title: Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1.
abstract: BACKGROUND
  We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.
  
  
  METHODS
  GC transactivation was measured using a Glucocorticoid Response Element (GRE)-Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα-Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.
  
  
  RESULTS
  TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.
  
  
  CONCLUSIONS
  Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.
authors.forename: Christine R
authors.initials: CR
authors.lastname: Keenan
authors.forename: Josephine Sl
authors.initials: JS
authors.lastname: Mok
authors.forename: Trudi
authors.initials: T
authors.lastname: Harris
authors.forename: Yuxiu
authors.initials: Y
authors.lastname: Xia
authors.forename: Saad
authors.initials: S
authors.lastname: Salem
authors.affiliation: Lung Health Research Centre, Department of Pharmacology and Therapeutics, University of Melbourne, Grattan St,, Parkville, VIC Australia. astew@unimelb.edu.au.
authors.forename: Alastair G
authors.initials: AG
authors.lastname: Stewart
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D005938:Glucocorticoids / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D020545:Respiratory Mucosa / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: D005938:Glucocorticoids; D016212:Transforming Growth Factor beta