Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence

Abstract

In the aging lung, the lung capacity decreases even in the absence of diseases. The progenitor cells of the distal lung, the alveolar type II cells (ATII), are essential for the repair of the gas-exchange surface. Surfactant protein production and survival of ATII cells are supported by lipofibroblasts that are peroxisome proliferator-activated receptor gamma (PPARγ)-dependent special cell type of the pulmonary tissue. PPARγ levels are directly regulated by Wnt molecules; therefore, changes in the Wnt microenvironment have close control over maintenance of the distal lung. The pulmonary aging process is associated with airspace enlargement, decrease in the distal epithelial cell compartment and infiltration of inflammatory cells. qRT-PCR analysis of purified epithelial and nonepithelial cells revealed that lipofibroblast differentiation marker parathyroid hormone-related protein receptor (PTHrPR) and PPARγ are reduced and that PPARγ reduction is regulated by Wnt4 via a β-catenin-dependent mechanism. Using a human in vitro 3D lung tissue model, a link was established between increased PPARγ and pro-surfactant protein C (pro-SPC) expression in pulmonary epithelial cells. In the senile lung, both Wnt4 and Wnt5a levels increase and both Wnt-s increase myofibroblast-like differentiation. Alteration of the Wnt microenvironment plays a significant role in pulmonary aging. Diminished lipo- and increased myofibroblast-like differentiation are directly regulated by specific Wnt-s, which process also controls surfactant production and pulmonary repair mechanisms.

Keywords: Wnt microenvironment; molecular biology of aging; pulmonary senescence.

Figure 1

Flow cytometric analysis of cell populations in (A): 1-month and (B): 24-month lungs of Balb/c mice (EpCAM1⁺, CD45⁺, EpCAM1^-CD45^-, EpCAM1⁺CD45⁺) (C): Bar chart of young and old mouse lung cells. The numbers of different cell subsets are shown in percentage (*P < 0.05).

Figure 2

(A): mRNA expression levels of adipose differentiation markers in 24-month-old Balb/c mouse lung epithelial and nonepithelial cells, compared with 1 month-old. Both the PPARγ and the ADRP are decreased at 24 month-old compared with the 1-month-old lung cell types. LipidTox staining of (B): 1-month-old and (C): 24-month-old Balb/c mouse lung sections showing nuclei staining, lipid staining (LipidTox), and EpCAM1-FITC staining individually then in a merged picture. (D): PPARγ protein levels were detected in 1-month-old and 24-month-old lung extracts by Western blotting. Equal protein loading was tested using anti-β-actin antibody. The blot is a representative of two individual experiments. (E): PTHrP receptor mRNA levels were measured in EpCAM-/CD45- cell populations of 1-month-old and 24-month-old Balb/c mouse lungs using qRT–PCR analysis. (The graph is a representative of three individual experiments).

Figure 3

(A): mRNA expression levels of different Wnt molecules in 24-month-old Balb/c mouse lung epithelial and nonepithelial cells were measured using qRT–PCR analysis and relative expression was determined to β-actin, then compared with Wnt expression in 1-month-old test animals. (B): Wnt4 protein levels were detected in 1-month-old and 24-month-old lung extracts by Western blotting. Equal protein loading was tested using anti-β-actin antibody. (The blot is a representative of two individual experiments). Immunofluorescent staining of Wnt5a in (C): 73-year and (D): 21-year human lung sections (The staining is representative of three separate experiments).

Figure 4

(A): PPARγ expression levels in 3D human lung tissue models, following 7-day exposure to control (ctrl) and Wnt4 supernatants of thymic epithelial cells (TEP1). PPARγ mRNA expression levels were determined by qRT–PCR analysis following 7-day exposure to control (ctrl) and Wnt4-enriched supernatants of TEP1, to 10 mm LiCl and to 1 μm IWR in (B): primary human lung fibroblast (NHLF) cells. (C): β-catenin protein levels were determined in NHLF cells after exposure to Wnt4-enriched supernatants of TEP1, 10 mm LiCl and 1 μm IWR for 7 days. Equal protein loading was determined using anti-β-actin antibody. (The blot is a representative of two individual experiments). The blots were then densitometrically scanned and plotted against the controls. Pro-SPC staining on (D): ctrl and E: Wnt4-enriched supernatant treated 3D human lung tissue model (red: pro-SPC, blue: DAPI stained nuclei) (The staining is representative of three separate experiments). F: Mean intensity differences in pro-SPC staining in Wnt4-treated and Ctrl 3D human lung tissue models. (The graph is a representative of three individual experiments).

Figure 5

Immunofluorescent staining of pro-surfactant protein C (pro-SPC) in (A): 21-year-old and (B): 73-years-old human lung tissue sections. (C): Confocal picture of intact 3D human lung tissue models infected with rAd-Ctrl-GFP and rAd-ICAT-GFP. SAEC are expressing GFP (green) within the 3D human lung tissue model. NHLF cells were prestained with Dil (red). (D): PPARγ mRNA expression levels were determined by qRT–PCR analysis in 3D human lung tissue model following 7-day suppression of β-catenin activity by ICAT specifically within the SAEC population using rAd gene delivery. (E): PPARγ mRNA expression levels were determined by qRT–PCR analysis in 3D human lung tissue model following suppression of β-catenin activity by ICAT specifically within the NHLF cell population using rL gene delivery. Immunofluorescent staining of pro-SPC in (F): control 3D human lung tissue model containing rAd-Ctrl-GFP SAEC and (G): ICAT overexpressing 3D human lung tissue model containing rAd-ICAT-GFP SAEC (the staining is representative of three separate experiments).

Figure 6

(A): Relative gene expression levels of S100A4 in Wnt4-enriched supernatant treated human lung tissue spheroids. qRT–PCR analysis of gene expression is presented as relative to controls (data is representative of two separate experiments). (B): Relative gene expression levels of IL1β and S100A4 in rhWnt5a-treated 3D human lung tissue models. qRT–PCR analysis of gene expression is presented as relative to untreated controls (data are representative of three separate experiments). (C): Schematic summary of molecular changes in the distal lung during senescence (TG: triglyceride; SPC: pro-surfactant protein C, PPAR: peroxisome proliferator-activated receptor gamma).