the aligned IPF matrix. Although cell intrinsic effects were observed, the aligned collagen matrix morphology had a larger effect on these metrics. Importantly, these biomimetic models of the lung cannot be synthesized by conventional fabrication methods. We suggest that the MPE image-based fabrication method will enable additional hypothesis-based testing studies of cell-matrix interactions in the context of tissue fibrosis.


Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/diagnóstico por imagem , Fibrose Pulmonar Idiopática/patologia , Processamento de Imagem Assistida por Computador , Tecidos Suporte/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno/farmacologia , Matriz Extracelular/efeitos dos fármacos , Humanos , Fótons , Polimerização , Ratos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo
16.
Tissue Eng Regen Med ; 16(6): 573-583, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31824820

RESUMO

Background: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process. Methods: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm2. After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of ß-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively. Results: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished. Conclusion: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Citocalasina D/farmacologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Osteocalcina/genética , Osteocalcina/metabolismo , beta Catenina/metabolismo
17.
PLoS One ; 14(12): e0226068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825988

RESUMO

Every year, more than 250,000 invasive candidiasis infections are reported with 50,000 deaths worldwide. The limited number of antifungal agents necessitates the need for alternative antifungals with potential novel targets. The 2-benzylidenebenzofuran-3-(2H)-ones have become an attractive scaffold for antifungal drug design. This study aimed to determine the antifungal activity of a synthetic aurone compound and characterize its mode of action. Using the broth microdilution method, aurone SH1009 exhibited inhibition against C. albicans, including resistant isolates, as well as C. glabrata, and C. tropicalis with IC50 values of 4-29 µM. Cytotoxicity assays using human THP-1, HepG2, and A549 human cell lines showed selective toxicity toward fungal cells. The mode of action for SH1009 was characterized using chemical-genetic interaction via haploinsufficiency (HIP) and homozygous (HOP) profiling of a uniquely barcoded Saccharomyces cerevisiae mutant collection. Approximately 5300 mutants were competitively treated with SH1009 followed by DNA extraction, amplification of unique barcodes, and quantification of each mutant using multiplexed next-generation sequencing. Barcode post-sequencing analysis revealed 238 sensitive and resistant mutants that significantly (FDR P values ≤ 0.05) responded to aurone SH1009. The enrichment analysis of KEGG pathways and gene ontology demonstrated the cell cycle pathway as the most significantly enriched pathway along with DNA replication, cell division, actin cytoskeleton organization, and endocytosis. Phenotypic studies of these significantly enriched responses were validated in C. albicans. Flow cytometric analysis of SH1009-treated C. albicans revealed a significant accumulation of cells in G1 phase, indicating cell cycle arrest. Fluorescence microscopy detected abnormally interrupted actin dynamics, resulting in enlarged, unbudded cells. RT-qPCR confirmed the effects of SH1009 in differentially expressed cell cycle, actin polymerization, and signal transduction genes. These findings indicate the target of SH1009 as a cell cycle-dependent organization of the actin cytoskeleton, suggesting a novel mode of action of the aurone compound as an antifungal inhibitor.


Assuntos
Antifúngicos/farmacologia , Benzofuranos/farmacologia , Candida albicans/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Antifúngicos/química , Benzofuranos/química , Candida albicans/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Farmacorresistência Fúngica/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Ontologia Genética , Humanos , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
18.
Nanotheranostics ; 3(4): 342-355, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31723548

RESUMO

Specific targeting of inflammation remains a challenge in many pathologies, because of the necessary balance between host tolerance and efficacious inflammation resolution. Here, we discovered a short, 4-mer peptide which possesses antagonist properties towards CC chemokine receptor 2 (CCR2), but only when displayed on the surface of lipid nanoparticles. According to BLAST analysis, this peptide motif is a common repeating fragment in a number of proteins of the CC chemokine family, which are key players in the inflammatory response. In this study, self-assembled, peptide-conjugated nanoparticles (CCTV) exhibited typical properties of CCR2 antagonism, including affinity to the CCR2 receptor, inhibition of chemotactic migration of primary monocytes, and prevention from CC chemokine ligand 2 (CCL2)-induced actin polymerization. Furthermore, CCTV ameliorated NFkB activation and downregulated the secondary, but not the primary, inflammatory response in cultured macrophages. When conjugated with gadolinium or europium cryptates, CCTV enabled targeted imaging (via magnetic resonance imaging and time-resolved fluorescence) of atherosclerosis, a chronic inflammatory condition in which the CCL2/CCR2 axis is highly dysfunctional. CCTV targeted CCR2hiLy6Chi inflammatory monocytes in blood and the atherosclerotic plaque, resulting in cell-specific transcriptional downregulation of key inflammatory genes. Finally, CCTV generated pronounced inflammasome inactivation, likely mediated through reactive oxygen species scavenging and downregulation of NLRP3. In summary, our work demonstrates for the first time that a short peptide fragment presented on a nanoparticle surface exhibit potent receptor-targeted antagonist effects, which are not seen with the peptide alone. Unlike commonly used cargo-carrying, vector-directed drug delivery vehicles, CCTV nanoparticles may act as therapeutics/theranostics themselves, particularly in inflammatory conditions with CCL2/CCR2 pathogenesis, including cardiovascular disease and cancer.


Assuntos
Imagem por Ressonância Magnética , Nanopartículas/química , Peptídeos/química , Receptores CCR2/antagonistas & inibidores , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Aterosclerose/diagnóstico por imagem , Aterosclerose/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Meios de Contraste/química , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamassomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Receptores CCR2/metabolismo
19.
FASEB J ; 33(12): 13476-13491, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570001

RESUMO

Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor ß (ERR-ß) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-ß protein products: ERR-ß short form (ERR-ßsf), ERR-ß2, and ERR-ß exon 10 deleted. Our prior studies have shown the ERR-ß2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-ßsf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-ß2 isoform in GBM. We show that the ERR-ß2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-ß2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-ß agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-ß agonist shifts isoform expression in favor of ERR-ß2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.


Assuntos
Neoplasias Encefálicas/prevenção & controle , Movimento Celular , Glioblastoma/prevenção & controle , Hidrazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Estrogênicos/metabolismo , Tiazóis/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Ciclo Celular , Proliferação de Células , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Isoformas de Proteínas , Receptores Estrogênicos/química , Receptores Estrogênicos/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
20.
Cell Physiol Biochem ; 53(4): 713-730, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31599538

RESUMO

BACKGROUND/AIMS: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR). METHODS: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy. RESULTS: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc). CONCLUSION: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR.


Assuntos
Angiotensina II/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Losartan/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Adesões Focais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vinculina/metabolismo
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