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-R alpha, 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-R alpha correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (alpha SMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects alpha SMA or modifies stimulation by transforming growth factor beta (TGF beta). Methods: Using flow cytometry we examined PDGF-R alpha, alpha SMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-R alpha expression. Using real-time RT-PCR we quantified alpha SMA mRNA in cultured MIg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGF beta. Results: The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-R alpha. At P4, more of the higher than lower PDGF-R alpha + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the alpha SMA + but not the alpha SMA-population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-R alpha + and alpha SMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-R alpha + and alpha SMA-whereas at P12, most Ki67+ fibroblasts were PDGF-R alpha- and alpha SMA-. More of the PDGF-R alpha + than - fibroblasts contained alpha SMA at both P4 and P12. In the lung, proximate alpha SMA was more abundant around nuclei in cells expressing high than low levels of PDGF-R alpha at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-R alpha-, but not in PDGF-R alpha + cells. In MIg fibroblasts, alpha SMA mRNA increased after exposure to TGF beta, but declined after treatment with PDGF-A. Conclusion: During both septal eruption (P4) and elongation (P12), alveolar PDGF-Ra may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate alpha SMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-R alpha more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGF beta may overshadow the antagonistic effects of PDGF-A/PDGF-R alpha signaling, enhancing alpha SMA-abundance in PDGF-R alpha-expressing fibroblasts.