Type VI collagen promotes lung epithelial cell spreading and wound-closure

Basement membrane (BM) is an essential part of the extracellular matrix (ECM) that plays a crucial role in mechanical support and signaling to epithelial cells during lung development, homeostasis and repair. Abnormal composition and remodeling of the lung ECM have been associated with developmental abnormalities observed in multiple pediatric and adult respiratory diseases. Collagen VI (COL6) is a well-studied muscle BM component, but its role in the lung and its effect on pulmonary epithelium is largely undetermined. We report the presence of COLVI immediately subjacent to human airway and alveolar epithelium in the pediatric lung, in a location where it is likely to interact with epithelial cells. In vitro, both primary human lung epithelial cells and human lung epithelial cell lines displayed an increased rate of “wound healing” in response to a scratch injury when plated on COL6 as compared to other matrices. For the 16HBE cell line, wounds remained >5-fold larger for cells on COL1 (p<0.001) and >6-fold larger on matrigel (p<0.001), a prototypical basement membrane, when compared to COL6 (>96% closure at 10 hr). The effect of COL6 upon lung epithelial cell phenotype was associated with an increase in cell spreading. Three hours after initial plating, 16HBE cells showed >7-fold less spreading on matrigel (p<0.01), and >4-fold less spreading on COL1 (p<0.01) when compared to COL6. Importantly, the addition of COL6 to other matrices also enhanced cell spreading. Similar responses were observed for primary cells. Inhibitor studies indicated both integrin β1 activity and activation of multiple signaling pathways was required for enhanced spreading on all matrices, with the PI3K/AKT pathway (PI3K, CDC42, RAC1) showing both significant and specific effects for spreading on COL6. Genetic gain-of-function experiments demonstrated enhanced PI3K/AKT pathway activity was sufficient to confer equivalent cell spreading on other matrices as compared to COL6. We conclude that COL6 has significant and specific effects upon human lung epithelial cell-autonomous functions.

基底膜（Basement membrane, BM）是细胞外基质（extracellular matrix, ECM）的核心组成部分，在肺发育、稳态维持与损伤修复过程中，对上皮细胞发挥关键的力学支撑与信号调控作用。肺细胞外基质的组成异常与重塑过程，与多种儿科及成人呼吸道疾病中观察到的发育异常密切相关。VI型胶原（Collagen VI, COL6）是研究较为充分的肌肉基底膜组分，但其在肺部的功能以及对肺上皮细胞的影响仍未明确。本研究证实，在儿科肺部的气道与肺泡上皮紧邻的下方区域存在COL6，其所处位置可与上皮细胞产生相互作用。体外实验显示，相较于其他基质，接种于COL6基质上的原代人肺上皮细胞与人肺上皮细胞系，在划痕损伤后均表现出更快的“创伤愈合”速率。以16HBE细胞系为例，与COL6基质组（10小时闭合率超96%）相比，COL1基质组的创面面积仍大于5倍（p<0.001），基质胶（一种典型基底膜基质）组的创面面积则大于6倍（p<0.001）。COL6对肺上皮细胞表型的影响与细胞铺展能力增强相关。接种3小时后，相较于COL6基质组，16HBE细胞在基质胶上的铺展程度低7倍以上（p<0.01），在COL1基质上的铺展程度低4倍以上（p<0.01）。值得注意的是，在其他基质中添加COL6同样可增强细胞铺展能力，原代细胞也表现出类似的响应。抑制剂实验表明，整合素β1的活性以及多条信号通路的激活，是所有基质上细胞铺展增强的必要条件；其中PI3K/AKT通路（包含PI3K、CDC42与RAC1）在COL6基质上的细胞铺展过程中，表现出显著且特异性的调控作用。基因功能获得性实验证实，增强PI3K/AKT通路的活性，足以使细胞在其他基质上获得与COL6基质组相当的铺展能力。综上，COL6对人肺上皮细胞的自主功能具有显著且特异性的调控作用。