RESUMO
Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4(+) lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRzeta protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRzeta. Importantly, the deficiency of the TCRzeta chain and of Lck and the compensatory up-regulation of FcepsilonRIgamma and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRzeta protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRzeta in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas rab4 de Ligação ao GTP/imunologia , Adolescente , Adulto , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Quinases/metabolismo , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR , Transfecção , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We investigated the localization of Rab4 in the rat parotid glands by Western blotting and light-microscopic immunohistochemistry. Rab4 was localized mainly on the intracellular membranes in the subapical-actin terminal web, but was not present in the basolateral region both in acinar and ductal cells. Actin, alpha-adaptin, Rab5A and aquaporin5 were detected in the Rab4-containing intracellular membrane fraction prepared using anti-Rab4 antibody covalently coupled to magnetic beads. Detection of actin indicated that the Rab4-containing intracellular membranes were attached to the actin filaments. Although alpha-adaptin was immunohistochemically distributed along the plasma membrane, this protein coexisted with Rab4 only at the apical region. Rab5A immunoreactivity was distributed all around the cytoplasm. These findings suggested that Rab4 participates in endocytosis at the apical membrane of parotid glands.