Abstract
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.
Highlights
There are 28 known families of collagens, with subtypes based on function and structure; fibrillar, FACIT (Fibril Associated Collagens with Interrupted Triple helices), beaded filament, anchoring fibril, transmembrane and network forming collagens [1]
Wound healing and spreading responses were characterized on matrices containing combinations of COL6 or COL1 with Matrigel, which contains many of the common basement membrane constituents
COL6 appears to be an essential component of many extracellular matrices, primarily localizing within or adjacent to basement membranes, where it appears to function to enhance cellmatrix interactions [29,33,39]
Summary
There are 28 known families of collagens, with subtypes based on function and structure; fibrillar, FACIT (Fibril Associated Collagens with Interrupted Triple helices), beaded filament, anchoring fibril, transmembrane and network forming collagens [1]. Fibrillar collagens I and III are the most abundant collagens in the lung parenchyma and provide most of the structure to the alveolar wall [2]. Several other collagens represent essential components of the lung ECM, including COL6, which can be found in the basement membrane in the lung parenchyma, airways and vasculature [3, 4]. It has been shown that deposition of the basement membrane and other ECM components is a critical event in alveolar septation during lung development [5]. The basement membrane is composed of collagen IV, laminin and entactin, and interacts with other collagens, heparin sulfate proteoglycans (HSPGs) and many other ECM components [6, 7]. Alterations in extracellular matrix composition and the expression of basement membrane components have been shown in many pulmonary disorders, including bronchopulmonary dysplasia (BPD), asthma, chronic obstructive pulmonary disorder (COPD) and idiopathic pulmonary fibrosis (IPF) [8]
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