RESUMO
Certain pathogenic strains of E. coli produce the cytotoxic necrotizing factors-1 or -2. Cytotoxic necrotizing factor-1 irreversibly activates the small GTPases of the Rho family Rho, Rac and Cdc42. Cytotoxic necrotizing factor-2 may have similar effects. Since the Rho proteins play an important role in the organization of the actin cytoskeleton and neuronal differentiation, we have investigated whether cytotoxic necrotizing factor-2 affects the morphology of cultured hippocampal neurons. The toxin indeed caused dendrite retraction and axon shortening. Within 4 h of application, cytotoxic necrotizing factor-2 induced a transient formation of short finger-like extensions. To study the role of the Rho proteins in the morphological changes caused by cytotoxic necrotizing factor-2, we transfected neurons with recombinant Rho proteins. Dominant-negative forms of Rac or Rho but not of Cdc42 prevented the formation of short extensions induced by cytotoxic necrotizing factor-2, indicating synergistic effects of Rac and Rho. In contrast, the retraction of dendrites induced by cytotoxic necrotizing factor-2 was only prevented by dominant-negative Rho. Analysis with pull-down assays showed that cytotoxic necrotizing factor-2 strongly activated Rac and Rho, whereas an effect on Cdc42 was not observed. Cytotoxic necrotizing factor-2 also diminished the total amount of Rac and Rho. The degradation of Rac was so pronounced that the increase in Rac activity was only transient. In organotypic cultures of the hippocampus, cytotoxic necrotizing factor-2 reduced the number of neurites per neuron, suggesting that neurons in the tissue context were also vulnerable. We conclude that cytotoxic necrotizing factor-2 has pronounced effects on neuronal morphology, which are due to activation of the GTPases Rho and Rac.
Assuntos
Toxinas Bacterianas/farmacologia , Citotoxinas/farmacologia , Proteínas de Escherichia coli , Escherichia coli/química , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Amidas/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Citocalasina D/farmacologia , Inibidores Enzimáticos/farmacologia , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Neurônios/citologia , Neurônios/ultraestrutura , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Transfecção , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/farmacologia , Quinases Associadas a rhoRESUMO
A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.