CARM1 is required for proper control of proliferation and differentiation of pulmonary epithelial cells

Abstract

Coactivator-associated arginine methyltransferase I (CARM1; PRMT4) regulates gene expression by multiple mechanisms including methylation of histones and coactivation of steroid receptor transcription. Mice lacking CARM1 are small, fail to breathe and die shortly after birth, demonstrating the crucial role of CARM1 in development. In adults, CARM1 is overexpressed in human grade-III breast tumors and prostate adenocarcinomas, and knockdown of CARM1 inhibits proliferation of breast and prostate cancer cell lines. Based on these observations, we hypothesized that loss of CARM1 in mouse embryos would inhibit pulmonary cell proliferation, resulting in respiratory distress. By contrast, we report here that loss of CARM1 results in hyperproliferation of pulmonary epithelial cells during embryonic development. The lungs of newborn mice lacking CARM1 have substantially reduced airspace compared with their wild-type littermates. In the absence of CARM1, alveolar type II cells show increased proliferation. Electron microscopic analyses demonstrate that lungs from mice lacking CARM1 have immature alveolar type II cells and an absence of alveolar type I cells. Gene expression analysis reveals a dysregulation of cell cycle genes and markers of differentiation in the Carm1 knockout lung. Furthermore, there is an overlap in gene expression in the Carm1 knockout and the glucocorticoid receptor knockout lung, suggesting that hyperproliferation and lack of maturation of the alveolar cells are at least in part caused by attenuation of glucocorticoid-mediated signaling. These results demonstrate for the first time that CARM1 inhibits pulmonary cell proliferation and is required for proper differentiation of alveolar cells.

Fig. 1.

Immunohistochemical analysis of CARM1 in embryonic and adult murine lung. (A-E) Lung sections from E18.5 (A-C) and 12-week-old (D,E) mice were stained with anti-CARM1. (A) Staining of CARM1 is observed throughout the embryonic lung. (B) High-magnification image showing that CARM1 is expressed in both the nucleus (left red arrow) and cytoplasm (right red arrow) of cuboidal AT2 cells in the spaces between alveolar sacs as well as in epithelial cells lining the terminal bronchioles (black arrows). (C) CARM1 staining is absent in mice with a targeted deletion of Carm1. (D,E) CARM1 expression is observed in cuboidal AT2 cells (D) and in the nucleus (E, left arrow) and cytoplasm (E, right arrow) of adult lung cells.

Fig. 2.

CARM1 is expressed in purified AT2 and BASC populations. (A,B) Double staining of CARM1 (pink) with SPC or CCSP (brown) in lung sections from E18.5 mice. Arrows identify cells expressing both CARM1 and SPC or CCSP. (C) Representative flow cytometric profile of AT2 and bronchioalveolar stem cells (BASCs) in lungs of 12-week-old wild-type mice stained with anti-CD45 (PTPRC) conjugated to tri-color (TC), anti-CD31-TC (pecam1), and anti-Sca-1 (LY6A) conjugated to FITC. AT2 cells are CD45^– CD31^– with high autofluorescence in the FITC channel and 8.1% of cells from whole lung are stained as AT2 (Kim et al., 2005). BASCs were identified as Sca-1⁺ CD45^– CD31^–. (D) qRT-PCR analysis for Carm1 expression was performed on whole lung tissue or isolated AT2 cells from five animals and the expression relative to Gapdh was averaged. ^*, P<0.05, based on a two-tailed Student's t-test for samples of unequal variance.

Fig. 3.

Loss of CARM1 disrupts development of the distal lung. (A) Whole lung homogenates were prepared at E18.5 from wild-type (lanes 1-3) or Carm1^Δ/Δ (lanes 4-6) mice and 25 μg of protein immunoblotted with polyclonal anti-CARM1. (B-E) Hematoxylin and Eosin (HE) staining of fixed lung tissue from wild-type (B,D) or Carm1^Δ/Δ (C,E) E18.5 lungs. Severe hypercellularity, thickening of the alveolar walls (black arrows), reduced air space, and dysmorphic cells (black arrows) are observed in Carm1^Δ/Δ lungs as compared with the wild type. The red arrow marks the presence of a terminal bronchiole in the Carm1^Δ/Δ lungs.

Fig. 4.

