RESUMO
A large body of data suggests that body weight influences puberty onset and adult reproduction. However, the underlying mechanism of how body weight influences puberty onset and fertility is not completely understood. The hypothalamic neuronal circuit regulating reproduction is restrained by inhibitory signals during childhood. At the time of puberty, these inhibitory signals are weakened and supplanted by stimulatory signals that, in turn, stimulate the release of gonadotropin-releasing hormone (GnRH) - a hypothalamic neuropeptide governing reproduction. A number of studies, however, suggest that puberty commencement occurs when body (fat) weight reaches a certain threshold, which is critical for the initiation of puberty and for support of the adult reproductive function. Previously, various signals have been studied which might link body (fat) weight-related information to the hypothalamic neuronal network regulating reproduction. However, the nature of the signal(s) that may link body fat and/or muscle mass with the hypothalamic neuronal network governing reproduction is still unclear. It has been intuitively speculated that augmentation of such signal(s) will cause a restriction of inhibitory input and activation of stimulatory input to GnRH secreting neurons at the time of puberty onset. Therefore, the unveiling of such signal(s) will greatly help in understanding the mechanism of puberty onset. Recently, it has been shown that expression of fibronectin type III domain containing-5 (FNDC5) mRNA in central and peripheral tissues upsurges during postnatal development, especially around the time of puberty onset. Moreover, the systemic level of irisin - one of the protein products of the FNDC5 gene that is secreted as myokine and adipokine - also rises during postnatal development and correlates with the timing of puberty onset. Therefore, we propose here that irisin might serve as a possible signal for linking body fat/muscle mass with the hypothalamic center governing reproductive function. We hypothesize that irisin acts as a trigger for the activation of the hypothalamic neuronal network monitoring the onset of puberty.
Assuntos
Fibronectinas/fisiologia , Hormônios/metabolismo , Puberdade , Tecido Adiposo , Animais , Peso Corporal , Fibronectinas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Hipotálamo , Modelos Biológicos , Modelos Teóricos , Neurônios/metabolismo , Maturidade Sexual/fisiologia , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD], a novel pyridone agent, showed potent antifibrotic properties. The aim of the present study was to investigate the effects of AKF-PD on diabetic nephropathy and kidney fibrosis, and to obtain an insight into its mechanisms of action. METHODS: We administered AKF-PD to diabetic db/db mice for 12 weeks. Moreover, we performed in vitro cultures using murine mesangial cells exposed to high ambient glucose concentrations. RESULTS: AKF-PD reduced renal hypertrophy, mesangial matrix expansion and albuminuria in the db/db mice. The upregulated expression of α1(I)- and α1(IV)-collagen and fibronectin mRNAs, transforming growth factor-ß1 (TGF-ß1), α-smooth muscle actin (α-SMA), and tissue inhibitors of metalloproteinase 1 (TIMP-1) mRNAs and proteins was inhibited by AKF-PD treatment in the renal cortex of db/db mice. The maximal effective dose of AKF-PD was about 500 mg/kg body weight. AKF-PD inhibited the upregulated expression of α1(I)- and α1(IV)-collagens, TGF-ß1, TIMP-1 and α-SMA induced by high glucose concentrations in cultured mesangial cells. CONCLUSIONS: Our data indicate that AKF-PD diminishes the abnormal accumulation of mesangial matrix through the inhibition of upregulated expression of TGF-ß target genes in kidneys of db/db mice, resulting in attenuation of renal fibrosis and amelioration of renal dysfunction despite persistent hyperglycemia. Therefore, AKF-PD, a potent antifibrotic agent, holds great promise in the treatment of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Hipoglicemiantes/farmacologia , Nefropatias/tratamento farmacológico , Rim/fisiopatologia , Piridonas/farmacologia , Albuminas/análise , Animais , Glicemia , Técnicas de Cultura de Células , Colágeno/fisiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Fibronectinas/fisiologia , Fibrose/patologia , Fibrose/fisiopatologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Hipoglicemiantes/uso terapêutico , Rim/patologia , Córtex Renal/fisiopatologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Piridonas/uso terapêutico , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
