The CCK-2/gastrin splice variant receptor retaining intron 4 transactivates the COX-2 promoter in vitro.
The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.
Huang
He
H
Laboratory for Molecular Gastroenterology, Dept. of Medicine I, St. Josef Hospital, Ruhr-University Bochum, Germany.
Ansorge
Nikolaus
N
Schrader
Henning
H
Banasch
Matthias
M
Yu
Hong-Gang
HG
Schmidt
Wolfgang E
WE
Höcker
Michael
M
Schmitz
Frank
F
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005755:Gastrins / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D005755:Gastrins; D020033:Protein Isoforms; D012333:RNA, Messenger; D043887:Receptor, Cholecystokinin B; D008156:Luciferases; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D015232:Dinoprostone