A Chronic Obstructive Pulmonary Disease Susceptibility Gene, FAM13A, Regulates Protein Stability of β-Catenin

Abstract

Rationale: A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown.

Objectives: To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility.

Methods: Fam13a null mice (Fam13a^−/−) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a^−/− and Fam13a^+/+ littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase–polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13A’s function.

Measurements and Main Results: In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a^−/−) were resistant to chronic cigarette smoke–induced emphysema compared with Fam13a^+/+ mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3β and β-catenin, inducing β-catenin degradation. Fam13a^−/− mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a β-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting β-catenin signaling. Moreover, human COPD lungs had decreased protein levels of β-catenin and increased protein levels of FAM13A.

Conclusions: We show that FAM13A may influence COPD susceptibility by promoting β-catenin degradation.

At a Glance of Commentary

Scientific Knowledge on the Subject

Genome-wide association studies (GWASs) have been successful in identifying genomic regions associated with chronic obstructive pulmonary disease. However, the identification of the key genes within those GWAS regions and their biologic impact requires further investigation.

What This Study Adds to the Field

Our research provides a functional study on FAM13A, a chronic obstructive pulmonary disease GWAS region gene, and reveals that the molecular mechanism by which FAM13A influences chronic obstructive pulmonary disease susceptibility is likely through interacting with PP2A/β-catenin, thus promoting degradation of β-catenin.

Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the United States (1, 2), is characterized by emphysematous destruction of the alveoli and thickening of the airway walls in response to chronic cigarette smoke (CS) exposure. Although the dose-response relationship between cigarette smoking and pulmonary function levels is well-established, there is considerable variability in the reduction in pulmonary function among smokers with similar smoking exposures (3, 4). The low percentage of variance in pulmonary function explained by smoking suggests that there could be differences in genetic susceptibility to the effects of cigarette smoking (5, 6). Genome-wide association studies (GWASs) provide an unbiased and comprehensive approach to search for common genetic susceptibility loci throughout the genome in thousands of subjects (7, 8). These studies have successfully identified susceptibility loci for many complex diseases (9, 10). The FAM13A gene (family with sequence similarity 13, member A) has been consistently associated with COPD by GWAS (11, 12), but the molecular mechanisms and biologic functions of FAM13A remain largely unexplored.

The central molecule in the Wnt pathway, β-catenin, is normally maintained at low levels in the cytosol because of its association with a destruction complex, including glycogen synthase kinase 3β (GSK-3β), which phosphorylates β-catenin for rapid degradation via ubiquitin-dependent proteolysis (13, 14). Upon stimulation by Wnt ligands, β-catenin is stabilized and then translocates to the nucleus, where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, activating the expression of Wnt target genes, promoting cell proliferation (15–17) and limiting cell differentiation (16, 18), thus contributing to both airway (18, 19) and alveolar epithelial repair (20–22) after injury. Consistent with this concept, activation of β-catenin signaling by lithium chloride attenuated elastase-induced experimental emphysema (21), suggesting that β-catenin signaling contributes to lung repair/regeneration in response to various injuries.

Herein, we used complementary in vivo and in vitro approaches coupled with studies of human COPD samples to provide evidence that FAM13A influences COPD susceptibility by promoting β-catenin degradation. Some of the results of this study have been previously reported in the form of an abstract (23).

Methods

Human Tissues

Studies of human samples were approved by the Partners Human Research Committee (Boston, MA). Sections of lung were obtained from lung biopsies or from explanted lungs from patients with COPD provided by the NHLBI-sponsored Lung Tissue Research Consortium (LTRC) or Pulmonary and Critical Care Medicine Human Biorepository at Brigham and Women’s Hospital. Deidentified specimens were used in all of our assays. The localization of FAM13A in human lungs was examined using immunofluorescence staining of formalin-fixed and paraffin-embedded lung sections. Expression levels of β-catenin and FAM13A were assessed in human lung sections as described previously (24). Subjects with very severe COPD had FEV₁ less than 40% predicted (see Table E2 in the online supplement). All of the demographic and clinical data were provided by the LTRC.

Generation of Fam13a^−/− Mice

All animal studies were approved by the Institutional Animal Care and Use Committee, Harvard Medical School. Murine embryonic stem cells (JM8.N4 bought from the knockout mouse project repository, now the International Mouse Phenotype Consortium, project #70561) transfected with a Fam13a gene-targeted mutant construct (25) were injected into C57BL/6 host embryos to generate Fam13a mutant chimeric mice that were then bred with C57BL/6 wild-type mice to obtain germ-line transmission of the mouse Fam13a mutant allele. All mice were housed in the animal facility of Harvard Medical School with a 12-hour light/12-hour dark cycle. Five Fam13a^−/− mice at 2 months of age were harvested and major organs were removed. Organ sections were assessed by a senior pathologist (L.K.) to determine whether there were any abnormalities in major organs (lung, kidney, intestine, liver, and heart) (see Figure E2).

