Differential Regulation of Extracellular Matrix and Soluble Fibulin-1 Levels by TGF-β1 in Airway Smooth Muscle Cells

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.

Fibulin-1 regulates the pathogenesis of tissue remodeling in respiratory diseases

“…Several types of mesenchymal cells are capable of producing Fbln1, and in the airways and lungs, there is likely not a single cellular source. In previous studies, we showed that ASM cells (13) and fibroblasts (14, 17) produce Fbln1. We also showed that Fbln1 is not upregulated by TGF-β (17), but TGF-β–stimulated fibroblasts can incorporate exogenously produced Fbln1 into the ECM.…”

“…In IPF, ECM proteins are increased in both plasma and lung tissue (15), and Fbln1c variant–specific peptide increases the proliferation of lung fibroblasts, the main producers of ECM, in COPD and IPF patients (14). Cytokines associated with lung diseases, such as TGF-β, that are known to induce ECM proteins (16) also stimulate Fbln1 production in ASM cells from COPD patients, further indicating roles for Fbln1 in airway remodeling (17). Nevertheless, the in vivo function of Fbln1 in tissue remodeling in chronic pulmonary diseases is poorly understood.…”

Effects of cigarette smoke extract on human airway smooth muscle cells in COPD

“…Cell culture and sample preparation Human ASM cells were obtained from human lung by a method modified from one described previously [16]. Human ASM cells were microdissected from approximately sixth-order or greater bronchi, and were initially cultured in growth medium comprised of DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (DKSH, Melbourne, Australia), 1% antibiotics (Invitrogen) and 25 mM Hepes (Invitrogen).…”

“…Human ASM cells were seeded in 96-well plates as described previously, and cells were stimulated with different concentrations of CSE or 10 ng?mL -1 of TGF-b1 for 72 h. Cell-free ECM plates were used to measure the deposition of protein in the ECM by ELISA according to the previously modified method [16]. Primary antibodies used for detecting ECM proteins were rabbit polyclonal anti-human COL5 antibody (2 mg?mL antibodies.…”

Calumenin and fibulin-1 on tumor metastasis: Implications for pharmacology

“…As an important extracellular adhesion molecule, fibulin-1 is deregulated in a lot of diseases, such as type II diabetes [35][36][37], cardiovascular diseases [38][39][40][41], respiratory diseases [42][43][44], liver diseases [45], bacterial infection [46], as well as cancer. However, the role of fibulin-1 in cancer is still elusive.…”