Deletion of LCMR1 in alveolar type II cells induces lethal impairment of lung structure and function in adult mice

Introduction

Lung cancer metastasis-related protein 1 (LCMR1) was originally identified by differential display polymerase chain reaction (PCR) between human large cell lung carcinoma cell lines 95C and 95D by our team in 2002 (Genbank accession number: AY148462) (1). Our previous work showed that LCMR1 is strongly overexpressed in non-small cell lung cancer and its expression is significantly associated with the clinical stage of patients (1). In addition, the knockdown of LCMR1 was reported to induce cell apoptosis in different cancer cells in vitro (2-4). LCMR1 is also known as a subunit of the Mediator complex, called MED19 (5). The Mediator is an evolutionarily conserved multisubunit protein complex that acts as a critical transcription cofactor conveying information between gene-specific transcription factors and RNA polymerase II general transcription machinery (6,7). The mammalian Mediator comprises up to 30 distinct subunits with unique biological functions (8,9). LCMR1 is a crucial facilitator of peroxisome proliferator-activated receptor gamma (PPARγ)-mediated gene expression that is required for adipogenesis and maintenance of white adipose tissue (WAT) mass in mice (10). LCMR1 also acts as a GATA cofactor and MED1 interactor in Drosophila (11). Additionally, LCMR1 and MED26 are synergistic functional targets of the RE1 silencing transcription factor (REST) in the epigenetic silencing of neuronal gene expression in non-neuronal cells (12). However, LCMR1 is still a relatively poorly characterized gene and the role of LCMR1 in normal lung tissue remains to be elucidated.

The lung is composed of air-conducting bronchi terminating in air-exchanging alveoli. The alveolar epithelium consists of squamous alveolar type I cells (AT1 cells), which are responsible for gas exchange, and cuboidal alveolar type II cells (AT2 cells), which serve as the only source of pulmonary surfactant, reducing surface tension and preventing lung collapse at end-expiration. AT2 cells also participate in modulation of immune responses and transportation of ions and fluids. In addition, AT2 cells have been recognized as alveolar stem cells with the capacity for self-renewal and differentiation into AT1 cells during lung homeostasis and injury repair (13,14). Specific subgroups of AT2 cells execute the progenitor role in repairing the alveoli, which preferentially re-enter the cell cycle, self-renew and regenerate mature AT1 and AT2 cells after acute lung injury (15,16). However, damaged or denuded alveolar epithelium often triggers defective or overwhelmed repair processes (17).

In this study, we generated a novel tamoxifen-inducible, AT2 cell-specific LCMR1 conditional knockout mouse model by breeding mice carrying floxed LCMR1 alleles with Sftpc-CreER^T2 mice, aiming to investigate the effects of AT2 cell-specific LCMR1 deletion on lung structure and function in adult mice. We demonstrated that AT2 cell-specific LCMR1 deletion caused increased weight loss, damaged lung structure, impaired lung function, and reduced survival of mice. The lethal phenotype we observed may result from severe AT2 cell depletion, pulmonary surfactant dysfunction, and alveolar structure destruction. These results indicate that LCMR1 may play an important role in the maintenance of AT2 cell homeostasis. We present the following article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-293/rc).

Methods

Mice

Mice carrying the floxed LCMR1 allele were generated in cooperation with Cyagen Biosciences Inc. (Suzhou, China) by combining CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and the Cre/loxP system. Five exons of the LCMR1 gene have been identified with the ATG start codon in exon 1 and the TAA stop codon in exon 5. Exons 2–4 were selected as the conditional knockout region. Briefly, to engineer the target vector, homology arms and a conditional knockout region were generated by PCR using bacterial artificial chromosome (BAC) clones RP24-220L15 and RP23-427H11 from the C57BL/6J library as templates. The donor vector containing loxP sites, guide RNAs (gRNA1: TTTAAAGGAATTCCACTCCCTGG; gRNA2: TTCACTGGGTTGTTCTAGGCAGG), and Cas9 messenger RNA (mRNA) were co-injected into fertilized mouse eggs to generate targeted conditional knockout mice.

Potential founders were identified by PCR and Sanger sequencing employing mouse tail DNA (data not shown) and then bred to WT mice. F1 offspring (LCMR1^flox/wt) were also identified by PCR with specific primers for the 5'-loxP site (F-CACGTAAACGGCCACAAGTTCGAG, R-CATGAGGTTAAGGAGAGTTGGGAC) and 3'-loxP site (F-GGTAACTAGGGAGGTCAGAGACATGAG, R-CGAAGTTATGTGCACAGTACTGTGG), respectively, and further confirmed via Sanger sequencing and Southern blot analysis (detailed methods not shown).

