Investigation of the Effects of Extracellular Matrix on TGF-β-induced Fibrotic Gene Expression

Abstract

Idiopathic pulmonary fibrosis (IPF) results from the excess accumulation of lung extracellular matrix (ECM). Fibronectin (FN) is a key ECM protein that is essential for processes like tissue development and repair and cell adhesion and migration. FN’s assembly into fibrils and its many binding sites for various ECM proteins provide a platform for other ECM proteins to bind. However, the dysregulation of secreted FN fibrils and excess accumulation of lung ECM can lead to IPF. Transforming growth factor-β (TGF-β) is a major signaling pathway that drives FN expression in normal human lung fibroblasts (NHLFs) and the progression of fibrosis. TGF-β has been shown to upregulate FN gene expression and matrix assembly, as well as a corresponding FN-binding protein signature consisting of a subset of FN-binding partners whose expression significantly increases upon the onset of IPF. It is unknown, however, whether the substrate in which cells grow induces increased expression, or whether potential substrate effects synergize with those of TGF-β to cause this upregulation of ECM genes characteristic of IPF. I performed immunoblots for FN and tenascin-C (TNC), a FN-binding ECM protein related to fibrosis, using SDS cell lysates of NHLFs grown on poly-D-lysine (PDL), a non-ECM control substrate, as well as FN and laminin-coated surfaces for 24 and 48 hours. On different ECM substrates without TGF-β treatment, no significant effects on protein production were detected. With addition of TGF-β for 48 hours, a slight increase in FN expression was detected in cells on bFN substrate and synergy between MG and TGF-β was suggested by a nearly two-fold increase in TNC expression. These results suggest that with extended treatment, TGF-β may have substrate-specific effects on NHLF ECM production.