PDGF-BB inhibits BMP-mediated contractile phenotype in PASMCs. (A) Human PASMCs were treated with 3 nM BMP-4 alone (+BMP4), both 3 nM BMP4 and 20 ng/ml PDGF-BB (+BMP4/PDGF-BB), or 20 ng/ml PDGF-BB alone (+PDGF-BB) for 24 h (for vSMC markers) or 6 h (for Id3), followed by qRT–PCR (top panel) and immunofluorescence staining (bottom panel). The relative mRNA levels of vSMC markers SMA, CNN, or SM22, as well as the BMP-target gene Id3, were examined by qRT–PCR. Results were normalized to GAPDH expression, and the relative mRNA levels are presented as means±s.e.m., with each experiment conducted in triplicate (n=3) (top panel). The difference between two results indicated by asterisks is statistically significant; ^*P<0.001. Immunofluorescence staining was performed with FITC-conjugated anti-SMA antibody (green) and by DAPI staining (blue) (bottom panel). (B) PASMCs were treated with 3 nM BMP4, 20 ng/ml PDGF-BB, or both for 24 h prior to being embedded to collagen gel lattices. Twenty-four hours after the collagen lattices were dissociated from the dish, gel contraction was photographed by using a digital camera (top panel). The area of the gel lattices was determined with the ImageJ software, and the relative lattice area was obtained by dividing the area at each time point by the initial area of the well (bottom panel). Experiments were performed three times. Data represent the means±s.e.m.; ^*P<0.01. (C) PASMCs were starved for 24 h, followed by treatment with BMP4, PDGF-BB, or both for 48 h. Cells were tryspinized and counted using a haemocytometer. The relative number of cells compared with untreated cells was plotted as means±s.e.m. (n=3); ^*P<0.01.

miR-24 leads to downregulation of Trb3. (A) A schematic representation of the miR-24-targeting site in the 3′-UTR Trb3 transcripts (top panel). The conserved 8-mer seed sequence is shown in grey. PASMCs were transfected with 1 nM non-specific (GFP) mimic (control) or increasing amounts (0.1, 0.3, 0.6, or 1 nM) of chemically modified, synthetic miR-24 oligonucleotides (miR-24 mimic). Twenty-four hours after transfection of mimic, cells were harvested and subjected to qRT–PCR analysis. Relative Trb3 mRNA level normalized to GAPDH (bottom left), as well as levels of mature miR-24 normalized to U6 snRNA (bottom right), is plotted as means±s.e.m. (n=3); ^*P<0.001 (versus the expression levels of control-transfected PASMCs). (B) PASMCs were transfected with 0.3 nM control mimic or 0.3 nM miR-24 mimic. Twenty-four hours after transfection of mimic, cells were stimulated with 20 ng/ml PDGF-BB for 6 h and subjected to immunoblot analysis with anti-hTrb3 antibody or anti-GAPDH antibody (loading control). The experiment was repeated three times, with similar results. (C) Cos7 cells were transfected with a construct carrying the human Trb3 cDNA construct with the 3′-UTR, which includes the miR-24 seed sequence (Trb3+3′-UTR) or deleted in the 3′-UTR (Trb3), with increasing amounts of miR-24 mimic (0.3 or 3 nM). Cells were harvested and subjected to qRT–PCR analysis (left panel) and immunoblot analysis (right panel). Relative Trb3 mRNA expression normalized to GAPDH is plotted (n=3); ^*P<0.001 (compared with no miR-24 mimic transfection (white bar) of each set). Immunoblot analysis was performed using anti-hTrb3 antibody and anti-GAPDH antibody used as loading control. The immunoblot presented is representative of three independent experiments. A full-colour version of this figure is available at The EMBO Journal Online.