Sftpc-CreER^T2 mice (Jackson Laboratory, Bar Harbor, ME, USA) express a tamoxifen-inducible Cre recombinase controlled by the Sftpc promoter, which can mediate the deletion of targeted sequences flanked by the loxP sites specifically in AT2 cells. LCMR1^flox/wt mice were mated to Sftpc-CreER^T2 mice, and the offspring were screened for the presence of floxed LCMR1 and the Sftpc-CreER^T2 transgene. The double heterozygous mice were then intercrossed to obtain Sftpc-CreER^+/−; LCMR1^flox/flox mice, which were used as breeding pairs to generate AT2 cell-specific LCMR1 conditional knockout mice (Sftpc-CreER^T2; LCMR1^flox/flox) and littermate control mice (LCMR1^flox/flox) for the following phenotype study.

Tamoxifen (20 mg/mL in corn oil; Sigma, T5648, St. Louis, MO, USA) was administered via intraperitoneal injection (50 mg/kg) to 6-week-old mice for 5 consecutive days, after which the Sftpc-CreER^T2; LCMR1^flox/flox mice were termed LCMR1^ΔAT2 mice. The control littermates (LCMR1^flox/flox) received the same doses of tamoxifen. The day of the last dose of tamoxifen administration was recorded as day 0. The mice were housed in individually ventilated cages with free access to food and water under specific pathogen-free (SPF) conditions. All mice were maintained in the C57BL/6J background. A protocol was prepared before the study without registration. All animal experiments were approved by the Institutional Animal Care Use Committee of Chinese PLA General Hospital (No. 2018-X14-25), in compliance with institutional guidelines for the care and use of animals.

Genotyping

Mouse tail genomic DNA (gDNA) was extracted using the DNA extraction kit (Takara, 9765, Shiga, Japan) according to the manufacturer’s protocol. The presence of the two loxP sites and the Sftpc-CreER^T2 transgene was determined by PCR with primers shown in Table S1. DNA of the sorted AT2 cells from lung tissue was also isolated to confirm LCMR1 deletion through PCR after tamoxifen administration. PCR was performed in a 50 µL reaction volume using Premix Taq (TaKaRa, RR902A) on a T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA), and PCR products were detected by agarose gel electrophoresis and visualized using a Molecular Imager ChemiDoc XR System (Bio-Rad) with Image Lab software, version 3.0 (Bio-Rad). The expected sizes of PCR products were also shown in Table S1.

Histopathological analysis

The lung tissues of the mice were fixed in 4% paraformaldehyde for 24 h at 4 ℃, dehydrated in a graded ethanol series, and then embedded in paraffin. The paraffin-embedded block was cut into 5-µm sections which were then deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) reagents (Soonbio, Beijing, China) according to the standard procedure. Collagen fibers in the lung sections were detected by Masson’s trichrome staining and Sirius red staining using standard procedures. The stained lung sections were examined under a light microscope (Olympus, Tokyo, Japan).

Immunofluorescence and TUNEL staining

For immunofluorescence staining, the 5-µm paraffin-embedded sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval. The sections were blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature and incubated with primary antibodies overnight at 4 ℃ including anti-prosurfactant protein C (proSP-C) (Millipore, AB3786, Temecula, CA, USA) and anti-α-smooth muscle actin (α-SMA) (Proteintech, 14395-1-AP, Wuhan, China). The sections were then incubated with a secondary antibody (Cy3-conjugated goat anti-rabbit IgG) for 1 h at room temperature. The nuclei were then stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min. The apoptosis of AT2 cells within lung sections was analyzed using proSP-C and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) co-staining, along with double immunofluorescence staining for proSP-C and cleaved caspase-3 (Servicebio, GB11532, Wuhan, China). Images were captured using an Olympus IX81 fluorescence microscope and a Nikon Eclipse Ti confocal microscope (Tokyo, Japan).

Transmission electron microscopy

Lung tissues were dissected, minced to approximately 1 mm³ pieces as soon as possible, and fixed overnight at 4 ℃ using 2.5% glutaraldehyde. The samples were then postfixed with 1% osmium tetroxide in cacodylate buffer, dehydrated in a graded ethanol series, and embedded in epoxy resin. Ultrathin sections (70 nm) were obtained using an Ultracut E ultramicrotome (Leica, Wetzlar, Germany), and stained with uranyl acetate and lead citrate. The sections were subsequently examined under an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) with an attached image capture software.

