RESUMEN
Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a "stressed UPR." Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in thetrappc11gene, which encodes a component of the transport protein particle (TRAPP) complex.trappc11mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation intrappc11mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen intrappc11mutants and is synthetically lethal withtrappc11mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction intrappc11mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with theTRAPPC11mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.
Asunto(s)
Oligosacáridos/metabolismo , Respuesta de Proteína Desplegada , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Atorvastatina/farmacología , Dolicoles/biosíntesis , Dolicoles/genética , Glicosilación , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Larva/efectos de los fármacos , Larva/metabolismo , Lípidos/química , Hígado/metabolismo , Hígado/patología , Mutación , Oligosacáridos/química , Terpenos/metabolismo , Terpenos/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Proteínas de Transporte Vesicular/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.
Asunto(s)
Hígado Graso/genética , Hígado Graso/patología , Respuesta de Proteína Desplegada/genética , Pez Cebra/genética , Animales , Brefeldino A/farmacología , Proteínas de Unión al ADN/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Hígado Graso/prevención & control , Glicosilación/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Factores de Transcripción del Factor Regulador X , Tapsigargina/farmacología , Factores de Transcripción/metabolismo , Tunicamicina , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
UNLABELLED: Many etiologies of fatty liver disease (FLD) are associated with the hyperactivation of one of the three pathways composing the unfolded protein response (UPR), which is a harbinger of endoplasmic reticulum (ER) stress. The UPR is mediated by pathways initiated by PRKR-like endoplasmic reticulum kinase, inositol-requiring 1A/X box binding protein 1, and activating transcription factor 6 (ATF6), and each of these pathways has been implicated to have a protective or pathological role in FLD. We used zebrafish with FLD and hepatic ER stress to explore the relationship between Atf6 and steatosis. A mutation of the foie gras (foigr) gene caused FLD and hepatic ER stress. The prolonged treatment of wild-type larvae with tunicamycin (TN), which caused chronic ER stress, phenocopied foigr. In contrast, acute exposure to a high dose of TN robustly activated the UPR but was less effective at inducing steatosis. The sterol regulatory element binding protein transcription factors were not required for steatosis in any of these models. Instead, depleting larvae of active Atf6 either through a membrane-bound transcription factor peptidase site 1 mutation or an atf6 morpholino injection protected them against steatosis caused by chronic ER stress, but exacerbated steatosis caused by acute TN treatment. CONCLUSION: ER stress causes FLD. A loss of Atf6 prevents steatosis caused by chronic ER stress but can also potentiate steatosis caused by acute ER stress. This demonstrates that Atf6 can play both protective and pathological roles in FLD.
Asunto(s)
Factor de Transcripción Activador 6/fisiología , Retículo Endoplásmico , Hígado Graso/etiología , Estrés Fisiológico , Animales , Hígado Graso/genética , Mutación , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
INTRODUCTION: In order to develop a model for investigating the genes that contribute to retinal degeneration, we examined the early graded photochemical stress response in the adult zebrafish (Danio rerio) retina and investigated the role of an NMDA inhibitor, thiokynurenate. METHODS: Following intravitreal injection of rose bengal (6 or 12 mg/mL), light (37x10(3) or 83x10(3) lx) was directed onto the central retina with and without 400 nM thiokynurenate. Histologic and electron microscopic analysis was performed at 2 and 4 h and gene expression analysis was carried out at 2, 4 and 6 h. RESULTS: Light and electron microscopy demonstrated a graded photochemical response in photoreceptor, nuclear, and ganglion cell layer thickness. Increased vacuolation of the inner plexiform layer was also observed. The inhibitor produced a distinct lesion pattern. Cellular stress genes were elevated in low and high lesions, while some homeobox gene expression was reduced with thiokynurenate. DISCUSSION: The phenotypic and genetic changes observed from this model can serve as a basis for understanding the pathology of retinal oxidative and cellular stress. These changes may aid our understanding of aging and macular degeneration.
Asunto(s)
Modelos Animales de Enfermedad , Degeneración Retiniana/inducido químicamente , Animales , Perfilación de la Expresión Génica , Inyecciones , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Luz/efectos adversos , Microscopía Electrónica , Estrés Oxidativo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Rosa Bengala , Cuerpo Vítreo , Pez CebraRESUMEN
UNLABELLED: Steatosis is the most common consequence of acute alcohol abuse and may predispose to more severe hepatic disease. Increased lipogenesis driven by the sterol response element binding protein (SREBP) transcription factors is essential for steatosis associated with chronic alcohol ingestion, but the mechanisms underlying steatosis following acute alcohol exposure are unknown. Zebrafish larvae represent an attractive vertebrate model for studying alcoholic liver disease (ALD), because they possess the pathways to metabolize alcohol, the liver is mature by 4 days post-fertilization (dpf), and alcohol can be simply added to their water. Exposing 4 dpf zebrafish larvae to 2% ethanol (EtOH) for 32 hours achieves approximately 80 mM intracellular EtOH and up-regulation of hepatic cyp2e1, sod, and bip, indicating that EtOH is metabolized and provokes oxidant stress. EtOH-treated larvae develop hepatomegaly and steatosis accompanied by changes in the expression of genes required for hepatic lipid metabolism. Based on the importance of SREBPs in chronic ALD, we explored the role of Srebps in this model of acute ALD. Srebp activation was prevented in gonzo larvae, which harbor a mutation in the membrane-bound transcription factor protease 1 (mbtps1) gene, and in embryos injected with a morpholino to knock down Srebp cleavage activating protein (scap). Both gonzo mutants and scap morphants were resistant to steatosis in response to 2% EtOH, and the expression of many Srebp target genes are down-regulated in gonzo mutant livers. CONCLUSION: Zebrafish larvae develop signs of acute ALD, including steatosis. Srebp activation is required for steatosis in this model. The tractability of zebrafish genetics provides a valuable tool for dissecting the molecular pathogenesis of acute ALD.
