Figure 1.
TNFα and TGFβ1 induce EMT in A549 cells.
(A) The combined treatment of TNFα (20 ng/ml) and TGFβ1 (10 ng/ml) for 72 h induced morphological alterations characterized as fibroblast-like cells. (B) TNFα and TGFβ1 treatment reduced expression of E-Cadherin at cell-to-cell contacts, and increased formation of F-actin stress fibers. Cells were immunostained with an anti-E-Cadherin antibody and counterstained F-actin and nuclei with rhodamine phalloidin and Hoechst 33342, respectively. (C) The redistribution of E-cadherin after TGFβ and TNFα treatment from the cell-to-cell contacts to cytosol was further demonstrated with confocal microscopy at higher maginification. (D) The 72 h treatment with TNFα and TGFβ1 decreased expressions of E-Cadherin and Occludin, epithelial markers, and increased expressions of Vimentin, a mesenchymal marker, in A549 cells. Surprisingly, the expression of Claudin 1, an epithelial marker, was increased as analyzed by western blotting.
Figure 2.
TNFα alone induces fibroblast-like morphology and Claudin 1 expression.
(A) TNFα alone induced fibroblast-like morphological alteration, reduced cell-to-cell contacts, and increased F-actin stress fibers. A549 cells treated with TNFα, TGFβ1 or TNFα and TGFβ1 together for 72 h were examined with light microscopy and stained with rhodamine phalloidin to visualize F-actin structures. TNFα-induced changes were similar as that of the TNFα and TGFβ1 treatment, whereas TGFβ1 alone did not induce these changes. (B) Protein levels of E-Cadherin were more effectively reduced by TGFβ1 than TNFα. TNFα alone increased Claudin 1 expression, whereas TGFβ1 had little effects on Claudin 1 after 24 h or 48 h treatment, and even increased it after 72 h treatment, as determined by western blotting. (C) In MDCK cells, TGFβ1 reduced Claudin 1 after 24, 48 or 72 h treatment. (D) Expression levels of Claudin 1 mRNA were significantly increased by TNFα in a time-dependent manner as measured by real-time quantitative RT-PCR. Mean ± SEM. n = 3 experiments. *p<0.05 (compared with control at 24 h). #p<0.05 (compared with control at 48 h).
Figure 3.
TNFα-induced Claudin 1 protein is mainly in the cytosolic fraction.
(A) At 24, 48 h after treating the cells with TNFα, expression of Claudin 1 protein was increased mainly in cytoplasm. A549 cells were immunostained with an anti-Claudin 1 antibody and counterstained F-actin and nuclei with rhodamine phalloidin and Hoechst 33342, respectively. (B) Confocal microscopy further confirmed the cytosolic distribution of Claudin 1 in both control and TNFα groups. (C) Claudin 1 and GAPDH were mainly found in the Triton X-100 soluble (S) cytoplasm fraction, whereas ZO-1 and Occludin were mainly in the Triton X-100 insoluble cytoskeletal pellets (P). A549 cells treated with or without TNFα (20 ng/ml for 24 h) were lyzed and separated into Triton X-100 soluble and insoluble fractions for immunoblotting.
Figure 4.
Down-regulation of Claudin 1 with siRNA significantly blocked TNFα-induced gene expression.
(A) Claudin 1 siRNA effectively reduced both basal and TNFα-induced gene expression of Claudin 1 (reading from microarray). (B) Principle Component Analysis (PCA) showed that the overall gene expression profiles are separated based on the Claudin 1 siRNA transfection and TNFα treatment. (C) Hierarchical clustering analysis demonstrates that gene expression patterns are highly dependent upon Claudin 1 siRNA transfection and TNFα treatments. A two-way ANOVA showed that 2,490 genes were significantly different. Red: up-regulated; blue: down-regulated. (D) Claudin 1 siRNA blocked 75% of the TNFα-induced gene expression changes. The gene expressions that were significantly changed by TNFα were defined with FDR q value less than 5.0% and fold of change greater than 1.3 by SAM analysis. Venn diagram shows that 75% of TNFα-induced expression changes were not shown in Claudin 1 siRNA transfected cells.
Figure 5.
Validation of gene expression by real-time quantitative RT-PCR.