Lung dissociation and AT2 cells isolation

Lung dissociation was performed as described previously with simple modification (18). The protease solution used for lung digestion in this study contained Collagenase Type I (450 U/mL; Sigma, C0130), Elastase (4 U/mL; Sigma, E1250), Dispase (5 U/mL; Sigma, D4693), and DNaseI (0.33 U/mL; Roche, 10104159001, Mannheim, Germany) dissolved in Dulbecco’s minimal essential medium/F12 (DMEM/F12) (Gibco, 11330021, Grand Island, NY, USA). The whole-lung suspension was blocked with TruStain FcX anti-mouse CD16/CD32 (BioLegend, 101319, San Diego, CA, USA) in the Dulbecco’s phosphate buffered saline (D-PBS) buffer with 10% fetal bovine serum (FBS), and then stained with anti-CD31-FITC (eBioscience, 11-0311-81), anti-CD45-FITC (eBioscience, 11-0451-81), and anti-CD326-APC (eBioscience, 17-5791-82). Fluorescence activated cell sorting (FACS) was performed on an SH800S Cell Sorter (Sony, San Jose, CA, USA).

Quantitative RT-PCR

Total RNA was isolated using RNASimple Total RNA Kit (Tiangen, DP451, Beijing, China) according to the manufacturer’s instructions. Complementary DNA (cDNA)was synthesized using the PrimeScript RT reagent Kit with a gDNA Eraser (Takara, RR047A). Quantitative RT-PCR (qPCR) was performed in a 20 µL reaction volume using KAPA SYBR FAST Universal (KAPA Biosystems, KK4601, Cape Town, South Africa) on MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primers used for amplification were listed in Table S2. The relative mRNA expression was assessed using the standard 2^−ΔΔCt method normalized by β-actin.

Western blot

The total protein were extracted using Radioimmunoprecipitation Assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Applygen, C1055, Beijing, China). Protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Applygen, P1511). Protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed using specific antibodies, including anti-MED19 (Invitrogen, PA5-78656, Carlsbad, CA, USA), anti-α-SMA (Proteintech), anti-SP-A (Proteintech, 11850-1-AP), anti-SP-B (Bioss, bs-22341R, Beijing, China), anti-SP-C (Proteintech, 10774-1-AP), anti-SP-D (Proteintech, 11839-1-AP), anti-P53 (CST, 2524S, Danvers, MA, USA), anti-B-cell lymphoma-2 (Bcl-2) (PTM Bio., PTM-5777, Hangzhou, China), anti-β-actin (Proteintech, 20536-1-AP), and anti-α-tubulin (PTM Bio., PTM-5442). The protein bands were visualized with a Chemiluminescent HRP Substrate (Millipore) using an automatic chemiluminescence imaging system (Tanon, Shanghai, China). The band intensity was quantified using ImageJ software, version 1.52 (National Institutes of Health) and normalized to α-tubulin or β-actin.

Analysis of bronchoalveolar lavage fluid

After the mice were anesthetized, their tracheas were cannulated and their lungs were lavaged three times with 1 mL of sterile saline. The recovered bronchoalveolar lavage fluid (BALF) was then centrifuged at 500 g for 10 min at 4 ℃. The supernatant was used to measure the total protein concentration using a BCA Protein Assay kit (Applygen) and cytokine levels using a LEGENDplex Mouse Inflammation Panel (BioLegend, 740446). The cell pellet was resuspended to determine the total cell numbers using a hemocytometer. Differential cell counts were determined manually from the cytospins of BALF cell pellets stained with H&E, including neutrophils, macrophages, and lymphocytes.

Lung wet/dry weight ratio

After the mice were anesthetized, the right lung was removed and weighed to obtain the wet weight. Subsequently, the samples were desiccated in an oven at 80 ℃ for 48 h and re-weighed to determine the dry weight. The lung wet/dry weight ratio was then calculated.

Pulmonary function measurement

Pulmonary function was measured using the AniRes2005 lung function testing system (Bestlab, Beijing, China). Briefly, the mice were anesthetized, cannulated via tracheotomy, placed into a sealed plethysmograph chamber, and then connected to a computer-controlled, mechanically ventilated system (AniRes2005). Five consecutive measurements were collected for each mouse, and the average value was used for further analyses of the forced vital capacity (FVC), dynamic lung compliance (Cdyn), expiratory resistance (Re), and inspiratory resistance (Ri).

Statistical analysis

Data were exhibited as the mean ± standard error of mean (SEM). Statistical analyses were performed using GraphPad Prism 5.0 software (National Institutes of Health). Comparisons between two groups were processed using the unpaired Student’s t-test. P<0.05 was considered statistically significant.

