LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor β activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1^−/−) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1^−/− mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume_0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1^−/− mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1^−/− mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.Supplementary key words: lipoprotein receptors, cell biology, ApoE, receptors, inflammation, animal models, epithelial cells, lung disease

The LDL receptor-related protein 1 (LRP1) is a member of the LDL receptor family and performs endocytic and signaling functions. LRP1 was originally identified as a receptor for chylomicron remnants and alpha-2 macroglobulin (1, 2), but many other ligands have been identified and their specific roles and regulation depend on cell type and tissue. Amongst others, extracellular LRP1 ligands include lipoproteins, matrix metalloproteases (MMPs), antiproteases such as tissue plasminogen activator, and extracellular matrix (ECM) components such as fibronectin. Intracellular LRP1 ligands include scaffolding and signaling proteins and membrane coreceptors (3). Some of the functions of LRP1 reflect its role as a lipoprotein receptor, whereas others are more in line with its anti-inflammatory actions mediated by interaction with serpins and tissue inhibitor of metalloproteinases (4, 5).In the human heart, LRP1 expression was greater with ischemic heart disease, and its activation with a synthetic ligand was cardioprotective in mice subjected to experimental acute myocardial infarction by ischemia-reperfusion (4). Genome-wide association study has linked SNPs in LRP1 with several diseases, such as abdominal aortic aneurysm, hyperlipidemia, and chronic obstructive pulmonary disease (COPD) (6, 7, 8, 9). Some of these SNPs decrease the mRNA stability and protein expression of LRP1. In smokers and patients with COPD, SNPs in LRP1 correlate with decreased lung function (9), but the role(s) of LRP1 in normal and pathological pulmonary physiology is relatively unexplored.Complete deletion of LRP1 in mice results in neonatal death (10), hence tissue-specific knockout mice have been used to decipher the functions of LRP1. Macrophage-LRP1 deficiency increased expression of proinflammatory mediators, whereas also decreasing VLDL uptake and lipid accumulation in vitro (11). In vivo, macrophage LRP1 deficiency worsened atherosclerotic lesions (12, 13). These data suggest that anti-inflammatory roles of LRP1 in macrophages are predominant over its lipid metabolic effects. Liver-specific Lrp1 knockout mice developed nonalcoholic fatty liver disease (14), an effect opposite to that expected for a receptor known to mediate uptake of chylomicron remnants. Adipose-specific deletion of Lrp1 delayed postprandial triglyceride clearance and decreased adipogenesis (15), suggesting that it primarily functions as a lipoprotein receptor in adipocytes.Although LRP1 is highly expressed in lungs and its actions affect inflammation and repair (reviewed in Ref. (16)), the specific cell processes responsible for these effects remain to be defined. One possibility is that LRP1 is required for alveolar macrophages to eliminate pathogens and cell debris (17). Another is that fibroblast LRP1 clears MMP-2 and MMP-9 and protects the integrity of the ECM (18). Another likely site of LRP1 actions in the lung is via its expression in epithelial cells, but the role of LRP1 specifically in airway epithelium has not been reported.We aimed at elucidating the role of LRP1 in airway epithelium. We found that LRP1 expression was increased primarily in bronchial epithelium of COPD patients and generated club cell-specific tamoxifen-inducible LRP1 knockout mice (club Lrp1^−/−). Club Lrp1^−/− mice had increased pulmonary inflammation, and in response to chronic cigarette smoke, they exhibited worse pulmonary compliance and greater fibrotic scoring than WT mice. Proteomic analysis of isolated club cells linked this phenotype with cytoskeleton signaling and oxidative damage. We found that club LRP1 expression increases resistance to smoke and likely explains why SNPs in this gene associate with greater COPD in humans.