BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

BMP9 is a pleiotropic growth factor cytokine with diverse roles in vascular development, homeostasis and disease. BMP9 regulates a broad array of vasoactive molecules that mediate endothelial and mural cell function, including ET-1, a potent vasoconstrictor, regulator of cell growth and fibrosis, and therapeutic target for pulmonary arterial hypertension (PAH). Consistent with its pleiotropic activities, BMP9 is unique in being able to recruit both BMP-responsive SMAD1/5/9 and TGF{beta}-responsive SMAD2/3 signaling effectors in endothelial cells, however, the physiologic significance of activating both pathways remains incompletely defined. We investigated the role of SMAD1/5/9 vs. SMAD2/3 signaling in BMP9-mediated regulation of ET-1, using primary and immortalized human and murine microvascular endothelial cells, with conditional knockout, small molecule inhibitor and siRNA strategies. BMP9-mediated expression of ET-1 requires coordinated activation of SMAD1/5/9 and SMAD2/3 effector pathways, both downstream of BMPR2, ALK1, and ENG. Analysis of the ET-1 promoter revealed that BMP9 requires, in addition to a SMAD3/4 binding site sufficient for TGF{beta}1-mediated transcription, a novel putative SMAD1/5 binding motif. BMP9-mediated regulation of endothelial ET-1 requires coordinated activation of both SMAD1/5 and SMAD2/3 downstream of ALK1, integrated at the promoter level, representing a non-canonical signaling motif linking BMP9 to a critical effector of vascular tone and remodeling in PAH and related vascular syndromes. Translational PerspectiveBMP9 has recently emerged as a novel therapeutic target with an ongoing Phase 2 clinical trial testing an anti-BMP9 antibody for efficacy in PAH (NCT06137742). The mechanisms by which BMP9 contributes to pulmonary vascular disease are thought to include its regulation of Endothelin-1 (ET-1), an established therapeutic target in PAH. We show that BMP9 potently regulates ET-1 transcription via transactivation of TGF{beta}1 effector SMAD3 via BMP receptors BMPR2 and ALK1, suggesting that pleiotropic effects of this growth factor cytokine result from non-canonical activation of BMP and TGF{beta} signaling pathways, with implications for its roles in physiology, and the rationale for its therapeutic modulation for PAH.