Results

Generation of AT2 cell-specific LCMR1 conditional knockout mice

To explore the role of LCMR1 in lung tissue, we generated a tamoxifen-inducible, AT2 cell-specific LCMR1 conditional knockout mouse model (Sftpc-CreER^T2; LCMR1^flox/flox) for which the gene targeting strategy was briefly illustrated in Figure 1A. Initially, we constructed a conditional knockout mouse line with exons 2–4 of the LCMR1 gene flanked by two loxP sites (LCMR1^flox/wt), which was identified and verified by PCR, Sanger sequencing, and Southern blot analysis (Figure S1A-S1C). To obtain the Sftpc-CreER^T2; LCMR1^flox/flox mice for AT2 cell-specific LCMR1 deletion, a subsequent breeding scheme was simply depicted in Figure 1B. The genotypic distribution of offspring was in accordance with Mendel’s law of inheritance (data not shown, only the genotype that we needed for further breeding is listed in the schematic diagram).

Figure 1 Generation and verification of tamoxifen-inducible, AT2 cell-specific LCMR1 conditional knockout mice. (A) Schematic diagram of the gene targeting strategy. Mice carrying the floxed LCMR1 allele in which exons 2–4 were flanked by loxP sites were constructed using CRISPR/Cas9 technology. Cre-mediated excision resulted in the deleted LCMR1 allele lacking exons 2–4 in AT2 cells after tamoxifen induction. (B) Schematic diagram of the breeding scheme. (C) Genotyping of mice by PCR for the floxed LCMR1 allele and Sftpc-CreER^T2 transgene to identify Sftpc-CreER^+/−; LCMR1^flox/flox mice. The floxed LCMR1 allele produced 265-bp (left panel) and 289-bp band (middle panel), indicating the two loxP sites respectively, while the wild-type allele produced 198-bp (left panel) and 214-bp band (middle panel), respectively. The expected size of the Sftpc-CreERT2 transgene was 210 bp (right panel). (D) Genotyping of mice by PCR for the presence of Sftpc-CreERT2 transgene. (E) Detection of the LCMR1 deletion band (192 bp) by PCR in AT2 cells sorted on day 7 from LCMR1^flox/flox (n=3) and LCMR1^ΔAT2 mice (n=4). (F) qPCR analysis of LCMR1 mRNA expression in AT2 cells sorted on day 7 from LCMR1^flox/flox and LCMR1^ΔAT2 mice, normalized to β-actin (n=4/group). (G) Western blot analysis of LCMR1 protein expression in AT2 cells sorted on day 7 from LCMR1^flox/flox and LCMR1^ΔAT2 mice, normalized to β-actin (n=3/group). Data are presented as the mean ± SEM. ****, P<0.0001 by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1. SEM, standard error of mean; PCR, polymerase chain reaction.

We first crossed LCMR1^flox/wt mice with Sftpc-CreER^T2 mice to obtain Sftpc-CreER^+/−; LCMR1^flox/wt mice, which were further bred for Sftpc-CreER^+/−; LCMR1^flox/flox mice (Figure 1C). Next, the Sftpc-CreER^+/−; LCMR1^flox/flox mice were used as breeding pairs to further generate Sftpc-CreER^T2; LCMR1^flox/flox mice together with LCMR1^flox/flox mice (Sftpc-CreER^T2 negative) as the littermate control. Based on Mendel’s law of inheritance, the offspring were all expected to harbor the homozygous floxed LCMR1 allele, and thus, genotyping was performed to detect the presence of the Sftpc-CreER^T2 transgene (Figure 1D). Once activated by tamoxifen administration, Cre recombinase can mediate LCMR1 deletion specifically in the AT2 cells of Sftpc-CreER^T2; LCMR1^flox/flox. Six-week-old mice were intraperitoneally injected with tamoxifen for 5 consecutive days, after which Sftpc-CreER^T2; LCMR1^flox/flox mice were termed LCMR1^ΔAT2 mice. The DNA of AT2 cells sorted on day 7 was extracted, and LCMR1 deletion was confirmed by PCR with a 192-bp band detected only in LCMR1^ΔAT2 mice (Figure 1E). The analysis of LCMR1 mRNA expression by qPCR demonstrated a dramatic reduction in the AT2 cells of LCMR1^ΔAT2 mice compared with LCMR1^flox/flox mice (Figure 1F). Western blot analysis showed that the LCMR1 protein expression was also significantly reduced in the AT2 cell of LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 1G). These results further verified that we successfully generated a tamoxifen-inducible, AT2 cell-specific LCMR1 conditional knockout mouse model.

Effects of AT2 cell-specific LCMR1 deletion on lung structure and permeability

The histopathology of lungs from LCMR1^ΔAT2 and LCMR1^flox/flox mice was examined to assess the effect of LCMR1 deletion on lung structure. H&E staining showed prominent structure damage in the lung sections of LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice on day 14 after tamoxifen administration (Figure 2A). Specifically, the AT2 cell-specific deletion of LCMR1 resulted in inflammatory cell infiltration, hemorrhage, edema, and alveolar wall thickening in the lung sections of LCMR1^ΔAT2 mice, which were rarely found in LCMR1^flox/flox mice. There was almost no normal alveolar structure in the areas with severe pathological damage in LCMR1^ΔAT2 mice.

