RESUMEN
The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.
Asunto(s)
Catarata/metabolismo , Conexinas/genética , Proteínas del Ojo/genética , Cristalino/metabolismo , Mutación , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 6/genética , Animales , Animales Recién Nacidos , Western Blotting , Proteínas de Unión al ADN/genética , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Fibroblastic preadipocyte cells are recruited to differentiate into new adipocytes during the formation and hyperplastic growth of white adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, is expressed at low levels in preadipocytes, and its levels increase dramatically and rapidly during the differentiation process. However, the mechanisms controlling the dynamic and selective expression of PPARγ in the adipocyte lineage remain largely unknown. We show here that the zinc finger protein Evi1 increases in preadipocytes at the onset of differentiation prior to increases in PPARγ levels. Evi1 expression converts nonadipogenic cells into adipocytes via an increase in the predifferentiation levels of PPARγ2, the adipose-selective isoform of PPARγ. Conversely, loss of Evi1 in preadipocytes blocks the induction of PPARγ2 and suppresses adipocyte differentiation. Evi1 binds with C/EBPß to regulatory sites in the Pparγ locus at early stages of adipocyte differentiation, coincident with the induction of Pparγ2 expression. These results indicate that Evi1 is a key regulator of adipogenic competency.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/genética , Proteína beta Potenciadora de Unión a CCAAT , Proteínas de Unión al ADN , PPAR gamma/genética , Proto-Oncogenes , Factores de Transcripción , Células 3T3-L1 , Adipocitos/citología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , PPAR gamma/metabolismo , Unión Proteica , Proto-Oncogenes/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.
Asunto(s)
Cristalino/embriología , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6 , Animales , Diferenciación Celular , Embrión de Mamíferos/metabolismo , Cristalino/citología , Ratones , Transducción de Señal , eIF-2 Quinasa/metabolismoRESUMEN
Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. Using two different mouse models, we show that the accumulation of collagen chains in the secretory pathway activates the stress signaling pathway termed unfolded protein response (UPR). Transgenic mice expressing ectopic Col4a3 and Col4a4 genes in the lens exhibited activation of IRE1, ATF6, and PERK associated with expansion of the endoplasmic reticulum and attenuation of general protein translation. The expression of the transgenes had adverse effects on lens fiber cell differentiation and eventually induced cell death in a group of transgenic fiber cells. In Col4a1(+/Deltaex40) mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results provide in vivo evidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum.
Asunto(s)
Autoantígenos/biosíntesis , Catarata/metabolismo , Colágeno Tipo IV/biosíntesis , Retículo Endoplásmico/metabolismo , Cristalino/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Autoantígenos/genética , Catarata/genética , Catarata/patología , Muerte Celular/genética , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Humanos , Cristalino/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transgenes/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Wingless (Wg) is a secreted ligand that differentially activates gene expression in target tissues. It belongs to the Wnt family of secreted signaling molecules that regulate cell-to-cell interactions during development. Activation of Wg targets is dependent on the ligand concentration in the extracellular milieu; cellular mechanisms that govern the synthesis, delivery and receipt of Wg are elaborate and complex. We have identified sprinter (srt), which encodes a novel, evolutionarily conserved transmembrane protein required for the transmission of the Wg signal. Mutations in srt cause the accumulation of Wg in cells that express it, and retention of the ligand prevents activation of its target genes in signal-receiving cells. In the absence of Srt activity, levels of Wg targets (including Engrailed in embryos lacking maternal and zygotic srt, and Senseless and Achaete in wing discs) are reduced. Activation of Wg targets in the receiving cells does not require srt. Hence, the function of Srt is restricted to events occurring within the Wg-producing cells. We show that srt is not required for any aspect of Hedgehog (Hh) signal transduction, suggesting specificity of srt for the Wg pathway. We propose that srt encodes a protein required for Wg secretion that regulates maturation, membrane targeting or delivery of Wg. Loss of srt function in turn diminishes Wg-pathway activation in receiving cells.