p38 MAPK in Epithelial-Mesenchymal Transition Figure 8. Expression of E-cadherin, CK, a-SMA, vimentin in HK-2 cells by Western blot. (A)a-SMA, (B)E-caderin, (C)CK, (D)vimentin were measured by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 5.5 mM glucose with p38 siRNA (lane 2), 5.5 mM glucose with AP-1 inhibitor (lane 3), 5.5 mM glucose+TGF-b1 (10 ng/ml) (lane 4), 5.5 mM glucose+TGF-b1(10 ng/ml) with p38 siRNA (lane 5), 5.5 mM glucose+TGF- b1(10 ng/ml) with the AP-1 inhibitor (lane 6). aP,0.05 vs. 5.5 mM glucose, bP,0.05 vs. 5.5 mM glucose+TGF-b1. doi:10.1371/journal.pone.0022806.g008 intrestingly, increased apoptosis were also observed in cells in high glucose conditions for 48 h (Figure S1). The underlying mecha- Figure 9. Fibronectin synthesis in HK-2 cells by ELISA. Fibronectin was measured by ELISA of the HK-2 cells 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) and 30 mM glucose+AP-1 inhibitor(lane 6). 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.g009 nism of this high glucose-induced apoptosis is to be further elucidated. Because MAPKs have been shown to play an important role in the up-regulation of AP-1 activation 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 the activation of AP-1 in HK-2 cells. AP-1 activity is rapidly induced by a broad range of stimuli, including growth factors, cytokines, stress signals, and infections, as well as oncogenic stimuli; therefore, AP-1 mediates many physiological processes, such as proliferation, survival, differentiation, apoptosis, and transformation [48]. The role of the AP-1 transcriptional complex in EMT has received little attention in the past and to our knowledge, data on the function of AP-1 in high glucose-induced EMT are lacking. The results of the present study provide strong evidence to support the role of AP-1 in high glucose-induced EMT. AP-1 has been shown to be a potential downstream therapeutic target in p38 MAPK-mediated diseases [49]. The findings in our study included the induction of AP-1 under high glucose conditions and its partial abrogation mediated by p38 siRNA. The levels of AP-1 binding activity were increased under high glucose conditions but were decreased after transfection with p38 siRNA. To further study AP-1 transactivation in high glucose- induced EMT, experiments demonstrated that [6]-Gingerol, PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22806