We further examined the ultrastructure of lung tissues through transmission electron microscopy (TEM). Consistent with the histological analysis, the TEM results demonstrated that the alveolar structure was disordered in LCMR1^ΔAT2 mice, while LCMR1^flox/flox mice exhibited a mostly normal structure (Figure 2B). The air-blood barrier was thicker in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice. Additionally, there were dysmorphic or electron-dense lamellar bodies in AT2 cells of LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice.

Figure 2 Effects of AT2 cell-specific LCMR1 deletion on lung structure and permeability. Lung tissues and BALF were collected from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration. (A) Representative images of H&E-stained lung sections. The insets (left) are shown magnified on the right. Scale bars, 200 µm (left panels) and 50 µm (right panels). (B) Representative transmission electron microscopy images of lung sections. Red arrows: air-blood barrier. Yellow arrows: lamellar bodies in AT2 cell. Nu: nucleus. Scale bars, left panels (10 µm, ×1,200), right panels (2 µm, ×6,800), middle panels (1 µm, ×9,300). (C) Representative images of the Sirius red-stained lung sections. The insets (left) are shown magnified on the right. Scale bars, 200 µm (left panels) and 20 µm (right panels). (D) Representative images of Masson’s trichrome-stained lung sections. The insets (left) are shown magnified on the right. Scale bars, 100 µm (left panels) and 20 µm (right panels). (E) Representative images of immunofluorescence staining for α-SMA (red) on lung sections. Nuclei were stained with DAPI (blue). Scale bars, 50 µm. (F) Western blot analysis of α-SMA protein expression in lung tissues, normalized to α-tubulin (n=3/group). (G) Quantification of lung wet/dry (W/D) weight ratio (n=4–5/group). (H) The concentration of total protein in BALF (n=5–6/group). *, P<0.05, ***, P<0.001, ****, P<0.0001 by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1. H&E, hematoxylin and eosin.

Sirius red staining of lung sections revealed enhanced collagen deposition mainly around the bronchioles or adjacent to the pleura in LCMR1^ΔAT2 mice compared with LCMR1^flox/flox mice (Figure 2C). Collagen deposition was similarly evidenced by Masson’s trichrome staining (Figure 2D). Furthermore, we also detected the expression of α-smooth muscle actin-positive (α-SMA), a marker of the myofibroblasts (19). The immunofluorescence staining results showed more α-smooth muscle actin-positive (α-SMA-positive) cells in the lung sections of LCMR1^ΔAT2 mice compared with those in LCMR1^flox/flox mice (Figure 2E). Additionally, western blot showed a significant increase in the whole-lung protein expression of α-SMA in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 2F). Besides the pathological changes, we found that the lung wet/dry weight ratio was significantly higher in LCMR1^ΔAT2 mice than that in LCMR1^flox/flox mice (Figure 2G), accompanied by a significant increase in BALF protein concentration collected from LCMR1^ΔAT2 mice compared with that from LCMR1^flox/flox mice (Figure 2H), which was indicative of higher lung permeability.

Effects of AT2 cell-specific LCMR1 deletion on body weight, pulmonary function, and survival

We monitored the health status of LCMR1^ΔAT2 mice and LCMR1^flox/flox mice daily after tamoxifen administration. Data on body weight changes were expressed as a percentage of body weight at day 0 (Figure 3A). LCMR1^ΔAT2 mice exhibited increased body weight loss as early as day 4 after tamoxifen administration, whereas all LCMR1^flox/flox mice were healthy and continued to gain weight throughout the observation period. There was a significant difference in the weight change of LCMR1^ΔAT2 mice and LCMR1^flox/flox mice after day 6, which increased with time. Despite prominent body weight loss, LCMR1^ΔAT2 mice exhibited shortness of breath, a hunched posture, and decreased activity, followed by death. Notably, the LCMR1^ΔAT2 mice showed significantly increased mortality compared with the LCMR1^flox/flox mice (Figure 3B). The survival of LCMR1^ΔAT2 mice was significantly decreased with the onset of mortality as early as day 10 after tamoxifen administration. There were no surviving LCMR1^ΔAT2 mice after day 17, with a median survival of 14 days post-tamoxifen, whereas all LCMR1^flox/flox mice were alive and healthy throughout the 30 days observed.

Figure 3 Effects of AT2 cell-specific LCMR1 deletion on body weight, pulmonary function, and survival. (A) Body weight of LCMR1^flox/flox and LCMR1^ΔAT2 mice after tamoxifen administration, shown as the percentage of the body weight on day 0 (n=5–7/group). (B) Survival curves of LCMR1^ΔAT2 mice and LCMR1^flox/flox mice (n=9/group). A log-rank test was used for statistical analysis of the survival curve. (C-F) Analysis of forced vital capacity (C), dynamic compliance (D), inspiratory resistance (E), and expiratory resistance (F) of LCMR1^ΔAT2 mice (n=5) and LCMR1^flox/flox mice (n=4) on day 14 after tamoxifen administration. Data are expressed as the mean ± SEM. *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001 by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1.

