Role for miR-204 in human pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Because microRNAs have been recently implicated in the regulation of cell proliferation and apoptosis, we hypothesized that these regulatory molecules might be implicated in the etiology of PAH. In this study, we show that miR-204 expression in PASMCs is down-regulated in both human and rodent PAH. miR-204 down-regulation correlates with PAH severity and accounts for the proliferative and antiapoptotic phenotypes of PAH-PASMCs. STAT3 activation suppresses miR-204 expression, and miR-204 directly targets SHP2 expression, thereby SHP2 up-regulation, by miR-204 down-regulation, activates the Src kinase and nuclear factor of activated T cells (NFAT). STAT3 also directly induces NFATc2 expression. NFAT and SHP2 were needed to sustain PAH-PASMC proliferation and resistance to apoptosis. Finally, delivery of synthetic miR-204 to the lungs of animals with PAH significantly reduced disease severity. This study uncovers a new regulatory pathway involving miR-204 that is critical to the etiology of PAH and indicates that reestablishing miR-204 expression should be explored as a potential new therapy for this disease.

Figure 1.

Correlation between miR-204 expression and PAH severity. (A) miR-204 is decreased in human, mouse, and rat PAH lungs. qRT-PCR analysis of miR-204 expression in human lungs with PAH (n = 8), mouse lungs with hypoxia-induced pulmonary hypertension (n = 6), and rat lungs with MCT-induced pulmonary hypertension (n = 5) compared with human (n = 8), mouse (n = 10) and rat (n = 5) control (Ctrl) lungs. (B) miR-204 is mainly expressed in the distal PAs. qRT-PCR analysis of miR-204 expression in several rat organs with MCT-induced pulmonary hypertension (n = 5) compared with control rats (n = 5). (C) miR-204 down-regulation correlates with PAH severity. qRT-PCR analysis of miR-204 expression in the lungs from healthy subjects (n = 8) and from patients with varying severity of PAH (n = 3), in mouse lungs with varying severity of hypoxia-induced pulmonary hypertension (n = 3), and in rat lungs with varying severity of MCT-induced pulmonary hypertension (n = 3) compared with control animals (n = 5 for both rats and mice; n = 3 experiments per patient or per animal for each pulmonary vascular resistance [PVR] or mean pulmonary arterial pressure [PAP] listed, and significance is compared with control group). In all experiments, the level of miR-204 is relative to the control RNA U6. Data are expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 2.

miR-204 modulates the Src–STAT3–NFATc2 pathway in PASMCs from a patient with PAH (PAH-PASMCs). (A) miR-204 regulates human PASMC apoptosis and proliferation. Analysis of PASMC proliferation (PCNA nuclear localization) and serum starvation–induced apoptosis (TUNEL staining) of PASMCs from PAH patients (n = 3 patients) and healthy controls (n = 5 individuals). Control (Ctrl) or miR-204 antagomirs (Inh) were added as indicated. (B and C) miR-204 down-regulation in PAH-PASMCs increases Src and STAT3 activation. Total and phosphorylated Src (B) and total and phosphorylated STAT3 (C) in PASMCs from three PAH and five control patients monitored by Western blots are shown. Control or miR-204 antagomirs or mimics were added when indicated. Smooth muscle actin (SM-actin) was used as a loading control. Representative Western blots are shown. (D) miR-204 down-regulation in PAH-PASMCs increases NFAT expression and activation in human PASMCs. NFATc2 mRNA expression (left) and activity (right) were measured by qRT-PCR (left) and luciferase assay (right) in PASMCs from control or PAH patients treated either with control or miR-204 antagomir. Error bars represent mean value ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

A primary STAT3 activation by circulating pro-PAH factors accounts for miR-204 down-regulation in PAH-PASMCs. (A) siSTAT3 increases miR-204 expression in PAH-PASMCs. miR-204 level measured by qRT-PCR in PAH treated with control siRNA (siRNA ctrl) or siSTAT3 as indicated (n = 3). (B) STAT3 activation and miR-204 expression are inversely correlated in PASMCs. Analysis of the correlation between STAT3 activation (measured by the pY705-STAT3/STAT3 ratio monitored by Western blot) and miR-204 expression (measured by qRT-PCR; n = 2 experiments/patient in three PAH and five control patients). (C) Pro-PAH factors decrease miR-204 and TRPM3 expression similarly by a STAT3-dependent mechanism in control PASMCs. miR-204 (top) and TRPM3 (middle) expression were measured by qRT-PCR performed on control cells treated with the pro-PAH factors PDGF, endothelin-1 (ET-1), or angiotensin II (AII) as indicated (n = 3 experiments/patient/in three PAH and five controls). (bottom) Analysis of the similarities between miR-204 and TRPM3 pattern of expression measured by qRT-PCR in control, PAH, and PAH treated with siSTAT3 as indicated. (D) p-STAT3–binding sites are detected downstream of the TRPM3 gene. ChIP-PCR experiments studying STAT3-binding sites upstream (Up1) and downstream (Dw1 and Dw2) on TRPM3 genes. The OR8J1 gene was used as a negative control, whereas the VEGF gene was used as a positive control. Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