Asunto(s)
Hígado Graso Alcohólico/fisiopatología , Hígado Graso/fisiopatología , Proteínas de Unión a los Elementos Reguladores de Esteroles/fisiología , Animales , Compuestos Azo , Colorantes , Modelos Animales de Enfermedad , Etanol/metabolismo , Hígado Graso Alcohólico/mortalidad , Hígado Graso Alcohólico/patología , Larva/metabolismo , Lípidos/fisiología , Análisis de Supervivencia , Pez CebraRESUMEN
OBJECTIVES: Fibroblast growth factors (FGFs) represent potent effectors and play essential roles in both normal development and many pathological processes. Little is known about their possible implication in retinoblastoma growth. We sought to examine FGF high- and low-affinity receptor (FGFR) expression, activation of FGFR1 by acidic FGF (FGF-1), and proliferative effects on Y79 cells. METHODS: Expression of FGFR1 to FGFR4 was screened in Y79 cells by means of immunochemical and reverse transcriptase polymerase chain reaction techniques. Tyrosine phosphorylation of FGFR1 induced by FGF was examined by immunoprecipitation after stimulation with FGF-1 in the presence or absence of heparin. Retinoblastoma proliferation was monitored by radiolabeled thymidine incorporation or a vital dye-based assay, after addition of FGF-1 with or without inclusion of a specific FGFR1 neutralizing antibody or FGFR1 antisense oligonucleotides. Low-affinity heparan sulfate proteoglycan coreceptors were blocked through sodium chlorate or heparinase treatment of Y79 cells. RESULTS: Y79 retinoblastoma expressed all 4 FGFRs, at both the protein and messenger RNA levels. The FGFR1 was differentially phosphorylated in a time- and heparin-dependent manner by FGF-1. Proliferation of Y79 cells induced by FGF-1 was entirely mediated by FGFR1, since inclusion of specific neutralizing antibodies or antisense oligonucleotides completely prevented tumor cell multiplication. Finally, FGF-1-induced proliferation was dependent on the presence and sulfation of heparan sulfate proteoglycan. CONCLUSIONS: Y79 retinoblastoma expresses all 4 FGFRs, but FGFR1 activation entirely accounts for FGF-1-driven cell proliferation. CLINICAL RELEVANCE: These studies demonstrate a role for the FGF-1/FGFR1 pathway in retinoblastoma proliferation, and may contribute to developing therapeutic strategies to limit retinoblastoma growth.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Transducción de Señal/fisiología , Western Blotting , Proliferación Celular/efectos de los fármacos , Factor 1 de Crecimiento de Fibroblastos/genética , Factor 1 de Crecimiento de Fibroblastos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteoglicanos de Heparán Sulfato/biosíntesis , Heparina/farmacología , Humanos , Oligorribonucleótidos Antisentido/farmacología , Fosforilación , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Neoplasias de la Retina/patología , Retinoblastoma/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Fibroblast growth factors (FGFs) are important regulators of retinal development and survival. We examined the expression and distribution of FGF9 and its preferred receptors FGFR2IIIc and FGFR3IIIc in this tissue. FGF9 transcripts in whole rat retina were detected by RT-PCR but were not present in purified cultured Muller glia. Transcripts appeared as 3.2-kb and 4.0-kb bands on Northern blots, and Western blotting of whole retina revealed FGF9-immunoreactive bands at 30 and 55 kDa. FGF9 mRNA demonstrated a biphasic expression profile, elevated at birth and adulthood, but relatively decreased during terminal retinal differentiation (4-14 days postnatal). Antibody labeling broadly reflected these findings: staining in vivo was observed mainly in the inner retina (and outer plexiform layer in adults) whereas FGF9 was not detectable in cultured Muller glia. In adults, FGF9 in situ hybridization also showed a detectable signal in inner retina. FGFR2IIIc and FGFR3IIIc were detected by RT-PCR, and Western blotting showed both FGFRs existed as multiple forms between approximately 100-200 kDa. FGFR2 and FGFR3 antibodies showed prominent labeling in the inner retina, especially in proliferating cultured Muller glia. Exogenous FGF9 elicited a dose-dependent increase in Muller glial proliferation in vitro. These data suggest a role for FGF9 in retinal differentiation and maturation, possibly representing a neuronally derived factor acting upon glial (and other) cells.