Gastrin-mediated interleukin-8 and cyclooxygenase-2 gene expression: differential transcriptional and posttranscriptional mechanisms.
OBJECTIVE
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 kappaB (NF-kappaB) 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 E(2) synthesis. In the presence of an NF-kappaB 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-kappaB. 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.
Subramaniam
Dharmalingam
D
Department of Internal Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Ramalingam
Satish
S
May
Randal
R
Dieckgraefe
Brian K
BK
Berg
Douglas E
DE
Pothoulakis
Charalabos
C
Houchen
Courtney W
CW
Wang
Timothy C
TC
Anant
Shrikant
S
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D015232:Dinoprostone / Q000096:biosynthesis; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005755:Gastrins / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016209:Interleukin-8 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D051058:Oncogene Protein v-akt; D020871:RNA Stability / Q000187:drug effects; D012334:RNA, Neoplasm / Q000235:genetics*; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014158:Transcription, Genetic / Q000187:drug effects*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D005755:Gastrins; D016209:Interleukin-8; D012334:RNA, Neoplasm; C013550:gastrin 17; D051546:Cyclooxygenase 2; D051058:Oncogene Protein v-akt; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone