RESUMEN
While it is now well-established that substrate stiffness regulates vascular endothelial growth factor-A (VEGF-A) mediated signaling and functions, causal mechanisms remain poorly understood. Here, we report an underlying role for the PI3K/Akt/mTOR signaling pathway. This pathway is activated on stiffer substrates, is amplified by VEGF-A stimulation, and correlates with enhanced endothelial cell (EC) proliferation, contraction, pro-angiogenic secretion, and capillary-like tube formation. In the settings of advanced age-related macular degeneration, characterized by EC and retinal pigment epithelial (RPE)-mediated angiogenesis, these data implicate substrate stiffness as a novel causative mechanism and Akt/mTOR inhibition as a novel therapeutic pathway.
Asunto(s)
Células Endoteliales/metabolismo , Mecanotransducción Celular/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Epitelio Pigmentado de la Retina/metabolismo , Serina-Treonina Quinasas TOR/genética , Factor A de Crecimiento Endotelial Vascular/genética , Fenómenos Biomecánicos , Línea Celular , Movimiento Celular , Proliferación Celular , Elasticidad , Células Endoteliales/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Neovascularización Patológica/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/metabolismo , Epitelio Pigmentado de la Retina/citología , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
High temperature requirement A1 (HtrA1) belongs to an ancient protein family that is linked to various human disorders. The precise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrA1 remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is implicated in unfolded protein response. In highly secretory cells of the retinal pigmented epithelia, endoplasmic reticulum (ER) stress upregulated HtrA1. HtrA1 co-localized with vimentin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions displayed reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mammalian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.
Asunto(s)
Estrés del Retículo Endoplásmico , Sustancias Protectoras/metabolismo , Proteínas/toxicidad , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Evolución Molecular , Técnicas de Silenciamiento del Gen , Humanos , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Filogenia , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Ubiquitina/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Vimentina/metabolismoRESUMEN
PURPOSE: Oxidative stress and metabolic dysregulation of the RPE have been implicated in AMD; however, the molecular regulation of RPE metabolism remains unclear. The transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1α to regulate RPE metabolic program and oxidative stress response. METHODS: Primary human fetal RPE (hfRPE) and ARPE-19 were matured in vitro using standard culture conditions. Mitochondrial mass of RPE was measured using MitoTracker staining and citrate synthase activity. Expression of PGC-1 isoforms, RPE-specific genes, oxidative metabolism proteins, and antioxidant enzymes was analyzed by quantitative PCR and Western blot. Mitochondrial respiration and fatty-acid oxidation were monitored using the Seahorse extracellular flux analyzer. Expression of PGC-1α was increased using adenoviral delivery. ARPE-19 were exposed to hydrogen peroxide to induce oxidative stress. Reactive oxygen species were measured by CM-H2DCFDA fluorescence. Cell death was analyzed by LDH release. RESULTS: Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1α gene expression. Overexpression of PGC-1α increased expression of OXPHOS and fatty-acid ß-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1α gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions. CONCLUSIONS: This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1α as a potent driver of RPE mitochondrial function and antioxidant capacity.