miR-24 expression is crucial for PDGF-BB downregulation of Trb3. (A) PASMCs were transfected with 50 nM control antisense (anti-GFP) or anti-miR-24 (anti-miR-24), followed by stimulation with or without 20 ng/ml of PDGF-BB (+PDGF) for 24 h. qRT–PCR analysis was performed and relative expression of Trb3 or SMA mRNA normalized to β-actin (left panel) and miR-24 expression normalized to U6 snRNA expression (right panel) was also examined and plotted as means±s.e.m. (n=3); ^*P<0.001 (compared with unstimulated sample transfected with the same anti-miR). (B) PASMCs were transfected with 50 nM non-targeting control siRNA (si-Control) or siRNA against human Trb3 (si-Trb3) for 24 h, followed by stimulation with 20 ng/ml PDGF-BB (+PDGF-BB) for 24 h. Relative expression of Trb3, SMA, or CNN normalized to GAPDH and miR-24 expression normalized to U6 snRNA is plotted (means±s.e.m., n=3; ^*P<0.001). (C) Rat PAC1 cells were transfected with a construct carrying the human Trb3 cDNA construct with the 3′-UTR (Trb3+3′-UTR) or deleted in 3′UTR (Trb3), followed by stimulation with 20 ng/ml PDGF-BB (+PDGF-BB) for 24 h. Percent inhibition of CNN or SM22 mRNA level upon PDGF treatment is plotted (top panel). Relative expression of both endogenous rat Trb3 and exogenous human Trb3 mRNA normalized to GAPDH is plotted (bottom panel) as means±s.e.m. (n=3); ^*P<0.001. (D) PASMCs were transfected with 50 nM si-Control or si-Trb3 for 24 h, followed by treatment with PDGF-BB for 48 h. Cells were tryspinized and counted using a haemocytometer. The relative number of cells compared with untreated cells is plotted as means±s.e.m. (n=3); ^*P<0.01.

miR-24 expression is essential for PDGF-BB inhibition of the BMP-mediated contractile phenotype in PASMCs. PASMCs were transfected with 50 nM anti-GFP (control) or anti-miR-24 for 24 h prior to stimulation with 3 nM BMP4, 20 ng/ml PDGF-BB, or both, as indicated. (A) qRT–PCR anaylses of SMA and CNN were performed. The fold induction of SMA mRNA (compared with no stimulation) is plotted; error bars represent s.e.m. (n=3); ^*P<0.001. (B) Cells were trypsinized and counted using a haemocytometer. The percentage of cell growth inhibition is plotted as means±s.e.m. (n=3); ^*P<0.001. (C) Cells were embedded in collagen gel lattices and treated with 3 nM BMP4 alone (+BMP4), or 3 nM BMP4 and 20 ng/ml PDGF-BB (+BMP4/PDGF-BB). Twenty-four hours after the collagen lattices were dissociated from the dish, percentage reduction of gel size as compared with the no stimulation condition was measured and is plotted (means±s.e.m., n=3; ^*P<0.001).

miR-24 antagonizes TGFβ signalling. (A) PASMCs were transfected with 0.3 nM Control mimic or 0.3 nM miR-24 mimic. Twenty-four hours after transfection of mimic, cells were stimulated with 100 pM TGFβ1 for 2 h and subjected to immunoblot analysis with anti-Trb3, anti-Smad2/3, anti-phospho-Smad2 (Pi-Smad2), or anti-GAPDH antibody (loading control). The experiment was repeated twice and produced similar results. (B) Mv1Lu cells were transfected with the TGFβ reporter construct SBE-Luc as well as with 3 nM control mimic, or 0.3 or 3 nM miR24 mimic. Cells were then stimulated with 100 pM TGFβ1 for 24 h and luciferase activities were measured. Results are plotted as fold induction upon TGFβ stimulation (means±s.e.m., n=3; ^*P<0.001), versus fold induction with control mimic). (C) Mv1Lu cells was transfected with SBE-Luc, 3 nM control mimic, or miR-24 mimic, as well as the Trb3 expression construct with the 3′-UTR containing the miR-24 seed sequence (Trb3+3′-UTR) or without the 3'UTR (Trb3). Cells were stimulated with 100 pM TGFβ1 for 24 h and luciferase activities were measured (means±s.e.m., n=3). Relative exogenous Trb3 mRNA normalized to GAPDH in samples transfected with control mimic were monitored by qRT–PCR (right panel). (D) PASMCs were transfected with 50 nM si-Control or si-Trb3, followed by 100 pM TGFβ1 treatment for 24 h. qRT–PCR analysis of SMA, CNN, SM22, PAI-1, or Trb3 mRNA was performed. The relative mRNA levels normalized to GAPDH are plotted as means±s.e.m. (n=3; ^*P<0.001). (E) PASMCs were transfected with 3 nM control mimic (control), or miR-24 mimic, followed by 100 pM TGFβ1 treatment for 24 h. qRT–PCR analysis of SMA, CNN, or Trb3 mRNA, or miR-21 or miR-24 was performed. Relative mRNA levels normalized to GAPDH and miR-21 expression normalized to U6 snRNA were plotted as means±s.e.m. (n=3; ^*P<0.001). (F) PASMCs were transfected with 50 nM anti-GFP (control) or anti-miR-24 for 24 h. Cells were then treated with 100 pM TGFβ1 alone (+TGFβ) or both100 pM TGFβ1 and 20 ng/ml PDGF-BB (+TGFβ/PDGF-BB) for 24 h. qRT–PCR analysis for SMA, CNN, or Trb3 was performed and data of fold induction of mRNA as compared with unstimulated was plotted as means±s.e.m. (n=3; ^*P<0.001). (G) A schematic representation of the mechanism of antagonism between PDGF and the TGFβ-signalling pathway mediated by miR-24.

