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American Journal of Respiratory and Critical Care Medicine logoLink to American Journal of Respiratory and Critical Care Medicine
. 2020 Nov 1;202(9):1225–1236. doi: 10.1164/rccm.201910-1958OC

Genome-Wide Association Study: Functional Variant rs2076295 Regulates Desmoplakin Expression in Airway Epithelial Cells

Yuan Hao 1,*, Samuel Bates 1,*, Hongmei Mou 2, Jeong H Yun 1, Betty Pham 1, Jiangyuan Liu 1, Weiliang Qiu 1, Feng Guo 1, Jarrett D Morrow 1, Craig P Hersh 1, Christopher J Benway 1, Lu Gong 1, Yihan Zhang 2, Ivan O Rosas 3, Michael H Cho 1, Jin-Ah Park 4, Peter J Castaldi 1, Fei Du 1, Xiaobo Zhou 1,✉
PMCID: PMC7605184  PMID: 32551799

Abstract

Rationale: Genetic association studies have identified rs2076295 in association with idiopathic pulmonary fibrosis (IPF). We hypothesized that rs2076295 is the functional variant regulating DSP (desmoplakin) expression in human bronchial epithelial cells, and DSP regulates extracellular matrix–related gene expression and cell migration, which is relevant to IPF development.

Objectives: To determine whether rs2076295 regulates DSP expression and the function of DSP in airway epithelial cells.

Methods: Using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 editing (including regional deletion, indel, CRISPR interference, and single-base editing), we modified rs2076295 and measured DSP expression in edited 16HBE14o- and primary airway epithelial cells. Cellular integrity, migration, and genome-wide gene expression changes were examined in 16HBE14o- single colonies with DSP knockout. The expression of DSP and its relevant matrix genes was measured by quantitative PCR and also analyzed in single-cell RNA-sequencing data from control and IPF lungs.

Measurements and Main Results: DSP is expressed predominantly in bronchial and alveolar epithelial cells, with reduced expression in alveolar epithelial cells in IPF lungs. The deletion of the DNA region-spanning rs2076295 led to reduced expression of DSP, and the edited rs2076295GG 16HBE14o- line has lower expression of DSP than the rs2076295TT lines. Knockout of DSP in 16HBE14o- cells decreased transepithelial resistance but increased cell migration, with increased expression of extracellular matrix–related genes, including MMP7 and MMP9. Silencing of MMP7 and MMP9 abolished increased migration in DSP-knockout cells.

Conclusions: rs2076295 regulates DSP expression in human airway epithelial cells. The loss of DSP enhances extracellular matrix–related gene expression and promotes cell migration, which may contribute to the pathogenesis of IPF.

Keywords: DSP, CRISPR/Cas9 genome-editing, bronchial epithelial cells, cell migration, rs2076295

At a Glance Commentary

Scientific Knowledge on the Subject

Noncoding variant rs2076295 is the most significant genetic variant at chromosome 6q24 associated with the susceptibility of idiopathic pulmonary fibrosis (IPF) in genome-wide association studies (GWAS). This variant is also associated with chronic obstructive pulmonary disease in the opposite direction. However, the causal gene and causal variant for the association in this locus are not entirely known yet.

What This Study Adds to the Field

Using a series of CRISPR (clustered regularly interspaced short palindromic repeat)-based genome-editing approaches, we demonstrated that rs2076295 regulates DSP (desmoplakin) expression in airway epithelial cells, which supported DSP as the causal gene in this GWAS locus implicated in IPF pathogenesis. We also showed that reduced levels of DSP, associated with the IPF risk allele in this GWAS locus, leads to increased expression of extracellular matrix genes and promotes the migration of airway epithelial cells. Our findings represent the critical and necessary first step on the post-GWAS functional studies, preceding future in-depth investigations on how the loss of DSP may promote pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive lung scarring and fibrosis in which alveolar architecture is destroyed and replaced by aberrant extracellular matrix (ECM) and activated myofibroblasts, eventually leading to respiratory failure and death (1). IPF affects more than 3 million people worldwide, with 2–3 years of median survival after diagnosis (2, 3). The etiology of IPF is complex, attributable to microenvironmental factors in subjects with genetic predispositions (4).

Genome-wide association studies (GWAS) have identified 14 disease susceptibility loci associated with IPF in an unbiased way (5), including a region at chromosome 6p24, with rs2076295 as the most significant variant (6–8). rs2076295 is a noncoding variant located in the intron 5 of DSP (desmoplakin) gene, encoding DSP that forms cell–cell adhesion complexes, enabling tissues to resist mechanical forces. Subsequent analysis of expression quantitative trait loci (eQTLs) identified rs2076295 as an eQTL SNP for DSP gene expression in human lungs (7, 9), suggesting that DSP is likely the causal gene in this locus.

