RESUMO
Dendritic polylysines (DPL) are highly branched nano-sized spherical polymer with positively charged primary amino groups on surface. This structural feature is useful for a delivery of antisense oligonucleotide or siRNA. In this study, we modified the surface of DPL with cyclic RGD (and iRGD) peptide by conjugation reaction generating RGD (and iRGD) peptide conjugated dendritic poly-lysines, RGD-DPL or iRGD-DPL. The prepared conjugates were evaluated for integrin receptor-mediated cellular delivery of antisense oligonucleotide. The conjugation of RGD or iRGD peptide on DPL was monitored by measuring the retention time in capillary zone electrophoresis and the absorbance at UV-Vis spectroscopy. Cellular delivery by DPL-RGD (or -iRGD)/antisense oligonucleotide complex was examined by antisense splicing correction assay on integrin alpha v/beta 3 positive A375B3-Luc cells, which were stably transfected with plasmid pLuc/705. DPL-RGD (or -iRGD)/antisense oligonucleotide complexes exhibited integrin receptor mediated uptake on A375B3 cells without inducing cellular toxicity. In addition, the delivery of antisense oligonucleotide was integrin receptor-dependent with moderate efficiency.
Assuntos
Dendrímeros/química , Oligonucleotídeos Antissenso/química , Oligopeptídeos/química , Polilisina/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/farmacocinética , Dendrímeros/toxicidade , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/toxicidadeRESUMO
The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Membranas Intracelulares/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Oligonucleotídeos/análise , RNA Interferente Pequeno/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing, they can interact with mRNA or pre-mRNA targets with high selectivity. As a result, they could precisely manipulate gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, and many candidates are already in clinical trials. However, a major impediment to the maturation of this field of oligonucleotide-based therapeutics remains: these relatively large and often highly charged molecules don't easily cross cellular membranes, making it difficult for them to reach their sites of action in the cytosol or nucleus. In this Account, we summarize some basic features of the biology of antisense and siRNA oligonucleotides. We then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Instead of focusing on the details of conjugation chemistry, we emphasize the pharmacological ramifications of oligonucleotide conjugates. In one important approach to improving delivery and efficacy, researchers have conjugated oligonucleotides with ligands designed to bind to particular receptors and thus provide specific interactions with cells. In another strategy, researchers have coupled antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Although both of these strategies have had some success, further research is needed before oligonucleotide conjugates can find an important place in human therapeutics.
Assuntos
Oligonucleotídeos/metabolismo , Animais , Carboidratos/química , Humanos , Lipídeos/química , Camundongos , MicroRNAs/química , MicroRNAs/metabolismo , Ácidos Nucleicos/química , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Peptídeos/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
A continuing problem in the area of oligonucleotide-based therapeutics is the poor access of these molecules to their sites of action in the nucleus or cytosol. A number of approaches to this problem have emerged. One of the most interesting is the use of ligand-oligonucleotide conjugates to promote receptor mediated cell uptake and delivery. Here we provide an overview of recent developments regarding targeted conjugates, including use of peptides, carbohydrates and small molecules as ligands. Additionally we discuss our own experience with this approach and point out both advantages and limitations.
Assuntos
Oligonucleotídeos/química , RNA Interferente Pequeno/química , Núcleo Celular/química , Humanos , Ligantes , Oligonucleotídeos/síntese químicaRESUMO
We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)ß(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)ß(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/química , Albumina Sérica/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Integrina alfaVbeta3/biossíntese , Melanoma/tratamento farmacológico , Melanoma/patologia , Micelas , Tamanho da Partícula , Peptídeos Cíclicos/química , Polietilenoglicóis/química , Propriedades de SuperfícieRESUMO
It has been known for some time that cell adhesion receptors, including members of the integrin family, play an important role in the biology of tumors. Until recently, most of the emphasis in this area of research has concerned the functioning of integrins as adhesive molecules in the invasive and metastatic behavior of malignant cells. Now it has become clear that integrins can function as true receptors capable of transducing signals to the cell interior. Tyrosine phosphorylation seems to be a key aspect of integrin-mediated signal transduction, and a new tyrosine kinase has been described that seems to be important in this process. Evidence is accumulating that integrin-mediated signals can induce gene expression and affect transit through the cell cycle. Thus, the role of integrins in cancer seems not only to involve cell adhesion events, but may also involve the regulation of tumor cell growth and differentiation.
Assuntos
Moléculas de Adesão Celular/fisiologia , Integrinas/fisiologia , Neoplasias/fisiopatologia , Animais , Linhagem Celular , Humanos , Integrinas/biossíntese , Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/fisiopatologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Signals from dynamic cellular interactions between the extracellular matrix and neighboring cells ultimately input into the cellular decision-making process. These interactions form the basis of anchorage-dependent growth. Recent advances have provided the mechanistic details behind the ability of integrins, and other cell adhesion molecules (CAMs), to regulate both early signal transduction events initiated by soluble factors and downstream events more proximally involved in cell cycle progression. These actions appear to depend on the ability of CAMs to initiate the formation of organized structures that permit the efficient flow of information.
Assuntos
Moléculas de Adesão Celular/fisiologia , Divisão Celular , Transdução de Sinais , Animais , Ciclo Celular , Diferenciação Celular , Integrinas/fisiologiaRESUMO
Integrins contribute to cell growth by providing a physical linkage between cytoskeletal structures and the extracellular matrix, and also by participating in various signal transduction processes. The interaction of integrins with matrix ligands can generate signals in and of itself, and can also modulate signals instigated by soluble factors such as peptide mitogens. Cellular events affected by integrin-mediated signaling include motility, cell division, differentiation and programmed cell death. Elucidation of how integrin-mediated cell adhesion controls cell growth is likely to be of fundamental importance in understanding complex biological processes, such as tissue morphogenesis and tumor progression.