PDGF-BB stimulation decreases Trb3 expression. (A) PASMCs were stimulated with 3 nM BMP-4 or 20 ng/ml PDGF-BB for 2, 4, 8, or 24 h and subjected to qRT–PCR analysis. Relative expression of Trb3 mRNA normalized to GAPDH is plotted. (B) PASMCs were treated with 20 ng/ml PDGF-BB for 4, 8, or 24 h. Cells were harvested and subjected to immunoblot analysis using anti-human Trb3 antibody and anti-GAPDH antibody used as loading control. The results are representative of three independent experiments. (C) PASMCs were treated with 20 ng/ml PDGF-BB for 24 h and then subjected to immunofluorescence staining using anti-hTrb3 antibody conjugated to Cy3 (red), anti-SMA antibody conjugated to FITC (green), and by DAPI staining (blue). (D) Whole-cell lysates from PASMCs treated with 3 nM BMP4 or 100 pM TGFβ1 for 0.5 h, 20 ng/ml PDGF-BB for 4 h, or stimulated with PDGF-BB for 4 h followed by BMP4 or TGFβ1 for 0.5 h were subjected to immunoblot analysis using anti-Smad1, anti-Smad2/3, anti-phospho-Smad-1/5 (Pi-Smad1/5), or anti-phospho-Smad2 (Pi-Smad2) antibody; anti-hTrb3 antibody; or anti-GAPDH antibody (loading control). The experiment was repeated twice, with similar results.

PDGF-BB induces miR-24 expression. (A) PASMCs were stimulated with 20 ng/ml of PDGF-BB for 1, 2, 4, 8, or 24 h, followed by qRT–PCR to determine the expression of miR-24, miR-221, and miR-222. The expression of individual miRNA normalized to U6 snRNA is plotted (means±s.e.m., n=3). (B) A schematic representation of the miR-24-1 gene cluster on human chromosome-9, which includes miR-23b and miR-27b, and the miR-24-2 gene cluster on human chromosome-19, which includes miR-27a and miR-23a. (C) PASMCs were treated with PDGF-BB for 2, 4, 6, or 24 h, followed by qRT–PCR analysis using primers for miR-23a, miR-27a, miR-27b, miR-23b, or miR-24. The time-course change in the expression of miRNA normalized to U6 snRNA after PDGF-BB stimulation is plotted (means±s.e.m., n=3). (D) PASMCs were treated with PDGF-BB for 2, 4, 6, or 24 h, followed by qRT–PCR analysis of primary transcripts of miR-24-1 (Pri-miR-24-1), primary transcripts of miR-24-2 (Pri-miR-24-2), intermediate products of miR-24-1 (Pre-miR 24-1), and intermediate products of miR-24-2 (Pre-miR-24-2). The time-course change in the expression of RNAs was normalized to GAPDH and plotted (means±s.e.m., n=3).