Despite multiple reports on the association of rs2076295 with IPF susceptibility, these following questions remain unaddressed: 1) Does rs2076295 directly regulate DSP expression? 2) What is the directional regulation of rs2076295 on the expression of DSP in the lung-relevant cell type? 3) What is the molecular function of DSP that may determine IPF susceptibility? The identification of functional variants in each GWAS locus is the important and necessary first step to understanding the biological function of GWAS loci (10). In this study, we identified rs2076295 as a functional variant at the 6q24 DSP GWAS locus by showing the allele-specific expression of DSP in isogenic human bronchial epithelial cell line (16HBE14o- cells) edited by CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9–based techniques targeting rs2076295. To determine the function of DSP in lung epithelial cells, we used the CRISPR/Cas9 method to generate knockout clones of DSP in 16HBE14o- cells that showed increased migration and reduced transepithelial electrical resistance (TEER). By RNA-sequencing (RNA-seq) in isogenic clones, we found that DSP knockout led to increased expression of MMP7 (matrix metalloproteinase 7) and MMP9, which in turn enhanced cell migration and might contribute to the pathogenesis of IPF. Expression levels of DSP and other ECM genes were measured by quantitative PCR and further explored using publicly available single-cell RNA-seq data in human lungs (normal and with IPF).

Methods

Cell Line, Primary Cells, and Human Lung Tissues

Human primary cells were isolated from healthy donor lungs or purchased from Lonza (normal human bronchial epithelial [NHBE] cells; CC-2540). The human lung tissue samples were approved by the institutional review board of Partners HealthCare. Detailed information on human IPF and control lung samples are shown in Table E1 in the online supplement.

CRISPR/Cas9 Guide RNA Delivery

Guide RNAs (gRNAs) used in CRISPR/Cas9-based methods targeting rs2076295 are summarized in Table E2. The gRNA was delivered into 16HBE14o- cells by transfection with Lipofectamine 3000 and into NHBE cells by the ribonucleoprotein method, using Amaxa 4D-Nucleofector electroporation.

Single-Cell RNA-Seq Dataset Analysis

The publicly available human single-cell RNA-seq dataset (GSE135893) (11) was reanalyzed for cell type–specific gene expression using Seurat version 3 (12).

Migration Assay

Cells were seeded in 96-well Oris Cell Migration Assay (Platypus Technologies) using Calcein-AM (Invitrogen) for live-cell fluorescence staining based on the manufacturer’s instructions.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 7 and R 3.6.2. All data were tested for normal distribution with the Shapiro-Wilk normality test before we used an unpaired Student’s t test or the Mann-Whitney test for comparisons. Details on statistical analyses are described in the figure legends and the online supplement.

Detailed methods are described in online supplement.

Results

rs2076295 Regulates the Expression of DSP

Regulatory regions near functional SNPs are typically characterized as open chromatin regions harboring DNase hypersensitivity peaks or assay for transposase-accessible chromatin using sequencing (ATAC-Seq) peak. Indeed, based on data from the Roadmap Epigenomics Project and ENCODE Project (13–16), strong DNase hypersensitivity signals were found near rs2076295 in bronchial epithelial cells, in contrast to lung endothelial cells and lung fibroblasts (Figure E1A). Consistently, in our assay for transposase-accessible chromatin sequencing analysis, we detected open chromatin peaks near exon 5 of DSP around rs2076295 (Figure E1B) in both human primary airway epithelial cells and alveolar type II epithelial cells isolated from healthy donors. These results suggested human bronchial epithelial cells as the relevant cell type for studying the function of rs2076295 in the DSP GWAS region.

In human primary bronchial epithelial cells (NHBE) obtained by bronchoscopy brushing (17), the samples with GG homozygous genotype at rs2076295 exhibited a lower expression of DSP compared with the TT homozygous samples (Figure 1A), prioritizing DSP as a potential candidate gene associated with rs2076295.

Figure 1.

Figure 1.