CS Exposure of Mice

Female Fam13a^−/− and Fam13^+/+ (∼10 wk old) mice were exposed to mixed main-stream and side-stream CS from 3R4F Kentucky Research cigarettes for 5 days/week in Teague TE 10z (Teague Enterprises, Woodland, CA) chambers (tobacco smoke pollution ∼100–200 mg/m³ and CO levels ∼6 parts per million). As a control, mice were exposed to filtered air (air) for the same time period. At the end of the exposure period, mice were killed by CO₂ narcosis and cervical dislocation before lung removal. Mice exposed to CS or air for 1 month and 6 months were harvested in a blinded way for subsequent analysis. We excluded two mice in the Fam13a^+/+ air group and one mouse in Fam13a^−/− CS group because of poor inflation for mean chord length analysis. Quality of lung inflation was judged by a senior investigator blinded to the experimental condition (26, 27).

Cell Lines and Plasmids

All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human bronchial epithelial cells (16HBE cells) and embryonic kidney epithelial cells (HEK 293) were cultured in Eagle's minimal essential medium or Dulbecco's modified Eagle medium, respectively, supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). For isolation of murine airway epithelial cells, mice were killed followed by immediate trachea removal. The trachea was then incubated in 15% pronase solution (Roche GmbH, Indianapolis, IN) overnight at 4°C. The next day, the digestion buffer was neutralized by 10% fetal bovine serum before three times of washing with Ham’s F12 media. Cell suspensions were centrifuged and then incubated with DNase-I (0.5 g/L) for 5 minutes on ice. Cells were then seeded on a 10-cm cell culture plate coated with rat-tail type I collagen (50 µg/cm²; BD Sciences, Franklin Lakes, NJ) and cultured in bronchial epithelial cell medium (ATCC) supplemented with bronchial epithelial cell growth kit (ATCC). Cells younger than passage 2 were used for experiments. Testing for Mycoplasma infection was performed routinely using Mycoalert Detection Kit from Lonza (LT07–218; Lonza, Hopkinton, MA) in cells used in this study.

Full-length human FAM13A (NP_001252507.1) and β-catenin (NP_001091679.1) were cloned into pCMV6-Entry Vector (Origene, Rockville, MD) with C-terminus FLAG and c-MYC tags and pmCherry.C1 vector with N-terminal mCherry tag. TOPFLASH and TK-rennila reporter plasmids were gifts from Dr. Ningzhi Xu (Chinese Academy of Medical Sciences and Peking Union Medical College) (28). The constitutively active mutant β-catenin (β-catenin::CS2) construct was a gift from Dr. Xingbin Ai (Harvard Medical School) (http://sitemaker.umich.edu/dlturner.vectors/home).

Statistical Methods

Statistical analysis for all pair-wise comparisons other than cell growth curve and β-catenin half-life measurement was performed using unpaired Student’s t test with equal variances for all measurements. If normality assumption is violated, then Wilcoxon rank sum test was used.

For cell growth curve, we performed linear regression for each growth curve with cell count as outcome and day as predictor. We then tested if the slope of one growth curve is equal to that of another growth curve using a Z test constructed based on the estimated slopes and their standard errors. In addition, we checked the log-transformed growth curves and found growth curves on the original scale were closer to linear than log transformed growth curves. Hence, we used the original scale in our analyses. Reductions in β-catenin levels in nontargeting control short hairpin RNA (NT-shRNA) and FAM13A shRNA cells in Figure 2G were compared based on linear mixed effects model. Detailed Methods are available in the online supplement.

Figure 2.

FAM13A interacts with PP2A/β-catenin complex and promotes degradation of β-catenin. (A) Affinity purification of cellular protein complexes associated with Flag-tagged FAM13A (FL) in HEK 293 cells as revealed by silver staining. Vector-transfected cells (V) were used as a control. The * indicates Flag-tagged FAM13A. Immunoprecipitation (IP) of FAM13A (B) and reverse immunoprecipitation (IP) targeting β-catenin (short for β) (C) was performed in HEK 293 cells. Examination of the levels and phosphorylation of β-catenin in human bronchial epithelial (16HBE) cells transfected with either increasing amount of FAM13A (D) or short hairpin RNAs (shRNAs) targeting FAM13A (E). NT = nontarget control hairpins; columns B, C, and D = three independent hairpins targeting FAM13A. (F) Expression of FAM13A and β-catenin in cells measured by reverse transcriptase–polymerase chain reaction. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. (G) Half-life of β-catenin was measured in 16HBE cells stably infected with nontargeting control shRNA (NT) or FAM13A shRNA B and treated with cycloheximide (CHX, 5 nM) for various time periods. Means ± SD were from two independent repeats normalized to α-tubulin. Linear mixed effects model was used to compare differences in slope of β-catenin reductions in NT-shRNA and FAM13A shRNA–transfected cells (P < 0.01).

Results

FAM13A Determines Susceptibility to Emphysema in Murine Models

FAM13A has been repeatedly identified in COPD GWA studies. However, the molecular mechanism by which FAM13A determines COPD susceptibility remains unclear. First, we stained FAM13A in human COPD lung sections to determine the tissue localization of FAM13A. Human airway cells positive for mucin 5AC (MUC5AC, mucosal cells), Club cell 10 KD protein (Club cells), or pan-cytokeratin (airway epithelial cells) and alveolar cells positive for surfactant protein-C (alveolar type II cells) express FAM13A in healthy smokers and COPD subjects (Figure 1A; see Figure E1A). Furthermore, FAM13A staining was also detected in CD14-positive alveolar macrophages (see Figure E1A). Immunofluorescence staining in murine lungs demonstrated the localization of Fam13a in airway epithelial cells staining positively for Club cell 10 KD protein and alveolar type II epithelial cells staining positively for surfactant protein-C (see Figure E1B). Both of these cell types are important for lung repair/regeneration (29, 30).

