Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.
Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR). Our aim was to determine the effects of dexamethasone treatment on wild-t...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3521767?pdf=render |
| _version_ | 1818016276695482368 |
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| author | Luiz Felipe M Prota Liudmila Cebotaru Jie Cheng Jerry Wright Neeraj Vij Marcelo M Morales William B Guggino |
| author_facet | Luiz Felipe M Prota Liudmila Cebotaru Jie Cheng Jerry Wright Neeraj Vij Marcelo M Morales William B Guggino |
| author_sort | Luiz Felipe M Prota |
| collection | DOAJ |
| description | Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR). Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression.Dose-response (1 nM to 10 µM) and time course (3 to 48 h) curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM) showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM) induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml) at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90) binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70) binding decreased 0.30 fold in an immunoprecipitation assay.Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR. |
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| id | doaj.art-15fe5662c62349278a73f19385200a54 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T07:10:00Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-15fe5662c62349278a73f19385200a542022-12-22T02:06:27ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01712e4740510.1371/journal.pone.0047405Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.Luiz Felipe M ProtaLiudmila CebotaruJie ChengJerry WrightNeeraj VijMarcelo M MoralesWilliam B GugginoDexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR). Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression.Dose-response (1 nM to 10 µM) and time course (3 to 48 h) curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM) showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM) induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml) at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90) binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70) binding decreased 0.30 fold in an immunoprecipitation assay.Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.http://europepmc.org/articles/PMC3521767?pdf=render |
| spellingShingle | Luiz Felipe M Prota Liudmila Cebotaru Jie Cheng Jerry Wright Neeraj Vij Marcelo M Morales William B Guggino Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. PLoS ONE |
| title | Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. |
| title_full | Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. |
| title_fullStr | Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. |
| title_full_unstemmed | Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. |
| title_short | Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90. |
| title_sort | dexamethasone regulates cftr expression in calu 3 cells with the involvement of chaperones hsp70 and hsp90 |
| url | http://europepmc.org/articles/PMC3521767?pdf=render |
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