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Genetic variant rs2076295 regulates DSP (desmoplakin) expression. (A) Expression of DSP in human bronchial epithelial cells collected from brushing with various genotypes of rs2076295. n = 7–11 subjects/genotype group, as indicated by numbers inside each column. (B) Expression of DSP in edited 16HBE14o- cells targeting rs2076295 for regional deletion by CRSIPR/Cas9. Means ± SEM are from six biological replicates. (C) Upper: Sequencing results in empty vector-transfected wild-type (WT) and three clonal cell lines with CRISPR (clustered regularly interspaced short palindromic repeat) (CRISPR)/Cas9–edited indel mutations targeting rs2076295. The inserted DNA bases are displayed in red, with the number of deleted (−) or inserted (+) bases for each clone shown on the right. Lower: Measurements on the expression of DSP in three indel single clones of 16HBE14o- cells and one WT control are shown. Means ± SEM are from at least four biological replicates. (D) Upper: Expression of DSP in 16HBE14o- cells edited by CRISPR interference (CRISPRi) targeting rs2076295. Means ± SEM are two biological replicates. Location of three single guide RNAs designed for CRISPRi targeting rs2076295 are indicated below. (E) Expression of DSP in 16HBE14o- lines (GT) edited by CRISPR/Cas9 method into homozygous (TT or GG) at rs2076295. Means ± SEM are from four to nine biological replicates of each isogenic line. (F) Relative luciferase activity with either G or T allele of rs2076295 as measured in reporter assay with endogenous DSP promoter cloned in pGL3 basic vector and transfected into 16HBE14o- cells. Means ± SEM are from two biological replicates representative of four biological repeats. (G) Expression of DSP in primary normal human bronchial epithelial cells with or without rs2076295 regional deletion by CRISPR/Cas9. Means ± SEM are from three biological replicates. WT control cells are cells transfected with empty vector. *P < 0.05, unpaired t test (for A–E and G) and Mann-Whitney test (for F). NHBE = normal human bronchial epithelial; sgRNA = single guide RNA.

Given the pervasive nature of eQTLs, many SNPs that are associated with both a disease phenotype and gene expression are not necessarily functional variants for GWAS. Nevertheless, the pattern of GWAS association in the 6q24 region (with statistical colocalization) (9) implicates SNP rs2076295 as the likely functional variant in this region. To experimentally confirm this, we performed four sets of CRISPR/Cas9-based gene editing experiments in human bronchial epithelial cells (16HBE14o- cells). First, we designed two gRNAs and generated a 55 bp deletion of nucleotide region-spanning rs2076295. This resulted in >90% deletion efficiency, as shown by Droplet Digital PCR (Figure E1C), accompanied by a 30% reduction in DSP expression (Figure 1B) in a mixed population of 16HBE14o- cells. Second, we designed a single gRNA targeting rs2076295 for the generation of small indels, and three single colonies were confirmed with variably sized indels near or spanning rs2076295. These resulting clonal cells exhibited significantly reduced expression of DSP (20–45%) (Figure 1C). Third, we applied CRISPR interference (CRISPRi) method (18), in which dCas-9 is coupled to the transcriptional repressor Krüppel–associated box to determine the regulatory effects of rs2079265 on the expression of DSP without chromatin cutting. Deactivating rs2076295 using the CRISPRi approach significantly decreased the expression of DSP to 50% in mixed cell populations (Figure 1D). All these results support the notion that the DNA region near rs2076295 is likely an enhancer that promotes the expression of DSP in 16HBE14o- cells. Lastly, we designed gRNA for CRISPR/Cas9-mediated homology-directed repair targeting rs2076295 in heterozygous 16HBE14o- cells (Figure 1E). This resulted in single colonies homozygous for the TT or GG genotype at rs2076295, which was confirmed by Sanger sequencing. By quantitative PCR analysis, cells with the GG genotype showed lower expression of DSP compared with those with the TT genotype, the same trend as seen in human primary bronchial epithelial cells (Figure 1A).

In addition to applying the CRISPR/Cas9 method to determine endogenous regulation of DSP expression by rs2076295, we also examined the allele-specific regulation of the DSP promoter by rs2076295 by luciferase reporter assays in 16HBE14o- cells. Indeed, the DNA constructs containing the T allele of rs2076295 led to higher DSP promoter activity compared with matched G allele constructs (Figure 1F).

Consistently, in primary NHBE cells, significantly decreased expression of DSP (∼20%) was detected (Figure 1G) after CRISPR/Cas9-mediated gRNA delivery targeting rs2076295, with more than 99% deletion efficiency shown by Droplet Digital PCR (Figure E1D).

Therefore, our results, obtained from a series of CRISPR/Cas9 genome-editing approaches and reporter assays, strongly support the idea that rs2076295 regulates DSP expression, with IPF risk allele GG decreasing expression of DSP in human bronchial epithelial cells.

DSP Is Localized in Both Human Airway and Alveolar Epithelial Cells

The expression of DSP was detected primarily in human lung epithelial cells (Figure E2A). By immunohistochemistry staining, DSP is detected in both airway and alveolar epithelial cells in normal human lungs (Figure 2A). Specifically, DSP showed a punctate staining pattern in the cell boundary between alveolar type I (AT1; marked with T1α) and type II cells (AT2; marked with ABCA3) in alveoli as well as the apical and lateral sides of airway epithelial cells, including ciliated cells and club cells (Figure 2B). The expression of DSP in airway basal cells (marked with Krt5) was also detected (Figure 2B). However, airway smooth muscle cells and lung fibroblasts showed no DSP staining, as expected (Figure E2B). In addition, in NHBE cells (Figure 2C) as well as in mouse tracheal epithelial cells differentiated at the air–liquid interface (Figures E2C and E2D), DSP demonstrated strong expression at the apical surface of the airway epithelium, suggesting that the desmosomes formed by DSP may be important for maintaining airway epithelial integrity.

