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.

Figure 1. High-dose FK506, the main activator of BMP signaling found in the high-throughput screen, recapitulates BMP4 signaling and function.

(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.

Figure 2. Low-dose FK506 induces pSMAD1/5/8 and ID1 in human mvPAECs related to FKBP12 interaction.

(A) Id1 mRNA, normalized to 18S mRNA, in human unstimulated mvPAECs (CON) or 1 hour after the addition of BMP4 (10 ng/ml) or FK506 (0.2 ng/ml and 2.0 ng/ml). (B) Representative immunoblot and densitometric analysis of ID1 protein relative to β-actin at time points and doses indicated in A. (A and B) (n = 3; *P < 0.05; **P < 0.01 vs. CON, 1-way ANOVA, Dunnett’s post test). (C) BRE-luciferase activity in C2C12 cells after stimulation with the BMPR2 ligand BMP6 (50 ng/ml) and FK506 (1 μg/ml) for 24 hours, with or without preincubation for 30 minutes with kinase inhibitor LDN-193189 (120 nM) (n = 6; ***P < 0.001 vs. CON; ^§§§P < 0.001 vs. BMP6, 1-way ANOVA, Bonferroni’s multiple-comparison test). (D) BRE-luciferase activity in C2C12 cells treated with nontargeting siRNA (NTsi), BMPR2 siRNA (BMPR2si), BMPR2si plus Activin2A siRNA (ActA2si), and BMPR2si plus Activin2B siRNA (ActBsi) and stimulated with FK506 (1 μg/ml) (n = 6; ^§§P < 0.01 vs. BMPR2si and FK treatment; ^##P < 0.01 vs. BMPR2si-Act2Asi, 1-way ANOVA, Bonferroni’s multiple-comparison test). (E) Immunoprecipitation with an antibody to FLAG and immunoblot for HA and FLAG in 293T cells transfected with plasmids HA-ALK1, HA-ALK2, and HA-ALK3 as well as FKBP12-FLAG and stimulated with FK506 (100 ng/ml for 30 minutes). (F–H). BRE-luciferase activity in C2C12 cells treated with NTsi, ALK1 siRNA (ALK1si), ALK2si, and ALK3si and stimulated with (F) BMP4 (250 pM), (G) FK506 (2 μg/ml), and (H) rapamycin (n = 8; ^##P < 0.01 vs. NTsi plus BMP4, NTsi plus FK506, NTsi plus rapamycin, 1-way ANOVA, Bonferroni’s multiple-comparison test). Mean ± SEM.

Figure 3. Low-dose FK506 rescues IPAH mvPAEC dysfunction.

(A) Representative immunoblot and densitometry showing BMPR2 and ID1/β-actin in human PAECs stimulated with BMP4 (10 ng/ml) or FK506 (15 ng/ml) following knockdown of BMPR2 with BMPR2si compared with NTsi as control (n = 3; *P < 0.05 vs. CON, 2-way ANOVA). (B) Representative immunoblot and densitometry of mvPAECs from 3 patients with IPAH showing ID1/β-actin at baseline (CON) or at various time points after stimulation with BMP4 (10 ng/ml) or FK506 (15 ng/ml) (n = 3; *P < 0.05 vs. CON, 2-way ANOVA). (C) ID1/18S mRNA 4 hours after and apelin/18S mRNA 8 hours after stimulation with BMP4 (10 ng/ml) and low-dose FK506 (0.2 ng/ml) in the same IPAH mvPAECs as in B (n = 3; *P < 0.05; **P < 0.01, vs. CON, 1-way ANOVA, Dunnett’s post test). (D) Representative images and quantitative analysis of tube number and length of tubes formed in Matrigel with mvPAECs from a patient with IPAH 4 hours after unstimulated CON, VEGF (10 ng/ml), BMP4 (10 ng/ml), and FK506 (0.2 ng/ml) (n = 6; ***P < 0.001; **P < 0.01 vs. CON, 1-way ANOVA, Dunnett’s post test; scale bar: 100 μm). (E) Proposed model of activation of BMPR2 in the presence of a subactivating dose of BMPs and an activating dose of BMPs and mutated or dysfunctional BMPR2 receptor with an activating dose of BMPs or with a subactivating dose of BMPs and FK506. Mean ± SEM.

Figure 4. Low-dose FK506 prevents the development of PAH in mice with a deletion of BMPR2 in ECs.

