title: Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells.
abstract: We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H(2)O(2) production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H(2)O(2) production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca(2+)-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.
authors.affiliation: Department of Medicine, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Hong
authors.forename: Murray
authors.initials: M
authors.lastname: Resnick
authors.forename: Jose
authors.initials: J
authors.lastname: Behar
authors.forename: Jack
authors.initials: J
authors.lastname: Wands
authors.forename: Ronald A
authors.initials: RA
authors.lastname: DeLellis
authors.forename: Weibiao
authors.initials: W
authors.lastname: Cao
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000704:Analysis of Variance; D001471:Barrett Esophagus / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004938:Esophageal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006851:Hydrochloric Acid / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; C107953:RAC1 protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D020830:rac1 GTP-Binding Protein; C506943:ATP2A1 protein, human; C506946:ATP2A2 protein, human; D006851:Hydrochloric Acid