D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts

Abstract

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated “M10,” with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10

“…We have discovered a ten amino acid peptide, “M10”, which is derived from the C-terminal part of the MET receptor tyrosine kinase via naturally occurring caspase-3 mediated cleavage [ 5 , 6 ]. We showed that M10 demonstrates profound antifibrotic effects in the bleomycin-induced mouse model of pulmonary fibrosis and in primary human lung and skin fibroblasts isolated from patients with scleroderma-associated pulmonary fibrosis [ 5 ].…”

“…To develop an indirect ELISA for M10 quantification, we used a commercially available anti-Met C12 antibody generated against the last 12 amino acids of MET, which recognizes M10 as an antigen but does not recognize a scrambled peptide used as a control [ 5 , 6 ]. We have established an ELISA method using synthetic M10 as a coating agent that yielded a sensitivity of 9.6 ng/ml and analytical recoveries between 89.9% and 112.9%.…”

“…Although mice do not generate endogenic M10, synthetic M10 demonstrates intense antifibrotic effects in a mouse model of bleomycin-induced pulmonary fibrosis [ 5 ]. Previously, we showed that cleavage of MET and generation of endogenous M10 occurs in the lungs of patients with scleroderma associated pulmonary fibrosis [ 6 ]. We speculate that concentrations of endogenous M10 could be high enough to modify disease severity.…”

“…We recently discovered a 10 amino acid antifibrotic peptide (M10) with antifibrotic properties in vitro and in a murine model of lung fibrosis, which is a natural cleavage product by caspase-3 of the cytoplasmic tail of the c-MET receptor tyrosine kinase [ 5 , 6 ]. Following binding of hepatocyte growth factor (HGF), c-MET undergoes auto phosphorylation at tyrosine residues in its cytoplasmic domain and initiates a cascade of signal transduction events leading to specific cellular responses implicated in embryonic development and tissue regeneration after injury [ 7 – 10 ].…”

Critical Role of LMCD1 in Promoting Profibrotic Characteristics of Lung Myofibroblasts in Experimental and Scleroderma‐Associated Lung Fibrosis

“…Lung fibroblasts were isolated as previously described (33,34) and used between the third and sixth passages in all experiments. LMCD1 siRNA (150 pmoles per 100-mm dish, 75 pmoles/well for 6-well plates, and 15 pmoles/well for 24-well plates) from Santa Cruz Biotechnology was transfected into cells as previously described (35).…”