Figure 1.

Depletion of Fam13a confers resistance to emphysema development in murine models. (A) FAM13A is localized in human airway cells including mucosal cells (MUC5AC), Club cells (CC10), pan-cytokeratin (PanCK, airway epithelial cells), and alveolar cells positive for surfactant protein-C (SP-C, alveolar type II cells). Representative images are from patients with very severe chronic obstructive pulmonary disease (n = 3) (FEV₁ <40%). Goat = anti-goat; Ms = anti-mouse; Rb = anti-rabbit. (B) Representative hematoxylin and eosin staining images (200×) in Fam13a null mice (Fam13a^−/−) and wild-type littermate control mice (Fam13a^+/+) exposed to cigarette smoke (CS) or room air (Air) for 6 months. (C) Mean alveolar chord length measurement in four groups of mice. Data shown are means ± SEM derived from 11–22 mice per group (the number of mice studied in each group is indicated inside each column). (D) Total leukocytes and leukocyte subsets in bronchoalveolar lavage (BAL) samples from Fam13a^−/− and Fam13a^+/+ mice exposed to CS or air for 1 month. Two-way analysis of variance tests were used to analyze the effects of treatment and genotype in (C) and (D). Unpaired Student’s t test, **P < 0.01.

To determine the in vivo function of FAM13A, we generated Fam13a null mice (Fam13a^−/−) by targeting exon 5 of the murine Fam13a gene on a C57BL/6J background. We detected no expression of Fam13a in murine lungs from Fam13a^−/− mice at both mRNA and protein levels (see Figures E2A and E2B). Fam13a^−/− mice are viable and showed no gross defects in major organs at 2 months of age (see Figure E2C).

To model human emphysema, we chronically exposed Fam13a^−/− mice and Fam13a^+/+ mice to CS or room air (air) for 6 months. Airspace size was assessed in four groups of mice. As previously reported (31), CS-exposed wild-type C57BL/6 Fam13a^+/+ mice developed significant although moderate airspace enlargement (15% increase) (Figures 1B and 1C; P < 0.05). However, Fam13a^−/− mice were resistant to CS-induced emphysema and had similar distal airspace size as air-exposed Fam13a^−/− mice, suggesting that the loss of Fam13a conferred resistance to CS-induced emphysema in murine lungs. Interestingly, CS exposure led to a trend, although insignificant, toward reduced levels of Fam13a mRNA in murine lungs (see Figure E3B). When respiratory mechanics were assessed, Fam13a^−/− mice showed no differences in their lung compliance and pressure-volume loops compared with those obtained on Fam13a^+/+ mice when either exposed to air or CS for 6 months (see Figure E3C), consistent with the mild emphysema that develops in C57BL/6 mice when exposed to CS chronically (32).

CS-induced chronic lung inflammation is a crucial determinant of emphysema development (33–36). Proteinases and proinflammatory mediators released by CS-activated lung leukocytes and epithelial cells can also amplify lung inflammation and destruction (37–40). Therefore, we compared lung inflammatory responses in Fam13a^−/− and Fam13a^+/+ mice exposed to CS for 1 month. Total leukocyte counts increased in bronchial alveolar lavage (BAL) samples after CS exposure, but no significant difference was observed in CS-exposed Fam13a^−/− and Fam13a^+/+ mice (Figure 1D). This suggested that FAM13A promotes emphysema development without significantly altering the lung inflammatory response to CS.

FAM13A Interacts with Protein Phosphatase 2A and β-Catenin and Also Negatively Regulates β-Catenin Protein Stability

To identify signaling pathways related to FAM13A, we performed affinity purification followed by mass spectrometry in HEK293 cells transfected with FLAG/MYC–tagged human FAM13A (Figure 2A) to assess the full spectrum of cellular interacting partners of FAM13A. Among the identified FAM13A-interacting proteins, 11 proteins consisted of different subunits of Protein Phosphatase 2A (PP2A). Pathway analysis on FAM13A interacting proteins revealed enrichment for the “β-catenin phosphorylation cascade” and “degradation of β-catenin by the destruction complex” pathways (corrected P < 0.01 by Reactome pathway analysis). These results suggested that FAM13A may regulate PP2A-mediated β-catenin degradation. Because the β-catenin/Wnt pathway was previously shown to be essential for COPD/emphysema development (21), we decided to focus our investigation on this pathway. Additional pathways enriched in FAM13A interacting proteins are listed in Table E1.

PP2A is a heterotrimeric substrate-specific serine/threonine protein phosphatase, consisting of a catalytic subunit (PP2Ac), a structural subunit (PP2Aa), and a variable regulatory subunit (PP2Ab). The association of the PP2Ab subunit with different adaptor proteins facilitates recognition of specific substrates by PP2A (41). As a known substrate of PP2A, β-catenin prevented experimental emphysema development in a murine model (21). Therefore, we tested the hypothesis that FAM13A recruits PP2A and thus regulates the stability of β-catenin. Immunoprecipitation targeting FLAG-tagged FAM13A pulled down PP2A and β-catenin (Figure 2B). Of note, FAM13A robustly coimmunoprecipitated with the structural PP2Aa subunit and the regulatory PP2Ab subunit, but showed weak interaction with its catalytic subunit (PP2Ac). Also, β-catenin coimmunoprecipitated with FAM13A, PP2Ab, and PP2Ac (Figure 2C), suggesting that FAM13A-PP2Ab-β-catenin form one protein complex.

