p38 MAPK in Epithelial-Mesenchymal Transition Figure 10. Activity of AP-1 in HK-2 cells by EMSA. (A)AP-1 activity was analyzed by EMSA of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h (lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5).(B) AP-1 activity was analyzed by EMSA of the HK-2 cells cultured in 5.5 mM glucose(lane 1), 30 mM glucose (lane 2) and 30 mM glucose+AP-1 inhibitor(lane 3). Values represent the mean 6 SD, aP,0.05 vs. 5.5 mM glucose, bP,0.05 vs. 30 mM glucose. doi:10.1371/journal.pone.0022806.g010 which is a chemical inhibitor of AP-1, inhibited high glucose- induced activation of AP-1. With high glucose treatment, HK-2 Figure 11. Expression and phosphorylation of p38 MAPK in HK-2 cells by Western blot. Expression and phosphorylation of p38 MAPK under 5.5 mM glucose (lane 1), 30 mM glucose(lane 2) and 30 mM glucose+AP-1 inhibitor were determined by. aP,0.05 vs. 5.5 mM glucose. doi:10.1371/journal.pone.0022806.g011 cells underwent striking morphological alterations, including membrane ruffling, numerous pseudopodial protrusions, increased cell motility and anchorage-independent (invasive) growth. These changes were inhibited by pre-treatment with [6]-Gingerol. The Snail family members have been shown to be involved in the regulation of EMT during embryonic development and tumor progression [50]. Snail possesses DNA binding capacity and recognizes the E-box elements in the promoter region of its target genes, which include E-cadherin. Binding of Snail to its cognate E- box often leads to the suppression of gene transcription [51,52]. Earlier studies showed that induction of Snail, which commonly occurs in malignant carcinomas, suppresses epithelial E-cadherin expression [53,54]. In this study, Snail expression was promoted in HK-2 cells under high glucose conditions, but knockdown of p38 MAPK and pretreatment with [6]-Gingerol reversed the increased Snail expression, which suggests that Snail expression in vitro is dependent on the p38/AP-1 signaling pathway. The expression of Snail suppressed the expression of E-cadherin protein. Although the underlying mechanism of Snail inhibition remains to be elucidated, the finding that p38/AP-1 is able to target Snail, which is a key EMT-regulatory gene, highlights a fundamental step in the p38/AP-1 signaling pathway in high glucose-induced EMT, which may prove its role in TIF. This study provides a novel insight into the role of the p38/AP- 1 signaling pathway, which was shown to be a crucial step in high glucose-induced EMT in HK-2 cells. The results indicate that the striking changes in TECs were induced by high glucose; these changes include the following: the activation of phosphorylated PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22806