Figure 2.

Figure 2.

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Localization of DSP (desmoplakin) in human lungs. (A) Representative images and enlarged images (in the black squares) of immunohistochemistry staining for DSP in the bronchi and alveoli in human lung tissue. Scale bars, 10 μm. (B) Representative images and enlarged images (in the white squares) of immunofluorescence staining of DSP (green) and various cell-type markers (red), including T1α, ABCA3, acetylated tubulin, CC10, and keratin 5, with DAPI (blue) in human lung tissue. Scale bars, 20 μm. The white dashed line indicates the basal membrane. (C) Images of immunofluorescence staining of DSP in differentiated human airway epithelial cells at the air–liquid interface after 7 days of differentiation. The three dashed lines (a–c) indicate the levels where images were taken (the top, middle, and bottom areas of the luminal epithelial cells at cross-section). Scale bars, 50 μm. ABCA3 = ATP-binding cassette subfamily A member 3; ACTTUB = acetylated tubulin; ALI = air–liquid interface; IHC = immunohistochemistry; Krt5 = keratin 5; T1α = lung type I cell membrane–associated glycoprotein.

Knockout of DSP Reduces TEER, Increases Epithelial–Mesenchymal Transition, and Promotes Cell Migration in Epithelial Cells

Using gRNA targeting exon 2 of DSP, we generated three DSP-knockout clonal lines in 16HBE14o- cells (Figures 3A and 3B). In DSP-knockout clonal lines, the loss of punctate staining of DSP between cell boundaries is accompanied by the perinuclear accumulation of keratin 5 (in red), in contrast to a normal filament network pattern of keratin 5 attached to desmosomes in wild-type cells (Figure 3C) (19). As a key component of the desmosome complex, DSP enables epithelial cells to resist mechanical stress, which is required to maintain the integrity of the epithelial barrier (20, 21). To directly examine the role of DSP in maintaining cellular integrity and permeability of bronchial epithelial cells cultured at monolayers, we assessed TEER in DSP-knockout clones. Indeed, decreased TEER was detected in the DSP-knockout clones compared with wild-type cells after monolayer culture in transwells (Figures 3D and 3E).

Figure 3.

Figure 3.

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Generation of DSP (desmoplakin)-knockout (KO) lines in 16HBE14o- human bronchial epithelial cell line. (A) Expression of DSP in three single colonies of 16HBE14o- cells with DSP-KO (KO1, KO2, and KO3) using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 targeting the coding region of DSP. Means ± SEM are from four biological replicates. (B) The levels of DSP as measured in wild-type (WT) and DSP-KO 16HBE14o- cells by Western blotting. Vinculin was applied as a loading control. (C) The representative immunofluorescence staining of DSP (green) and keratin 5 (red) in DSP-KO cells and control cells. Nuclei are counterstained with DAPI (blue). Scale bars, 10 μm. (D) The transepithelial electrical resistance (TEER) measurement in WT and three DSP-KO 16HBE14o- cells cultured on transwells for 18 continuous days. Means ± SEM are shown from triplicate wells within each line. One representative result is shown from two biological replicates. A linear mixed effects model with Dunnett’s post hoc test used to compare difference among four groups demonstrated significant difference among groups over time. Significantly reduced TEER was found in at least two KO lines compared with WT cells, starting from day 7 to Day 18. (E) TEER measurements on Day 18 in D were shown in WT and DSP-KO cells. *P < 0.05, unpaired t test. WT control cells are cells transfected with empty vector. Krt5 = keratin 5.

Epithelial cells in IPF lungs usually have epithelial–mesenchymal transition (EMT) pathological changes (22). Indeed, significant downregulation of epithelial markers (E-cadherin, Syndecan-1, Claudin-1, and COL4A1) and upregulation of mesenchymal markers (Snail, vimentin, S100A4, N-cadherin, and Slug) were detected in DSP-knockout clones compared with in wild-type (empty vector-transfected) 16HBE14o- cells (Figures 4A and 4B). Similarly, decreased E-cadherin and increased vimentin were also detected by immunofluorescence staining in DSP-knockout cells (Figure 4C), indicating that the loss of DSP may promote EMT in human bronchial epithelial cells. Consistent with EMT gene expression changes, 16HBE14o- cells with reduced expression of DSP by either knockout or rs2076295 indel mutation exhibited increased cell migration (Figures 4D and 4E), a key feature of fibrotic cells in IPF lungs (23). We also confirmed increased cell migration upon DSP knockout in primary airway epithelial cells isolated from DSP flox/flox mice treated with AAV-Cre ex vivo (Figures 4F and 4G), in which the knockout efficiency of DSP was more than 80% (Figure 4H). In contrast, cell death and cell proliferation are comparable between control and DSP-knockout 16HBE14o- clonal lines (Figures E3A and E3B).

