Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene

https://doi.org/10.1016/j.bbrc.2007.04.103Get rights and content

Abstract

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D3. Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5′-flanking sequence of this gene. A series of deletion constructs between nucleotides −1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides −548 and −294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5′-untranslated region did not appear to play a major role in the vitamin D response.

Section snippets

Materials and methods

Preparation of deletion constructs of the CYP24 gene’s upstream sequence. Human genomic DNA was isolated from skin-derived fibroblasts TIG-112 (Health Science Research Resources Bank (HSRRB), Osaka, Japan), using DNeasy tissue kit (Qiagen) according to the manufacturer’s instruction. Various regions of the CYP24 gene’s upstream sequence were amplified by polymerase chain reaction (PCR). The reaction was carried out in a 50 μl mixture containing 1 U KOD-Plus DNA polymerase (TOYOBO), 5 μl of (10×)

Deletion analysis of the upstream sequence of the human CYP24 gene

Various regions of the upstream sequence of the human CYP24 gene were inserted into the promoter site of the luciferase gene in the expression vector pGL3-basic. These constructs were transfected into TIG-112 cells, and transient expression of the luciferase gene was monitored 12 h after addition of 1,25(OH)2D3 (Fig. 1A). Although the construct −294/+209 includes both of the two VDREs, it was not enough to fully respond to 1,25(OH)2D3. An extension of the insert to nucleotide −548 increased the

Discussion

We have described deletion analyses of the 5′-flanking sequence as well as 5′-UTR of the human CYP24 gene. Most of our effort was directed to the analyses of the region between nucleotides −548 and −294, which is located immediately upstream of the two VDREs, and was not fully studied previously. We found that this region contains sequence elements that synergistically function with the nearby two VDREs. Neither of the sequences was dispensable for the expression of the CYP24 gene. The sequence

Acknowledgment

This work was supported in part by a grant awarded to M.R. from the Hiroshima Prefectural Government in Japan.

Cited by (24)

  • Effects of 25-hydroxyvitamin D<inf>3</inf> on cathelicidin production and antibacterial function of human oral keratinocytes

    2013, Cellular Immunology
    Citation Excerpt :

    25-hydroxyvitamin D3 (25VD3), the inactive form of vitamin D3 requires the activating enzyme 1α-hydroxylase (CYP27B1) to catalyze the conversion into its active form, 25-dihydroxyvitamin D3 (1,25VD3) [8]. 24-hydroxylase (CYP24A1), the inactiving enzyme of vitamin D3, can ubiquitously prevent the biological effects of 1,25VD3 [9]. The biological effects of active vitamin D3 result from altered gene expression via the nuclear receptor and transcription factor, VDR [10].

  • CYP24A1 regulation in health and disease

    2011, Vitamin D: Two-Volume Set
View all citing articles on Scopus
1

Present address: Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, 7-6-8, Saito, Asagi, Ibaraki, Osaka 567-0085, Japan.

View full text