We then tested whether FAM13A regulates β-catenin levels in airway epithelial cells. In the HBE cell line, 16HBE cells, the overexpression of FLAG-tagged human FAM13A increased phosphorylation of β-catenin but decreased total β-catenin protein levels (Figure 2D), whereas silencing of FAM13A led to reduced phosphorylation of β-catenin but increased total cellular β-catenin protein levels (Figure 2E) without altering its mRNA levels (Figure 2F). Consistent with these results, silencing of FAM13A extended the half-life of the β-catenin protein in 16HBE cells (Figure 2G). Similarly, the half-life of β-catenin was also extended in alveolar epithelial cells from Fam13a^−/− mice compared with those from Fam13a^+/+ mice (see Figure E4A). Our findings support that endogenous human FAM13A coprecipitates with the PP2A and β-catenin complex and promotes phosphorylation and degradation of β-catenin. This is inconsistent with findings from a recent study showing that overexpressing murine Fam13a promotes degradation of Axin 1 thus leading to accumulation of β-catenin in A549 cells. Therefore, we assessed Axin 1 levels in 16HBE cells in which human FAM13A was overexpressed or silenced. No significant changes in Axin 1 protein levels were detected (see Figure E4B). This result was also confirmed in alveolar epithelial cells from Fam13a^−/− mice (see Figure E4C). Thus, regulation of Axin 1 protein levels by FAM13A may be cell type specific.

β-catenin is normally sequestered in membrane through binding with E-cadherin, and thus is protected from degradation by its degradation complex. However, under FAM13A deficiency, enhanced interaction between β-catenin and E-cadherin was detected (see Figure E5A), indicating that FAM13A might disassociate β-catenin from the E-cadherin/β-catenin complex. Thus, overexpression of FAM13A led to reduced levels of membrane-bound β-catenin (see Figure E5B). However, CS extract treatment disrupted the cytoplasmic interaction between FAM13A and β-catenin in 16HBE cells (see Figure E5A), suggesting a dynamic interaction between FAM13A and β-catenin.

FAM13A Regulates β-Catenin Activity and Promotes Cell Proliferation

Accumulated β-catenin molecules can translocate into the cell nucleus where they bind to the TCF4/LEF transcription factor to activate the canonical Wnt pathway (42, 43). To identify the mechanism by which FAM13A promotes degradation of β-catenin and shed light on the composition of the FAM13A/PP2Ab/β-catenin complex, we applied the TOPFLASH reporter assay in 16HBE cells to monitor transcriptional activity of β-catenin regulated by FAM13A (Figure 3A). Cells transfected with constitutively active mutant β-catenin (β-catenin::CS2) (44) were used as positive controls and cells treated with an inhibitor of β-catenin, PKF118-310 (7.5 μg/ml), were used as negative controls in this assay. In 16HBE cells, depletion of FAM13A led to an approximately threefold increase in luciferase activity, and this effect was attenuated by overexpression of FAM13A (Figure 3A), suggesting that FAM13A regulates the activity of the Wnt pathway. Activation of the Wnt pathway can increase cell proliferation (45) and modulate lung injury/repair responses during CS-induced emphysema development (21). Consistent with this paradigm, silencing of FAM13A increased cell proliferation in 16HBE cells, which was reversed by transfection of FAM13A (Figure 3B), suggesting that FAM13A regulates cell proliferation.

Figure 3.

FAM13A down-regulates β-catenin activity and regulates cell proliferation. (A) Assessments of the β-catenin activity by TOPFLASH reporter assay in human bronchial epithelial (16HBE) cells transfected with full-length human FAM13A. Cells treated with β-catenin inhibitor PKF118-310 (7.5 μg/ml) overnight were used as negative controls and cells transfected with constitutively active β-catenin mutant (β-catenin::CS2) as positive controls in this reporter assay. Statistical comparisons in controls were not shown. Means ± SD were from triplicate assays with separately transfected and treated wells per condition. *P <  0.05, **P < 0.01. Unpaired Student’s t test was applied. One representative repeat from three independent biologic repeats was shown here. The other two repeats were shown in Figure E4D. (B) Cell growth curve in 16HBE cells transfected with different constructs. Z-test was used to compare the slopes between the curves using linear regression model. The significance was indicated right to the last time point of each curve: *Comparison between FAM13A shRNA-pCMV and NT shRNA-pCMV, P < 0.05; ^##Comparison between FAM13A shRNA-pCMV and FAM13A shRNA-FL, P < 0.05. FL = full-length FAM13A; NT shRNA = nontargeting shRNA; pCMV = empty vector as controls; shRNA = short hairpin RNA.

