RESUMEN
Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Portadoras/genética , Proteínas de Choque Térmico , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Factores de Transcripción/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Variación Genética , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Presenilina-1 , Presenilina-2 , Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción CHOP , Regulación hacia ArribaRESUMEN
Mature single-positive (SP) T lymphocytes enter a "resting" state in which they are proliferatively quiescent and relatively resistant to apoptosis. The molecular mechanisms regulating this quiescent phenotype were unknown. Here it was found that the expression of a Kruppel-like zinc finger transcription factor, lung Kruppel-like factor (LKLF), is developmentally induced during the maturation of SP quiescent T cells and rapidly extinguished after SP T cell activation. LKLF-deficient T cells produced by gene targeting had a spontaneously activated phenotype and died in the spleen and lymph nodes from Fas ligand-induced apoptosis. Thus, LKLF is required to program the quiescent state of SP T cells and to maintain their viability in the peripheral lymphoid organs and blood.
Asunto(s)
Interfase , Linfocitos T/citología , Linfocitos T/inmunología , Transactivadores/fisiología , Dedos de Zinc , Animales , Apoptosis , Linfocitos B/metabolismo , Supervivencia Celular , Quimera , Proteína Ligando Fas , Eliminación de Gen , Marcación de Gen , Factores de Transcripción de Tipo Kruppel , Ganglios Linfáticos/citología , Activación de Linfocitos , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Bazo/citología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Transactivadores/biosíntesis , Transactivadores/genética , Transfección , Receptor fas/biosíntesisRESUMEN
The transcriptional programs that regulate blood vessel formation are largely unknown. In this paper, we examine the role of the zinc finger transcription factor LKLF in murine blood vessel morphogenesis and homeostasis. By in situ hybridization and immunohistochemistry, we show that LKLF is expressed as early as embryonic day 9.5 (E9.5) in vascular endothelial cells throughout the developing mouse embryo. To better understand the function of LKLF, we used homologous recombination in embryonic stem (ES) cells to generate LKLF-deficient (LKLF-/-) mice. Both angiogenesis and vasculogenesis were normal in the LKLF-/- mice. However, LKLF-/- embryos died between E12.5 and E14.5 from severe intra-embryonic and intra-amniotic hemorrhaging. This bleeding disorder was associated with specific defects in blood vessel morphology. Umbilical veins and arteries in the LKLF-/- embryos displayed an abnormally thin tunica media and aneurysmal dilatation before rupturing into the amniotic cavity. Similarly, vascular smooth muscle cells in the aortae from the LKLF-/- animals displayed a cuboidal morphology and failed to organize into a compact tunica media. Consistent with these findings, electron microscopic analyses demonstrated endothelial cell necrosis, significant reductions in the number of vessel-wall pericytes and differentiating smooth muscle cells, and decreased deposition of extracellular matrix in the LKLF-/- vessels. Despite these defects, in situ hybridization demonstrated normal expression of platelet-derived growth factor B, Tie1, Tie2, transforming growth factor beta, and heparin-binding epidermal growth factor in the vasculature of the LKLF-/- embryos. Therefore, LKLF defines a novel transcriptional pathway in which endothelial cells regulate the assembly of the vascular tunica media and concomitant vessel wall stabilization during mammalian embryogenesis.