Differential regulation of extracellular matrix and soluble fibulin-1 levels by TGF-β₁ in airway smooth muscle cells.

Ling Chen,Qi Ge,Linhong Deng,Brian G G Oliver,Judith L Black,Janette K Burgess,Zhongjun Zhou

doi:10.1371/journal.pone.0065544

Abstract

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β1) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×104 cells/cm2, and stimulated with or without TGF-β1 (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β1 decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β1 did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β1 induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β1 induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β1 is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.

Highlights

Chronic obstructive pulmonary disease (COPD), a common preventable and treatable disease, is characterised by airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases [1]
As the deposition of FBLN-1 into the extracellular matrix (ECM) was increased by transforming growth factor beta 1 (TGF-b1), cellular FBLN-1 was assessed by western blotting to confirm this up regulation
After 72 hours of stimulation with 10 ng/ml TGF-b1, the FBLN-1 was significantly up regulated in both COPD (p,0.001, n = 8) and non-COPD (p,0.05, n = 7) airway smooth muscle (ASM) cells, and there was no difference between the COPD and non-COPD groups in the fold change of cellular FBLN-1 induced by TGF-b1 (Fig. 1B, 1C)

Summary

Introduction

Chronic obstructive pulmonary disease (COPD), a common preventable and treatable disease, is characterised by airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases [1]. It is known that the extracellular matrix (ECM) is altered in the airway walls of patients with COPD [2,3]. The ECM is a complex structured network of secreted macromolecules and proteolytic enzymes that provide the basis of cell-cell and cell-matrix interactions. ECM components fundamentally influence the structure and function of airways. Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with ECM formation and rebuilding. We have previously found elevated FBLN-1 in the serum and lung washing fluid (bronchoalveolar lavage) of people with asthma, and shown FBLN-1 to regulate airway smooth muscle (ASM) cell proliferation, highlighting the potential role of FBLN-1 in airway wall remodelling [13]

Objectives

Methods

Results

Conclusion