SHP2 up-regulation by miR-204 promotes activation of the Src–STAT3–NFAT axis in PAH-PASMCs. (A) SHP2 is up-regulated by miR-204 in PAH-PASMCs. Total and phosphorylated JAK2 (left), SHC1 (middle), and SHP2 (right) protein expression was monitored by Western blot (n = 3 independent experiments) in PASMCs from three PAH and five control patients. (right) miR-204 antagomir (Inh) and mimic along with appropriate controls (Ctrl) were added as indicated. SM-actin, smooth muscle actin. (B) miR-204 directly targets the SHP2 3′ UTR. (left) Binding sites of miR-204 found in the 3′ UTR of SHP2. Mutations introduced into the luciferase reporter are shown in red. (right) Relative firefly luciferase activity derived from the SHP2 3′ UTR and SHP2 3′ UTR mutated reporter constructs monitored after transfection in control PASMCs (n = 5). Control and miR-204 inhibitor (n = 3) were added as indicated. L.U., luciferase unit. (C) STAT3 regulates NFATc2 expression. NFATc2 mRNA level (left) relative to 18S measured by qRT-PCR in PASMCs from control and PAH patients treated when indicated with control or STAT3 siRNA (n = 3 qRT-PCR/patient in three PAH and five control patients). ChIP-PCR experiments (right) studying STAT3 binding on genes encoding the indicated NFAT isoforms (NFATc1, -c2, and -c3). The OR8J1 gene was used as a negative control, whereas the VEGF gene was used as a positive control. Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01).

Figure 5.

miR-204 restoration decreases [Ca²⁺]_i and depolarizes mitochondrial membrane potential. (A–C) Analysis of [Ca²⁺]_i (Fluo3-AM) and cell proliferation (PCNA nuclear localization) of PASMCs from PAH and control (Ctrl) patients. miR-204 antagomir (Inh) and mimics, VIVIT (NFAT competitor peptide), and PP2 (Src inhibitor) compared with PP3, its negative control, STAT3 siRNA, SHP2 siRNA, and appropriate controls for each treatment were added as indicated (n = 50–150 cells/patient in three PAH and five control patients). (D–F) Analysis of the mitochondrial membrane potential (ΔΨ_m; TMRM) and serum starvation–induced apoptosis (TUNEL staining) of PASMCs from PAH and control patients. miR-204 antagomir (Inh) and mimics, VIVIT (NFAT competitor peptide), PP2 (Src inhibitor), STAT3 siRNA, SHP2 siRNA, and appropriate controls for each treatment were added as indicated (n = 50–150 cells/patient in three PAH and five control patients). F.U., fluorescence unit. Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 6.

miR-204 level decreases within distal PAs of rats injected with MCT during the fourth week of PAH development. (A) miR-204 expression relative to U6 measured by qRT-PCR in distal PAs of rats. (B) SHP2 protein expression was quantified in distal PAs by immunofluorescence (F.U., fluorescent unit) on lung sections (n = 5 measurements by rat in five rats per time point). (C and D) STAT3 and NFAT activation were measured by the percentage of cells presenting p-STAT3 and NFAT nuclear localization, respectively, in distal PAs of rats (n = 5 measurements by rat in 10 rats per time point). (E) Mean PA pressure measured by right catheterization in closed chest rats (n = 5 rats per group). Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 7.

Increasing the level of miR-204 by nebulization reverses MCT-PAH. (A) miR-204 restoration decreases mean PA pressure. Mean PA pressure measured by right catheterization in closed chest rats (n = 5 rats per group) is shown. miR-204 antagomir (Inh) and mimic along with appropriate controls (Ctrl) as indicated were intratracheally nebulized after 2 wk of MCT injection. (B) miR-204 restoration decreases PA wall thickness. PA remodeling was measured by the percentage of media wall thickness on lung sections stained by hematoxylin and eosin (n = 5 measurements/rat in 10 rats). The pictures shown illustrate representative distal arteries, and the graph represents mean values. (C) By decreasing SHP2, STAT3, and NFAT activation, miR-204 restoration increases apoptosis and decreases proliferation in MCT-rat PASMCs. SHP2 protein expression (left) was quantified in distal PAs by immunofluorescence (F.U., fluorescent unit) on lung sections; STAT3 and NFAT activation (middle) measured by the percentage of cells presenting p-STAT3 and NFAT nuclear localization, respectively, in distal PAs of rats; apoptosis and proliferation measured by the percentage of cells presenting TUNEL and PCNA nuclear localization, respectively, in distal PAs of rats. Control and miR-204 mimic were intratracheally nebulized after 2 wk of MCT injection as indicated (n = 5 measurements by rat in five rats per group). Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).