Effect of epidermal growth factor signal pathway on integrin alpha5 beta1 in prostate cancer cell line DU145 / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of epidermal growth factor (EGF) signal pathway on the expression of integrin alpha5 beta1 in prostate cancer cell line DU145.</p><p><b>METHODS</b>Using flow-cytometry, the effects of EGF and the mitogen-activated protein kinase (MAPK) signal pathway inhibitor PD98059 on the expression of integrin alpha5 and beta1 subunits on DU145 cell surface were analyzed. RT-PCR and Western blot methods were used to examined the expression of mRNA and cell total protein of integrin alpha5 and beta1 subunits. And the metastatic phenotypes in DU145 cell were investigated.</p><p><b>RESULTS</b>The expression levels of integrin alpha5 beta1, which was the receptor for fibronectin, were changed. EGF up-regulated the protein and mRNA expression of beta1 subunit on DU145 cell surface, 231% and 248% (P < 0.01) compared to the control respectively, and it could significantly promote the ability of DU145 cell adhesion to fibronectin and migration. However PD98058, which was the inhibitor of MAPK signal pathway, down-regulated the protein and mRNA expression of beta1 subunit, 60% and 63% (P < 0.01) compared to the control respectively, and it had the contrary function on the adhesion and migration ability of DU145 cell. But both had no effect the expression of alpha5 subunit.</p><p><b>CONCLUSIONS</b>EGF might promote the metastatic ability mainly by up-regulating the expression of beta1 subunit by activating MAPK signal pathway in DU145 cells. Their regulation effects are on the mRNA transcriptional level.</p>

Expressão e papel funcional de receptores de fibronectina no timo de camundongos / Expression and functional paper of fibronectin receptors in the thymus of mice

Nesta tese, objetivamos estudar a expressão e papel funcional de receptores de fibronectina no timo incluindo aspectos fisiológicos e patológicos desse órgão. Ao avaliarmos o papel de duas proteínas de ECM (fibronectina e laminina) em interações envolvendo células fagocitárias do retículo tímico (PTR) e timócitos, vimos que as PTR expressam ambas as proteínas de ECM e seus respectivos receptores, e que a formação de rosetas de timócitos às PTR dependem de interações dos pares ligante/receptor correspondentes. Por outro lado, experimentos in vivo, nos quais procuramos bloquear a saída de recém emigrantes do timo pelo uso de peptídeos bloqueadores de receptores de fibronectina, sugerem que o endereçamento de recentes emigrantes tiímicos (RTE)para o baço aparentemente depende, ainda que de forma parcial, de interação entre fibronectina e a5b1. O modelo patológico investigado nesta tese foi uma linhagem de camundongos (C57BL10ScSnmdx, ou mdx) que desenvolvem uma distrofia muscular semelhante à distrofia muscular de Duchenne a qual ocorre em humanos. Estes animais apresentaram uma intensa atrofia tímica, representada pela baixa celularidade e por uma queda expressiva nas proporções de timócitos corticais de fenótipo CD4+CD8+. Vimos ainda uma diminuição significativa na expressão do receptor de fibronectina a5b1 em timócitos de mdx quando comparados aos controles. Interessantemente, testes funcionais demonstraram que os timócitos dos camundongos mdx apresentam diminuição tanto de sua capacidade de adesão a células epiteliais tímicas, quanto da migração induzida por fibronectina. Em conjunto, os dados aqui apresentados mostram que as interações intratímicas mediadas por fibronectina e seus receptores a4b1 e a5b1 abrangem diferentes componentes do microambiente tímico, e participam do controle de fenômenos de adesão/migração de timócitos. Vimos ainda que alterações das interações fibronectina/a5b1, podem levar a distúrbios no padrão de migração, cujo significado patológico especificamente no que diz respeito à fisiopatologia da distrofia muscular observada nos camundongos mdx, necessita ser avaliada.

Interaction of leukemic cells with proteins of the extracellular matrix / Interações de células leucêmicas com proteínas da matriz extracelular

The interaction of neoplastic cells with basement membrane molecules is the first step for the dissemination of tumor cells in vivo. Leukemic cells have a great ability to spread in the host, since cells are released from the bone marrow to the circulation. In this study we analysed whether CEM, U937, K562 and HL-60 cells were able to attach to different concentrations of laminin and/or fibronectin and/or type IV collagen. Attachment to type IV collagen was low, but it increased with the addition of laminin and occurred in all four leukemic cell lines. On the other hand, attachment to fibronectin was higher, but it decreased with the addition of laminin in the assays using U937 and HL-60 cells. The combination of type IV collagen and fibronectin was a good substratum for cellular attachment. However, the addition of laminin to this substratum impaired its attachment activity in U937, HL-60 and K562. These data suggest that laminin may control cellular attachment to the extracellular matrix during leukemic dissemination in hosts in different ways.