BACKGROUND AND PURPOSE Matrix metalloproteinase (MMP) production from monocyte/macrophages is implicated in matrix remodelling and modulation of inflammation. However, knowledge of the patterns and mechanisms of gene regulation of MMPs and their endogenous tissue inhibitors (TIMPs) is fragmentary. MMP up-regulation may be a target for cyclooxygenase (COX) and prostaglandin (PG) receptor inhibition, but the extent and mechanisms of COX-independent MMP up-regulation are unclear. EXPERIMENTAL APPROACH We studied MMP mRNA expression and selected protein levels in human peripheral blood monocytes before and after adhesion, upon stimulation with bacterial lipopolysaccharide (LPS), PGE(2) or forskolin and after culturing with monocyte colony-stimulating factor on plastic or human fibronectin for up to 7 days. KEY RESULTS Monocyte adherence for 2 h transiently up-regulated COX-2, MMP-1, MMP-7 and MMP-10 mRNAs, and persistently up-regulated MMP-2, MMP-9, MMP-14 and MMP-19 mRNAs. LPS, PGE(2) or forskolin selectively increased MMP-1, MMP-9, MMP-10, MMP-12 and MMP-14 mRNAs. LPS increased PGE(2) production through COX but up-regulated MMP levels independently of COX. Differential dependence on inhibition of p42/44 and p38 mitogen-activated protein kinases, c-jun N-terminal kinase and inhibitor of κB kinase2 paralleled the diverse patterns of MMP stimulation by LPS. Differentiation on plastic increased mRNA levels of MMP-7, MMP-9, MMP-12 and MMP-14 and TIMP-2 and TIMP-3 independently of COX; fibronectin accelerated MMP but not TIMP up-regulation. CONCLUSIONS AND IMPLICATIONS Adhesion, LPS stimulation and maturation of human monocytes lead to selective, COX-independent MMP and TIMP gene regulation, which is a potential target for selective inhibition by signalling kinase inhibitors.
Assuntos
Adesão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Metaloproteases/fisiologia , Monócitos/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Colforsina/farmacologia , Dinoprostona/metabolismo , Fibronectinas/fisiologia , Humanos , Inflamação/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/metabolismo , Monócitos/enzimologia , Subunidade p52 de NF-kappa B/antagonistas & inibidores , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Fitoterapia , Preparações de Plantas/farmacologia , Plásticos/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/análise , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Inibidores Teciduais de Metaloproteinases/biossíntese , Inibidores Teciduais de Metaloproteinases/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Malignant tumors remain a significant health threat, with death often occurring as a result of metastasis. Cell adhesion is a crucial step in the metastatic cascade of tumor cells, and interruption of this step is considered to be a logical strategy for prevention and treatment of tumor metastasis. Celastrol [3-hydroxy-24-nor-2-oxo-1(10),3,5,7-friedelatetraen-29-oic acid], a quinone methide triterpene from the medicinal plant Tripterygium wilfordii, possesses antitumor activities, whereas the underlying mechanism(s) remains elusive. Here, we found that celastrol inhibited cell-extracellular matrix (ECM) adhesion of human lung cancer 95-D and mouse melanoma B16F10 cells. This inhibition was achieved through suppressing beta1 integrin ligand affinity and focal adhesion formation, accompanied by the reduced phosphorylation of focal adhesion kinase (FAK). In understanding the underlying mechanisms, we found that celastrol activated p38 mitogen-activated protein kinase (MAPK) by phosphorylation before the decrement of phosphorylated FAK and that this action was independent of the presence of fibronectin. Using 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of p38 MAPK, the effects of celastrol on beta1 integrin function, cell-ECM adhesion, and phosphorylation of FAK were partially attenuated. In addition, focal adhesion-dependent cell migration and invasion were both inhibited by treatment with celastrol. Finally, the antimetastatic activity of celastrol was examined in vivo using the B16F10-green fluorescent protein-injected C57BL/6 mouse model, as indicated by decreased pulmonary metastases in celastrol-administrated mice. Taken together, these data demonstrate for the first time that celastrol exerts potent antimetastatic activity both in vitro and in vivo, and they provide new evidence for the critical roles of p38 MAPK in the regulation of integrin function and cell adhesion.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Matriz Extracelular/fisiologia , Integrina beta1/fisiologia , Metástase Neoplásica/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ativação Enzimática , Fibronectinas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Metástase Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
In implantology it is known that fibronectin affects cell-substrate adhesion, consequently, the structure and composition of the initially adsorbed fibronectin layer to a large extent determines the biological response to a biomaterial implanted into the body. In this study we have used neutron reflectometry and quartz-crystal microbalance with dissipation to investigate the amount of fibronectin adsorbed, the layer density, thickness and structure of films adsorbed to polished silicon oxide surfaces. We have cultured MG63 osteoblast-like cells on surfaces coated and uncoated with fibronectin and monitored the cellular response to these surfaces. The results show that at fibronectin concentrations in the range 0.01 to 0.1 mg/ml a single highly hydrated layer of fibronectin approximately 40-50 Å in thickness adsorbs to a polished silicon oxide surface and is likely to correspond to one diffuse monolayer of fibronectin arranged side-on. Cells cultured on this fibronectin layer have dramatically different morphology and growth to those grown on bare surfaces. Using a model silicon oxide surface has enabled us to study the substrate/protein interface, together with the impact of a fibronectin layer on the cellular response using consistent experimental conditions across a unique set of experimental techniques.
