title: The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.
abstract: The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1 (Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Adrian
authors.initials: A
authors.lastname: O'Hara
authors.forename: Alice
authors.initials: A
authors.lastname: Howarth
authors.forename: Andrea
authors.initials: A
authors.lastname: Varro
authors.forename: Rod
authors.initials: R
authors.lastname: Dimaline
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D005755:Gastrins / Q000494:pharmacology*; D055785:Gene Knockdown Techniques; D006801:Humans; D051847:Lithostathine / Q000235:genetics* / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D005755:Gastrins; D051847:Lithostathine; D017396:Plasminogen Activator Inhibitor 2; D021122:Protein Subunits; C497616:Reg1a protein, rat; C577981:PSMB1 protein, human; D046988:Proteasome Endopeptidase Complex
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