Abstract
Here we investigated the regulatory mechanism of lipocalin-type prostaglandin D synthase (L-PGDS) gene expression in human TE671 (medulloblastoma of cerebellum) cells. Reporter analysis of the promoter region from -730 to +75 of the human L-PGDS gene demonstrated that deletion or mutation of the N-box at -337 increased the promoter activity 220-300%. The N-box was bound by Hes-1, a mammalian homologue of Drosophila Hairy and enhancer of split, as examined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Functional expression of the Notch intracellular domain significantly increased Hes-1 expression and decreased L-PGDS expression level in TE671 cells. Moreover, knock-down of Hes-1 mRNA by RNA interference significantly enhanced the L-PGDS mRNA level, indicating that the L-PGDS gene expression is repressed by the Notch-Hes signaling. When the AP-2 element at -98 of the promoter region was deleted or mutated, the promoter activity was drastically decreased to approximately 10% of normal. The AP-2 element was bound by AP-2beta dominantly expressed in TE671 cells, according to the results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay. L-PGDS expression was induced by 12-O-tetradecanoylphorbol-13-acetate in TE671 cells, and this induction was inhibited by a protein kinase C inhibitor. Stimulation of TE671 cells with 12-O-tetradecanoylphorbol-13-acetate or transfection with protein kinase Calpha expression vector induced phosphorylation of Hes-1, inhibition of DNA binding of Hes-1 to the N-box, and activation of the AP-2beta function to up-regulate L-PGDS gene expression. These results reveal a novel transcriptional regulatory mechanism responsible for the high level expression of the human L-PGDS gene in TE671 cells.
Highlights
Biochemical, physiological, and genetic properties of lipocalin-type prostaglandin D synthase (L-PGDS) have been studied extensively in mammals [3,4,5]
Human L-PGDS mRNA was highly expressed in TE671 cells, weakly in MCF-7 cells, and negligibly in 1321-N1 cells (Fig. 1A, upper panel), whereas the expression level of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA, as the internal control, was almost identical in these three types of cells (Fig. 1A, lower panel)
No efficient reporter activities were detected in any of the cells transfected with the Ϫ90/ϩ75 construct. These results indicate that the region from Ϫ730 to Ϫ90 contained the ciselements responsible for the cell type-specific regulation of human L-PGDS gene expression in TE671 cells
Summary
Cell Culture—Human TE671 (medulloblastoma) cells were kindly provided by Dr David M. The resultant PCR products were digested with XhoI and HindIII and inserted into the upstream site of the luciferase reporter gene of the pGL3-Enhancer vector (Promega, Madison, WI). A fragment carrying the promoter region from Ϫ730 to ϩ75 was cloned into a pGL3-Enhancer vector to construct “Ϫ730/ϩ75.”. The cells were transfected with each promoter-luciferase reporter construct (0.3 g) together with pRL-CMV (0.1 g; Promega) carrying the Renilla luciferase gene under the control of the cytomegalovirus promoter by using Effectene (Qiagen, Hilden, Germany) or FuGENE 6 (Roche Diagnostics) according to the manufacturer’s instructions. After 24 h of transfection, the cells were washed with phosphate-buffered saline and cultured for another 4 h in phosphate-free Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and 0.5 Ci/ml [32P]orthophosphate (Amersham Biosciences) in the presence or absence of TPA (100 ng/ml).
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