We also examined whether the observed histopathological changes have any effect on lung function through invasive pulmonary function testing. The forced vital capacity (FVC) was significantly decreased in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 3C). Additionally, the dynamic compliance (Cdyn) was significantly lower in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 3D). We also found increases in the inspiratory resistance (Ri) and expiratory resistance (Re) of LCMR1^ΔAT2 mice compared with that of LCMR1^flox/flox mice (Figure 3E,3F). These data suggested increased airway resistance and decreased lung compacity and compliance in LCMR1^ΔAT2 mice.

Effects of AT2 cell-specific LCMR1 deletion on cellularity and inflammatory cytokines in BALF

To evaluate the effect of LCMR1 deletion on the inflammatory response in the lung, we detected the total cell counts, differential cell counts, and typical cytokine levels in BALF collected from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration. BALF from LCMR1^ΔAT2 mice exhibited a substantial increase in total cell counts compared with that from LCMR1^flox/flox mice (Figure 4A). We further performed differential cell counts, which revealed significantly increased neutrophils from LCMR1^ΔAT2 mice compared with LCMR1^flox/flox mice (Figure 4B). Similar trends were also observed in the numbers of macrophages and lymphocytes in BALF (Figure 4C,4D).

Figure 4 Effect of AT2 cell-specific LCMR1 deletion on cellularity and inflammatory cytokines in BALF. (A-D) Counts of total cells (A), neutrophils (B), macrophages (C), and lymphocytes (D) in BALF collected from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration (n=4/group). (E-H) Levels of TNF-α (E), IL-1β (F), IL-6 (G), and IL-10 (H) in BALF collected from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 10 after tamoxifen administration (n=4/group). Data are expressed as the mean ± SEM. **, P<0.01, ***, P<0.001, ****, P<0.0001; NS, not significant by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1.

Regarding typical cytokines, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10 were also detected in BALF from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration. We also observed significantly elevated levels of proinflammatory cytokines in BALF from LCMR1^ΔAT2 mice compared with that from LCMR1^flox/flox mice, including TNF-α, IL-1β, and IL-6 (Figure 4E-4G). The level of the typical anti-inflammatory cytokine, IL-10, also trended higher in LCMR1^ΔAT2 mice compared with LCMR1^flox/flox mice but the increase was not significantly different between the two groups (Figure 4H). Alterations of cell and cytokine profiles in BALF indicated an enhanced inflammatory response in the lungs of LCMR1^ΔAT2 mice, which was consistent with the histological changes.

Effects of LCMR1 deletion on the AT2 cell number and pulmonary surfactants

Next, we assessed the impact of LCMR1 on the number and function of AT2 cells. Immunofluorescence staining for the AT2 cell marker proSP-C (20) was performed on lung sections from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration, which revealed an apparent reduction of proSP-C-positive (proSP-C⁺) cells in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice, indicating a loss of AT2 cells (Figure 5A). As AT2 cells synthesize and secrete surfactant proteins, we then analyzed the expressions of SP-A, SP-B, SP-C, and SP-D in lung tissue by qPCR and western blot analysis. qPCR analysis showed a significant reduction in the mRNA expressions of SP-A, SP-B, and SP-C in the lung tissues of LCMR1^ΔAT2 mice compared with those of LCMR1^flox/flox mice, while there was no significant change in whole-lung SP-D mRNA expression in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 5B-5E).

Figure 5 Effects of LCMR1 deletion on the AT2 cell number and pulmonary surfactant proteins. (A) Representative images of immunofluorescence staining for proSP-C (red) in lung sections from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration. The nuclei were stained with DAPI (blue). Scale bars, 50 µm. (B-E) qPCR analysis of the mRNA expressions of SP-A (B), SP-B (C), SP-C (D), and SP-D (E) in the lung tissue from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration, normalized to β-actin (n=4/group). (F,G) Western blot (F) and quantitative analysis (G) of the protein expressions of SP-A, SP-B, SP-C, and SP-D in lung tissues from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration, normalized to α-tubulin (n=3/group). Data are presented as the mean ± SEM. *, P<0.05, **, P<0.01, ***, P<0.001; NS, not significant by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1; SEM, standard error of mean.