Figure 4.

Figure 4.

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Influences of DSP (desmoplakin)-knockout (KO) on epithelial–mesenchymal transition, and bronchial epithelial cell migration. (A) Expression of epithelial markers (CDH1, COL4A1, and SDC1) and mesenchymal markers (SNAI1, VIM, and S100A4) in control and DSP-KO 16HBE14o- cells measured by quantitative PCR. Means ± SEM are from four biological replicates. GAPDH was applied as a reference gene. *False discovery rate–adjusted P value < 0.05, unpaired t tests with false discovery rate correction for multiple testing. (B) The Western blotting result of mesenchymal and epithelial markers in control and DSP-KO 16HBE14o- cells. α-Tubulin was applied as a loading control. (C) The representative images and enlarged images of immunofluorescence staining of E-cadherin (green) and vimentin (red) with DAPI (blue) staining in wild-type (WT) and DSP-KO 16HBE14o- cells. Scale bars, 50 μm. (D) The representative images of cell migration assays in WT, DSP-KO, and rs2076295 indel mutant 16HBE14o- cells at 0 and 24 hours after migration initiation. The leading edges of the migrated cells were outlined by black lines. (E) Quantification on cell migration rate by fluorescence measurement with Calcein-AM dye. Means ± SEM are from three biological replicates. (F) The representative images of cell migration assays in WT and two ex vivo induced DSP-KO mouse tracheal basal cells at 0 and 48 hours after migration initiation. The leading edges of the migrated cells are outlined by black lines. *P < 0.05, unpaired t test. (G) Quantification on cell migration rate by fluorescence measurement with Calcein-AM dye. Means ± SEM are from triplicate wells in the tracheal basal cells isolated from WT (DSPfl/fl) and two ex vivo induced DSP-KO mice (DSPfl/fl+Cre). *P < 0.05, unpaired t test. (H) Expression of DSP in mouse tracheal basal cells used in G. Means ± SEM are from duplicate wells. WT control cells in A–E are cells transfected with empty vector. E-cad = E-cadherin; Indel = insertions or deletions of three or multiples of three base pairs; VIM = vimentin.

Differentially Expressed Genes in DSP-Knockout 16HBE14o- Cells Include Differentially Expressed Genes Found in Human IPF Lungs

To further dissect the signaling pathways regulated by DSP, RNA-seq was performed in DSP-knockout and wild-type 16HBE14o- cells. A total of 209 genes exhibited significantly decreased expression, and 50 genes exhibited significantly increased expression in DSP-knockout 16HBE14o- cells compared with wild-type cells (Table E3).

Given that the IPF risk allele G at rs2076295 (24) is associated with reduced expression of DSP (Figures 1A and 1E), we hypothesized that the knockout of DSP in 16HBE14o- cells might lead to gene expression changes that resemble transcriptomic signatures of IPF lungs. Therefore, we compared previously published RNA-seq data in human IPF lungs (25) with RNA-seq data in DSP-knockout clones (Figure E4). Overlapped genes include four upregulated ECM-related genes (COL6A3, MMP7, MFAP2, and DNAH3) and six downregulated genes (SEMA3E, KLF15, PTX3, MGAM, TNNC1, and BDNF), supporting the idea that the downregulation of DSP may trigger similar pathways to those altered in human IPF lungs.

DSP Modulates Bronchial Epithelial Cell Migration via Regulating ECM-related Genes

Pathway analysis on differentially expressed genes in DSP-knockout cells demonstrated that genes with decreased expression in knockout clones were involved in cell–cell adhesion and cell junction, whereas genes with increased expression were involved in ECM remodeling. We then validated the expression changes of upregulated ECM genes in cells with either DSP knockout or indel mutation at rs2076295 (Figure 5A), including MFAP2, ADAM19, SRGN, MMP7, MMP9, COL6A3, and COL13A1 by quantitative PCR (Figures 5B and 5C). Among these seven genes, five of them, including MFAP2, ADAM19, MMP7, MMP9, and COL6A3, showed consistently increased expression when either DSP was knocked out or rs2076295 was modified with an indel. Furthermore, increased expression of MMP7, MMP9, and ADAM19 was confirmed in NHBE cells cultured at the air–liquid interface (Figure 5D) after DSP knockout by CRISPR/Cas9 as well as primary EpCAM+ lung epithelial cells from DSP flox/flox mice with ex vivo treatment of Adeno-Cre in three-dimensional organoid models (Figure 5E). These results suggest that DSP represses the expression of ECM genes in both murine and human primary lung epithelial cells.

