Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-β-induced alveolar epithelial to mesenchymal transition

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-β-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-β-dependent signaling in AECs.

Figure 1

Rat organ homogenates were prepared and analyzed the RAGE expression by Western blot analysis. RAGE expression was predominantly high in the lung compared with other tissues.

Figure 2

Characteristics of rat alveolar type II epithelial cells and expression of RAGE. (A) Phase-contrast micrographs after seeding isolated rat alveolar type II epithelial cells (magnification × 400). (B) Alveolar type II epithelial cells were stained with alkaline phosphatase (magnification × 400). (C) A lot of alveolar type II epithelial cells were stained with an antibody to RAGE (magnification × 200). (D) Electron micrograph of isolated alveolar type II epithelial cells (magnification × 10,000).

Figure 3

Quantitative real time RT-PCR analysis of α-SMA and E-cadherin in rat alveolar type II epithelial cells. Total RNA was isolated from epithelial cells in each treatment group and subjected to quantitative RT-PCR using a iQ5 cycler instrument. The mean point for the α-SMA and E-cadherin product was normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Advanced glycation end product (AGE) alone had no effect on the epithelial-mesenchymal transition (EMT) but decreased the TGF-β-mediated EMT; decreased the α-SMA mRNA and increased the E-cadherin mRNA. RAGE siRNA transfection reversed the inhibitory effects of AGE on the TGF-β-mediated EMT. Statistical analysis was performed using student's t test; ^*P < 0.05, ^**P < 0.01.

Figure 4

Western blot analysis of α-SMA and E-cadherin in rat alveolar type II epithelial cells. Rat alveolar epithelial cells were stimulated with TGF-β (10 ng/ml), AGE (200 µg/ml) for 72 hours and total cell lysates were taken for immunoblot. Addition of AGE reduced the TGF-β-mediated α-SMA expression and increased the E-cadherin expression. Transfection of RAGE siRNA to the rat alveolar epithelial cells reversed the inhibitory effects of AGE on the TGF-β induced EMT. Values given are the mean ± SEM. ^*P < 0.05, ^**P < 0.01 by student t test.

Figure 5

Effects of AGE on alveolar type II cell morphology and expression of α-SMA and E-cadherin. Immunoreactivity for α-SMA (green) and E-cadherin (red) was assessed by immunofluorescence 72 h after AGE treatment. Nuclei were stained with DAPI (blue). α-SMA was highly expressed in TGF-β treated alveolar type II epithelial cells but was disappeared in AGE treatment. E-cadherin expression was decreased by TGF-β but was increased by AGE treatment. AGE siRNA transfection reversed these effects of AGE on the TGF-β-mediated EMT. Results shown are representative of three independent experiments. Original magnification × 400.

Figure 6

AGE decreased the TGF-β-mediated Smad2 phosphorylation in rat alveolar type II epithelial cells. Alveolar epithelial cell lysates were subjected to Western blotting using an anti-pSmad2 and total Smad2 antibody. Blots were analyzed by densitometry and the ratios of pSmad2 to Smad2 were compared with control. Statistical analysis was performed using student's t test; ^*P < 0.05, ^**P < 0.01.

Figure 7

AGE reversed the TGF-β-mediated down-regulation of Smad7 expression. Alveolar epithelial cell lysates were subjected to Western blotting using an anti-Smad7 antibody. RAGE siRNA reversed the effects of AGE on the TGF-β mediated down-regulation of Smad7. Blots were analyzed by densitometry and the expression of Smad7 was compared to control values. Statistical analysis was performed using student's t test; ^*P < 0.05, ^**P < 0.01

Figure 8

AGE or anti-TGF-β antibody inhibited the TGF-β mediated ERK1/2 phosphorylation. RAGE siRNA did not reverse the inhibitory effects of AGE on the TGF-β mediated ERK1/2 phosphorylation. Blots were analyzed by densitometry and the ratio of pERK1/2 to ERK1/2 was compared to control ratio. Statistical analysis was performed using student's t test; ^*P < 0.05, ^**P < 0.01.