RESUMO
Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1-/- mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1-/- mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1-/- mice, and growth hormone production was slowed in SERP1-/- pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1-/- pancreas. In basal conditions, the Sec61 complex in SERP1-/- microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.
Assuntos
Retículo Endoplasmático/metabolismo , Deleção de Genes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Animais , Células Cultivadas , Retículo Endoplasmático/química , Retículo Endoplasmático/patologia , Regulação da Expressão Gênica no Desenvolvimento , Glucose/metabolismo , Intolerância à Glucose , Homeostase , Humanos , Insulina/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipófise/citologia , Transporte Proteico , Vesículas Secretórias/metabolismoRESUMO
Mutations of NPHS1, the gene encoding the kidney glomerular filtration barrier protein nephrin, cause congenital nephrotic syndrome of the Finnish type. Nephrin is a component of the interpodocyte-spanning slit diaphragm: it mediates outside-in signaling and forms a nexus for homo- and heterotypic molecular interactions. When studying the nephrin-deficient mouse line generated by random insertional mutagenesis we unexpectedly discovered an endogenous antisense transcript originating from the nephrin-encoding locus. Further evidence of the antisense transcript (Nphs1as) was obtained by searching for Nphs1-like expressed sequence tags. Surprisingly, one clone showed exact complementarity in the antisense orientation. Nphs1as is expressed in the brain, thymus, and peripheral lymph nodes as well as in the embryonic stem cells. However, the mesenteric lymph nodes and the main sites of nephrin expression, the kidney and pancreas, were negative. Nphs1as is a continuous, polyadenylated mRNA that spans Nphs1 exons from 7 to 12 in the reverse orientation. The relative amounts of sense and antisense mRNAs as well as nephrin protein were determined by semiquantitative RT-PCR and immunoblotting, respectively, in various mouse tissues. These results suggest that Nphs1as may be important for the regulation of the appropriate tissue- and cell-type-specific expression of nephrin.
Assuntos
Proteínas/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , Animais , Sequência de Bases , Northern Blotting , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/genética , Éxons/genética , Etiquetas de Sequências Expressas , Expressão Gênica , Genoma , Íntrons/genética , Rim/metabolismo , Glomérulos Renais/metabolismo , Linfonodos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Pâncreas/metabolismo , Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timo/metabolismoRESUMO
Development of the pharyngeal region depends on the interaction and integration of different cell populations, including surface ectoderm, foregut endoderm, paraxial mesoderm, and neural crest. Mice homozygous for a hypomorphic allele of Fgfr1 have craniofacial defects, some of which appeared to result from a failure in the early development of the second branchial arch. A stream of neural crest cells was found to originate from the rhombomere 4 region and migrate toward the second branchial arch in the mutants. Neural crest cells mostly failed to enter the second arch, however, but accumulated in a region proximal to it. Both rescue of the hypomorphic Fgfr1 allele and inactivation of a conditional Fgfr1 allele specifically in neural crest cells indicated that Fgfr1 regulates the entry of neural crest cells into the second branchial arch non-cell-autonomously. Gene expression in the pharyngeal ectoderm overlying the developing second branchial arch was affected in the hypomorphic Fgfr1 mutants at a stage prior to neural crest entry. Our results indicate that Fgfr1 patterns the pharyngeal region to create a permissive environment for neural crest cell migration.