Figure 5.

Figure 5.

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DSP (desmoplakin) regulates extracellular matrix (ECM)-related gene expression and cell migration in bronchial epithelial cells. (A) The heatmap of RNA-sequencing results showing differentially expressed ECM-related genes in DSP-knockout (KO) 16HBE14o- cells compared with wild-type (WT) control cells. (B) Relative expression of DSP-regulated ECM-related genes identified from RNA-sequencing results in WT and three clones of DSP-KO 16HBE14o- cells (KO1, KO2, and KO3). Means ± SEM are from two biological replicates for each individual clone. (C) Relative expression of DSP-regulated ECM-related genes in rs2076295 indel mutation cells (Indel) versus WT control. Means ± SEM are from two biological replicates. (D) Relative expression of DSP- and ECM-related genes in WT and DSP-KO primary human bronchial epithelial cells cultured at the air–liquid interface for 7 days. Means ± SEM are from duplicate wells. (E) Relative expression of ECM-related genes in EpCAM+ lung epithelial cells from a DSP flox/flox mouse treated with Adeno-Cre followed by subsequent culture at three-dimensional organoid models. Means ± SEM are from two independent replicates. *False discovery rate–adjusted P value < 0.05, unpaired t tests with false discovery rate correction for multiple testing for B–E. (F) The representative images of WT and DSP-KO 16HBE14o- cells with or without knockdown of MMP7 and MMP9 in migration assay at 24 hours after migration initiation. The leading edges of the migrated cells were outlined in black. (G) The cell migration rate in WT and DSP-KO 16HBE14o- cells after knockdown of individual ECM-related genes by siRNAs. Means ± SEM are from three biological replicates. * or §P < 0.05, two-way ANOVA with Dunnett’s post hoc test comparing cells transfected with siRNA targeting various genes to cells transfected with negative control within DSP-KO or WT groups, respectively. #P < 0.05, unpaired t test comparing between WT and DSP-KO cells. WT control cells are cells transfected with empty vector. ALI = air–liquid interface; EpCAM = epithelial cell adhesion molecule; NHBE = normal human bronchial epithelial cells.

To determine whether these DSP-regulated ECM genes contribute to DSP-mediated cell migration, we knocked down five ECM-related genes individually in wild-type and DSP-knockout 16HBE14o- cells and measured cell migration. The silencing of MMP7 or MMP9 reversed the increased migration in DSP-knockout cells (Figures 5F and 5G), indicating that MMP7 and MMP9 may mediate DSP-regulated cell migration.

Cell Type–Specific Expression Changes of DSP in Human IPF Lungs

To determine the expression changes of DSP and its regulated genes in human IPF lungs, we performed a series of analyses in human IPF lung samples. First, the expression of DSP showed an approximately threefold increase at the mRNA level and a twofold increase at the protein level in total IPF lungs compared with that found in normal control lungs (Figures 6A–6C). Moreover, reduced concentrations of the epithelial cell marker E-cadherin and increased concentrations of the mesenchymal cell marker vimentin were detected in IPF lungs, which is consistent with increased EMT in IPF lungs (Figures 6D and 6E), as reported previously (22, 26). Second, decreased expression of E-cadherin and increased staining of vimentin were evident in airway regions from IPF lungs (Figure 6F). However, we were unable to detect EMT changes in distal lungs because of the complete loss of normal alveolar structure in the end stage of IPF. Lastly, publicly available single-cell RNA-seq data in normal and IPF human lungs(11) were analyzed for cell type–specific expressions of DSP and its regulated genes. Despite the increased expression in whole lungs, DSP showed significantly reduced expression in alveolar epithelial cells, including AT1, AT2, and ciliated cells (Figures 6G and 6H). Among the nine EMT marker genes we examined, seven genes showed detectable expression (Figure 6I) in at least one lung epithelial cell type. As an important cell type for IPF, AT2 cells showed significant downregulation of epithelial genes (CDH1) and upregulation of mesenchymal genes (VIM and FN1) as well as ECM-related genes (MMP7 and SRGN). In summary, human IPF lungs demonstrated reduced expression of epithelial cells genes including DSP and increased expression of mesenchymal and ECM genes in AT2 cells, as we observed in DSP-knockout 16HBE14o- cells.

Figure 6.

Figure 6.