Similarly, western blot analysis revealed decreased protein levels of SP-A and SP-B, as well as SP-C which was nearly undetectable in the lung tissues of LCMR1^ΔAT2 mice compared with that of LCMR1^flox/flox mice (Figure 5F,5G). On the contrary, the whole-lung SP-D protein level was significantly increased in LCMR1^ΔAT2 mice compared with that in LCMR1^flox/flox mice (Figure 5F,5G). These results indicated that LCMR1 deficiency led to the loss of AT2 cells and the dysfunction of pulmonary surfactants.

AT2 cell-specific LCMR1 deletion promotes apoptosis in AT2 cells

Since LCMR1 knockdown was reported to promote apoptosis in lung cancer cells (2), we hypothesized that apoptosis may also play an important role in the loss of AT2 cells in LCMR1^ΔAT2 mice. To detect AT2 cell apoptosis, we performed double staining for proSP-C and TUNEL on lung sections from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 10 after tamoxifen administration (Figure 6A), which showed more proSP-C and TUNEL double positive (proSP-C⁺TUNEL⁺) cells in LCMR1^ΔAT2 mice compared with those in LCMR1^flox/flox mice. Likewise, double staining for proSP-C and cleaved caspase-3 on lung sections also showed more apoptotic AT2 cells in LCMR1^ΔAT2 mice than those in LCMR1^flox/flox mice (Figure 6B). Additionally, elevated apoptosis in AT2 cells was further confirmed by western blot analysis for p53 and Bcl-2 protein expression, which exhibited upregulated p53 expression paralleled by downregulated Bcl-2 expression in AT2 cells from LCMR1^ΔAT2 mice compared with that from LCMR1^flox/flox mice (Figure 6C-6E).

Figure 6 AT2 cell-specific LCMR1 deletion promotes apoptosis in AT2 cells. (A) Representative images of proSP-C (red) and TUNEL (green) double staining of lung sections from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration. Nuclei were stained with DAPI (blue). Scale bars, 50 µm. (B) Representative images of proSP-C (red) and cleaved caspase-3 (green) double staining of lung sections from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 10 after tamoxifen administration. Scale bars, 50 µm. (C-E) Western blot (C) and quantitative analysis of the protein expressions of p53 (D) and Bcl-2 (E) in AT2 cells from LCMR1^flox/flox and LCMR1^ΔAT2 mice on day 14 after tamoxifen administration, normalized to β-actin (n=3/group). Data are presented as the mean ± SEM. **, P<0.01 by unpaired Student’s t-test. AT2, alveolar type II; LCMR1, lung cancer metastasis-related protein 1; SEM, standard error of mean.

Discussion

As LCMR1 was first identified from human large-cell lung carcinoma cell lines, previous studies of LCMR1 mainly focused on its relationship with cancer, especially regarding cell growth, apoptosis, invasion, and metastasis in different cancer types (2-4,21-25). LCMR1 down-regulation can induce laryngocarcinoma cancer cell apoptosis via activation of apoptotic protease activating factor 1 (Apaf-1) (4). LCMR1 can also promote melanoma cell invasion through activating Tetraspanin 8 (Tspan8) protein expression (21). Additionally, LCMR1 can promote breast cancer progression by regulating the EGFR/MEK/ERK signaling pathway (22). Simultaneously, given its role as a subunit of the Mediator complex, LCMR1 is also expected to function as a co-activator of RNA polymerase II enzyme to regulate gene transcription in normal tissues and cells, which has rarely been reported. In this study, we generated the first tamoxifen-inducible, AT2 cell-specific LCMR1 conditional knockout mouse model and preliminarily characterized its phenotype. We found prominent structural abnormalities in the lung tissues of LCMR1^ΔAT2 mice, including inflammatory cell infiltration, collagen deposition, alveolar hemorrhage, and edema. Consistently, we also observed impaired pulmonary function, including increased airway resistance and decreased lung compacity and compliance, in LCMR1^ΔAT2 mice. All LCMR1^ΔAT2 mice died within 17 days after tamoxifen administration. Collectively, these results showed that AT2 cell-specific LCMR1 deletion could induce lethal lung structure and function alterations in adult mice.

