Increased expression of bone morphogenetic protein-7 and its related pathway provides an anti-fibrotic effect on silica induced fibrosis in vitro

Abstract

Objective: to better understand the effect and mechanism of modulating endogenous BMP-7 expression on silica induced fibrosis, and to investigate whether the fibrotic processes were regulated by pretreatment with small organic molecules in vitro. Methods: we established BMP-7 over-expressing (BMP-7(+)/RLE-6TN) and BMP-7 silent (BMP-7(−)/RLE-6TN) cell lines by lentivirus mediated gene transduction in RLE-6TN, a kind of alveolar type II epithelial cells. For inhibition studies, the inhibitor of the TGF-β/activin type I receptor (SB-431542) or BMP receptor (LDN-193189) was used to pretreat the RLE-6TN cells. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp). Using qPCR and western blotting, the mRNA and protein levels of BMP-7, fibronectin (FN), collagen I and collagen III were detected. The Smad signaling pathway proteins, including phosphorylated Smad1/5 (P-Smad1/5) and phosphorylated Smad2/3 (P-Smad2/3) were detected using western blotting. In addition, the MTT assay was used to explore the toxic effects of SB-431542 and LDN-193189. Results: results showed that pretreating with SB-431542 or over-expressed BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3 and fibrosis-related markers (collagen I, collagen III, and FN). On the contrary, using LDN-193189 or small hairpin RNA (shRNA) against BMP-7 could promote the secretion of Hyp and fibrosis-related mRNA and protein expression by increasing P-Smad2/3 and decreasing P-Smad1/5. Conclusion: BMP-7 exerts an inhibitory effect on silica induced fibrosis in RLE-6TN cells via a blockade of TGF-β signaling and a restoration of BMP signaling. BMP-7 can encourage the development of new therapeutic agents in silica induced fibrosis.