Background/Purpose

Systemic sclerosis (SSc) is an irreversible fibrotic disorder with interstitial lung disease (ILD) being a major complication and leading cause of mortality. African American SSc patients exhibit higher prevalence of ILD and worse outcomes than those of other races. We previously reported that a cell-protective and antifibrotic factor, hepatocyte growth factor (HGF), is downregulated in bronchoalveolar lavage fluid and plasma from African American SSc-ILD patients compared with white SSc-ILD patients. Here we report a newly identified C-terminal fragment of the HGF receptor, designated as “M10”, as a peptide with robust antifibrotic properties that is lacking in certain African American SSc-ILD patients.

Methods

Lung tissue was collected postmortem from SSc patients with ILD and RNA was extracted from lung fibroblasts. The coding exons of the HGF receptor, MET (mesenchymal-epithelial transition factor), were amplified by PCR, and sequences were analyzed. Adenoviruses carrying wild type MET gene or the D1398G mutant observed in African American patients were generated. Lung fibroblasts were infected by either type of adenovirus and treated with HGF, transforming growth factor-β (TGF-β), and the M10 peptide. MET phosphorylation, connective tissue growth factor (CTGF, CCN2) and collagen expression was examined by immunoblotting.  Potential peptide-protein interactions were modulated in-silico.

Results

We have identified the D1398G mutation in African American SSc-ILD patients whose MET signaling is impaired. When we compared MET phosphorylation following HGF treatment between normal lung fibroblasts expressing wild type or the D1398G MET, we found that the D1398G mutant showed reduced MET phosphorylation. Additionally, normal fibroblasts expressing the D1398G mutant exhibited a diminished reduction in CTGF and collagen expression following HGF treatment compared with wild type. Sequence analysis revealed that the D1398G mutation is located at the terminal amino acid sequence of “DEVD” which is a Caspase-3 cleavage motif, suggesting that the D1398G MET mutant is incapable of generating the terminal 10-amino-acid-fragment of MET, M10. We found that synthetic M10 effectively reduces, in a dose-dependent manner, collagen and CTGF in SSc fibroblasts. Computational molecular modeling based on the peptide-binding sites from protein surfaces predicts that M10 may negatively regulate TGF-β signaling by an interaction with protein domains identified as 2KXQ and 1U7V (Protein Data Bank).

Conclusion

A D1398G mutation in MET is associated with impaired phosphorylation and reduced HGF signaling in African American SSc-ILD lung fibroblasts. On the other hand, the M10 peptide generated from wild type MET signaling demonstrates strong antifibrotic effects and should be considered as a potential therapeutic agent for SSc-ILD and other fibrosing diseases.

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« Back to ACR/ARHP Annual Meeting 2014

ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-fibrotic-effects-of-a-newly-discovered-hgf-receptor-carboxy-terminal-fragment-in-systemic-sclerosis/