Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio). (7th October 2014)

• Record Type: Journal Article
• Authors: Chun, Rene F.; Blatter, Elizabeth; Elliott, Stephanie; Fitz‐Gibbon, Sorel; Rieger, Sandra; Sagasti, Alvaro; Adams, John S.; Hewison, Martin
• Is Part Of: Cell biochemistry and function. Volume 32:Number 8(2013:Dec.)
• Page Start: 675
• Page End: 682
• Publication Date: 2014-10-07
• Subjects: Cytochemistry -- Periodicals; Cell metabolism -- Periodicals; Biochemistry -- Periodicals; Cytology -- Periodicals; 572
• DOI: 10.1002/cbf.3071
• Languages: English
• ISSNs: 0263-6484

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Activation of precursor 25‐hydroxyvitamin D<sub>3</sub> (25D) to hormonal 1, 25‐dihydroxyvitamin D<sub>3</sub> (1, 25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used <italic>Danio rerio</italic> (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (<italic>cyp27b1</italic>). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1, 25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene <italic>cyp24a1</italic> relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish <italic>cyp27b1</italic> cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a c<italic>yp27b1</italic> expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1, 25D resulting in greater than twofold induction of <italic>CYP24A1</italic> mRNA expression and a 25‐fold increase in 1, 25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish <italic>CYP27B1</italic>, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of <italic>cyp27b1</italic> expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.</p> </abstract>