FAM13A Facilitates the Interaction between PP2A and β-Catenin, Thus Promoting β-Catenin Degradation

Because FAM13A forms a complex with both PP2A and β-catenin, we next asked whether this interaction contributes to the regulation of FAM13A on the protein stability of β-catenin. Degradation of β-catenin relies on the activation of the kinase GSK-3β, which can be achieved through dephosphorylation of GSK-3β at Ser 9 by PP2A. Therefore, we assessed PP2A phosphatase activity in the PP2A/β-catenin /GSK-3β complex regulated by FAM13A. Interestingly, although total cellular PP2A catalytic activity remained intact in 16HBE cells depleted of FAM13A (Figure 4A), we observed significantly reduced phosphatase activity of PP2Ac in the β-catenin protein complex (Figure 4B). Furthermore, Western blotting on the immunoprecipitated complex demonstrated reductions in the amount of PP2Ab and PP2Ac associated with the β-catenin complex in the absence of FAM13A (Figures 4C and 4D). These results suggest that FAM13A may facilitate the binding between PP2Ab and β-catenin/GSK-3β, which dephosphorylates and activates GSK-3β that promotes degradation of β-catenin (Figure 4E).

Figure 4.

FAM13A is required for the interaction between β-catenin, and PP2A thus promotes degradation of β-catenin. (A and B) Phosphatase activity of PP2A in 16HBE cells stably infected with nontargeting control shRNA (NT) or FAM13A shRNA B (B) was measured using PP2A Immunoprecipitation Phosphatase Assay Kit (Millipore). Cells treated with okadaic acid (OA) for 18 hours were used as negative controls in this assay. Cellular proteins were immunoprecipitated with PP2Ac antibody (A) or β-catenin antibody (B) separately. Note that the same amount of cellular proteins was used for immunoprecipitation (IP) in NT cells as in B cells. (C) Detection of β-catenin and PP2Ab associated with PP2Ac in NT cells and B cells. (D) Detection of PP2Ac and PP2Ab associated with β-catenin in NT cells and B cells. **P < 0.05. Unpaired Student’s t test was applied. (E) Schematic illustration on how FAM13A regulates β-catenin protein stability through interacting with PP2A. 16HBE = human bronchial epithelial cells; GSK-3β = glycogen synthase kinase 3β; ns = not significant; shRNA = short hairpin RNA.

Increased β-Catenin Signaling in Fam13a^−/− Mice

We further assessed β-catenin signaling changes and cell proliferation that may contribute to the resistance of Fam13a^−/− mice to CS-induced emphysema using lungs from Fam13a^−/− mice exposed to CS for 1 or 6 months. First, in line with the previously mentioned in vitro model, we detected decreased β-catenin S33/37/T41 phosphorylation (S33/37/T41P), increased β-catenin levels, and increased GSK-3β S9 phosphorylation in whole lung samples from Fam13a^−/− mice compared with Fam13a^+/+ mice (Figure 5A; see Figures E6A and E7). Expression levels of known target genes of β-catenin, TCF7 (46) and WISP2 (47), were significantly increased in lung samples from Fam13a^−/− mice compared with Fam13a^+/+ mice (see Figure E6B). However, CS exposure also showed genotype-dependent regulation on expression of these two genes. Taken together, these results indicate that loss of Fam13a results in activation of β-catenin signaling.

Figure 5.

Depletion of Fam13a leads to activation of β-catenin signaling in murine lungs. (A) Quantification of total and phosphorylated β-catenin and GSK-3β by Western blotting in murine lungs from the same groups of mice exposed to air or cigarette smoke (CS) for 6 months as in Figure 6A. Representative Ki67 staining images (200× magnification with 600× magnification in insets) (B) and quantifications (C) in lung sections from Fam13a^−/− and Fam13a^+/+ mice exposed to air or cigarette smoke for 6 months. Cells positive for Ki67 staining were counted in six randomly acquired fields per mouse. Data are mean ± SEM from four mice per group (number of mice in each group is indicated inside each column). *P < 0.05; **P < 0.01, unpaired Student’s t test analysis in A and C. GSK3β = glycogen synthase kinase 3β.

We also assessed cell proliferation in murine lung sections by staining them with Ki67. There was increased staining for Ki67 in the lungs of Fam13a^−/− mice compared with Fam13a^+/+ mice exposed to air for 6 months (Figures 5B and 5C). CS induced increases in Ki67 staining in Fam13a^+/+ lungs, mainly in alveolar septal cells. However, Ki67 staining did not increase further in alveolar septal cells in CS- versus air-exposed Fam13a^−/− mice (Figures 5B and 5C). We hypothesize that Fam13a^+/+ mice develop a compensatory response in the lung to CS exposure (Figures 5B and 5C). This notion is supported by the protective role of β-catenin activation in the elastase-induced emphysema model (21). Taken together, these results indicate that loss of Fam13a resulted in activation of β-catenin signaling and increased cell proliferation (Figures 5B and 5C).

FAM13A Determines Susceptibility to Emphysema by Regulating β-Catenin Signaling

Next, we directly tested the hypothesis that activation of β-catenin is required to protect Fam13a^−/− mice from developing emphysema by evaluating the effects of a β-catenin inhibitor in Fam13a^+/+ versus Fam13a^−/− mice with emphysema. We used a porcine pancreatic elastase (PPE)-induced emphysema murine model to test this hypothesis because β-catenin has been shown to protect mice from elastase-induced emphysema (21); and the β-catenin transcriptional inhibitor we studied (PKF118-310) produces hepatic toxicity, which precludes its use in chronic model systems, such as the 6-month CS exposure model of emphysema. We coadministered PKF118-310 (48, 49) and PPE to Fam13a^+/+ and Fam13a^−/− mice and measured emphysema development (50). The optimal dose of PKF118-310(0.25 mg/kg) tested was chosen based on the significant inhibition of Axin2 expression associated with minimal liver toxicity after acute administration (see Figures E8A–E8C).