Assuntos
Fibronectinas/análise , Difração de Nêutrons/métodos , Osteoblastos/fisiologia , Técnicas de Microbalança de Cristal de Quartzo/métodos , Adsorção , Adesão Celular , Células Cultivadas , Fibronectinas/química , Fibronectinas/fisiologia , Fibronectinas/ultraestrutura , Cinética , Osteoblastos/citologia , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.
Assuntos
Fibronectinas/genética , Fibronectinas/fisiologia , Proteínas de Plasma Seminal/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/isolamento & purificação , Expressão Gênica , Ligação Genética , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas de Plasma Seminal/fisiologia , Homologia de Sequência de Aminoácidos , SuínosRESUMO
During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.
Assuntos
Adjuvantes Imunológicos/fisiologia , Comunicação Celular/imunologia , Matriz Extracelular/enzimologia , Matriz Extracelular/imunologia , Glucuronidase/fisiologia , Proteínas Recombinantes/farmacologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Adjuvantes Imunológicos/metabolismo , Adesão Celular/imunologia , Comunicação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/imunologia , Colágeno Tipo IV/fisiologia , Matriz Extracelular/fisiologia , Fibronectinas/metabolismo , Fibronectinas/fisiologia , Quinase 2 de Adesão Focal , Glucuronidase/metabolismo , Humanos , Ácido Hialurônico/fisiologia , Interfase/imunologia , Migração e Rolagem de Leucócitos/imunologia , Ativação Linfocitária , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/imunologia , Especificidade por Substrato/imunologia , Subpopulações de Linfócitos T/enzimologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The aim of this study was to evaluate the effect of astilbin on concanavalin A (Con A)-induced hepatitis, a T cell-dependent model of liver injury. Con A administration resulted in a severe liver injury in mice, with a strong increment in spleen cell adhesion and liver infiltration of T cells, as well as in tumour necrosis factor (TNF)-alpha production. Against this liver injury, astilbin significantly inhibited the elevation in transaminase activity, reduced the TNF-alpha production, and improved the histological changes, including inflammatory infiltration, hepatocyte necrosis and degeneration and Kupffer cell hyperplasia. In addition, astilbin inhibited the adhesion of spleen cells and purified T lymphocytes isolated from the liver-injured mice to fibronectin, laminin and type IV collagen.Moreover, the adhesion of human Jurkat T cells to endothelial cell line ECV-304 was also inhibited by astilbin. These results suggest that the improvement of the T cell-mediated liver injury by astilbinmay be related to the reduction in TNF-alpha production and in T cell adhesion to extracellular matrices and endothelial cells.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Flavonóis/farmacologia , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Adesão Celular , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno Tipo IV/fisiologia , Concanavalina A , Matriz Extracelular/fisiologia , Feminino , Fibronectinas/fisiologia , Flavonóis/isolamento & purificação , Humanos , Células Jurkat , Laminina/fisiologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Extratos Vegetais/química , Smilax , Linfócitos T/fisiologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Fibronectin (FN) is an extracellular matrix (ECM) protein involved in tumor growth and metastasis. Five human FN cDNA segments encoding for FN fragments, all starting with the II1 repeat and ending with different C-terminal extensions, have been stably expressed in chick embryo fibroblasts (CEF). These FN cDNAs induce the formation of an organized ECM in CEF as long as they retain a sequence coding for a 13-amino acid stretch (FN13), with collagen binding activity, localized between type II2 and I7 repeats. An FN13 synthetic peptide induces in control CEF the assembly of an FN-ECM comparable with that observed in CEF-expressing FN fragments. The activity of FN13 is specific for its amino acid sequence, although the cysteine present in the 6th position can be substituted with a polar serine without affecting the induction of a fibrillar FN-ECM. A less fibrillar matrix is induced by FN13-modified peptides in which the cysteine is methylated or substituted by a non-polar alanine. FN13 induces the assembly of an FN-ECM also in Rous sarcoma virus-transformed CEF lacking the ECM and in hepatoma (SK-Hep1) and fibrosarcoma (HT-1080) human cell lines. FN13 also promotes the adhesion of CEF and Rous sarcoma virus-CEF at levels comparable with those obtained with purified intact FN. Finally, FN13 inhibits the migratory and invasive properties of tumorigenic cells, whereas intact FN favors their migration. All FN13-modified peptides show similar effects, although with reduced efficiency. None of these activities is supported by a scrambled peptide. These data suggest a possible role of FN13 in tumor growth and metastasis inhibition and its possible use as anti-tumorigenic agent.
