Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells

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.

The Role of Caffeine in Noninvasive Respiratory Support

“…These effects include improving lung compliance and airway resistance, 28,29 increasing minute ventilation 28,30 , increasing diaphragm muscle contractility 31,32 decreasing lung inflammation 33–35 and improving airway remodeling 36 . Although it is unclear which of these potential mechanisms are responsible for the clinical benefits of caffeine observed in large clinical trials, it is likely that multiple mechanisms, beyond a reduction in apnea, are responsible for the benefits of caffeine on pulmonary health.…”

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts

“…We recently demonstrated that caffeine is able to antagonize TGF-β1 induced upregulation of CTGF on the transcriptional and translational level [47] and that gene expression-related additive and synergistic effects exist for caffeine in combination with dexamethasone [48, 49]. At higher concentrations, caffeine may act as an unspecific inhibitor of PDEs increasing intracellular levels of cAMP.…”

A new target for caffeine in the developing lung: endoplasmic reticulum stress?

“…The effects of caffeine on limiting inflammation and other physiological processes in adult animals in response to proinflammatory stimuli such as hyperoxia (18), LPS application (29), and ischemia-reperfusion injury (8) have also been described. Further information has been gleaned from in vitro studies, where in epithelial cells, caffeine promotes cell-cycle arrest (14,36), drives surfactant protein production (11)(12)(13), promotes apoptosis (37), and modulates TGF-␤ signaling (14,28). In lung fibroblasts and fibroblast-like cell lines, caffeine impacts peroxynitrite-mediated matrix metalloproteinase production (33), modulates glucocorticoid effects in the lungs (12), and modulates TGF-␤ signaling (12,28).…”