Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2023346 | Regulatory Peptides | 2007 | 9 Pages |
Abstract
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. 5â²deletions 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, p38MAPK and JNK were examined by using immunoblotting. Prostaglandin E2 (PGE2) 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 p38MAPK 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 10Â h after stimulation. PGE2 secretion from gastrin-treated cells increased significantly 8Â h after stimulation. Treatment with gastrin also stimulated PGE2 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.
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Biochemistry
Authors
He Huang, Nikolaus Ansorge, Henning Schrader, Matthias Banasch, Hong-Gang Yu, Wolfgang E. Schmidt, Michael Höcker, Frank Schmitz,