Assuntos
Matriz Extracelular/metabolismo , Fibronectinas/fisiologia , Alanina/química , Aminoácidos/química , Animais , Adesão Celular , Movimento Celular , Embrião de Galinha , Clonagem Molecular , Colágeno/química , Colágeno/metabolismo , DNA Complementar/metabolismo , Fibronectinas/química , Humanos , Hibridização In Situ , Metilação , Microscopia de Fluorescência , Peptídeos/química , Ligação Proteica , Coelhos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais CultivadasRESUMO
The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.
Assuntos
Adesão Celular/efeitos dos fármacos , Fibronectinas/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Laminina/efeitos dos fármacos , Extratos Vegetais/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fibronectinas/fisiologia , Imunofluorescência , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Laminina/análise , Sensibilidade e Especificidade , Regulação para Cima/efeitos dos fármacosRESUMO
Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.
Assuntos
Movimento Celular/fisiologia , Substância Própria/fisiologia , Fibroblastos/fisiologia , Fibronectinas/fisiologia , Tenascina/fisiologia , Anticorpos Bloqueadores/fisiologia , Adesão Celular , Sulfatos de Condroitina/fisiologia , Colágeno/fisiologia , Géis , Humanos , Integrinas/fisiologia , Sulfato de Queratano/fisiologia , Microscopia EletrônicaRESUMO
Electrical stimulation has been used to promote wound healing. The mechanisms by which such stimulation could interact with biological systems to accelerate healing have not been elucidated. One potential mechanism could involve stimulation of macrophage migration to the site of a wound. Here we report that oscillatory electric fields induce human macrophage migration. Macrophages exposed to a 1 Hz, 2 V/cm field show an induced migration velocity of 5.2+/-0.4 x 10(-2) microm/min and a random motility coefficient of 4.8+/-1.4 x 10(-2) microm2/min on a glass substrate. Electric field exposure induces reorganization of microfilaments from ring-like structures at the cell periphery to podosomes that are confined to the contact sites between cell and substrate, suggesting that the cells are crawling on glass. Treatment of cells with monoclonal antibodies directed against beta2-integrins prior to field exposure prevents cell migration, indicating that integrin-dependent signaling pathways are involved. Electric fields cause macrophage migration on laminin or fibronectin coated substrates without inducing podosome formation or changes in cellular morphology. The migration velocity is not significantly altered but the random movement is suppressed, suggesting that cell movements on a laminin- or fibronectin-coated surface are not mediated by cell crawling. It is suggested that electric field-induced macrophage migration utilizes several modes of cell movement, including cell crawling and possibly cell rolling.
Assuntos
Movimento Celular/fisiologia , Terapia por Estimulação Elétrica/métodos , Integrinas/fisiologia , Ativação de Macrófagos/fisiologia , Transdução de Sinais/fisiologia , Fibronectinas/fisiologia , Humanos , Laminina/fisiologia , Microscopia Confocal , Microscopia de Vídeo , Modelos Biológicos , Cicatrização/fisiologiaRESUMO
The Arg-Gly-Asp (RGD) tripeptide and ajoene were used for studying the role of adhesive receptors in the respiratory burst. Activation of the respiratory burst was examined by using luminol-dependent and lucigenin-dependent chemiluminescence. Recently, it was shown that ajoene, (E, Z)-4,5,9-trithiadodeca-1,6,11-trien-9-oxide, a substance isolated from garlic extract, inhibits the binding of fibrinogen to activated platelets by direct interaction with fibrinogen receptor (Apitz-Castro, R., Lederma, E., Escalante, J. and Jain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150). Taking into consideration the structural and functional similarity of integrins, it would be reasonable to assume that ajoene as well as RGD can inhibit adhesive interactions of human neutrophils. We have shown that the effect of various activators on the respiratory burst was abolished by ajoene or RGD treatment. The inhibitory effect of RGD and ajoene was dose-dependent. The treatment of neutrophils with antiserum against human plasma fibronectin inhibited the respiratory burst in response to formyl-methionyl-leucylphenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). This effect is dose-dependent and reversible with the addition of fibronectin. These data indicate that the respiratory burst in human neutrophils is mediated by the integrin family of receptors and that interactions between the extracellular matrix fibronectin and cells are necessary for the respiratory burst.
