TLR2 activation induced by H. pylori LPS promotes the differential expression of claudin-4, -6, -7 and -9 via either STAT3 and ERK1/2 in AGS cells

Gastric carcinogenesis has been associated to H. pylori virulence factors that induce a chronic inflammation process. Lipopolysaccharides play a role in chronic inflammatory responses via TLR2- and TLR4-dependent signaling pathways. Similarly, cellular invasiveness, metastatic potential and prognosis are usually associated to claudin-4, -6, -7 and -9 expression in gastric carcinogenesis. Therefore, the aim of this study was to determine if H. pylori LPS exerts an influence on carcinogenesis-related claudin expression and if it was directly regulated through the TLR2 pathway. Human antrum gastric adenocarcinoma AGS cells exposed or not to H. pylori LPS were used. Polyclonal anti-claudin-4, -6, -7 and -9, anti-TLR2, anti-pERK1/2 as well as rabbit monoclonal anti-pNFκB p65 and mouse monoclonal anti-CdX2 were used. ERK1/2 inhibitor UO126 and STAT3 inhibitor Stattic were also used. Western blot, immunofluorescence and confocal experiments were performed in whole cells as well as total protein, nuclear and cell membrane fractions. The results showed that H. pylori LPS increased the expression of TLR2 in a time dependent bi-phasic manner (<12 and >12 h exposure). Immunofluorescence using AGS monolayers corroborated the double phase TLR2 expression mainly on the cell membrane but a detectable signal was also determined in the cytoplasm of the cells. Activation of NFkB was downstream and depended on TLR2 expression as a statistically significant increase in pNFkB, that followed a pattern highly similar to the TLR2 expression was observed on the cell membrane fraction. The increase in TLR2 expression was accompanied by dramatically increased claudin-4 expression in cultures exposed from 30 m to 8 h to LPS. Increased expression of claudin-6, -7 and -9 also increases in >12 h LPS exposure times. The increase in claudins expression was also dependent on NFkB activation. The results also showed an increase in pSTAT3 that followed a bi-phasic pattern that began 30 min after stimulation and was compatible with the increase in TLR2 expression. The expression of the claudin-4 related CDX2 transcription factor did not followed the biphasic pattern. The results also showed that claudin-4 expression was STAT3 dependent whereas claudin-6, 7 and 9 expressions was ERK1/2 dependent. Our results suggest that H. pylori LPS induces TLR2 expression in the AGS cells, and that the longer the exposure to LPS, the greater the expression of TLR2 in the cell membrane. Consequently the expression of claudin-4, -6, -7 and -9 also increases.