title: Identification of a renal proximal tubular cell-specific enhancer in the mouse 25-hydroxyvitamin d 1alpha-hydroxylase gene.
abstract: The active form of vitamin D is synthesized by 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase), which is expressed predominantly in renal proximal tubular cells. To clarify the mechanism of cell-specific gene expression of this enzyme, the 5'-flanking region of the mouse 1alpha-hydroxylase gene was investigated. Investigation began with mRNA expression of 1alpha-hydroxylase in cultured cells, including LLC-PK1, NIH/3T3, HepG2, MDCK, and OK cells. Expression of 1alpha-hydroxylase mRNA was restricted in LLC-PK1 cells. Several lengths of the 5'-flanking region of 1alpha-hydroxylase gene were linked to a pGL3-basic luciferase vector and introduced into these cells. Only LLC-PK1 cells had a substantial luciferase activity. Deletion analyses revealed that luciferase activity was detected in constructs extending from the transcription initiation site to -1652 to -105 bp, whereas further deletion to -80 bp resulted in a marked decrease in activity. The region from -105 to -80 bp contained two ternary complex factor-1 (TCF-1) sites, and mutations in the proximal TCF-1 site decreased the activity. Electrophoretic mobility shift assay demonstrated binding of LLC-PK1 nuclear proteins to this region. Tests of enhancer function in LLC-PK1 cells indicated that the 26-bp fragment behaved as a classical enhancer, i.e., independently of position and orientation. Moreover, a decoy oligonucleotide corresponding to this region substantially inhibited the promoter activity of 1alpha-hydroxylase gene. This study suggests that the -105 to -80 bp element of mouse 1alpha-hydroxylase gene contains an enhancer to be necessary for renal proximal tubular cell-specific expression.
authors.affiliation: Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Jun
authors.initials: J
authors.lastname: Yoshino
authors.forename: Matsuhiko
authors.initials: M
authors.lastname: Hayashi
authors.forename: Takao
authors.initials: T
authors.lastname: Saruta
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics*; D016475:3T3 Cells; D000818:Animals; D004285:Dogs; D004742:Enhancer Elements, Genetic; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D051379:Mice; D011401:Promoter Regions, Genetic; D013552:Swine
chemical_list: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase