PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

BACKGROUND Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Ralpha, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Ralpha correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (alphaSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects alphaSMA or modifies stimulation by transforming growth factor beta (TGFbeta). METHODS Using flow cytometry we examined PDGF-Ralpha, alphaSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Ralpha expression. Using real-time RT-PCR we quantified alphaSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFbeta. RESULTS The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Ralpha. At P4, more of the higher than lower PDGF-Ralpha + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the alphaSMA + but not the alphaSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Ralpha + and alphaSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Ralpha + and alphaSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Ralpha- and alphaSMA-. More of the PDGF-Ralpha + than - fibroblasts contained alphaSMA at both P4 and P12. In the lung, proximate alphaSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Ralpha at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Ralpha-, but not in PDGF-Ralpha + cells. In Mlg fibroblasts, alphaSMA mRNA increased after exposure to TGFbeta, but declined after treatment with PDGF-A. CONCLUSION During both septal eruption (P4) and elongation (P12), alveolar PDGF-Ralpha may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate alphaSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Ralpha more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFbeta may overshadow the antagonistic effects of PDGF-A/PDGF-Ralpha signaling, enhancing alphaSMA-abundance in PDGF-Ralpha-expressing fibroblasts.