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Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the sam...

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Autores principales: Zadjali, Fahad, Kumar, Prashant, Yao, Ying, Johnson, Daniel, Astrinidis, Aristotelis, Vogel, Peter, Gross, Kenneth W., Bissler, John J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084746/
https://www.ncbi.nlm.nih.gov/pubmed/32138326
http://dx.doi.org/10.3390/ijms21051729
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author Zadjali, Fahad
Kumar, Prashant
Yao, Ying
Johnson, Daniel
Astrinidis, Aristotelis
Vogel, Peter
Gross, Kenneth W.
Bissler, John J.
author_facet Zadjali, Fahad
Kumar, Prashant
Yao, Ying
Johnson, Daniel
Astrinidis, Aristotelis
Vogel, Peter
Gross, Kenneth W.
Bissler, John J.
author_sort Zadjali, Fahad
collection PubMed
description The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.
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spelling pubmed-70847462020-03-24 Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content Zadjali, Fahad Kumar, Prashant Yao, Ying Johnson, Daniel Astrinidis, Aristotelis Vogel, Peter Gross, Kenneth W. Bissler, John J. Int J Mol Sci Article The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies. MDPI 2020-03-03 /pmc/articles/PMC7084746/ /pubmed/32138326 http://dx.doi.org/10.3390/ijms21051729 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zadjali, Fahad
Kumar, Prashant
Yao, Ying
Johnson, Daniel
Astrinidis, Aristotelis
Vogel, Peter
Gross, Kenneth W.
Bissler, John J.
Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title_full Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title_fullStr Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title_full_unstemmed Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title_short Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content
title_sort tuberous sclerosis complex axis controls renal extracellular vesicle production and protein content
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084746/
https://www.ncbi.nlm.nih.gov/pubmed/32138326
http://dx.doi.org/10.3390/ijms21051729
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