CRISPR/Cas9 gene editing technology can effectively induce precise cleavage at target sites in the genome based on short RNA-guided DNA recognition (26,27). Due to its high efficiency and precision, CRISPR/Cas9 has been used to manipulate genes in vitro and in vivo to investigate phenotypic changes and underlying mechanisms of respiratory diseases (28,29). CRISPR/Cas9 can also be used for drug screening, target validation and novel target discoveries, especially in lung cancer (30,31). The Cre/loxP system can efficiently act to direct tissue-specific, site-specific, and heritable chromosomal DNA recombination events in transgenic mice (32). Therefore, combining CRISPR/Cas9 and the Cre/loxP system makes it possible to precisely investigate gene functions in specific cells or tissues, which largely avoids the unspecific side effects of systemic gene knockdown (33). Additionally, with the fusion of Cre recombinase to the ligand-binding domain of estrogen receptor (Cre-ER), the deletion of floxed genes can be induced at the desired time by tamoxifen administration (34). Taking advantage of these sophisticated tools, we constructed a mouse strain carrying the conditional LCMR1 allele with exons 2–4 flanked by loxP sites. We could achieve conditional gene inactivation of LCMR1 in different tissue or cell types by crossing the LCMR1^flox mice with mice expressing specific Cre recombinase. In the present study, we bred LCMR1^flox mice with Sftpc-CreER^T2 mice harboring tamoxifen-inducible Cre activity specifically in AT2 cells. To our knowledge, this is the first reported AT2 cell-specific LCMR1 conditional knockout mouse model to date.

Surprisingly, all of the LCMR1^ΔAT2 mice died within 17 days after tamoxifen administration in the absence of additional damaging agents, while all of the control mice were healthy and alive. We found massive AT2 cell loss in the lung tissues of LCMR1^ΔAT2 mice through immunofluorescence staining for the AT2 cell marker, proSP-C. LCMR1 knockdown was reported to promote cell apoptosis in human osteosarcoma, laryngocarcinoma, and lung cancer cells in vitro (2-4). In addition, adipose-specific MED19 knockout in adult mice can cause lipodystrophy due to increased apoptosis and macrophage infiltration (12). Therefore, we hypothesized that AT2 cell loss in LCMR1^ΔAT2 mice may be related to increased apoptosis. ProSP-C and TUNEL co-staining demonstrated more apoptotic AT2 cells in LCMR1^ΔAT2 mice compared with those in LCMR1^flox/flox mice, as evidenced simultaneously by proSP-C and cleaved caspase-3 double staining. Furthermore, the elevated apoptosis in AT2 cells was confirmed by upregulated p53 protein and downregulated Bcl-2 protein expression. The regulation of AT2 cell apoptosis has been reported in some studies but the main molecular mechanism remains uncertain (35,36). Therefore, further research is needed to determine the specific molecular mechanism through which LCMR1 deletion results in AT2 cell loss.

Here, we highlight two possible causes of death for LCMR1^ΔAT2 mice. On the one hand, extensive AT2 cell loss resulting from LCMR1 deletion destroyed the integrity of the alveolar architecture, as evidenced by histopathological examinations. As essential components and stem cells of the alveolar epithelium, AT2 cells are important in alveolar maintenance and repair in the adult lung (14). Targeted injury of AT2 cells at a high level can cause lung injury, fibrosis, and even death in mice (37,38). On the other hand, the depletion of AT2 cells may also disrupt the homeostasis of pulmonary surfactant and weaken its function to lower the surface tension at the air-liquid interface. Despite the quantitative reduction of AT2 cells, we found dysmorphic or electron-dense lamellar bodies in AT2 cells through TEM, which may aggravate pulmonary surfactant dysfunction as lamellar bodies are functionally specialized organelles synthesizing and storing pulmonary surfactants in AT2 cells. SP-B and SP-C are the critical surfactant proteins involved in surfactant organization and its surface-active properties (39). We found significantly reduced expression of SP-B and SP-C in the lung tissues of LCMR1^ΔAT2 mice compared with that of LCMR1^flox/flox mice, especially SP-C, which was almost undetectable. Nevertheless, the expression of SP-D was significantly increased in the lung tissues of LCMR1^ΔAT2 mice. One possible cause may be that AT2 cells are not the only source of SP-D which can also be synthesized and secreted by Clara cells and macrophages (40). Additionally, the changes in histopathology, permeability, and the inflammatory response observed in the lung tissues of LCMR1^ΔAT2 mice produced a phenotype with features resembling lung injury. It was previously reported that SP-D expression was increased in the lung tissues of animals with acute lung injury (41,42).

Conclusions

In conclusion, this study demonstrated for the first time that the conditional knockout of LCMR1 specifically in the AT2 cells of adult mice results in a lethal phenotype with impaired lung structure and function. The main causes for this could lie in extensive AT2 cell loss, alveolar integrity destruction, and pulmonary surfactant deficiency. Our data indicate that LCMR1 is essential for AT2 cell integrity and maintenance. These findings also highlight the critical role of AT2 cells in maintaining lung homeostasis.

Acknowledgments

We thank the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science for assistance with transmission electron microscopy.

Funding: This work was supported by the National Natural Science Foundation of China (Nos. 30070335 and 30370616) and the China Postdoctoral Science Foundation (No. 20090461438).

Footnote

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-293/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments performed in this study were approved by the Institutional Animal Care Use Committee of Chinese PLA General Hospital (No. 2018-X14-25), in compliance with institutional guidelines for the care and use of animals.

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