(A and B) LacZ staining documenting BMPR2 deletion in (A) distal PAECs and (B) capillary ECs in EC-Bmpr2^–/– mice. Scale bar: 100 μm. (C) Reduced BMPR2 in whole lung lysates by Western immunoblot comparing wild-type mice with tamoxifen (TAM) with floxed Bmpr2 Cre-ER^T mice with or without tamoxifen. (D) Absence of Bmpr2 mRNA in ECs harvested from EC-Bmpr2^–/– (KO) vs. wild-type mice (n = 3; ***P < 0.001 WT vs. EC-Bmpr2^–/–, 1-way ANOVA, Dunnett’s). (E) RVSP in wild-type and EC-Bmpr2^–/– mice treated with vehicle or FK506 (0.05 mg/kg/d) in normoxia or hypoxia (10% O₂) for 3 weeks (n = 8 per group; **P < 0.01 compared with vehicle normoxia; ^§§P < 0.01 vehicle hypoxia wild-type vs. EC-Bmpr2^–/–, 2-way ANOVA, Dunnett’s). (F) RVH (Fulton index, weight of RV/LV and septum) in same groups as in E. (G) Representative histology of barium-injected lungs of wild-type and EC-Bmpr2^–/– mice after normoxia/hypoxia with or without vehicle or FK506. The decreased number of vessels in hypoxia is ameliorated by FK506 (arrows). Scale bar: 100 μm. (H) Percentage muscularized alveolar wall and alveolar duct arteries per total arteries in wild-type and EC-Bmpr2^–/– mice after normoxia/hypoxia with or without vehicle or FK506 (n = 8 mice;mean of 5 ×20 fields per mouse; **P < 0.01; ***P < 0.001 vs. normoxia wild-type and EC-Bmpr2^–/–, 2-way ANOVA, Dunnett’s). (I) Arteries per 100 alveoli in same animals as in H. (J) Apelin/18S mRNA and (K) eNOS/18S mRNA in mvPAECs from EC-Bmpr2^–/– mice, preconditioned in 1% hypoxia and harvested 8 hours after BMP4 (10 ng/ml) or FK506 (0.2 ng/ml) vs. vehicle (CON) (n = 3; *P < 0.05 vs. CON, 1-way ANOVA, Dunnett’s). Mean ± SEM.

Figure 5. Low-dose FK506 reverses monocrotaline-induced PAH in rats, inhibits proliferation, and induces apoptosis in PASMCs.

(A) RVSP (mmHg) in rats 21 days after monocrotaline (MCT) treatment (60 mg s.c. once), with or without 3-week treatment with vehicle (VEH) or low-dose FK506 (n = 6–8; **P < 0.01 compared with VEH treated; ^#P < 0.05 compared with day 21 monocrotaline; ^§§§P < 0.001 compared with day 42 monocrotaline vehicle, 1-way ANOVA, Bonferroni post test). (B) RVH (Fulton index) in same group as above (***P < 0.001 compared with vehicle treated; ^§P < 0.05 compared with day 42 monocrotaline vehicle, 1-way ANOVA, Bonferroni post test). (C) Represented lung histology (Movat stain) (scale bar: 100 μm). (D) MTT proliferation assay with PASMCs in the presence or absence of PDGF (20 ng/ml) 72 hours after stimulation with vehicle (CON), BMP4 (10 ng/ml), or FK506 (0.2 and 2.0 ng/ml) (n = 5; **P < 0.01, ***P < 0.001, BMP4 vs. CON [in the presence of PDGF], 1-way ANOVA, Dunnett’s). (E) Assessment of apoptosis with measurement of caspase-3/7 luminescence under 24-hour serum starvation or treatment with BMP4 (10 ng/ml) or FK506 (0.2, 1.0, 2.0, and 15 ng/ml) (n = 5; *P < 0.05 1-way ANOVA, Dunnett’s post test). (F) Luciferase NFAT reporter activation assay in PASMCs stimulated with PDGF (30 ng/ml) and doses of FK506 between 0.2 and 15 ng/ml. (^##P < 0.01 and ^###P < 0.001, vs. stimulation by PDGF alone, 1-way ANOVA with Bonferroni’s multiple pairs test). (G) Il2/Gapdh mRNA expression in whole lungs of rats 21 days after monocrotaline treatment (60 mg s.c. once), with or without 3-week treatment with vehicle or low-dose FK506 (n = 6–8; nonsignificant, 2-way ANOVA). Mean ± SEM.

Figure 6. Low-dose FK506 reverses established PAH and neointima formation in rats.

(A) Timeline of experiments. (B) RVSP and RVH (Fulton index) in rats in normoxia with or without vehicle (VEH) or FK506 and in rats after SUGEN with 3 weeks hypoxia and 5 weeks normoxia, sacrificed at 8 weeks and at 11 weeks following treatment with vehicle or FK506 (n = 8; *P < 0.05; ***P < 0.001 vs. same condition in normoxia, 2-way ANOVA, Bonferroni post test). (C) Representative images (scale bar: 100 μm) and percentage vessels with neointima in hypoxia/SUGEN/normoxia rats at 8 and 11 weeks treated with vehicle or FK506 (n = 8; **P < 0.01 vs. vehicle, 1-way ANOVA, Bonferroni post test). (D) Number of alveoli per mm² in normoxia (with or without vehicle or FK506) and in rats after hypoxia/SUGEN/normoxia, sacrificed at 11 weeks (with or without 3-week treatment of vehicle or FK506) (n = 8; *P < 0.05 vs. untreated normoxia condition, 2-way ANOVA, Dunnett’s post test). (E) Mean number of arteries per 100 alveoli (5 fields ×20) in same groups as in D (n = 8, ^#P < 0.05, 1-way ANOVA, selected pairs post test, vehicle vs. FK506). (F) Apelin/β-actin mRNA expression in total lung lysate in same group as in D (n = 8; *P < 0.05, 1-way ANOVA, Dunnett’s post test). (G) Il2/Gapdh mRNA expression in whole lungs of rats in normoxia (with or without vehicle or FK506) and in rats after hypoxia/SUGEN/normoxia, sacrificed at 11 weeks (with or without 3-week treatment of vehicle or FK506) (n = 8; *P < 0.05 vs. vehicle normoxia, ^#P < 0.05 vs. FK normoxia, nonsignificant trend in decrease of IL-2 with FK506 treatment, 2-way ANOVA, Bonferroni multiple-comparison test). Mean ± SEM.