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

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....

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Main Authors:	Fehrholz, Markus, Speer, Christian P., Kunzmann, Steffen
Format:	Online
Language:	English
Published:	Public Library of Science 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020861/

id	pubmed-4020861
recordtype	oai_dc
spelling	pubmed-40208612014-05-21 Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells Fehrholz, Markus Speer, Christian P. Kunzmann, Steffen Research Article 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. Public Library of Science 2014-05-14 /pmc/articles/PMC4020861/ /pubmed/24828686 http://dx.doi.org/10.1371/journal.pone.0097357 Text en © 2014 Fehrholz et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Fehrholz, Markus Speer, Christian P. Kunzmann, Steffen
spellingShingle	Fehrholz, Markus Speer, Christian P. Kunzmann, Steffen Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
author_facet	Fehrholz, Markus Speer, Christian P. Kunzmann, Steffen
author_sort	Fehrholz, Markus
title	Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
title_short	Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
title_full	Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
title_fullStr	Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
title_full_unstemmed	Caffeine and Rolipram Affect Smad Signalling and TGF-β1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells
title_sort	caffeine and rolipram affect smad signalling and tgf-β1 stimulated ctgf and transgelin expression in lung epithelial cells
description	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.
publisher	Public Library of Science
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020861/
_version_	1612088948810579968

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

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