Gastrin-Mediated Interleukin-8 and Cyclooxygenase-2 Gene Expression: Differential Transcriptional and Posttranscriptional Mechanisms

Dharmalingam Subramaniam, Satish Ramalingam, Randal May, Brian K. Dieckgraefe, Douglas E. Berg, Charalabos Pothoulakis, Courtney W. Houchen, Timothy C. Wang, Shrikant Anant

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

Abstract

Background & Aims: Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin-dependent gene expression. Methods: AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor κB (NF-κB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. Results: Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3′ kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2-mediated prostaglandin E2 synthesis. In the presence of an NF-κB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-κB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. Conclusions: Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively.

Original languageEnglish
Pages (from-to)1070-1082
Number of pages13
JournalGastroenterology
Volume134
Issue number4
DOIs
StatePublished - Apr 2008

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