Increased proliferation of pulmonary cells in the absence of CARM1. (A,B) Wild-type (A) and Carm1^Δ/Δ (B) E18.5 mouse lungs were fixed and stained for Ki-67, a marker of cell division. Increased Ki-67 staining is observed in Carm1^Δ/Δ lungs as compared with the wild type. (C) Double staining of Ki-67 (pink) and SPC (brown) in lung sections from E18.5 Carm1^Δ/Δ mice. Thick arrows identify cells expressing both Ki-67 and SPC. Thin arrows identify cells expressing Ki-67 but not SPC.

Fig. 5.

CARM1 is required for differentiation to AT1 cells in the developing lung. (A,B) Lungs from wild-type (A) and Carm1^Δ/Δ (B) E18.5 mice were fixed and processed for TEM (see Materials and methods). (A) Lamellar bodies, small patches of cytoplasmic glycogen and AT1 cells are visible in wild-type lung. By contrast, Carm1^Δ/Δ lungs (B) contain an overabundance of glycogen (G) and visible lamellar bodies (L), but no AT1 cells. The presence of lamellar bodies indicates some AT2 development that is blocked before differentiation to AT1 cells. Scale bars: 1 μm. (C-F) IHC of lungs from wild-type (C,E) and Carm1^Δ/Δ (D,F) E18.5 mice with anti-AQP5 shows reduced staining and abnormal morphology of AT1 cells in Carm1^Δ/Δ lungs. (G) qRT-PCR analysis of markers of AT1 cells indicates loss of this cell type in Carm1^Δ/Δ lungs. qRT-PCR was performed using RNA isolated from five wild-type and five Carm1^Δ/Δ E18.5 lungs. Mean and s.d. are expressed as a percentage of Gapdh expression. ^**, P <0.01; ^#, P=0.075.

Fig. 6.

Altered AT2 differentiation in the absence of CARM1. (A-D) Immunohistochemical analysis of fixed lung tissue from E18.5 wild-type (A,C) or Carm1^Δ/Δ (B,D) mice. (A,B) Increased staining by anti-SPC in Carm1^Δ/Δ lungs demonstrates that the increased cellularity is of AT2 origin. (C,D) Staining of CCSP, a marker of Clara epithelial cells, reveals no difference in cellular distribution. (E) qRT-PCR analysis of surfactant protein (Sftpa1, Sftpb, Sftpc and Sftpd) expression was performed using RNA isolated from five wild-type and five Carm1^Δ/Δ E18.5 lungs. Mean and s.d. are expressed as a percentage of Gapdh expression. ^*, P<0.05.

Fig. 7.

Gene expression analysis reveals dysregulation of cell cycle-related genes in Carm1^Δ/Δ lungs. (A) Gene expression analysis demonstrates increases in pathways regulating the metaphase checkpoint, the cell cycle, cellular division and cytoskeletal remodeling. (B) qRT-PCR analysis validates the microarray results, showing decreased expression of Gadd45g, Scn3b, Nkd1, Klf9, Ace, and Sphk1, and increased expression of Cpa3 and Cdc6. Mean and s.d. are expressed as a percentage of Gapdh expression. ^*, P<0.05.

Fig. 8.

CARM1, GR and p53 interact with the proximal promoter of the Scn3b gene. (A) Putative p53 and glucocorticoid receptor (GR) binding sites in the proximal promoter of Scn3b, shown forming a complex with CARM1. Horizontal arrows indicate the positions of the primers used in PCR in the ChIP assays. (B) ChIP assay (Southern blot shown) demonstrating binding of GR and p53 to the proximal promoter of Scn3b (gi:149259969) in lung cell isolates from wild-type mice. Note that CARM1 immunoprecipitation detects Scn3b, suggesting that a complex forms between CARM1, p53 and GR. (C) Knockdown of CARM1 by siRNA. Human BEAS-2B cells were transfected with Carm1 siRNA (siCarm1), non-target siRNA (non-target), or left untransfected (control) for 48 hours. RNA was isolated and expression of Carm1 analyzed by qRT-PCR. Error bars indicate s.d. (n=5). (Inset) Expression of CARM1 as detected by western blot analysis. (D) Upregulation of SCN3B is suppressed by CARM1 knockdown. BEAS-2B cells transfected with siRNAs were incubated in the presence of ethanol (vehicle control) or 500 nM dexamethasone for 72 hours. RNA was isolated and expression of SCN3B mRNA was analyzed by qRT-PCR. The fold change in SCN3B expression was calculated by comparing dexamethasone-treated and vehicle control-treated cells. Error bars indicate s.d. (n=5). ^***, P <0.001; NS, not significant.