Caffeine and rolipram affect Smad signalling and TGF-β1 stimulated CTGF and transgelin expression in lung epithelial cells

PLoS One. 2014 May 14;9(5):e97357. doi: 10.1371/journal.pone.0097357. eCollection 2014.

Abstract

Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-β1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGF-β1-inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGF-β1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-β1 induced up-regulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An understanding of these mechanisms might help to explain the protective effects of caffeine in prevention of BPD and suggests rolipram to be a potent replacement for caffeine.

MeSH terms

  • Airway Remodeling / drug effects
  • Airway Remodeling / genetics
  • Caffeine / pharmacology*
  • Cell Line, Tumor
  • Connective Tissue Growth Factor / genetics*
  • Down-Regulation / drug effects
  • Down-Regulation / genetics
  • Epithelial Cells / drug effects*
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / genetics
  • Humans
  • Lung / drug effects*
  • Microfilament Proteins / genetics*
  • Muscle Proteins / genetics*
  • Promoter Regions, Genetic / drug effects
  • Rolipram / pharmacology*
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Smad Proteins / genetics*
  • Transforming Growth Factor beta1 / genetics*
  • Up-Regulation / drug effects
  • Up-Regulation / genetics

Substances

  • CCN2 protein, human
  • Microfilament Proteins
  • Muscle Proteins
  • Smad Proteins
  • Transforming Growth Factor beta1
  • transgelin
  • Connective Tissue Growth Factor
  • Caffeine
  • Rolipram

Grant support

The authors have no support or funding to report.