Assuntos
Substâncias de Crescimento/fisiologia , Integrinas/fisiologia , Transdução de Sinais/fisiologia , Animais , Divisão Celular/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Humanos , Modelos BiológicosRESUMO
Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Here we show that inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signalling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signalling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction.
Assuntos
Adesão Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Células 3T3 , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocalasina D/farmacologia , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/metabolismo , Quinases Ativadas por p21RESUMO
DLC-1 was originally identified as a potential tumor suppressor. One of the key biochemical functions of DLC-1 is to serve as a GTPase activating protein (GAP) for members of the Rho family of GTPases, particularly Rho A-C and Cdc 42. Since these GTPases are critically involved in regulation of the cytoskeleton and cell migration, it seems clear that DLC-1 will also influence these processes. In this review we examine basic aspects of the actin cyoskeleton and how it relates to cell motility. We then delineate the characteristics of DLC-1 and other members of its family, and describe how they may have multiple effects on the regulation of cell polarity, actin organization, and cell migration.
Assuntos
Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Movimento Celular , Humanos , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoCRESUMO
Antisense oligonucleotides with sequences complementary to a given genetic target can enter cells in sufficient quantities to selectively inhibit gene expression. Thus, they have a potential therapeutic use in preventing undesirable gene expression in diseases such as cancer and AIDS. However, it is remarkable that these molecules, which have high molecular weights and are often charged, gain entry to cells at all. In this article, we review the possible mechanisms by which oligonucleotides enter cells and their subsequent intracellular fates. We also discuss current approaches for improving cellular uptake and delivery of antisense nucleic acids to their intended targets.
RESUMO
We have investigated the adhesion and detachment properties of wild-type Chinese hamster cells and of variant lines, which possess altered cell surface glycoproteins as detected by galactose oxidase-[3H]borohydride labeling. The wild-type and variant lines tested all adhered to protein-coated glass surfaces at the same rate; however, the variant cells differed from wild type and from each other in terms of the ease with which they were detached by trypsinization. Morphological differences between the various lines were also apparent. Our results suggest that the carbohydrate moieties of the terminal region of surface glycoproteins are not directly involved in the initial phase of cell-to-substratum attachment, but that they may modulate the proteolytic susceptibility of surface components which are involved in cell detachment.
Assuntos
Adesão Celular , Proteínas de Membrana/análise , Boroidretos , Linhagem Celular , Membrana Celular/fisiologia , Galactose Oxidase , Cinética , MutaçãoRESUMO
Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.
Assuntos
Adesão Celular , Membrana Celular/fisiologia , Colágeno , Fibronectinas , Animais , Sangue , Linhagem Celular , Separação Celular , Concanavalina A , Cricetinae , Feminino , Mutação , Ovário , PolilisinaRESUMO
We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides.
Assuntos
Adesão Celular , Fibroblastos/análise , Receptores Imunológicos/análise , Âmnio , Animais , Anticorpos Monoclonais , Antígenos de Superfície/fisiologia , Moléculas de Adesão Celular , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Fibronectinas/fisiologia , Glioblastoma , Humanos , Linfócitos , Ovário , Receptores de Fibronectina , Receptores Imunológicos/fisiologiaRESUMO
Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.
Assuntos
Adesão Celular/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Linhagem Celular , Concanavalina A/farmacologia , Fibronectinas , Integrinas , Cinética , Polilisina/farmacologia , Receptores Imunológicos/efeitos dos fármacosRESUMO
It has become widely accepted that adhesion receptors can either directly activate, or significantly modulate, many of the signaling cascades initiated by circulating growth factors. An interesting recent development is the realization that adhesion receptors and their cytoskeletal partners can regulate the trafficking of signaling proteins between the cytoplasm and nucleus. Cell adhesion molecule control of nucleocytoplasmic trafficking allows adhesion to influence many cell decisions, and highlights the diversity of nuclear import and export mechanisms.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Moléculas de Adesão Celular/fisiologia , Citoesqueleto/fisiologia , Animais , Adesão Celular , Núcleo Celular/metabolismo , Modelos Biológicos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM-dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride-galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.
Assuntos
Adesão Celular , Espaço Extracelular/fisiologia , Fibronectinas/fisiologia , Glicoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Animais , Linhagem Celular , Cricetinae , Feminino , Peso Molecular , Mutação , Ovário , Trombina/metabolismo , Tripsina/metabolismoRESUMO
Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression. The plethora of integrin alpha and beta subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways. Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin alpha5 subunit, inhibits cell motility, and alters actin filament organization. Nischarin is primarily a cytosolic protein, but clearly associates with alpha5beta1, as demonstrated by coimmunoprecipitation. Overexpression of Nischarin markedly reduces alpha5beta1-dependent cell migration in several cell types. Rat embryo fibroblasts transfected with Nischarin constructs have "basket-like" networks of peripheral actin filaments, rather than typical stress fibers. These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases. In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter. Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement.
Assuntos
Antígenos CD/metabolismo , Movimento Celular/fisiologia , Integrinas/metabolismo , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células CHO , Compartimento Celular , Cricetinae , Citoesqueleto/ultraestrutura , Biblioteca Gênica , Receptores de Imidazolinas , Integrina alfa5 , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Proteínas/genética , Ratos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Técnicas do Sistema de Duplo-Híbrido , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos CD/metabolismo , Receptores de Fibronectina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Adesão Celular , Movimento Celular , Células Clonais , Cricetinae , Fibronectinas/metabolismo , Citometria de Fluxo , Variação Genética , Vetores Genéticos , Humanos , Integrina alfa5 , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosfotirosina , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Fibronectina/biossíntese , Receptores de Fibronectina/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.