Nemo-like kinase regulates the expression of vascular endothelial growth factor (VEGF) in alveolar epithelial cells

Abstract

The canonical Wnt signaling can be silenced either through β-catenin-mediated ubiquitination and degradation or through phosphorylation of Tcf and Lef by nemo-like kinase (NLK). In the present study, we generated NLK deficient animals and found that these mice become cyanotic shortly before death because of lung maturation defects. NLK-/- lungs exhibited smaller and compressed alveoli and the mesenchyme remained thick and hyperplastic. This phenotype was caused by epithelial activation of vascular endothelial growth factor (VEGF) via recruitment of Lef1 to the promoter of VEGF. Elevated expression of VEGF and activation of the VEGF receptor through phosphorylation promoted an increase in the proliferation rate of epithelial and endothelial cells. In summary, our study identifies NLK as a novel signaling molecule for proper lung development through the interconnection between epithelial and endothelial cells during lung morphogenesis.

Figure 1. Generation of NLK deficient animals.

(A) The insertion of the stop cassette was shown by genotyping analysis of wild type and NLK knockout mice (Left top panel). RT and qRT-PCR of total RNA from mouse embryonic fibroblast isolated from E 13.5 wild type and NLK knockout mice. GADPH was used as a loading control. (B,C) Protein and mRNA expression levels of NLK in wild type mouse tissues including lung, brain, skin, kidney, and liver. The blot in figure (C) has been cut in two pieces and blotted against NLK or actin under the same experimental conditions. (D) Protein and mRNA expression levels of NLK expressed in lung tissues at birth (P1) of wild type and NLK knockout mice. The blot in this figure has been cut in two pieces and blotted against NLK or actin under the same experimental conditions.

Figure 2. NLK deficient animals have a severe phenotype with a short lifespan.

(A) The survival rate of wild type (n = 74), heterozygous (n = 123) and NLK knockout mice (n = 59) between postnatal day 1 and day 3. (B) Images of NLK+/+ (WT) and NLK−/− (KO) mouse 24 hours (left panel) and 36 hours (right panel) after birth. (C) Relative birth weight of wild type NLK and NLK−/− pups.

Figure 3. Smaller alveoli with irregular morphology and hyperplastic in NLK−/− compared to wild type mouse.

(A) H&E-stained lung sections from E18.5, E 20.5, and P1 isolated form NLK+/+ and NLK−/− mice. (B) H&E-stained lung sections show the different appearance of alveolar septum between NLK+/+ and NLK−/− mice at P1. (C) The mean chord length of alveoli in NLK+/+ and NLK−/− mice at P1. (D) The thickness of the alveolar capillary wall in NLK+/+ and NLK−/− mice at P1.

Figure 4. Increased cell proliferation in NLK−/− lung sections compared to wild type animals.

(A) TUNEL staining and quantification of the number of positive cells in NLK+/+ and NLK−/− lung tissues at P1. (B) Cyclin D1 staining and quantification of the number of positive cells in NLK+/+ and NLK−/− lung tissues at P1. (C) Ki67 staining and quantification of the number of positive cells in NLK+/+ and NLK−/− lung tissues at P1. (D) Measurement of the growth rate of NLK+/+ and NLK−/− primary murine alveolar epithelial (left panel) and endothelial cells (right panel) over a period of 24–72 hours using MTS assay. (E) Double staining of PCNA and CD31 or PCNA and proSP-C in NLK+/+ and NLK−/− lung tissues at P1. (F) Immunofluorescence staining of total Lef1 and phospho-Lef1 positive cells in the lung sections isolated from NLK+/+ and NLK−/− at P1. (G) Quantification of the number of phospho-Lef1 positive cells among Lef1 positive cells in the lung sections isolated from NLK+/+ and NLK−/− at P1. (H) Quantification of the number of Ki67 positive cells among phospho-Lef1 positive cells in the lung sections isolated from NLK+/+ at P1. (I) The levels of total Lef1 and phospho-Lef1 in NLK+/+ and NLK−/− primary murine alveolar epithelial and endothelial cells. The blot in this figure has been blotted against pLef1. The membrane has been stripped and used again for blotting against total Lef1 under the same experimental conditions.

Figure 5. Hyperthickening of the lung vascularization and VEGF-A expression.

(A) H&E-stained lung sections from showing hyperthickening of the lung vascularization in lung sections from NLK+/+ and NLK−/−. (B) The levels of VEGF-A at the mRNA levels in NLK+/+ and NLK−/− lung tissues isolated from E18.5 and P1. (C) The levels of secreted VEGF in the culture media of NLK+/+ and NLK−/− primary epithelial cells. (D) The levels of secreted VEGF in the culture media of NLK+/+ and NLK−/− primary pulmonary endothelial cells. (E) Immunofluorescence staining and quantification of phosphorylated VEGF receptor 2 (VEGFR2) in lung sections isolated from NLK+/+ and NLK−/− at P1. Heat-mediated antigen retrieval in high pH buffer was performed before labeling. Data is given as fraction labeling (%); percentage positively labeled area of the total lung tissue area.

Figure 6. Recruitment of Lef1 to the promoter of VEGF-A.

(A) Luciferase activity in human lung epithelial cells P2GH (left panel) and P2G (right panel) using deletion mutants of VEGF promoter. Control (Ctrl): cells without transfection with luciferase expression plasmid. (B) VEGF promoter luciferase activity (1,6 kb) in primary isolated mouse lung epithelial cells. Control (Ctrl): cells without transfection with luciferase expression plasmid. (C) Luciferase activity in human lung epithelial cells P2GH (left panel) and P2G (right panel) using deletion mutant of VEGF promoter (1,6 kB) in cells transfected with Full length (FL-NLK), catalytically inactive mutant of NLK (KM-NLK), or empty expression vector (Ctrl). (D) Recruitment of Lef1 to the VEGF-A promoter by ChIP using human lung epithelial cells (P2GH) transfected with Full length (FL-NLK), catalytically inactive mutant of NLK (KM-NLK), or empty expression vector (Ctrl). IP: Lef1 indicates immunoprecipitation (IP) using polyclonal antibodies. IP: IgG, IP using pre-immune serum. Input: 10% of the cell lysate used for the IP is shown. (E) Model of how deletion of NLK leads to elevated levels of VEGF expression causing aberrant proliferation of pulmonary epithelial and endothelial cells (left). In the wildtype epithelial cells, NLK mediated-phosphorylation of Lef1 prevents the binding of Lef1 to the promoter of VEGF (right).