Krüpple-like-factor 4 Attenuates Lung Fibrosis via Inhibiting Epithelial-mesenchymal Transition

Abstract

Epithelial-mesenchymal transition (EMT) plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Krüpple-like-factor 4 (KLF4), has been suggested to play an important role in the phenotype transition. However, its function in pulmonary fibrosis and EMT of human alveolar epithelial cells (AECs) remains unclear. This study aimed to examine the role of KLF4 in pulmonary fibrosis and EMT. Decreased expression of KLF4 was first observed in human IPF lung tissues and models of bleomycin-induced pulmonary fibrosis. Transgenic mice with overexpression of KLF4 were subjected to bleomycin-induced pulmonary fibrosis model and showed attenuated lung fibrosis and EMT compared to wild type group. Furthermore, the effects overexpression and knockdown of KLF4 on TGF-β1-induced EMT were examined in AECs. Adenovirus-mediated overexpression of KLF4 attenuated TGF-β1-induced EMT and activation of Smad2/3 and Dvl in AECs. Conversely, knockdown of KLF4 promoted the activation of pathways above mentioned and TGF-β1-induced EMT. Our results demonstrates that KLF4 plays an important role in bleomycin-induced lung fibrosis through suppressing TGFβ1-induced EMT. Thus, it may serve as a potential target for the treatment of pulmonary fibrosis.

Figure 1

KLF4 expression was downregulated in human IPF lung tissues and mouse models of bleomycin-induced pulmonary fibrosis. Representative microphotographs of immunohistochemistry staining of KLF4 in cancer adjacent normal lung tissue (A, left column) and fibrotic tissues (A, right column) of IPF patients, and mouse models of bleomycin-induced pulmonary fibrosis (B, right column) or control saline group (B, left column). KLF4 expression level in sections of IPF patients or control group (C), and bleomycin-induced fibrosis model or control group (D) were shown by immunoreactive system (IRS) and analyzed.Scale bar was as indicated in the figure. qPCR and western blotting were performed to evaluate the expression of KLF4 in mouse lung tissues of bleomycin-induced pulmonary fibrosis and control group (E,F). The values were expressed as mean ± SD. N = 4 for each group. ***P < 0.001 by t-test.

Figure 2

Overexpression of KLF4 inhibited bleomycin-induced pulmonary fibrosis in vivo. Overexpression of KLF4 in the transgenic mice were confirmed with PCR (A) and western-blotting (B). Mice were intratracheally intubated and injected with bleomycin to induce pulmonary fibrosis Saline was used as a control, in overexpression of KLF4 mice group and wild-type group. H&E staining (C) and Masson staining (D) were used to detect collagen depositions. Ashcroft scores showed the degree of fibrosis (E). The lung tissues of mice were harvested and assayed for hydroxyproline analysis (F). The expression of E-cadherin and fibronectin was evaluated by western blotting (G) in KLF4 overexpressing group and wild-type group. Representative images were from five mice per group. Scale bar = 200 μm. The values were expressed as mean ± SD from N = 5 in each group. *P < 0.05 and **P < 0.01 by t-test.

Figure 3

Overexpression of KLF4 attenuated EMT in bleomycin-induced lung fibrosis in vivo. Mice were intratracheally intubated and given bleomycin or saline as control to induce pulmonary fibrosis overexpression in KLF4 transgenic mice group and wild-type group. Double-labelled immunofluorescent staining were performed to examine the expression of E-cadherin (green) and fibronectin (red) as indicated by the white arrow. Scale bar = 50μm and 25μm as indicated in the figure (A). The percentage of double-label positive cells was calculated in each group (B). Double-labelled immunofluorescent staining was detected E-cadherin (green) and α-SMA (red). Scale bar = 25 μm (C). Representative image of three independent experiments were shown. The values were expressed as mean ± SD from five samples in each group were shown. **P < 0.01 by t-test.

Figure 4

Overexpression of KLF4 attenuated TGF-β1-induced EMT of alveolar epithelial cells. (A) KLF4 mRNA level expression in AECs^(a) and lung tissues from mice^(b) was assessed with PCR. Immunocytochemistry was performed using a primary antibody against KLF4 to show the expression of KLF4 in AECs^(c) and lung tissue of mice^(d). Scale bar = 50 μm for (A). (B) AECs were co-infected with AdKLF4 and AdtTA (20 MOI) and maintained in the medium with or without tetracycline (Tc; 0.1 μg/mL). Nuclear protein lysates were immunoblotted with antibodies against KLF4 or Histone as an internal control. (C) F-actin staining was performed to show the morphological changes in AECs upon TGF-β1 stimulation. (D) AECs were co-infected with AdKLF4 and AdtTA in the medium with or without tetracycline and then treated with or without TGF-β1 (5 ng/mL) for 48 hours.The TGF-β1-stimulated morphological change in AECs with or without overexpression of KLF4 was observed under phase contrast light microscopy. (E) Total proteins were immunoblotted with antibodies against E-cadherin and fibronectin. β-actin was used as a loading control. Data shown are representative of three independent experiments. (F) Immunochemistry staining of E-cadherin and fibronectin in TGF-β1-induced EMT in AECs with and without overexpression of KLF4. (G) Cells were fixe F-actin staining (Green) was shown. Nuclei were counterstained with DAPI (Blue). ((A) Scale bar = 50 μm. (B) Scale bar = 20 μm). Data shown are representative of three independent experiments.

Figure 5

Knockdown of KLF4 promoted TGF-β1-induced EMT in alveolar epithelial cells. AECs were transfected with KLF4 siRNAs or control siRNA (100 nM) for 48 h. KLF4 expression level was assessed with qRT-PCR (A). AECs transfected with siRNA or control siRNA were stimulated with TGF-β1 for 48 hours. The TGF-β1 induced morphological changes were shown (B). Total proteins were immunoblotted with antibodies against E-cadherin and fibronectin. β-actin was used as loading control (C). Immunocytochemistry staining of E-cadherin and fibronectin in KLF4 knock down and control AECs (D). (E) RNA interference of KLF4 F-actin staining (Green) was shown. Nuclei were counterstained with DAPI. ((A) Scale bar = 50 μm. (B). Scale bar = 20 μm). Data shown are representative of three independent experiments.

Figure 6

KLF4 inhibited TGF-β1-induced phosphorylation of Smad2/3 and Dvl. AECs were co-infected with AdKLF4 and AdtTA in the medium with or without tetracycline and then stimulated with or without TGF-β1 (5 ng/mL) for the indicated time (A,E). AECs were transfected with KLF4 siRNAs or control siRNA (100 nM) and then were stimulated with TGF-β1 (5 ng/mL) for indicated time (B,F). Total protein lysate was immunoblotted with antibodies against Smad2/3, their phosphorylated forms, DVL-2 and β-actin. Densitometry analysis of figure A and B was performed with imageJ (C,D). Similar results were obtained in three independent experiments.