FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension
Edda Spiekerkoetter, … , Peter ten Dijke, Marlene Rabinovitch
Edda Spiekerkoetter, … , Peter ten Dijke, Marlene Rabinovitch
Published July 15, 2013
Citation Information: J Clin Invest. 2013;123(8):3600-3613. https://doi.org/10.1172/JCI65592.
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Research Article Vascular biology

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Abstract

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Authors

Edda Spiekerkoetter, Xuefei Tian, Jie Cai, Rachel K. Hopper, Deepti Sudheendra, Caiyun G. Li, Nesrine El-Bizri, Hirofumi Sawada, Roxanna Haghighat, Roshelle Chan, Leila Haghighat, Vinicio de Jesus Perez, Lingli Wang, Sushma Reddy, Mingming Zhao, Daniel Bernstein, David E. Solow-Cordero, Philip A. Beachy, Thomas J. Wandless, Peter ten Dijke, Marlene Rabinovitch

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Figure 1

High-dose FK506, the main activator of BMP signaling found in the high-throughput screen, recapitulates BMP4 signaling and function.

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High-dose FK506, the main activator of BMP signaling found in the high-t...
(A) BRE-luciferase activity in different numbers of C2C12 cells at different concentrations of BMP4. BMP4 at the EC-20 concentration of 250 pM for 1,500 cells per 60 μl per well permitted identification of coactivators. (B) Percentage BRE-luc activation, compared with 250 pM BMP4, in 6-point serial dilution for FK506 and rapamycin. (C) BRE-luciferase activity in C2C12 cells comparing FK506 (FK; 2 μg/ml), rapamycin (Rapa; 4 μg/ml), cyclosporine (Cyclo; 2 μg/ml), and Shield-1 (1 μg/ml) (n = 8; ***P < 0.001 vs. CON, ###P < 0.001 vs. FK506, §§§P < 0.001 vs. cyclosporine, 1-way ANOVA, Bonferroni multiple-comparison test). (D) In human PAECs, ID1 expression relative to B2M mRNA 24 hours after stimulation with vehicle (CON), FK506 (15 ng/ml), or rapamycin (10 ng/ml) (n = 3; **P < 0.01 vs. vehicle, 1-way ANOVA, Dunnett’s post test). (E and F) Representative Western immunoblot and relative densitometric analysis of PAECs showing (E) pSMAD1/5/8 and (F) ID1 relative to β-actin following stimulation with BMP4 (10 ng/ml) or FK506 (15 ng/ml) (n = 3; *P < 0.05; **P < 0.01 vs. CON, 2-way ANOVA). (G) Assessment of apoptosis with measurement of caspase-3/7 luminescence 24 hours after serum starvation and treatment with BMP4 (10 ng/ml) or FK506 (15 ng/ml). (n = 5; *P < 0.05; **P < 0.01 vs. CON, 1-way ANOVA, Dunnett’s post test). (H) Representative images from a Matrigel tube formation assay. Tube number and length were assessed after 8 hours in unstimulated cells (CON) or following stimulation with VEGF (20 ng/ml), BMP4 (10 ng/ml), or FK506 (15 ng/ml). (n = 6; *P < 0.05; **P < 0.01 vs. CON, 1-way ANOVA, Dunnett’s post test). Scale bar: 100 μm. Mean ± SEM.