Dataset Information

AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.

ABSTRACT: ?B-crystallin (?B) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of ?B has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that ?B is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of ?B in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize ?B in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, ?-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of ?B is predominantly apical. Using OptiPrep gradients we demonstrate that ?B resides in the DRM fraction. The secretion of ?B is inhibited by the cholesterol-depleting drug, methyl ?-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.

SUBMITTER: Gangalum RK

PROVIDER: S-EPMC3030331 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.

Gangalum Rajendra K RK Atanasov Ivo C IC Zhou Z Hong ZH Bhat Suraj P SP

The Journal of biological chemistry 20101119 5

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epitheli ...[more]

PMID: 21097504

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Project description:Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-? and tumour necrosis factor (TNF)-? are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-? and/or 5 ng/ml TGF-?2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-? alone (Exo-TNF) and co-stimulated with TNF-? and TGF-?2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-?2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.

Project description:?B crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether ?B crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular ?B crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that ?B crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of ?B crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced ?B crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, ?B crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that ?B crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of ?B crystallin to the basolateral side. In normal mouse retinal sections, ?B crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous ?B crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. ?B Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for ?B crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of ?B crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for ?B crystallin in ocular diseases.

Dataset Information

AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.

Publications

AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.

Similar Datasets

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