PPE treatment of mice did not induce significant changes in Fam13a expression in lungs (see Figure E9A). PKF118-130 treatment alone did not affect distal airspace size (see Figures E9B–E9C). Similar to the protective effect of loss of Fam13a in CS-exposed mice, PPE-treated Fam13a^−/− mice developed minimal airspace enlargement, but this protection was reversed by coadministration of PKF118-310 (Figures 6A and 6B). Furthermore, treating Fam13a^+/+ mice with PPE reduced Ki67 staining in alveolar septal cells (Figure 6C), likely reflecting the acute and destructive effects of PPE on the lung, in contrast to the milder effects of chronic CS exposure on the lung (51, 52). Interestingly, coadministration of PPE and PKF118-310 to Fam13a^−/− mice reduced Ki67 staining in Fam13a^−/− lungs. These results suggest that increased alveolar septal cell proliferation in Fam13a^−/− mice relies on activation of β-catenin, which may contribute to reduced emphysema development. These data also further supported a protective role for β-catenin in emphysema development (21).

Figure 6.

Inhibition on transcriptional activity of β-catenin reverted the resistance to emphysema in Fam13a^−/− mice in elastase-induced experimental emphysema. (A) Airspace size assessment in Fam13a^+/+ and Fam13a^−/− mice treated with phosphate-buffered saline, porcine pancreatic elastase (PPE), or β-catenin inhibitor PKF118-310 (200× magnification). (B) Mean chord length was quantified on randomly selected images in a blinded fashion. Data shown are means ± SEM derived from n = 6–8 mice per group (number in each column represents the number of mice in each group). (C) Ki67 staining measurement in lung tissue from mice treated with PPE. Data shown are mean ± SEM from four mice per group with six randomly acquired images of lung sections stained for Ki67. (D) Total leukocytes counted in bronchoalveolar lavage (BAL) samples from Fam13a^−/− and Fam13a^+/+ mice treated with PPE with or without PKF118-310. *P < 0.05; **P < 0.01, unpaired Student’s t test in B–D.

BAL leukocytes increased to a similar extent in PPE-treated Fam13a^+/+ and Fam13a^−/− mice. Coadministration of PPE and PKF118-310 reduced BAL leukocyte counts only modestly in Fam13a^+/+ mice but had no effects on BAL leukocyte counts in Fam13a^−/− mice (Figure 6D). These results suggest that Fam13a-regulated β-catenin signaling does not play a major role in regulating the inflammatory response in the PPE-induced emphysema model.

Decreased β-Catenin and Increased FAM13A Protein Levels in Human COPD Lungs

Next, we measured FAM13A protein levels in lungs from subjects with COPD and healthy ex-smokers (control subjects) by Western blotting. Increased FAM13A and reduced β-catenin protein levels were detected in very severe COPD lungs (Global Initiative for Chronic Obstructive Lung Disease stage IV) compared with control lungs (Figures 7A and 7B; see Figure E10), consistent with a previous report that the Wnt signaling pathway is reduced in human COPD lungs (53). However, no significant changes were detected in FAM13A or CTNNB1 mRNA levels (Figures 7C and 7D). These findings in human lung samples indicate that increased FAM13A (Figure 7A) and decreased β-catenin protein levels (Figure 7B) are associated with very severe COPD. These patients may have impaired lung repair that could contribute to their increased risk of emphysema development with CS exposure.

Figure 7.

Increased FAM13A levels and reduced β-catenin levels are associated with human emphysema. (A) Increased levels of FAM13A protein and decreased levels of β-catenin protein (B) in human chronic obstructive pulmonary disease lungs compared with healthy ex-smokers. Data shown are mean ± SEM from six subjects for each group. **P < 0.01, unpaired Student’s t tests were used to analyze the data. (C and D) Measurements on FAM13A (C) and β-catenin (D) mRNA levels in human chronic obstructive pulmonary disease lungs by reverse transcriptase polymerase chain reaction. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; S = healthy ex-smokers; SC = very severe COPD.

Discussion

GWASs have demonstrated a consistent association with COPD susceptibility at the FAM13A locus. However, GWAS alone is unable to address whether the FAM13A gene is protective or whether it accelerates COPD development, or even if FAM13A is the key gene at that GWAS locus. Herein, we demonstrated that Fam13a null mice (Fam13a^−/−) were resistant to chronic CS or elastase-induced emphysema development. FAM13A also promotes the assembly of the PP2Ab/β-catenin complex leading to degradation of β-catenin. It is noteworthy that activation of β-catenin signaling contributes to airway (18) and alveolar (21, 54) epithelial repair/regeneration after various injuries. Hence, we connect FAM13A, located in one of the most consistently associated COPD GWAS regions, with β-catenin signaling, a potentially important repair pathway that determines COPD development and/or progression.

