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. 2012;7(12):e47405.
doi: 10.1371/journal.pone.0047405. Epub 2012 Dec 13.

Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90

Luiz Felipe M Prota  1 , Liudmila Cebotaru, Jie Cheng, Jerry Wright, Neeraj Vij, Marcelo M Morales, William B Guggino
Affiliations

Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90

Luiz Felipe M Prota et al. PLoS One. 2012.

Abstract

Background: 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.

Methods and results: 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.

Conclusion: 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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of dexamethasone on CFTR and αENaC mRNA expression.
Graphs represent the mean ± SE for mRNA relative expression of CFTR and αENaC acquired by real-time PCR. Dose–response curves for dexamethasone on (A) CFTR and (B) αENaC mRNA expressions. *Significant difference compared with the control (C) group #significant difference compared with the group treated with 1 nM dexamethasone; p<0.05, n = 5. Time course of (C) CFTR and (D) αENaC mRNA expression in response to treatment with 10 nM dexamethasone for 3, 24, and 48 h. *Significant difference compared with the control group; §significant difference compared with the group treated for 3 h; p<0.05, n = 4.
Figure 2
Figure 2. Glucocorticoid and mineralocorticoid receptor inhibition by mifepristone and spironolactone, with or without dexamethasone (Dx) following 24 h of treatment.
(A) Graphs representing the densitometric values of protein expression from the blots normalized by GAPDH and the respective blots showing CFTR and GAPDH protein expression in the experimental groups (B). *p<0.05, n = 4.
Figure 3
Figure 3. Total protein expression and surface protein biotinylation following treatment with dexamethasone (Dx, 10 nM) 24 h.
(A) Graphs represent the means ± SE for densitometric values of CFTR total (A) and surface (B) protein expression and respective blots showing CFTR and GAPDH total and surface protein expression for the control, C (total), and after treatment with dexamethasone (10 nM), Dx (total), for 24 h. *p<0.05, n = 3 (pooled with 2 samples per N).
Figure 4
Figure 4. Inhibition of protein synthesis by cycloheximide and treatment with dexamethasone for 24 h in Calu-3 cells.
(A) Graphs representing means ± SE for densitometric values of respective blots showing CFTR and GAPDH protein expression in C, control group; Dx, dexamethasone 10 nM; CHX, cycloheximide (50 µg/ml) treated group; and CHX+Dx, cycloheximide (50 µg/ml) plus dexamethasone (10 nM) treated group. *Significantly different from the C group; #significantly different from the Dx group; p<0.05, n = 4.
Figure 5
Figure 5. CFTR protein expression in Calu-3 cells in the presence of cycloheximide with or without dexamethasone.
Calu-3 cells were treated (A) with (+) or (B) without (−) 10 nM Dx for 24 h, and then incubated in the presence of 100 µg/ml cycloheximide (CHX) for the indicated times (0.5, 1, 2, 4, and 8 h). In the graphs, CFTR protein expression is represented by the densitometric values of each blot using GAPDH as a housekeeper protein. For CHX+Dx, the protein expression was normalized by the group treated with dexamethasone for 24 h and for CHX the normalization was related to the C group. *Significantly different from the respective 24-h group; p<0.05, n = 4.
Figure 6
Figure 6. CFTR protein expression in Calu-3 using [35S]methionine pulse-labeling analysis.
Pulse labeling with 35S for 0, 5, and 15 min after a 24-h cell treatment with or without 10 nM dexamethasone. (A) Blot representing the CFTR band (B) protein labeled with 35S on a radiographic film. The graphs represent the densitometric values of the bands shown in the blot for CFTR. n = 3, *p<0.05.
Figure 7
Figure 7. Immunoprecipitation assay for CFTR protein followed by Western blot for HSP90 or HSP70 in Calu-3 cells.
Cells were treated with (Dx) or without (C group) dexamethasone or with mifepristone (Dx+Mif) for 24 h and total cell lysate. (A) Immunoprecipitation with CFTR Ab and subsequent Western blot for HSP90 or HSP70. The graphs represent the densitometric values of the bands shown in the blots for (B) CFTR, (C), HSP90, and (D) HSP70. Results are presented as the average and SE. n = 4, p<0.05.
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