A Case of Ullrich's Disease

Ullrich's disease is a congenital muscular dystrophy clinically characterized by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. All the patients develop rigidity of spine, often assoicated with scoliosis, failure to thrive, and early and severe respiratory involvement, irrespective of their levels of motor function. Intellectual development is normal. The biopsied muscles show dystrophies including remarkable variation in the fiber size, notably proliferated endomysial connective tissues, and a lot of degenerated and regenerated fibers. The expression of merosin and dytrophin is normal. Recent studies have demonstrated that collagen VI is deficient in the muscles of the patients with Ullrich's disease, and some result from recessive mutations of the collagen VIalpha 2 gene(COL6A2). And a marked reduction of fibronectin receptors in the extracellular matrix of skin and cultured fibroblasts of these patients is also reported. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that any abnormalities of cell adhesion may be involved in the pathogenesis of the disease. A case of Ullrich's disease has not been reported yet in Korea. So, we describe a male patient with Ullrich's disease with a brief review of the literature.

Aspectos biomoleculares del carcinoma basocelular y su relación con la metástasis / Biomolecular aspects of basal cell carcinoma and its relation with metastasis

El carcinoma basocelular es una neoplasia cutánea de crecimiento lento. Rara vez produce metástasis, siendo un modelo ideal para analizar los factores que condicionan la misma. Se han identificado numerosos factores de riesgo: radiación ultravioleta, la cual lesiona directamente al ADN induciendo una inmunodepresión selectiva, algunas cepas del virus papiloma humano y trastornos autosómicos recesivos, donde se identificaron mutaciones del gen supresor del crecimiento tumoral. Actualmente, numerosas líneas de investigación están abocadas al estudio de la baja incidencia de metástasis de éste tumor. En éste carcinoma no existe una disminución en las caderinas epiteliales, las cuales intervienen en la adherencia celular. Asimismo, existe disminución de los receptores de laminina y fibronectina. En la matriz extracelular no se ha observado un aumento de enzimas proteolíticas que la degraden. La inmunovigilancia tumoral está muy activada por medio de un aumento de linfocitos T citolíticos y la producción de interferón gamma. Concluimos que no sólo los queratinocitos neoplásicos intervienen en la metástasis, sino que la matriz extracelular juega un rol preponderante en la escasa incidencia de diseminación de éste tumor, por lo que un estudio exhaustivo de la misma permitiría ser aplicado a otras neoplasias y otorgar una mayor sobrevida

A avaliaçäo da adesäo, migraçäo e proliferaçäo da linhagem celular CHO=K1 frente à mutante CHO-745 deficiente na biossíntese de glicosaminoglicanos sulfatados: amostra que a integrina alfa-5beta-1 é um proteoglicano envolvido na migraçäo celular / The role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of CHO cells and CHO-K1 cells and a mutant CHO-745 which is deficient in syntesis of proteoglycans due to lack of activity of xylosyl transferase

Na presente tese examinamos o papel dos proteoglicanos e glicosaminoglicanos na divisao celular: adesao e proliferaçao de células em cultura CHO ("chinese hamster ovary") e sua mutante CHO-745 que é deficiente na síntese de glicosaminoglicanos devido a falta da enzima xilosiltransferase. Usando diferentes quantidades de células (selvagem e mutante) pouca adesao pode ser observada na presença de laminina e colágeno tipo I. Entretanto quando fibronectina e vitronectina foram usadas como substratos houve um aumento de adesao dos dois tipos celulares. Sómente a CHO selvagem mostrou adesao em funçao do tempo sobre colágeno tipo IV. Estes resultados sugerem que as duas linhagens celulares possuem diferentes propriedades de adesao. Ensaios de adesao usando colágeno tipo IV com células CHO cultivadas na presença de xilosídeo ou clorato, mostraram reduçao nos níveis de adesao, confirmando a importancia dos glicosaminoglicanos neste fenômeno. Várias evidências experimentais sugerem que os proteoglicanos de hepara sulfato estao envolvidos na adesao celular como moduladores positivos da proliferaça celular e como composto chave no processo de divisao celular. Ensaios de proliferaçao e ciclo celular demonstraram que uma diminuiçao das quantidades do proteoglicano nao inibem a proliferaçao da mutante CHO-745 quando comparadas com a célula selvagem pois os dois tipos celulares entram na fase S ao redor das 8 horas. A inteiraçoes celulas-matriz estao implicadas em uma grande variedade d funçoes. Estas inteiraçoes sao principalmente mediadas por integrinas que sa receptores da superfície celular e estao aparentemente envolvidas em adesao, migraçao e diferenciaçao. Por exemplo, a asj3, integrina está envolvida na migraçao e adesao da células sobre fibronectina. Também neste estudo, usando marcaçao radioativa com sulfato e imunoprecipitaçao...(au)

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with Alpha 4+ and Alpha 5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were alpha 6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections

Detection of post-translational sulfation of alpha-5 beta-1 integrin and its role in integrin-fibronectin binding

Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha(5)beta(1) integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation an a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.