Pathologic changes in human COPD lungs include emphysema (destruction of alveolar walls and reduced alveolar surface areas), small airway remodeling (mainly composed of small airway fibrosis and destruction), and chronic bronchitis. Herein, we focused on emphysema development that may result from a combination of increased vulnerability to CS-induced injury in alveolar epithelial cells and failure to adequately repair. Our results demonstrate that Fam13a does not regulate the lung inflammatory response to either CS or PPE. Rather, our data support the notion that the resistance of Fam13a^−/− mice to PPE-induced airspace enlargement is likely caused (at least in part) by more efficient lung repair in Fam13a^−/− mice, possibly because of accumulation of β-catenin in alveolar epithelial cells (see Figure E4A), enhanced β-catenin signaling in lungs (see Figures E6 and E7), and increased cell proliferation (Figure 6C) in the absence of Fam13a.

Different types of epithelial cells in the respiratory tract may have similar or different responses to CS, which lead to destruction of the alveolar walls and/or abnormal repair processes in the small airways associated with deposition of extracellular matrix. Therefore, the molecular mechanisms by which FAM13A contributes to COPD pathogenesis identified in HBE cells (16HBE cells) were also assessed and confirmed in vivo using lung samples (Figures 5 and 6) with experimental emphysema and primary alveolar epithelial cells (see Figure E4A) from Fam13a^+/+ and Fam13a^−/− mice. Fam13a^−/− mice were protected from PPE-induced emphysema. This protection was reversed by treating Fam13a^−/− mice with a β-catenin inhibitor, which led to reduced alveolar septal cell proliferation in Fam13a^−/− mice that were treated with PPE and the β-catenin inhibitor (Figure 6C), suggesting that β-catenin activation is required for Fam13a-regulated cell proliferation and reduced emphysema susceptibility in Fam13a^−/− mice.

Using murine models to recapitulate human COPD pathologic changes has been challenging because of anatomic and physiologic differences between humans and mice. The chronic CS exposure model produces relatively mild emphysema (with only ∼15% increases in airspace size), but it is still so far the best approximation to human COPD because it uses the same environmental risk factor, and induces lung inflammation and airspace enlargement by broadly similar mechanisms as those that contribute to human COPD (52). The elastase-induced emphysema model is an acute emphysema model. Although it lacks a number of the pathologic features of human COPD, the elastase model is an efficient way to test various preventative and therapeutic strategies for emphysema (51). Therefore, the approach to use both models herein enabled us to gain a more comprehensive picture of the mechanisms by which FAM13A promotes susceptibility to emphysema development through regulating β-catenin activity.

The lung inflammatory response in Fam13a^−/− mice to 1 month of CS exposure was similar to that of Fam13a^+/+ mice. Although activation of β-catenin signaling was shown to repress the inflammatory response of alveolar epithelial cells through TLR signaling in vitro (55), conditional depletion of β-catenin in AT II cells did not significantly alter lung inflammatory responses to bleomycin (56), similar to our results in CS- or elastase-treated Fam13a^−/− mice (Figure 6D). Therefore, it is more likely that enhanced epithelial repair caused by activation of β-catenin signaling in Fam13a^−/− mice contributed to their resistance to CS-induced emphysema. However, it is noteworthy that chronic CS exposure in Fam13a^+/+ mice increased cell proliferation, whereas acute instillation of PPE reduced cell proliferation in Fam13a^+/+ murine lungs but not in Fam13a^−/− mice (Figure 6C). These results indicate differences in the mechanisms, severity, and/or duration, of injury in these two emphysema models. Chronic CS exposure over 6 months induces a milder and less acute form of airspace enlargement associated with a less intense inflammatory and injury response in the lung. Also, low concentrations of CS extract increased cell proliferation in vitro (57, 58), consistent with our findings in vivo. However, PPE potently degrades lung extracellular matrix proteins that are crucial for the survival of lung structural cells, thus leading to reduced cell proliferation as reported previously (59, 60). Therefore, it was not entirely surprising that PPE treatment in mice reduced cell proliferation.

The FAM13A interacting partner PP2A is one major Ser/Thr phosphatase in mammalian cells that forms stable complexes with a large number of regulatory subunits. Different regulatory b subunits of PP2A determine the substrate specificity of PP2A, which regulates a variety of cell biologic processes and signaling pathways, including mitosis, apoptosis, and DNA damage signaling (reviewed in Reference 61). Importantly, these regulatory subunits also regulate Akt, Wnt, and c-Myc signaling by interacting with different adaptor proteins. Our data suggested that FAM13A may facilitate recruitment of PP2Ab and β-catenin and promote degradation of β-catenin, consistent with the inhibition of PP2Ab on the Wnt pathway in Xenopus (62) possibly by promoting degradation of β-catenin (63). Given that there are four families of PP2Ab subunits encoded by 15 human genes (reviewed in Reference 62), additional studies are needed to identify which subunits of PP2Ab are recruited by FAM13A to the β-catenin degradation complex. Nevertheless, our studies linked PP2A to human COPD by FAM13A promoting degradation of β-catenin. Another report also linked PP2A to COPD by showing increased activity of PP2A in murine lungs exposed to CS for 3 days (64). When the expression of FAM13A is silenced in 16HBE cells, we detected reductions in PP2A activity only in β-catenin-associated PP2A (Figures 4A and 4B). The overall contributions of PP2A activity induced by CS to emphysema development need further investigation.