miR-24 inhibits BMP signalling in vSMCs through downregulation of Trb3 (A). PASMCs were transfected with 0.3 nM Control mimic or 0.3 nM miR-24 mimic. Twenty-four hours after transfection of mimic, cells were stimulated with 3 nM BMP4 for 2 h and subjected to immunoblot analysis using anti-Trb3, anti-Smad1, anti-phospho-Smad1/5 (Pi-Smad1/5), or anti-GAPDH antibody (loading control). The experiment was repeated twice and produced similar results. (B) PASMCs were transfected with 0.1 nM miR-24 mimic or control mimic, followed by stimulation with 3 nM BMP4 (+BMP4) for 6 h (Id3) or for 24 h (all other markers). qRT–PCR was then performed. Relative expression of SMA, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; ^*P<0.001). (C) PASMCs were transfected with 0.3 nM miR-24 mimic or control mimic, followed by adenoviral transduction of the Trb3 expression construct (Trb3 (WT) or Trb3 (ΔK)), or adenoviral transduction of GFP expression construct as control. Twenty-four hours later stimulation with 3 nM BMP4 was performed for 6 h for Id3 or 24 h for SMA. qRT–PCR was then performed and relative expression of SMA, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; ^*P<0.001). (D) PASMCs were transfected with 0.3 nM control mimic or miR-24 mimic (left panel) for 24 h, followed by stimulation with 3 nM BMP4 (+BMP4) for 4 h. qRT–PCR was then performed to determine relative expression of miR-21 normalized to U6 snRNA and Trb3 expression normalized to GAPDH (means±s.e.m., n=3; ^*P<0.001). (E) PASMCs were transfected with 50 nM si-Control or si-Trb3 (right panel) for 24 h, followed by stimulation with 3 nM BMP4 (+BMP4) for 4 h. qRT–PCR was then performed to determine relative expression of miR-21 normalized to U6 snRNA and Trb3 expression normalized to GAPDH (means±s.e.m., n=3; ^*P<0.001). (F) PASMCs were transfected with 3 nM control mimic (control) or miR-24 mimic (miR-24 mimic) for 24 h, placed in starvation media for 24 h, followed by stimulation with BMP4 (+BMP4) for 48 h. Cells were trypsinized and counted using a haemocytometer. The relative number of cells is plotted means±s.e.m. (n=3); ^*P<0.001 (compared with unstimulated). (G) PASMCs were transfected with 3 nM control mimic (control) or miR-24 mimic (miR-24 mimic) for 24 h, followed by stimulation with BMP4 (+BMP4) for 24 h or left unstimulated (none). PASMCs were then embedded to collagen gel lattices with continued stimulation. Twenty-four hours after the collagen lattices were dissociated from the dish, gel contraction was photographed by using a digital camera (left panel). The relative size of collagen gel was quantitated using ImageJ and is plotted (means±s.e.m., n=3; ^*P<0.001, compared with unstimulated condition (right panel)). (H) A clone of P19 cells, stably transfected with the BMP reporter construct BRE-Luc, was transfected with 3 nM control mimic, 0.3 nM or 3 nM miR-24 mimic, or 50 nM si-Trb3, followed by 24-h BMP4 treatment. Luciferase activity was then measured and fold induction of the activity after BMP4 treatment is plotted (means±s.e.m., n=3; ^*P<0.001, compared with control mimic). (I) C2C12 cells were transfected with 0.003 nM control mimic (control) or 0.003 nM miR-24 mimic (miR-24) for 24 h followed by stimulation with BMP4 (+BMP4) for 2 h, or left unstimulated (none). qRT–PCR was then performed. Relative expression of ALP, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; ^*P<0.001).

Modulation of Trb3 and miR-24 expression in a rat PAH model (A). qRT–PCR analysis of lung samples from rats after 17-day hypoxia or normoxia treatment. Relative expression of Trb3, SMA, CNN, and SM22 (top panel); or miR-24, miR-221, and miR-100 (bottom panel) was plotted by normalizing the level of expression of normoxia samples as 1 (means±s.e.m., n=3; ^*P<0.001). (B) Histological examination of PAs from rats after 17-day hypoxia or normoxia treatment with anti-SMA, anti-Smad1, anti-phospho Smad1/5 (Pi-Smad1/5), or anti-Trb3 antibodies (brown, × 400) and by haematoxylin staining (blue, × 400). The media (M) and adventitia (A) of the PAs are indicated. (C) Sections of PAs were probed with either anti-miR-24 (top two panels) or control (bottom panel) conjugated to FITC (green). Cells were counterstained with DAPI (blue). The media (M) and adventitia (A) of the PAs are indicated. Relative expression of miR-24 was quantitated using the ImageJ software and imaged (n=3; ^*P<0.001).