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1.
Histochem Cell Biol ; 144(3): 223-35, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25990946

RESUMO

To evaluate the role of actin cytoskeleton in the regulation of NF-κB transcription factor, we analyzed its involvement in the intracellular transport and nuclear translocation of the NF-κB RelA/p65 subunit in A431 epithelial cells stimulated with fibronectin and EGF. Live cell imaging and confocal microscopy showed that EGF activated the movement of RelA/p65 in the cytoplasm. Upon cell adhesion to fibronectin, RelA/p65 concentrated onto stress fibers, and EGF stimulated its subsequent allocation to membrane ruffles, newly organized stress fibers, and discrete cytoplasmic actin-rich patches. These patches also contained α-actinin-1 and α-actinin-4, vinculin, paxillin, α-tubulin, and PI3-kinase. Cytochalasin D treatment resulted in RelA/p65 redistribution to actin-containing aggregates, with the number of cells with RelA/p65-containing clusters in the cytoplasm increasing under the effect of EGF. Furthermore, EGF proved to induce RelA/p65 accumulation in the nucleus after cell pretreatment with actin-stabilizing and actin-destabilizing agents, which was accompanied by changes in its DNA-binding activity after either EGF stimulation or cytochalasin D treatment. Thus, EGF treatment of A431 cells results in simultaneous nuclear RelA/p65 translocation and cytoplasmic redistribution, with part of RelA/p65 pool forming a very tight association with actin-rich structures. Apparently, nuclear transport is independent on drug stabilization or destabilization of the actin.


Assuntos
Actinas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fibronectinas/metabolismo , Fator de Transcrição RelA/metabolismo , Actinas/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , NF-kappa B/biossíntese , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fator de Transcrição RelA/efeitos dos fármacos , Translocação Genética/efeitos dos fármacos
2.
Cell Biol Int ; 37(4): 387-96, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23408724

RESUMO

NF-kB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-kB activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-kB proteins RelA/p65 and p50 in relation to the inhibitor IkB-a, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-kB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NFkB was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IkB-a. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IkB-a in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IkB-a in the membrane. Furthermore, EGF stimulation for 30 min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-kB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IkB-a. Moreover, we discovered the existence of a dynamic, IkB-a-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-kB stimulation.


Assuntos
Fator de Transcrição RelA/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/fisiologia , Fibronectinas/metabolismo , Humanos , Transporte Proteico
3.
Cell Biol Int ; 35(12): 1189-93, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22070413

RESUMO

The prospect of developing transport systems using histones for site-specific delivery of therapeutic agents that have poor penetration characteristics through cellular membranes and tissue barriers has been investigated. Histones immobilized on microspheres can also be used to modify surfaces intended for cell cultivation, facilitating adhesion, proliferation and network formation by interactions of cells through contacts with several microspheres. They can be applied to three-dimensional pore matrices that are designed for producing tissue-like structures in vitro.


Assuntos
Estruturas Celulares/fisiologia , Histonas/química , Animais , Adesão Celular , Comunicação Celular , Proliferação de Células , Estruturas Celulares/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Microesferas , Engenharia Tecidual/métodos
4.
Biochem Biophys Res Commun ; 397(2): 192-6, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20519146

RESUMO

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.


Assuntos
Actinina/metabolismo , Núcleo Celular/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Eur J Cell Biol ; 87(11): 905-20, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18762352

RESUMO

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimento Celular , Pseudópodes/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pseudópodes/genética , Ratos , Tropomiosina/genética
7.
Prosthet Orthot Int ; 39(6): 477-86, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25249382

RESUMO

BACKGROUND: Direct skeletal attachment of limb prostheses is associated with high rate of transcutaneous infection and loosening of the fixture in the medullary canal prompting for careful assessment of various means for enhancing the skin-device and bone-device interface. The skin and bone integrated pylon system constitutes a technological platform for different modifications being evaluated previously. OBJECTIVES: The current study assessed the combination of nano-treatment skin and bone integrated pylon with its pre-seeding with dermal fibroblasts. We hypothesized that this combination will enhance cell interaction with skin and bone integrated pylon compared to nano-treatment and the fibroblast seeding when done separately. STUDY DESIGN: The feasibility and safety of in-bone implantation of the skin and bone integrated pylon with nanotubes was investigated in vitro and in vivo in the animal model. METHODS: TiO2 nanotubes were fabricated on the skin and bone integrated pylon, and the fibroblasts taken from rabbit's skin were cultured on the pylons before implantation. RESULTS: The in vitro experiments demonstrated higher cellular density in the samples with a nanotubular surface than in the non-modified pylons used as control. There were no postoperative complications in any of the animals during the 6-month observation period. Subsequent scanning electron microscopy of the pylon extracted from the rabbit's femur showed the stable contact between the pylon and soft tissues in comparison to control samples where the patchy fibrovascular ingrowth was detected. CONCLUSION: The promising results prompt further investigation of the integrative properties of the nanotextured skin and bone integrated pylon system seeded with dermal fibroblasts and its optimization for clinical application. CLINICAL RELEVANCE: The study is devoted to the development of more safe and efficient technology of direct skeletal attachment of limb prostheses aimed in improving quality of life of people with amputations.