Another group recently showed that overexpression of murine FAM13A interacts with PP2Ab, which regulated the translocation of FAM13A from the nucleus to the cytoplasm (65). However, in either murine/human lung sections (Figure 1; see Figure E1) or in 16HBE cells transfected with tagged human FAM13A, we found little if any FAM13A in the nucleus (see Figures E11A and E11B). Therefore, further investigations are warranted to assess the biologic function of nuclear FAM13A under physiologic and pathologic conditions. In contrast, it was also reported that overexpression of myc-tagged murine FAM13A increased protein levels of exogenous myc-tagged β-catenin in A549 cells (65), a human lung adenocarcinoma cell line. This was attributed to reduced protein levels of Axin1, a key molecule in the β-catenin degradation complex. However, we detected no accumulation of AXIN1 in FAM13A-deficient human 16HBE cells or primary alveolar epithelial cells isolated from Fam13a^−/− mice. The discrepancy in the results in our study versus the prior report could be explained by use of different cell lines, and also by gain-of-function approaches (overexpression of murine protein in human cells) in the prior report versus loss-of-function approaches in our study. Future studies will address whether there is dynamic and complex regulation of β-catenin by FAM13A under different pathologic conditions, such as lung cancer and COPD.

Cellular levels of β-catenin are tightly controlled by its destruction complex including the crucial kinase GSK-3β that phosphorylates β-catenin for proteasome-dependent degradation. Although GSK-3β Ser9 phosphorylation was shown to be dispensable for Wnt3a-mediated β-catenin accumulation in HEK 293 cells (66), several other independent studies have challenged this conclusion using various models (67, 68). The Ser9 site of GSK-3β is a well-accepted target for PP2A. We observed increased Ser9 phosphorylation accompanied by accumulation of β-catenin in the absence of FAM13A in vivo, suggesting that GSK-3β Ser9 phosphorylation correlates with FAM13A-regulated β-catenin degradation. In contrast, a transient increase of GSK-3B Ser9 was detected in murine lungs after 1 month of CS exposure (see Figure E7), but no significant increases were detected after 6 months of CS exposure. These results indicate that CS-induced rapid inactivation of GSK-3β through Ser9 phosphorylation may contribute to increases in β-catenin level after acute CS exposure.

Activation of β-catenin signaling has been reported to contribute to the repair/regeneration processes triggered by lung injury in adulthood (69). Stabilized β-catenin molecules translocate to the nucleus, where they activate the expression of Wnt target genes that promote cell proliferation (15–17) and limit cell differentiation (16, 18). Therefore, activation of β-catenin signaling contributes to both airway (18, 19) and alveolar epithelial cell repair (20–22) after various injuries (18, 19, 21, 56). Our data suggest that reduced levels of FAM13A may promote lung repair by enhancing β-catenin signaling in CS-exposed or PPE-treated lungs to promote epithelial proliferation and thereby limit emphysema development.

Compared with ex-smoker control lungs, we found that β-catenin protein levels were significantly reduced in COPD lungs in contrast to a previous report showing similar β-catenin mRNA and protein levels in COPD and control lungs (21). There are several possibilities that could contribute to such discordant results. First, previous measurements of β-catenin levels in COPD lungs were performed using rejected transplant donor lungs as controls, whereas we used lungs from ex-smokers with normal lung function as controls. Second, the COPD lungs were obtained from different sources in these two studies. We included a small number of lung tissue samples obtained in subjects that were characterized through the LTRC. COPD is a heterogeneous disease, and patients have varying combinations of emphysema, and large and small airway disease. It is not clear which of these COPD phenotypes were included in either study because chest computed tomography scan analyses were unavailable on these subjects. In addition, the COPD cases in our study had very severe COPD at a relatively early age. Thus, studying patients with different COPD phenotypes could explain, in part, the differences between these two studies.

In human lungs, expression quantitative trait locus analysis suggested that COPD risk alleles at the FAM13A locus may be associated with increased expression of FAM13A (70), which could potentially lead to a more effective lung repair program in lung alveolar epithelial cells after CS exposure. Importantly, FAM13A protein levels are increased and β-catenin protein levels are decreased in human COPD lung tissues compared with ex-smokers without COPD, suggesting that FAM13A possibly promotes the development of human and murine emphysema by inhibiting the β-catenin-mediated lung repair pathway. Admittedly, the impact of functional variants for human complex diseases on gene expression is likely more moderate than complete depletion of genes in murine knockout models. We recognize this limitation due to differential perturbations on FAM13A gene expression in our post-GWAS functional assessment. However, the knock-out mouse model is necessary and instrumental to elucidate biologic functions of FAM13A (and other genes implicated by GWAS) in the pathogenesis of COPD.

Further studies are warranted to identify the functional genetic variants at the FAM13A locus that influence COPD susceptibility and to determine their mechanism of action. The combination of the knock-out mouse model with functional variants identification will elucidate a complete mechanism by which genetic variations at the FAM13A locus determine the susceptibility to develop COPD. Genetic variants within the FAM13A region, although in a different part of this genomic region than the COPD GWAS variants (12), have also been associated with idiopathic pulmonary fibrosis (71). Thus, additional work is required to determine whether any of the pathways identified in our COPD work are also relevant for interstitial lung disease.

In summary, our data provide in vitro and in vivo evidence to demonstrate activities for the FAM13A-PP2Ab-β-catenin axis in the development of human and murine emphysema and a molecular mechanism by which FAM13A possibly influences susceptibility to human COPD development.