Identification of a renal proximal tubular cell-specific enhancer in the mouse 25-hydroxyvitamin D 1α-hydroxylase gene

Tadashi Yoshida, Jun Yoshino, Matsuhiko Hayashi, Takao Saruta

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Abstract

The active form of vitamin D is synthesized by 25-hydroxyvitamin D 1α-hydroxylase (1α-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 1α-hydroxylase gene was investigated. Investigation began with mRNA expression of 1α-hydroxylase in cultured cells, including LLC-PK1, NIH/3T3, HepG2, MDCK, and OK cells. Expression of 1α-hydroxylase mRNA was restricted in LLC-PK1 cells. Several lengths of the 5′-flanking region of 1α-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. More-over, a decoy oligonucleotide corresponding to this region substantially inhibited the promoter activity of 1α-hydroxylase gene. This study suggests that the -105 to -80 bp element of mouse 1α-hydroxylase gene contains an enhancer to be necessary for renal proximal tubular cell-specific expression.

Original languageEnglish
Pages (from-to)1455-1463
Number of pages9
JournalJournal of the American Society of Nephrology
Volume13
Issue number6
DOIs
Publication statusPublished - 2002 Jan 1

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ASJC Scopus subject areas

  • Nephrology

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