Background Lung diseases related to collagen disorder are often accompanied by fibrosis and TGF-β is one of the essential mediators in tissue fibrosis. Leucine rich alpha-2 glycoprotein (LRG) is a novel serum protein that is increased in patients with inflammatory diseases, such as rheumatoid arthritis. LRG expression is increased in inflamed tissues, suggesting that the molecule is involved in the pathogenesis of the diseases. Since LRG has recently been reported as a modulator of TGF-β signals  , LRG may play a role in tissue fibrosis.
Objectives In this study, we aimed to investigate the involvement of LRG in a murine model of lung fibrosis.
Methods Wild type mice and LRG knockout (KO) mice were intratrachally treated with bleomycin and fibrosis were evaluated 21 days after the treatment. At the time of sacrifice, the lungs were harvested and processed for histological examination using hematoxylin and eosin staining, detection of collagen fibers using azan staining, and immunohistochemistry. Furthermore, the effect of LRG on TGF-β signaling was investigated using L929 mouse fibroblast cell line.
Results Intratracheal administration of bleomycin increased LRG protein levels in bronchial alveolar lavage fluids (BALF) and lung tissues. When compared to wild type mice, fibrosis was markedly inhibited in LRG knockout (KO) mice, as evalulated by azan staining and by hydroxyproline assay. Although there was no significant difference in BALF TGF-β levels between WT and LRG KO mice, phosphorylation and nuclear translocation of Smad2 protein were attenuated in the lung of LRG KO mice. In vitro experiments using L929 revealed that LRG enhanced TGF-β-induced Smad2 phosphorylation and the expression of downstream genes, such as plasminogen activator inhibitor-1 (PAI-1) and alpha smooth muscle actin (SMA). Furthermore, TGF-β-induced Smad2 phosphorylation was enhanced by exogenous LRG protein in a dose-dependent manner. Although LRG has been reported to interact with endoglin, an accessory receptor of TGF-β, knockdown of endoglin showed no effect on LRG-induced enhancement of Smad2 activation.
Conclusions These data demonstrate that LRG enhances TGF-β-induced activation of Smad2 cascade in fibroblasts leading to promote fibrosis in murine model of lung fibrosis.
Nature 2013 Jul 18; 499 (7458):306–11
Oncotarget 2015 May 10; 6 (13):11009–22
Acknowledgement We wish to thank K. Omori and Y. Maeda for their advice and expertise, E. Harada and R. Nishi for their technical assistance and the staff of Animal Bioscience Department of the Nagahama Institute of Bio-Science and Technology for their help in histopathological evaluation.
Disclosure of Interest None declared
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