Assuntos
Adesão Celular , Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Neutrófilos/fisiologia , Adulto , Adesão Celular/efeitos dos fármacos , Dissulfetos/farmacologia , Fibronectinas/farmacologia , Alho , Interações Ervas-Drogas , Humanos , Técnicas In Vitro , Cinética , Medições Luminescentes , Luminol , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Extratos Vegetais/farmacologia , Plantas Medicinais , Receptores Imunológicos/efeitos dos fármacos , Sulfóxidos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system whereas no proliferation was observed when CD4 cells were cultured with anti-CD3 alone or fibronectin alone. In addition, anti-CD29 (integrin beta 1) as well as anti-VLA-5 (human fibronectin receptor) antibodies blocked this CD4 cell activation in this system. Although anti-CD3 alone or fibronectin alone cannot induce IL-2 message by CD4 cells, the combination of anti-CD3 plus fibronectin induced IL-2 message by CD4 cells. In an analysis of the molecular mechanism by which IL-2 message was generated, we showed that a fibronectin-VLA-5 fibronectin receptor interaction may contribute an independent signal distinct from the CD3 pathway of activation by the induction of an AP-1 transcriptional factor. Thus the VLA-5 fibronectin receptor on CD4 cells can play a complementary role in CD3-TCR-mediated signal transduction through its interaction with fibronectin.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/biossíntese , Fibronectinas/fisiologia , Ativação Linfocitária , Receptores de Antígeno muito Tardio/fisiologia , Fatores de Transcrição/biossíntese , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/fisiologia , Complexo CD3 , Expressão Gênica , Humanos , Técnicas In Vitro , Interleucina-2/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-jun , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Fibronectina , Receptores Imunológicos/fisiologia , Receptores de Interleucina-2/metabolismoRESUMO
A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.
Assuntos
Fibronectinas/fisiologia , Agregação Plaquetária/fisiologia , Anticorpos Monoclonais , Ligação Competitiva , Plaquetas/metabolismo , Fibronectinas/metabolismo , Humanos , Immunoblotting , Substâncias Macromoleculares , Serotonina/metabolismo , Trombina/farmacologiaRESUMO
To study the interaction of migrating newt epidermal cells with purified extracellular matrix (ECM) molecules we have developed an in vitro migration assay using pieces of newt skin explanted onto culture dishes coated with various ECM molecules and cultured for 18 h in defined serum-free medium. Newt epidermal cells migrate out from explants placed on dishes coated with either collagen, vitronectin, fibronectin, or fibrinogen but not on albumin-coated or uncoated dishes. Explant outgrowth on collagen was best in CEM 2000 medium diluted to 60% of mammalian osmolarity. Other media such as RPMI 1640 or Ex-Cell 300, diluted similarly, may also be used although in our hands CEM 2000 always allowed more migration. We found no migration on collagen when skin explants were incubated in Holtfreter's solution (an amphibian saline solution that we have previously shown allows reepithelialization on amputated newt limbs). Supplementation of Holtfreter's solution with glucose did not improve its ability to support migration. By testing various supplement combinations in conjunction with CEM 2000 and RPMI 1640 we found that neither serum, insulin, selenium, transferrin, nor L-glutamine is required for explant outgrowth. Of the additives tested, outgrowth was stimulated only by insulin. Epidermal cell outgrowth on collagen was inhibited by both puromycin and cycloheximide, indicating the necessity for protein synthesis in this system. Whether the effects of these protein synthesis inhibitors are specifically on migration-related events or on general metabolic requirements is not clear. Inasmuch as there was no correlation (r = -0.227) between DNA synthesis (measured by incorporation of tritiated thymidine) and the amount of outgrowth, we believe that our assay is a measure of cell migration alone rather than a combination of mitosis and migration. This explant outgrowth system represents a new and relatively simple assay that can be used in the study of cell-substrate interactions during newt epidermal cell migration over extracellular matrix molecules in a defined serum-free environment.