The expression of six genes (BAAT, TM4SF4, CCL5, TNFAIP3, IL-6, and IL-8) in Claudin 1 siRNA transfected cells were compared with control siRNA transfected cells treated with or without TNFα. The microarray results are plotted in the left column. The qRT-PCR results normalized to the level of SDHA are plotted in the right column. N = 4, Mean ± SEM.
Table 1.
Top 20 up-regulated genes induced by TNFα in Control-siRNA or Claudin 1-siRNA transfected A549 cells.
Table 2.
Top 10 down-regulated genes induced by TNFα in Control-siRNA or Claudin 1-siRNA transfected A549 cells.
Figure 6.
Knocking down Claudin 1 with siRNA reduced gene expression related to inflammation and cell migration.
Ingenuity Pathway Analysis was performed on 353 genes which were significantly changed by TNFα treatment in Control siRNA transfected cells, but not in Claudin 1 siRNA transfected cells (highlighted area with dots on Venn diagram in Fig. 4D). (A) Signal network related to inflammation. Note that NFκB is located at the center of the network. (B) Signal network related to cell movement. Note MMP-9 is located at the center of the network.
Table 3.
Top Bio Functions of TNFα-Induced Genes Blocked by Claudin 1 siRNA, as analyzed by Ingenuity Pathway analysis.
Figure 7.
TNFα promotes migration of A549 cells.
(A) TNFα enhanced cell migration in a wound healing assay. After mechanical wounding, confluent A549 cells were treated with TNFα (20 ng/ml), TGFβ1 (10 ng/ml) or TNFα and TGFβ1 together. Representative photomicrographs of the wounded cell monolayer are shown. (B) Percentage of cell free area in each condition was calculated. n = 4. Mean ± SEM. *p<0.05 (compared with control at the same time point). (C) TNFα and/or TGFβ1 did not affect cell proliferation determined by MTS assay. (D) TNFα treatment for 24 h increased the gene expression of MMP-9 (as determined by microarray and qRT-PCR) and the level of active MMP-9, while the levels of both pro- and active-MMP 2 had no dramatic changes, as determined by gelatin zymography assay.
Figure 8.
Claudin 1 expression levels affect cell migration.
(A) Down regulation of Claudin 1 with siRNA reduced spontaneous as well as TNFα-enhanced migration of A549 cells. The control or Claudin 1 siRNA transfected A549 cells were cultured until confluent, mechanically wounded, and then treated with or without 20 ng/ml TNFα. Representative photomicrographs of wounded cell monolayer are shown. (B) Percentage of cell free area in each condition was calculated. n = 4. Mean ± SEM. *p<0.05 (compared with control siRNA transfected cells at the same time point). (C) Claudin 1 siRNA effectively reduced both basal and TNFα induced expression levels of Claudin 1 in A549 cells. Control or Claudin 1 siRNA transfected cells were treated with or without TNFα (20 ng/ml for 24 h) and harvested for western blotting. (D) Over-expression of Claudin 1 enhanced cell migration. (E) Percentage of cell free area in each condition was calculated. n = 4. Mean ± SEM. *p<0.05, compared with empty vector transfected cells at the same time point. (F) Claudin 1-pcDNA effectively increased the expression level of Claudin 1. A549 cells were transfected with empty vector or Claudin 1-pcDNA for 24 h, and harvested for western blotting.
Figure 9.
Reducing Claudin 1 protein levels enhanced serum and/or EGF induced A549 cell migration, and Claudin 1 siRNA reduced TNFα-induced morphological changes of A549 cells.
Cells were transfected with Claudin 1 or control siRNA. The confluence monolayers were serum starved, mechanically wounded and then stimulated with serum (10% FBS) and/or EGF (50 ng/ml). The wounded areas at 12 h were quantified. N = 4, Mean ± SEM. *P<0.05 vs. control siRNA treated group. (B). Claudin 1 siRNA clearly reduced Claudin 1 protein levels as shown by Western blotting. (C). Claudin 1 siRNA reduced TNFα- and TNFα plus TGFβ-induced morphological changes as shown by F-actin staining at 48 h. Similar results were also found after 24 h or 72 h of TNFα treatment.