p38 MAPK in Epithelial-Mesenchymal Transition Figure 4. Expression of E-cadherin, CK, a-SMA, vimentin in HK-2 cells by immunochemistry. (A–C)The HK-2 cells under 30 mM glucose(B) or 30 mM glucose+cont siRNA(C) showed a loss of CK and E-cadherin and an increase of a-SMA and vimentin expression compared to that of the cells under 5.5 mM glucose condition(A).(D–F)These changes were prevented by exposed to 30 mM glucose+p38siRNA for 24 h(D) or 48 h(E) or+AP-1 inhibitor(F). doi:10.1371/journal.pone.0022806.g004 detected fibronectin synthesis in HK-2 cells that were cultured in high glucose for 48 hours. Secretion of fibronectin was signifi- cantly up-regulated by the stimulation of high glucose (P,0.05), whereas the level of secretions was markedly decreased after pre- treatment with p38 siRNA (P,0.05) (Figure 9). The activation of AP-1 in high-glucose-treated HK-2 cells Because MAPKs have been shown to play a critical role in the up-regulation of AP-1 activity in various types of cultured cells via multiple mechanisms, we attempted to examine the possible involvement of p38 MAPK, which is the major upstream kinase, in AP-1 activation in HK-2 cells. The levels of AP-1 binding activity increased after treatment with high glucose but decreased after transfection with p38 siRNA (P,0.05) (Figure 10). To further study the transactivation of AP-1 in high-glucose-treated HK-2 cells, experiments using [6]-Gingerol [33], which is a chemical inhibitor of AP-1, demonstrated that it could partially inhibit high- glucose-induced activation of AP-1, but was irrelevant to the regulation of p38 expression and phosphorylation. (P,0.05) (Figure 10 and 11). The effect of AP-1 on E-cadherin, cytokeratin, vimentin, and a-SMA expression under high glucose conditions To determine the role of AP-1 in the EMT, E-cadherin, CK, vimentin, and a-SMA expression was assessed. Compared to cells that were incubated with 5.5 mM glucose, the expression of E- cadherin and CK was significantly decreased under high glucose conditions or low glucose with TGF-b1 but was increased after pre-treatment with the AP-1 inhibitor, protein expression was increased (P,0.05) (Figure 4, 8, 12). It was further confirmed that the CK mRNA levels were markedly diminished under high glucose conditions; however, the levels were notably increased by pre-treatment with the AP-1 inhibitor (P,0.05) (Figure 12). a-SMA and vimentin were not detected in cells that were incubated with 5.5 mM glucose. After culture with high glucose or low glucose with TGF-b1, the expression of a-SMA and vimentin were markedly increased but decreased with pre-treatment with the AP-1 inhibitor (P,0.05) (Figure 4, 8, 12). The levels of vimentin mRNA were increased when the cells were treated with high glucose, whereas they were significantly decreased after pre- treatment with the AP-1 inhibitor (P,0.05) (Figure 12). The effect of AP-1 on fibronectin synthesis in HK-2 cells In the culture media from the cells treated with high glucose, the concentration of fibronectin was significantly greater than that of the media from the cells treated with 5.5 mM glucose; the concentration of fibronectin after pre-treatment with the AP-1 inhibitor was significantly decreased compared to the concentra- tion after treatment with high glucose (Figure 9). The role of p38 MAPK/AP-1 in Snail expression under high glucose conditions Snail is a member of the Snail family of zinc finger transcription factors that induce the EMT initially identified as a gene controlling cell migration in the neural crest and developing mesoderm in the chick embryo [34]. Snail mRNA represses CDH1/E-cadherin and other epithelial genes, and induces the expression of mesenchymal genes in epithelial cells of diverse origin [35–37]. Therefore, we observed the effect of p38 MAPK and AP-1 on Snail mRNA expression in high glucose-induced EMT. In our study, Snail mRNA expression was amplified by the stimulation of high glucose compared to 5.5 mM glucose (P,0.05), whereas Snail mRNA expression was significantly decreased after pre-treatment with p38 siRNA or the AP-1 inhibitor (P,0.05) (Figure 13). PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22806