Mechanotransduction and Lung Alveolar Differentiation

描述(申请人提供)：由重复的呼吸运动和液体膨胀在子宫中产生的机械力对哺乳动物肺的发育是必不可少的。然而，肺细胞感知和传递机械信号的机制在很大程度上尚不清楚。长期目标是确定机械力如何促进肺成熟。表皮生长因子受体(EGFR)对胎肺发育至关重要。尽管过去的研究表明EGFR对肺切除后II型细胞的分化和伸展介导的代偿性生长很重要，但EGFR被激活的机制尚不清楚。我们的研究已经确定了EGFR和特定的整合素在牵张诱导胎儿II型细胞分化中的潜在作用。进一步的研究表明，这一过程可能是通过强迫诱导EGFR配体的释放来实现的，因为当EGFR的配体结合域被中和抗体阻断时，菌株诱导的II型细胞分化明显受到抑制。我们还表明，当加入非拉伸细胞时，来自拉伸细胞的条件培养液促进了II型细胞的分化。在胎羊身上进行的实验进一步支持了这一假说，这些实验表明，气管结扎后的肺液成分，而不仅仅是肺内压力的增加，对加速肺的生长和分化至关重要。因此，这一应用的特定假设是，应变诱导的胎儿II型细胞的分化是通过自分泌释放膜锚定的EGFR配体来介导的。我们的具体目标是：1)鉴定肺上皮细胞在机械应激下释放的促进II型细胞分化的EGFR配体。2)分析机械牵张诱导EGFR配体释放的信号机制。3)探讨外力诱导的可溶性生长因子释放在器官型上皮细胞模型中的生理作用。利用Flexpercell单位装置、细胞信号技术、通过诱导或敲除表达对建议的关键信号蛋白的修饰以及EGFR和ADAM17基因敲除小鼠，我们将检测促进肺分化的胎儿II型细胞中强制释放的EGFR配体。我们将分析ADAM17作为在整合素受体机械刺激后裂解膜锚定的EGFR配体的蛋白酶的作用。最后，我们将使用体外胎肺移植模型来验证我们的体外研究结果。基于机械力在胎儿肺正常发育中所起的关键作用，识别胎儿肺中由应变激活的关键调节通路可能提供一个独特的机会来挽救表型，并促进在肺发育受损的临床条件下促进肺成熟的新方法的开发，包括肺发育不良、支气管肺发育不良和极低出生体重婴儿的出生后肺发育。

DESCRIPTION (provided by applicant): Mechanical forces generated in utero by repetitive breathing movements and by fluid distension are essential to mammalian lung development. However, the mechanisms by which pulmonary cells sense and transduce mechanical signals are largely unknown. The long-term goals are to define how mechanical forces promote lung maturation. Epidermal growth factor receptor (EGFR) is critical for fetal lung development. Although past studies indicate that EGFR is important for differentiation of type II cells and stretch-mediated compensatory growth after pneumonectomy, the mechanisms by which EGFR is activated are not known. Our investigations have identified potential roles for the EGFR and specific integrins in stretch-induced fetal type II cell differentiation. Further studies have shown that this process may be mediated by force-induced release of EGFR ligands, since strain-induced type II cell differentiation was markedly inhibited when the ligand-binding domain of the EGFR was blocked with neutralizing antibodies. We also showed that conditioned medium from stretched cells promoted type II cell differentiation when added to unstretched cells. This hypothesis is further supported by experiments performed in fetal lambs, which demonstrate that lung fluid composition after tracheal ligation, and not just increase in intrapulmonary pressure, is critical to accelerate lung growth and differentiation. Therefore, the specific hypothesis of this application is that strain-induced differentiation of fetal type II cells is mediated via autocrine release of membrane-anchored EGFR ligands. Our Specific Aims are: 1) To identify EGFR ligands released by lung epithelial cells in response to mechanical strain that promote type II cell differentiation. 2) To analyze the signaling mechanisms by which mechanical stretch induces EGFR ligand release. 3) To demonstrate the physiological role of force-induced release of soluble growth factors in organotypic models of epithelial strain. Using the Flexercell Strain Unit apparatus, cell signaling techniques, modification of the proposed key signaling proteins by inducible or knockdown expression and EGFR and ADAM17 knockout mice, we will examine EGFR ligands released by force in fetal type II cells that promote lung differentiation. We will analyze the role of ADAM17 as the protease that cleaves membrane-anchored EGFR ligands after mechanical stimulation of integrin receptors. Finally, we will validate our in vitro findings using ex vivo fetal lung explant models. Based on the critical role played by mechanical forces during normal fetal lung development, the identification of key regulatory pathways activated by strain in fetal lungs may provide a unique opportunity to rescue the phenotype and to facilitate development of new approaches to accelerate lung maturation in clinical conditions where lung development is impaired, including pulmonary hypoplasia, bronchopulmonary dysplasia and postnatal lung growth in extremely-low-birth-weight infants.