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Detection of DSP (desmoplakin) and its regulated genes in human idiopathic pulmonary fibrosis (IPF) lungs. (A) Expression of DSP in human IPF (n = 16) and control lungs (n = 16). *P < 0.05, unpaired t test. (B) Protein levels of DSP, E-cadherin, and vimentin measured in healthy control and IPF lungs by Western blotting. β-actin was applied as a loading control. (C–E) The quantification of protein levels for DSP, E-cadherin, and vimentin in the lung of healthy control (n = 15) and IPF (n = 14) lungs. Means ± SEM are shown. *P < 0.05, unpaired t test (for C) or Mann-Whitney test (for D and E). (F) The representative images of immunofluorescence staining of E-cadherin (green), vimentin (red), and DAPI (blue) in human control and IPF lung tissue. Scale bars, 20 μm. (G) Heatmap showing average expression of DSP in control and IPF lungs using data from single-cell RNA-sequencing in human IPF lungs. (H) Heatmap showing the average expression of DSP within various lung epithelial subtypes. (I) Heatmap showing average log2 fold difference in gene expression between IPF and control lungs by cell type categories. Genes with an adjusted P value lower than 0.10 were plotted. AT1 = type I alveolar epithelial cells; AT2 = alveolar type II epithelial cells; avg_diff = average log2 fold difference; E-Cad = E-cadherin; ECM = extracellular matrix; VIM = vimentin.

Discussion

Multiple publications have consistently reported the association of rs2076295, an intronic variant within the DSP gene, with IPF susceptibility by GWAS (14, 27), yet no functional studies have been conducted on this variant (7, 28). Using comprehensive CRISPR/Cas9-based methods in both cell line and primary NHBE cells, we demonstrate for the first time 1) the direct causal link between rs2076295 and DSP expression, 2) the directional regulation of rs2076295 on the expression of DSP in human airway epithelial cells, 3) the location of DSP at the apical surface of airway epithelial cells, and 4) that the loss of DSP in lung epithelial cells leads to reduced epithelial features (reduced barrier function) with transitional changes toward fibroblasts (increased expression of EMT and ECM genes with increased cell migration). Our findings represent the important and necessary first step for functional studies on the DSP GWAS locus in IPF.

The IPF risk allele rs2076295G is genetically associated with decreased expression of DSP (8, 24); however, increased expression of DSP at both RNA and protein levels in human IPF lungs has been reported (8), as we observed as well (Figures 6A–6C). One possible reason that explains such seemingly conflicting changes is the stiffness-induced expression of DSP in IPF lungs, which have a significantly increased number of myofibroblasts and stiffness of lung matrix (29). Therefore, increased expression of DSP in total lungs is a consequence instead of a driving force of IPF development. Second, elevated expression levels of DSP in total IPF lungs may represent a mixture of altered gene expression in multiple lung cell types, as supported by single-cell RNA-seq analysis (Figures 6G and 6H). We found that in healthy lungs, ciliated cells have the most DSP expression compared with other lung epithelial cells, whereas in IPF lungs, aberrant basal cells (30) have significantly increased expression of DSP (Figure 6H), suggesting that basal cells may be the most sensitive to matrix stiffness–induced expression of DSP. Meanwhile, alveolar epithelial cells, including AT1 and AT2 cells, have reduced expression of DSP, supporting the theory that reduced DSP expression increased the risk of IPF. Therefore, although altered expression levels of a given GWAS gene in human diseased tissues can support its relevance to human disease, the directional impacts of the GWAS gene on disease susceptibility and progression are more reliably inferred by the combined analysis of genetic association studies, eQTL analysis in relevant tissues, and CRISPR/Cas9-based genome-editing approaches in relevant cell types.

In addition to IPF, rs2076295 is also associated with chronic obstructive pulmonary disease (COPD) in GWAS but with an opposite direction of the effects for the risk allele (9), similar to other COPD/IPF-overlapped GWAS loci such as FAM13A and MAPT loci, where the fibrosis risk alleles are protective for COPD (24). It would be intriguing to determine how these overlapped GWAS loci may divert the disease susceptibility toward either IPF or COPD in future animal models. Despite lacking in vivo evidence to conclude the roles of DSP in the development of IPF and COPD, our studies have provided a series of solid evidence to improve our understanding of the function of this GWAS locus. First, the functional variant rs2076295, as the top GWAS SNP for both COPD and IPF, regulates the expression of DSP (supported by comprehensive CRISPR/Cas9 genome-editing approaches). Second, DSP is localized in various lung epithelial cell types that are relevant for both COPD and IPF. Third, in a loss-of-function model, DSP regulates ECM gene expression and inhibits cell migration, an important cellular function for both COPD and IPF pathogenesis.

