Background Mutations in BMP type II receptor (BMPR-II) account for 70% of heritable pulmonary arterial hypertension (PAH) cases, but low penetrance (~20%) in mutation carriers implies a ‘second hit’ is required for disease initiation. Inflammation has been implicated, yet the molecular mechanisms by which it influences pathology are unclear. Tumour necrosis factor alpha (TNFα) has been reported to influence BMPR-II expression, yet the molecular interplay remains unclear.
Methods Human pulmonary arterial smooth muscle cells (PASMCs) were stimulated with TNFα (1ng/mL). Biochemical (immunoprecipitation, western blotting), molecular biology (quantitative PCR, plasmid DNA transfection, site-directed mutagenesis, RNA interference) and pharmacological (metalloprotease, proteasome and lysosome inhibitors) approaches were used to assess the impact of TNFα on BMPR-II and BMP signalling pathways. To assess this in vivo, two animal models of PAH were utilised: a mouse model overexpressing TNFα specifically in the lung as well as a moncrotaline induced PAH (MCT-PAH) rat model.
Results TNFα stimulation reduced BMPR-II mRNA and protein expression, leading to loss of BMP signalling, as evidenced by abrogated Smad 1/5 and ID1 activation. Notably, a low molecular weight form of BMPR-II accumulated in PASMC lysates following prolonged TNFα exposure: identified as a C-terminal cleavage product of BMPR-II. Furthermore, the N-terminal ectodomain of BMPR-II could be immunoprecipitated from PASMC conditioned media and was quantified by ELISA. TNFα increased expression of two A Disintegrin and Metalloproteinase Domain-containing proteins (ADAMs); ADAM10/17. Pharmacological blockade and RNA interference revealed both proteases were capable of BMPR-II cleavage. Mutation of the putative cleavage site restored BMP signalling. The cleaved ectodomain acted as a ligand trap, sequestering BMP ligands and inhibiting their signalling capacity. Proliferation assays revealed loss of BMP2/4 induced PASMC anti-proliferation in the presence of BMPR-II ectodomain. Finally, both animal models revealed reduced BMPR-II and c-terminal cleavage product in lung tissue - highlighting this event occurs in vivo.
Conclusions We identified a novel mechanism by which TNFα impairs BMP signalling and promotes PASMC proliferation in the lung vasculature. TNFa may provide the critical link between inflammation and disease initiation in PAH. Our in vivo observations highlight TNFα as a potential therapeutic target in PAH.
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