Background/Purpose: We recently identified M10, a caspase cleavage product of the hepatocyte growth factor receptor, as an anti-fibrotic peptide that interacts with Smad2 and inhibits TGFβ-induced Smad2 phosphorylation and collagen production in human lung fibroblasts [1]. While Smad2/3 signaling pathway is mainly activated by TGFβ1 through type I receptor activin receptor-like kinase (ALK) 5, Smad1/5 signaling pathway is mainly activated by bone morphogenetic proteins (BMPs), which are other members of TGFβ superfamily, through ALK1/2/3/6 receptors. Recently, BMP9 has been identified as a pro-fibrotic ligand in mouse embryo fibroblasts [2]. In SSc fibroblasts, ALK1/Smad1/5 pathway is suggested to play a pivotal role in the regulation of fibrosis [3]. The aims of this study are to investigate the role of BMP9 in SSc lung fibroblasts and to examine the additional anti-fibrotic mechanisms of M10.

Methods: Fibroblasts were derived from lung tissues obtained at autopsy from SSc patients and from age- race-, and sex-matched normal subjects. MRC5 human fetal lung fibroblasts were purchased from Sigma. Potential peptide-protein interactions were modeled in-silico using PepSite [4]. Smad phosphorylation, type I collagen, and smooth muscle α-actin (α-SMA) expression were determined by immunoblotting and RT-PCR.

Results: Using a computational modulation approach available from PepSite, we found a statistically significant (p < 0.0001) potential interaction of M10 with the BMP9/ALK1/activin receptor type II B (ActRIIB) complex. The most probable binding sites are located at β-turn motifs in BMP9. We demonstrate that when recombinant BMP9 is added to the medium, phosphorylation of Smad1/5/8 is rapidly induced in MRC5 cells and SSc lung fibroblasts in a dose-dependent manner. The expression of type I collagen is upregulated after 48 hours treatment with BMP9 in MRC5 cells and SSc lung fibroblasts as well as normal lung fibroblasts. The expression of α-SMA is increased in normal lung fibroblasts in a dose dependent manner. Intriguingly, the anti-fibrotic peptide M10 significantly downregulated BMP9-induced type I collagen expression and α-SMA expression as well as Smad1/5/8 phosphorylation in lung fibroblasts.

Conclusion: We demonstrate that BMP9 shows pro-fibrotic effects in human fetal lung fibroblasts and in adult lung fibroblasts obtained from SSc patients and matched normal subjects. M10 peptide has a potential to bind to BMP9 and to inhibit BMP9-induced collagen production and epithelial-mesenchymal transition through the Smad1/5/8 signaling pathway.

References:

[1] Atanelishvili I, et al. Transl Res. 2016;170:99-111.

[2] Munoz-Felix JM, et al. Cell Signal. 2016;28:1252-61.

[3] Munoz-Felix JM, et al. Cytokine Growth Factor Rev. 2013;24:523-37.

[4] Trabuco LG, et al. Nucleic Acids Res. 2012;40:W423-7.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/m10-a-caspase-cleavage-product-of-the-hepatocyte-growth-factor-receptor-downregulates-bone-morphogenetic-protein-9-induced-smad158-phosphorylation-and-collagen-production-in-human-lung-fibroblasts/