Assuntos
Membros Artificiais , Fibroblastos/transplante , Osseointegração/fisiologia , Desenho de Prótese/métodos , Alicerces Teciduais/química , Titânio/química , Animais , Bioengenharia/métodos , Osso e Ossos/cirurgia , Estudos de Viabilidade , Fibroblastos/citologia , Modelos Animais , Nanotubos , Implantação de Prótese/métodos , Coelhos , Sensibilidade e Especificidade , Pele
8.
J Biomed Mater Res A ; 102(9): 3033-48, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24115308

RESUMO

Angio- and osteogenesis following the two-stage (TS) implantation of the skin- and bone-integrated pylon seeded with autologous fibroblasts was evaluated. Two consecutive animal substudies were undertaken: intramedullary subcutaneous implantation (15 rabbits) and a TS transcutaneous implantation (12 rabbits). We observed enhanced osseointegrative properties of the intramedullary porous component seeded with fibroblasts induced into osteoblast differentiation, as compared to the untreated porous titanium pylon. The three-phase scintigraphy and subsequent histological analysis showed that the level of osteogenesis was 1.5-fold higher than in the control group, and significantly so (p < 0.05). The biocompatibility was further proved by the absence of inflammatory response or encapsulation and sequestration on the histology assay. Treatment of the transcutaneous component with autologous fibroblasts was associated with nearly a 2-fold decrease in the period required for the ingrowth of dermal and subdermal soft tissues into the implant surface, as compared to the untreated porous titanium component. Direct dermal attachment to the transcutaneous implant prevented superficial and deep periprosthetic infections in rabbits in vivo.


Assuntos
Membros Artificiais , Fibroblastos/transplante , Osseointegração , Osteoblastos/citologia , Alicerces Teciduais/química , Animais , Fibroblastos/citologia , Masculino , Osteogênese , Desenho de Prótese , Coelhos , Titânio/química
9.
Oncotarget ; 4(2): 362-72, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23482348

RESUMO

ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65- dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65.


Assuntos
Actinina/genética , Fator de Transcrição RelA/genética , Actinina/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Multimerização Proteica , Fator de Transcrição RelA/metabolismo , Ativação Transcricional
10.
Exp Cell Res ; 314(5): 1030-8, 2008 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-18215660

RESUMO

The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.


Assuntos
Actinina/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas dos Microfilamentos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular Tumoral , Galinhas , Citoplasma/química , Humanos , Ligação Proteica , Fibras de Estresse
11.
Cell Biol Int ; 31(4): 360-5, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17336103

RESUMO

Alpha-actinins are actin-binding proteins of non-muscle cells, which can participate in the regulation of transcription factor activity. We describe the distribution of alpha-actinin-1 and -4 depending on different actin cytoskeleton formed as a result of cell adhesion to extracellular matrix proteins, such as fibronectin and laminin 2/4. Immunofluorescent studies show a difference in the distribution of alpha-actinin and -4. Both isoforms localise along stress-fibres, but alpha-actinin-1 localises in the perinuclear region more abundantly than alpha-actinin-4. Western blot analysis demonstrated existence of truncated forms of both isoforms. Truncated alpha-actinin-1 appears in cells spread on fibronectin or laminin. Cell spreading also correlated with more tight association of alpha-actinin-4 with chromatin. Basing on our previous finding of an interaction of alpha-actinin-4 with p65 subunit of the NF-kappaB, we checked the possible influence of immobilised ligands on its redistribution in nuclear complexes containing p65. alpha-Actinin-4 seems to be present in some but not all nuclear complexes containing p65. Immobilised ligands may affect the interaction of alpha-actinin-4/p65 complexes with chromatin. The data suggest that adhesion to extra-cellular matrix may interfere in cellular reactions mediated by alpha-actinin-1 and -4.


Assuntos
Actinina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Western Blotting , Adesão Celular , Citoplasma , Humanos , Imunoprecipitação , NF-kappa B/metabolismo , Frações Subcelulares , Células Tumorais Cultivadas
12.
J Rehabil Res Dev ; 44(5): 723-38, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17943684

RESUMO

This article presents results of the further development and testing of the "skin and bone integrated pylon" (SBIP-1) for percutaneous (through skin) connection of the residual bone with an external limb prosthesis. We investigated a composite structure (called the SBIP-2) made of titanium particles and fine wires using mathematical modeling and mechanical testing. Results showed that the strength of the pylon was comparable with that of anatomical bone. In vitro and in vivo animal studies on 30 rats showed that the reinforcement of the composite pylon did not compromise its previously shown capacity for inviting skin and bone cell ingrowth through the device. These findings provide evidence for the safe and reliable long-term percutaneous transfer of vital and therapeutic substances, signals, and necessary forces and moments from a prosthetic device to the body.


Assuntos
Membros Artificiais , Osso e Ossos/cirurgia , Procedimentos Cirúrgicos Dermatológicos , Osseointegração , Amputação Cirúrgica , Amputados/reabilitação , Animais , Fenômenos Biomecânicos , Osso e Ossos/citologia , Modelos Animais de Doenças , Masculino , Modelos Teóricos , Porosidade , Desenho de Prótese , Ratos , Ratos Wistar , Pele/citologia , Fenômenos Fisiológicos da Pele
13.
Cell Biol Int ; 30(10): 784-92, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16891133

RESUMO

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Integrina alfa2beta1/fisiologia , Integrina alfa3beta1/fisiologia , Integrina alfa6beta4/fisiologia , Laminina/metabolismo , Receptores de Laminina/fisiologia , Animais , Anticorpos/farmacologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Humanos , Integrina alfa2beta1/imunologia , Integrina alfa3beta1/imunologia , Integrina alfa6beta4/imunologia , Camundongos , Neoplasias Cutâneas/metabolismo , Células Tumorais Cultivadas
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