PDGF-D and PDGF-C expression in lung fibrosis induced by silica.

<p>RNA samples were harvested from lungs of three control mice and four silica-treated mice. (A) PDGF-D and (B) PDGF-C gene expression was measured by quantitative PCR and reported to the expression of actin. ANOVA analysis revealed that the effect of treatment was highly significant (p-value: <2.2e-16, data not shown).</p

Growth factors regulate PDGF-D and PDGF-C expression.

<p>MRC5 cells were treated with TGFβ 4 ng/ml, EGF 50 ng/ml, FGF-4 10 ng/ml (in the presence of heparin 10 µg/ml), PDGF-BB 20 ng/ml alone or all together for 24 h and PDGF-D (A) and PDGF-C (B) gene expression was measured by RT-qPCR. (C) AG01523 fibroblasts were treated with TGFβ 4 ng/ml, IL-1β 10 ng/ml or both for 24 h and PDGF-D gene expression was measured by q-PCR. The average of 3 experiments is shown with S.E.M, * p<0.05, ** p<0.005, *** p<0.0005.</p

PDGF-D and PDGF-C regulation by TGFβ at the mRNA and protein levels.

<p>(A) and (D), MRC5 human fibroblasts were pre-treated with TGFβ receptor inhibitor SB431542 (10 µM) 30 min before treatment with TGFβ (4 ng/ml) for 24 h. The control condition was treated with DMSO alone. PDGF-D and PDGF-C mRNA expression was measured by RT-qPCR and normalized with the housekeeping gene RPLP0. (B) and (E) MRC5 cells were treated with TGFβ (4 ng/ml) for the indicated period of time. PDGF-D and PDGF-C protein expression was analyzed by western blotting. Blots were re-hybridized with an anti-β-actin antibody as control. (C) Active PDGF-DD levels were measured by ELISA in the culture supernatant of MRC5 cells treated or untreated with TGFβ 4 ng/ml for 24 h. The average of 3 experiments is shown with S.E.M, ** p<0.005, *** p<0.0005.</p

TGFβ represses PDGF-D at the transcriptional level through an IKK-dependent pathway.

<p>We used a 1.3 kb fragment of the PDGF-D promoter cloned upstream of the luciferase reporter gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Liu1" target="_blank">[31]</a>. (A) Gamma-2A cells were co-transfected with the PDGF-D promoter construct or the CAGA12 promoter construct (positive control), with either empty vector or constitutively activated Alk5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Pierreux1" target="_blank">[33]</a> and with a control β-galactosidase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108656#pone.0108656-Demoulin1" target="_blank">[28]</a>. Cells were treated with TGFβ 4 ng/ml or left untreated for 24 h. (B) Gamma-2A cells were co-transfected with PDGF-D promoter construct and with β-galactosidase. They were treated with BMS-345541 (10 µM) for 30 min and then TGFβ was added for 24 h. In all experiments, luciferase and β-galactosidase activities were measured and the luciferase activity was normalized by dividing by the β-galactosidase activity. The mean of three independent experiments is shown.</p