Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio)

Rene F. Chun, Elizabeth Blatter, Stephanie Elliott, Sorel Fitz-Gibbon, Sandra Rieger, Alvaro Sagasti, John S. Adams, Martin Hewison

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (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 Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5-150nM) or active 1,25D (0.1-10nM) induced dose responsive expression (15-95-fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 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 cyp27b1 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 CYP24A1 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 CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism.

Original languageEnglish (US)
Pages (from-to)675-682
Number of pages8
JournalCell Biochemistry and Function
Volume32
Issue number8
DOIs
StatePublished - Dec 1 2014
Externally publishedYes

Keywords

  • CYP24A1
  • CYP27B1
  • Cytochrome P450
  • Metabolism
  • Vitamin D

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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