Si Jin, Fu Xiaoying, Behar Jose, Wands Jack, Beer David G, Souza Rhonda F, Spechler Stuart J, Lambeth David, Cao Weibiao
Department of Medicine, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903, USA.
J Biol Chem. 2007 Jun 1;282(22):16244-55. doi: 10.1074/jbc.M700297200. Epub 2007 Apr 2.
We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid-induced increase in prostaglandin E2 (PGE2) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE2 production. Knockdown of NOX5-S or NF-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE2 production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE2 production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.
我们已经表明，NADPH氧化酶NOX5-S可能通过增加细胞增殖和减少细胞凋亡，在从巴雷特食管进展为食管腺癌（EA）的过程中发挥重要作用。然而，酸诱导的NOX5-S介导的细胞增殖增加的机制尚不清楚。我们发现，在SEG1 EA细胞中，酸诱导的前列腺素E2（PGE2）生成增加是由环氧化酶-2（COX2）的激活介导的，而不是由COX1介导的。酸处理增加了细胞内Ca2+，细胞内Ca2+增加的阻断抑制了酸诱导的COX2表达和PGE2生成增加。通过小干扰RNA敲低NOX5-S或NF-κB1 p50可显著抑制SEG1细胞中酸诱导的COX2表达和PGE2生成。当用NF-κB体内激活报告质粒pNF-κB-Luc转染SEG1细胞时，酸处理显著降低了IκBα并增加了荧光素酶活性。在一个过表达NOX5-S的新型巴雷特细胞系中，当这些巴雷特细胞用pNF-κB-Luc转染时，IκBα显著降低，荧光素酶活性增加。巴雷特细胞中NOX5-S的过表达显著增加了H2O2生成、COX2表达、PGE2生成和胸苷掺入。COX2抑制剂或小干扰RNA显著降低了在过表达NOX5-S的巴雷特细胞中发生的胸苷掺入增加或SEG1 EA细胞中酸处理诱导的胸苷掺入增加。我们得出结论，酸诱导的COX2表达和PGE2生成取决于SEG1细胞中胞质Ca2+的增加以及NOX5-S和NF-κB的顺序激活。COX2衍生的PGE2生成可能有助于SEG1细胞中NOX5-S介导的细胞增殖。
