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Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts

AIM: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agent...

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Autores principales: Ruan, Shaobo, Rody, Wellington J., Patel, Shivani S., Hammadi, Lina I., Martin, Macey L., de Faria, Lorraine P., Daaboul, George, Anderson, Leif S., He, Mei, Holliday, L. Shannon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10629932/
https://www.ncbi.nlm.nih.gov/pubmed/37936884
http://dx.doi.org/10.20517/evcna.2023.38
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author Ruan, Shaobo
Rody, Wellington J.
Patel, Shivani S.
Hammadi, Lina I.
Martin, Macey L.
de Faria, Lorraine P.
Daaboul, George
Anderson, Leif S.
He, Mei
Holliday, L. Shannon
author_facet Ruan, Shaobo
Rody, Wellington J.
Patel, Shivani S.
Hammadi, Lina I.
Martin, Macey L.
de Faria, Lorraine P.
Daaboul, George
Anderson, Leif S.
He, Mei
Holliday, L. Shannon
author_sort Ruan, Shaobo
collection PubMed
description AIM: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. METHODS: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. RESULTS: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast’s cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. CONCLUSION: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.
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spelling pubmed-106299322023-11-07 Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts Ruan, Shaobo Rody, Wellington J. Patel, Shivani S. Hammadi, Lina I. Martin, Macey L. de Faria, Lorraine P. Daaboul, George Anderson, Leif S. He, Mei Holliday, L. Shannon Extracell Vesicles Circ Nucl Acids Article AIM: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. METHODS: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. RESULTS: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast’s cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. CONCLUSION: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs. 2023 2023-09-06 /pmc/articles/PMC10629932/ /pubmed/37936884 http://dx.doi.org/10.20517/evcna.2023.38 Text en https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Article
Ruan, Shaobo
Rody, Wellington J.
Patel, Shivani S.
Hammadi, Lina I.
Martin, Macey L.
de Faria, Lorraine P.
Daaboul, George
Anderson, Leif S.
He, Mei
Holliday, L. Shannon
Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title_full Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title_fullStr Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title_full_unstemmed Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title_short Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts
title_sort receptor activator of nuclear factor-kappa b is enriched in cd9-positive extracellular vesicles released by osteoclasts
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10629932/
https://www.ncbi.nlm.nih.gov/pubmed/37936884
http://dx.doi.org/10.20517/evcna.2023.38
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