Bone Morphogenetic Protein Receptor II Is a Novel Mediator of Endothelial Nitric-oxide Synthase Activation

Abstract

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS proteinprotein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6 -22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor L-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/ BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH. Pulmonary arterial hypertension (PAH)2 is a devastating disease, characterized by increasing pulmonary arterial pressures, leading to right heart failure and eventually, death (1, 2). Recent studies demonstrate an important role for BMPRII in the pathogenesis of the disease when mutations to the BMPR2 gene were identified as a major underlying cause of heritable or familial PAH (FPAH) (3-5). Approximately 144 different mutations in 210 patients with FPAH have now been identified (6 -8). Additionally, it is estimated that 20% of patients with idiopathic PAH (IPAH) harbor mutations to the BMPR2 gene. These mutations may be a previously undetected inherited mutation or arise from sporadic mutation of the gene.BMPRII is widely expressed in normal tissues. In the lung vasculature, BMPRII is most highly expressed on endothelial cells (9) and at lower levels in smooth muscle cells and fibroblasts. Expression of BMPRII is markedly reduced in the pulmonary vasculature of patients with FPAH and IPAH, regardless of whether they harbor mutations in the BMPR2 gene (9).Pulmonary artery smooth muscle cells (PASMCs) from patients with mutations in BMPRII have a reduced capacity to activate Smad1/5. This is coupled with a reduced ability of BMPs to inhibit proliferation of PASMCs (10). In contrast, the BMPRII ligands BMP2 and BMP4 promote proliferation, migration, and survival in PAECs (11, 12). The opposite effects of BMPs on PAECs and PASMCs provide a convincing model for pulmonary vascular damage in PAH, where a loss of BMP...

Genotype‐Phenotype Effects of Bmpr2 Mutations on Disease Severity in Mouse Models of Pulmonary Hypertension

“…These findings are consistent with the observation that BMP ligands enhance NO production and induce phosphorylation of Ser1177 eNOS in cultured PECs 34,54 and that there are decreased eNOS activity and pSer1177 eNOS levels in the pulmonary vasculature of Bmpr2 ΔEx2/+ mutant mice and in PECs from ΔEx2/+ (ΔE2/+) mice. A, Western blots of lung lysates, demonstrating phosphorylated S1177 and T495 (pS1177 and pT495, respectively) and total eNOS levels and β-actin.…”

“…25,54 However, these findings also suggest that the differential regulation of pSer1177 eNOS is unlikely to account for differences in PH severity in Bmpr2 +/− and Bmpr2…”

Human Primary Lung Endothelial Cells in Culture

“…Additionally, all glassware and plastics used for cell culture were opened in a sterile work environment. This protocol is detailed to give investigators the ability to study more reliably the role of pulmonary endothelial cells in lung vascular diseases (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

TGFβ Signaling and Cardiovascular Diseases

“…This was shown to be due to decreased endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2+/-but not Bmp4+/-mice. It was also shown that activation of BMPRII promotes pulmonary artery EC (PAEC) survival, proliferation, and migration by activating eNOS [125]. BMP2 and BMP4 were shown to stimulate eNOS phosphorylation resulting in increased eNOS activity.…”