Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3
Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promo...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-06-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/9/7/1589 |
| _version_ | 1797563686213124096 |
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| author | Mikel D. Haggadone Peter Mancuso Marc Peters-Golden |
| author_facet | Mikel D. Haggadone Peter Mancuso Marc Peters-Golden |
| author_sort | Mikel D. Haggadone |
| collection | DOAJ |
| description | Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs. |
| first_indexed | 2024-03-10T18:46:59Z |
| format | Article |
| id | doaj.art-f2c94451190e4852b50e892933b04942 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T18:46:59Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-f2c94451190e4852b50e892933b049422023-11-20T05:29:19ZengMDPI AGCells2073-44092020-06-0197158910.3390/cells9071589Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3Mikel D. Haggadone0Peter Mancuso1Marc Peters-Golden2Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USAGraduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USAGraduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USAExtracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.https://www.mdpi.com/2073-4409/9/7/1589alveolar macrophage (AM)extracellular vesicles (EVs)suppressor of cytokine signaling 3 (SOCS3)reactive oxygen species (ROS)proteasome |
| spellingShingle | Mikel D. Haggadone Peter Mancuso Marc Peters-Golden Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 Cells alveolar macrophage (AM) extracellular vesicles (EVs) suppressor of cytokine signaling 3 (SOCS3) reactive oxygen species (ROS) proteasome |
| title | Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 |
| title_full | Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 |
| title_fullStr | Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 |
| title_full_unstemmed | Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 |
| title_short | Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3 |
| title_sort | oxidative inactivation of the proteasome augments alveolar macrophage secretion of vesicular socs3 |
| topic | alveolar macrophage (AM) extracellular vesicles (EVs) suppressor of cytokine signaling 3 (SOCS3) reactive oxygen species (ROS) proteasome |
| url | https://www.mdpi.com/2073-4409/9/7/1589 |
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