Deficiency of Integrin β4 Results in Increased Lung Tissue Stiffness and Responds to Substrate Stiffness via Modulating RhoA Activity.

Yinxiu Chi,Yang Xiang,Chi Liu,Wenjie Huang,Mingxing Ouyang,Yu Chen,Xiangping Qu,Yan Pan,Jingjing Li,Xiaoqun Qin,Linhong Deng,Wang Jiang,Lei Liu,Huijun Liu

doi:10.3389/fcell.2022.845440

Abstract

The interaction between extracellular matrix (ECM) and epithelial cells plays a key role in lung development. Our studies found that mice with conditional integrin β4 (ITGB4) knockout presented lung dysplasia and increased stiffness of lung tissues. In accordance with our previous studies regarding the functions of ITGB4 in bronchial epithelial cells (BECs), we hypothesize that the decreased ITGB4 expression during embryonic stage leads to abnormal ECM remodeling and increased tissue stiffness, thus impairing BECs motility and compromising lung development. In this study, we examined lung tissue stiffness in normal and ITGB4 deficiency mice using Atomic Force Microscopy (AFM), and demonstrated that ITGB4 deficiency resulted in increased lung tissue stiffness. The examination of ECM components collagen, elastin, and lysyl oxidase (LOX) family showed that the expression of type VI collagen, elastin and LOXL4 were significantly elevated in the ITGB4-deficiency mice, compared with those in normal groups. Airway epithelial cell migration and proliferation capacities on normal and stiff substrates were evaluated through video-microscopy and flow cytometry. The morphology of the cytoskeleton was detected by laser confocal microscopy, and RhoA activities were determined by fluorescence resonance energy transfer (FRET) microscopy. The results showed that migration and proliferation of ITGB4 deficiency cells were noticeably inhibited, along decreased cytoskeleton stabilization, and hampered RhoA activity, especially for cells cultured on the stiff substrate. These results suggest that decreased ITGB4 expression results in increased lung tissue stiffness and impairs the adaptation of bronchial epithelial cells to substrate stiffness, which may be related to the occurrence of broncho pulmonary dysplasia.

Highlights

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease that inflicts upon premature infants due to complications arising from lung injury and mechanical ventilation
To avoid the lethal effect of Integrin beta 4 (ITGB4) null on mice, we constructed a conditional ITGB4 deficiency model known as CCSP–rtTAtg/−/TetOCretg/−/ITGB4fl/fl (Ceteci et al, 2012; Hsu et al, 2014), in which ITGB4 was deleted only in airway epithelial cells, following the protocols elaborated in previous studies
In the presence of Dox, responsive transactivator (rtTA) binds to the7 CMV promoter, which activates Cre-recombinase expression and deletes the ITGB4 gene (Liu et al, 2018). β4f/f was referred as the control group, and the β4ccsp.cre was referred as the experiment group

Summary

Introduction

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease that inflicts upon premature infants due to complications arising from lung injury and mechanical ventilation. One of the clinical characteristics of BPD is abnormal ECM stiffness, which stymies lung parenchymal growth (Mammoto et al, 2013). Abnormal ECM stiffness leads to deregulated proliferation and migration of cells, thereby facilitating the pathological progression (Lu et al, 2011). Lysyl oxidase proteins (LOXs) are a class of amine oxidases mostly distributed in fibroblasts and smooth muscle cells contributing to the formation of ECM in a copper dependent manner (Bryan and D’Amore, 2007; Finney et al, 2014). The Lysyl-oxidase (LOXs) gene family is comprised of LOX and four LOX-like proteins (including LOXL1, LOXL2, LOXL3, and LOXL4), and play a key role in organ development via modulating ECM structure and mechanics through cross-linking of collagen and elastin (Smith-Mungo and Kagan, 1998; Lucero and Kagan, 2006; Barker et al, 2012). Previous research reported that infants with BPD show increased collagen abundance in the lung, abnormal collagen scaffolding and collagen fibers (Thibeault et al, 2003), as well as altered level of elastin (Bruce et al, 1985)

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