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).