title: Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.
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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Luiz Felipe M
authors.initials: LF
authors.lastname: Prota
authors.forename: Liudmila
authors.initials: L
authors.lastname: Cebotaru
authors.forename: Jie
authors.initials: J
authors.lastname: Cheng
authors.forename: Jerry
authors.initials: J
authors.lastname: Wright
authors.forename: Neeraj
authors.initials: N
authors.lastname: Vij
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Morales
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
mesh_terms: D020816:Amino Acid Motifs; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D003513:Cycloheximide / Q000627:therapeutic use; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015735:Mifepristone / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
chemical_list: C505032:CFTR protein, human; D003470:Culture Media; D005938:Glucocorticoids; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015735:Mifepristone; D003907:Dexamethasone; D003513:Cycloheximide