Bronchial epithelial cells were used as the main cellular model for rs2076295 functional studies because of feasibilities and efficiency related with transfection experiments and CRISPR/Cas9-mediated genome-editing methods. Furthermore, open chromatin near rs2076295 in both bronchial epithelial cells and AT2 cells (Figures E1A and E1B) also supports airway epithelial cells as a relevant cellular model to study the effects of rs2076295. Admittedly, critical roles of alveolar epithelial cells for the pathogenesis of progressive fibrosis are nonnegligible, future open chromatin profiling studies on normal and IPF AT2 cells may illuminate more comprehensive and mechanistic insights into putative functional variants in all IPF GWAS loci.

DSP knockout led to an increased expression of genes related to the ECM (Figure 5A). The ECM not only provides a scaffold for epithelial cells, it also provides essential cues for tissue morphogenesis during development and for tissue repair during homeostasis and disease. The ECM composition is markedly changed in the pathogenesis of both COPD and IPF (31, 32). IPF is characterized by an accumulation of excess fibrous material in the lung, resulting from the excessive production and deposition of ECM (32), whereas emphysema, a major pathological manifestation of COPD, results from parenchymal destruction due to significant ECM loss and defective alveolar repair. We speculate that increased ECM gene expression under DSP deficiency may prevent emphysema development but promote fibrotic changes because we see that MFAP2, MMP7 and COL6A3 (Figures 5B and 5C) were all upregulated in human IPF lungs (Figure E4).

Furthermore, DSP-deficient 16HBE14o- cells have increased cell migration, as reported previously, and depletion of DSP in mice is associated with increased tumor invasion in pancreatic cancer (33). More importantly, we found that the silencing of MMP7 or MMP9 reversed DSP knockout–induced cell migration (Figures 5F and 5G). MMP7 and MMP9 belong to matrix metalloproteinases, a pivotal family of zinc enzymes capable of degrading ECM components, including basement membrane collagen, interstitial collagen, fibronectin, and various proteoglycans, during tissue remodeling and repair processes (34). Several matrix metalloproteinases (including MMP1, MMP7, MMP8, and MMP9) showed increased levels in human IPF lungs, possibly contributing to ECM remodeling and basement membrane disruption during IPF development (35–37). For example, MMP7 induced epithelial-to-mesenchymal transition, whereas MMP9 may promote abnormal epithelial cell migration and other aberrant repair processes (37), thus promoting the pathogenesis of fibrotic lungs. Admittedly, future mechanistic studies on the possible regulation of DSP on Wnt/β-catenin (38), TGFβ (39), and Notch pathways (40, 41) modulating cell migration will illuminate additional insights into DSP.

In conclusion, our study provides multiple evidences to elucidate the role of IPF-susceptibility variant rs2076295 in regulating DSP expression in human bronchial epithelial cells, with the risk allele G leading to lower DSP expression. DSP is normally expressed predominantly in lung epithelial cells, and reduced DSP expression promotes cell migration possibly by upregulating ECM-related genes, which may contribute to IPF development.

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Acknowledgments

Acknowledgment

The authors thank Dr. Scott H. Randell from the University of North Carolina at Chapel Hill for generously providing healthy human lung samples for primary lung epithelial cell isolation used in quantitative PCR and assay for transposase-accessible chromatin sequencing analysis. They also thank Dr. Xingbin Ai from Massachusetts General Hospital, Harvard Medical School, for her valuable comments on this study.

Footnotes

Supported by U.S. NIH grants R01HL127200, 1P01HL132825, R01HL148667 (X.Z.), and R01HL137927 and R01HL147148 (M.H.C. and X.Z.); Cystic Fibrosis Foundation grant MOU19G0 (H.M.); and a Child Health Research Award (H.M.) from the Charles H. Hood Foundation.

Author Contributions: Y.H., F.D., and X.Z. designed the study. Y.H. designed, conducted, and analyzed the biological experiment. S.B. and F.D. designed and conducted CRISPR/Cas9 genome-editing and RNA-sequencing experiments. Y.H. and S.B. performed immunofluorescence and cell migration experiment. J.H.Y. performed single-cell RNA-sequencing data analysis. B.P. performed quantitative PCR and luciferase reporter assay. H.M., Y.Z., and J.-A.P. performed air–liquid interface culture and identification. J.L., W.Q., C.J.B., and M.H.C. analyzed RNA-sequencing data. J.D.M. and C.P.H. analyzed gene expression data in the human brushing sample. L.G. performed online data mining on open chromatin status nearby rs2076295. F.G. isolated human alveolar type II epithelial cells. I.O.R. provided normal and idiopathic pulmonary fibrosis human lung tissue samples. Y.H., H.M., J.-A.P., P.J.C., F.D., and X.Z. participated in the manuscript writing.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.201910-1958OC on June 18, 2020

Author disclosures are available with the text of this article at www.atsjournals.org.

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