Gene expression profiling reveals novel TGFβ targets in adult lung fibroblasts

Elisabetta A Renzoni,Andrew G Nicholson,Sarah Howat,George Bou-Gharios,David J Abraham,Kenneth I Welsh,Jeremy D Pearson,Xu Shi-Wen,Srihari Veeraraghavan,Athol U Wells,Piersante Sestini,Roland M Du Bois,Andrew Leask,Carol M Black,Christopher P Denton

doi:10.1186/1465-9921-5-24

Abstract

BackgroundTransforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFβ on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown.MethodsWe used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFβ in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFβ-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold.ResultsExposure of fibroblasts to TGFβ had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFβ in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules.Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis.ConclusionsThis study identifies several novel TGFβ targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis.

Highlights

Transforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role
Cell culture Primary adult lung fibroblasts were cultured from three control samples and from open-lung biopsy samples of lung fibrosis patients, three with idiopathic pulmonary fibrosis (IPF) [8] and three with pulmonary fibrosis associated with the fibrotic disease systemic sclerosis [9]
We did not observe a substantial degree of difference in the response to TGFβ between the two fibrotic groups and control lung fibroblasts

Summary

Introduction

Transforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Transforming Growth Factor beta (TGFβ) is a multifunctional cytokine that regulates a variety of physiological processes, including cell growth and differentiation, extracellular matrix production, embryonic development and wound healing [1]. Animal models support a central role played by TGFβ in lung fibrosis. Intra-tracheal adenovirus-mediated TGFβ gene transfer causes severe lung fibrosis extending to the periphery of the lungs [5]. Mice lacking alphavbeta 6, an integrin which is crucial to the release of active TGFβ from latent extracellular complexes, develop lung inflammation but are strikingly protected from bleomycin-induced lung fibrosis [10]. Experimental inhibition of TGFβ with neutralizing antibodies, soluble receptors, or gene transfer of the TGFβ inhibitor Smad, inhibits fibrosis in animal models [12,13,14]

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