Mechanotransduction and Lung Alveolar Differentiation

描述（由申请人提供）：子宫内通过重复呼吸运动和液体扩张产生的机械力对哺乳动物肺发育至关重要。然而，肺细胞感知和识别机械信号的机制在很大程度上是未知的。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）对胎儿肺发育至关重要。尽管过去的研究表明EGFR对肺切除术后II型细胞的分化和牵张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和特异性整合素在牵张诱导的胎儿II型细胞分化中的潜在作用。进一步的研究表明，这一过程可能是由力诱导的EGFR配体释放介导的，因为当EGFR的配体结合结构域被中和抗体阻断时，菌株诱导的II型细胞分化被显著抑制。我们还表明，条件培养基从拉伸细胞促进II型细胞分化时，加入未拉伸的细胞。在胎羊中进行的实验进一步支持了这一假设，这些实验表明，气管结扎后的肺液组成，而不仅仅是肺内压的增加，对加速肺生长和分化至关重要。因此，本申请的具体假设是胎儿II型细胞的菌株诱导的分化是通过膜锚定的EGFR配体的自分泌释放介导的。我们的具体目标是：1）鉴定肺上皮细胞响应于机械应变而释放的促进II型细胞分化的EGFR配体。2)分析机械拉伸诱导EGFR配体释放的信号机制。3)证明力诱导的可溶性生长因子释放在上皮细胞株器官型模型中的生理作用。使用Flexercell应变单位装置，细胞信号传导技术，通过诱导或敲低表达以及EGFR和ADAM 17敲除小鼠对拟议的关键信号传导蛋白进行修饰，我们将研究在胎儿II型细胞中通过强制释放促进肺分化的EGFR配体。我们将分析ADAM 17作为蛋白酶在整合素受体的机械刺激后切割膜锚定的EGFR配体的作用。最后，我们将使用离体胎肺外植体模型验证我们的体外研究结果。基于机械力在正常胎肺发育期间所起的关键作用，对胎肺中由应变激活的关键调节途径的鉴定可以提供独特的机会来挽救表型并促进开发新的方法以在肺发育受损的临床病症（包括肺发育不全，极低出生体重儿的支气管肺发育不良和出生后肺生长。

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.