Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc).
Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3<sup>rd</sup> culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR.
CD31+CD102+ ECs isolated from SSc lungs expressed high levels of mesenchymal cell-specific genes (type I collagen, type III collagen, and fibronectin), EC-specific genes (type IV collagen and VE-cadherin), profibrotic genes (transforming growth factor β1 and connective tissue growth factor), and genes encoding EndoMT-related transcription factors (TWIST1 and SNAI2).
Compared to normal fibroblast (NF), the expression of collagen and fibronectin in SSc (SScF) dramatically increased, and this could be reduced by Astragaloside IV (AST) in a dose- or time-dependent manner at both protein and mRNA levels.
Our investigations revealed that roscovitine coordinately inhibited the expression of collagen, fibronectin, and connective tissue growth factor (CTGF) in normal and SSc fibroblasts.
Imatinib mesylate efficiently reduced basal synthesis of COL1A1, COL1A2, and fibronectin 1 messenger RNA in SSc and normal dermal fibroblasts, in a dose-dependent manner.
These results strongly suggest the contribution of p38 MAPK signaling to the TGF-beta-mediated regulation of the human alpha2(I) collagen gene in normal dermal fibroblasts and constitutive upregulated expression of type I collagen and fibronectin in SSc fibroblasts.
In the present study we analysed the contribution of transcriptional activity and post-transcriptional transcript stability to the increases in pro-alpha 1(I) collagen and fibronectin mRNA steady-state levels in activated (scleroderma) fibroblasts.
Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients.
The effects of TNF-alpha on collagens I, III and VI, fibronectin and collagenase gene expression by fibroblasts derived from normal individuals and patients with systemic sclerosis (SSc) were studied.
Our data confirm abnormal regulation of fibronectin gene expression in SSc fibroblasts and suggest increased sensitivity to TGF-beta by some SSc fibroblast strains.