Binding of Hypoxia-Induced Mitogenic Factor/RELM-β to Bone Morphogenetic Protein Receptor 2 Complex Promotes Pulmonary Hypertension

BACKGROUND: HIMF (hypoxia-induced mitogenic factor) induces pulmonary hypertension; however, the molecular nature of its extracellular membrane receptor(s) remains unknown. METHODS: A combination of cross-immunoprecipitation and immunoblotting, yeast 2-hybrid assays, and proteomics analysis was performed to screen and identify the candidate receptors. The interaction of HIMF with these candidate(s) was further evaluated using overexpression, silencing, point mutation, and blocking peptide strategies in chronic hypoxia and sugen/hypoxia pulmonary hypertension rat models to decipher the underlying pathophysiologic mechanisms. RESULTS: Cross-immunoprecipitation and immunoblotting identified HIMF interaction with the BMPR2 (bone morphogenetic protein receptor 2) complex. Yeast 2-hybrid revealed HIMF binding to the DTLPF motif (Asp-Thr-leucine-Pro-Phe [aspartic acid-threonine-leucine-proline-phenylalanine]) at the 54 to 58 amino acids (aa) in the extracellular domain of the BMPR1A (bone morphogenetic protein receptor 1A), one partner of the BMPR2 heterodimeric complex, but not BMPR2 itself. HIMF and human counterpart RELM (resistin-like molecule)-β binding to BMPR1A disrupted the BMPR1A/BMPR2 complex and decreased BMPR2 activity as revealed by reduced downstream events including ≈43% to 56% decline in Smad1/5/9 (Sma- and Mad-related protein 1/5/9) phosphorylation and ≈41% to 60% decrease in Id-1 (inhibitor of DNA binding 1) expression. This dynamic induced pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling leading to pulmonary hypertension. A mutated motif in rats or a blocking peptide targeting this motif restrained HIMF binding with BMPR1A, rescued BMPR2 activity by ≈25% increase in Smad/1/5/9 phosphorylation and ≈39% elevation in Id-1 expression, and attenuated chronic hypoxia or sugen/hypoxia-induced pulmonary hypertension. CONCLUSIONS: HIMF induced pulmonary hypertension by direct binding to BMPR1A ectodomain, subsequently disrupting its binding to BMPR2 and BMPR2 activity. This HIMF signaling pathway represents a potential therapeutic target by selectively interfering with BMPR1A binding.