ALK2 and BMPR2 knockdown and endothelin-1 production by pulmonary microvascular endothelial cells

BackgroundMany cases of pulmonary arterial hypertension (PAH) are heritable and related to gene mutations in bone morphogenic receptor-2 (BMPR2). These patients consequently may have a signaling imbalance within the transforming growth factor beta (TGFβ) receptor superfamily. The causes of increased endothelin-1 (ET-1), which contributes to PAH, are unknown, and we therefore studied the contribution of various BMPs and their receptors on ET-1 production in vitro, after knockdown of BMPR2 in human pulmonary microvascular endothelial cells (HMVEC-LBl).Methodology/principal findingsReceptor knockdown in HMVEC-LBl was performed using siRNA to BMPR2, and activin like-kinases 1 and 2 (ALK1, ALK2). ET-1 and TGFβ levels in the medium were measured by ELISA. In some experiments, cells were exposed to TGFβ or BMP7 or FK506 (tacrolimus). Using Western blotting, levels of BMPR2, endothelin ETB receptor, phosphorylated SMAD 2 (pSMAD 2), phosphorylated SMAD 1,5 (pSMAD 1,5), ALK1, ALK2, ALK5, TGFβ receptor 2, plasminogen activator inhibitor-1 (PAI-1) and ID1 were measured. BMPR2 knockdown significantly increased ET-1 levels. It did not affect ETB receptor or TGFβ levels. TGFβ increased ET-1 levels, with or without BMPR2 knockdown. BMPR2 knockdown did not affect TGFβ (pSMAD 2 and PAI-1) signaling. BMP7 increased ET-1 levels after BMPR2 knockdown but this was prevented by ALK2 knockdown as was the increase in ID1 caused by BMPR2 knockdown. FK506, which interacts with ALK2, increased ET-1 levels and ID1 levels, and this was blocked by ALK2 knockdown.Conclusion/significanceALK2 may be an important receptor in ET-1 production during BMPR2 knockdown.

► Knockdown of the BMPR2 stimulates ET-1 production in human pulmonary microvascular EC.
► Activin like kinase 2 (ALK2) is essential in the control of endothelin-1 production.
► Increased endothelin-1 and ID1 levels following FK506 exposure are dependent on ALK2.
► Knockdown of BMPR2 increases ID1 in microvascular endothelial cells.
► FK506 and knockdown of ALK2 both increase BMPR2 levels.