Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus
Abstract Nucleus pulposus (NP) cells reside in a physiologically hyperosmotic environment within the intervertebral disc. TonEBP/NFAT5 is an osmo-sensitive transcription factor that controls expression of genes critical for cell survival under hyperosmotic conditions. A recent report on NP and studi...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2017-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-04876-2 |
| _version_ | 1818429673049161728 |
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| author | Chao Liu Hyowon Choi Zariel I. Johnson Jiwei Tian Irving M. Shapiro Makarand V. Risbud |
| author_facet | Chao Liu Hyowon Choi Zariel I. Johnson Jiwei Tian Irving M. Shapiro Makarand V. Risbud |
| author_sort | Chao Liu |
| collection | DOAJ |
| description | Abstract Nucleus pulposus (NP) cells reside in a physiologically hyperosmotic environment within the intervertebral disc. TonEBP/NFAT5 is an osmo-sensitive transcription factor that controls expression of genes critical for cell survival under hyperosmotic conditions. A recent report on NP and studies of other cell types have shown that hyperosmolarity triggers autophagy. However, little is known whether such autophagy induction occurs through TonEBP. The goal of this study was to investigate the role of TonEBP in hyperosmolarity-dependent autophagy in NP. Loss-of-function studies showed that autophagy in NP cells was not TonEBP-dependent; hyperosmolarity did not upregulate autophagy as previously reported. NP tissue of haploinsufficient TonEBP mice showed normal pattern of LC3 staining. NP cells did not increase LC3-II or LC3-positive puncta under hyperosmotic conditions. Bafilomycin-A1 treatment and tandem mCherry-EGFP-LC3B reporter transfection demonstrated that the autophagic flux was unaffected by hyperosmolarity. Even under serum-free conditions, NP cells did not induce autophagy with increasing osmolarity. Hyperosmolarity did not change the phosphorylation of ULK1 by mTOR and AMPK. An ex vivo disc organ culture study supported that extracellular hyperosmolarity plays no role in promoting autophagy in the NP. We conclude that hyperosmolarity does not play a role in autophagy induction in NP cells. |
| first_indexed | 2024-12-14T15:21:15Z |
| format | Article |
| id | doaj.art-ec6850c9958649eb86cff7b63c20bb32 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T15:21:15Z |
| publishDate | 2017-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-ec6850c9958649eb86cff7b63c20bb322022-12-21T22:56:10ZengNature PortfolioScientific Reports2045-23222017-07-017111310.1038/s41598-017-04876-2Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposusChao Liu0Hyowon Choi1Zariel I. Johnson2Jiwei Tian3Irving M. Shapiro4Makarand V. Risbud5Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson UniversityDepartment of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson UniversityDepartment of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson UniversityDepartment of Orthopaedics, Shanghai General Hospital, Shanghai Jiao Tong University School of MedicineDepartment of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson UniversityDepartment of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson UniversityAbstract Nucleus pulposus (NP) cells reside in a physiologically hyperosmotic environment within the intervertebral disc. TonEBP/NFAT5 is an osmo-sensitive transcription factor that controls expression of genes critical for cell survival under hyperosmotic conditions. A recent report on NP and studies of other cell types have shown that hyperosmolarity triggers autophagy. However, little is known whether such autophagy induction occurs through TonEBP. The goal of this study was to investigate the role of TonEBP in hyperosmolarity-dependent autophagy in NP. Loss-of-function studies showed that autophagy in NP cells was not TonEBP-dependent; hyperosmolarity did not upregulate autophagy as previously reported. NP tissue of haploinsufficient TonEBP mice showed normal pattern of LC3 staining. NP cells did not increase LC3-II or LC3-positive puncta under hyperosmotic conditions. Bafilomycin-A1 treatment and tandem mCherry-EGFP-LC3B reporter transfection demonstrated that the autophagic flux was unaffected by hyperosmolarity. Even under serum-free conditions, NP cells did not induce autophagy with increasing osmolarity. Hyperosmolarity did not change the phosphorylation of ULK1 by mTOR and AMPK. An ex vivo disc organ culture study supported that extracellular hyperosmolarity plays no role in promoting autophagy in the NP. We conclude that hyperosmolarity does not play a role in autophagy induction in NP cells.https://doi.org/10.1038/s41598-017-04876-2 |
| spellingShingle | Chao Liu Hyowon Choi Zariel I. Johnson Jiwei Tian Irving M. Shapiro Makarand V. Risbud Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus Scientific Reports |
| title | Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| title_full | Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| title_fullStr | Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| title_full_unstemmed | Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| title_short | Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| title_sort | lack of evidence for involvement of tonebp and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus |
| url | https://doi.org/10.1038/s41598-017-04876-2 |
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