Molecular and Cellular Biology Ph.D. program, University of Iowa, Iowa City, Iowa, USA.
Respir Res. 2009 Nov 25;10(1):119. doi: 10.1186/1465-9921-10-119.
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).
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.
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.
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.
血小板衍生生长因子 A (PDGF-A) 仅通过 PDGF-Ralpha 传递信号，在肺泡发育过程中，对于成纤维细胞的增殖和转分化（成纤维细胞向肌成纤维细胞转化）是必需的，因为 pdgfa 基因敲除小鼠既缺乏肌成纤维细胞也缺乏肺泡。然而，这些 PDGF-A 介导的机制仍不完全明确。在出生后第 4 天和第 12 天（P4 和 P12），使用小鼠肺成纤维细胞，我们研究了（a）PDGF-Ralpha 如何与 ki67（增殖标志物）或 alpha-平滑肌肌动蛋白（alphaSMA，肌成纤维细胞标志物）表达相关，以及（b）PDGF-A 是否直接影响 alphaSMA 或改变转化生长因子β（TGFbeta）的刺激作用。
使用流式细胞术，我们检查了在表达绿色荧光蛋白（GFP）作为 PDGF-Ralpha 表达标志物的小鼠中 PDGF-Ralpha、alphaSMA 和 Ki67 的表达。使用实时 RT-PCR，我们定量了 PDGF-A 处理后培养的 Mlg 新生小鼠肺成纤维细胞中的 alphaSMA mRNA。
GFP 荧光强度使我们能够区分具有缺失、较低或较高 PDGF-Ralpha 水平的三组成纤维细胞。在 P4 时，较高 PDGF-Ralpha+的成纤维细胞中包含更多的 Ki67（Ki67+），并且 Ki67+成纤维细胞在 alphaSMA+而不是 alphaSMA-群体中占主导地位。到 P12 时，Ki67+成纤维细胞在 PDGF-Ralpha+和 alphaSMA+群体中均占少数。在 P4 时，大多数 Ki67+成纤维细胞是 PDGF-Ralpha+和 alphaSMA-，而在 P12 时，大多数 Ki67+成纤维细胞是 PDGF-Ralpha-和 alphaSMA+。在 P4 和 P12 时，大多数 PDGF-Ralpha+的成纤维细胞都含有 alphaSMA。在肺中，在 P4 和 P12 时，在表达高水平 PDGF-Ralpha 的细胞中，邻近的 alphaSMA 比表达低水平 PDGF-Ralpha 的细胞更丰富。在 PDGF-Ralpha-细胞中，核 SMAD 2/3 从 P4 到 P12 下降，但在 PDGF-Ralpha+细胞中没有下降。在 Mlg 成纤维细胞中，暴露于 TGFbeta 后 alphaSMA mRNA 增加，但用 PDGF-A 处理后下降。
在间隔爆破（P4）和伸长（P12）期间，肺泡 PDGF-Ralpha 可能增强成纤维细胞转分化的倾向，而不是直接刺激 alphaSMA，alphaSMA 优先定位于非增殖成纤维细胞。相应地，PDGF-Ralpha 在 P4 时比在 P12 时更主要地影响成纤维细胞的增殖。在肺中，TGFbeta 可能会掩盖 PDGF-A/PDGF-Ralpha 信号的拮抗作用，增强 PDGF-Ralpha 表达的成纤维细胞中 alphaSMA 的丰度。
