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ZFIN ID: ZDB-PUB-141008-3
Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio)
Chun, R.F., Blatter, E., Elliott, S., Fitz-Gibbon, S., Rieger, S., Sagasti, A., Adams, J.S., Hewison, M.
Date: 2014
Source: Cell biochemistry and function   32(8): 675-82 (Journal)
Registered Authors: Rieger, Sandra, Sagasti, Alvaro
Keywords: CYP24A1, CYP27B1, cytochrome P450, metabolism, vitamin D
MeSH Terms:
  • Animals
  • Cell Line
  • Cloning, Molecular*
  • Gene Expression Profiling
  • Humans
  • Kidney/metabolism
  • Models, Animal
  • Myocardium/metabolism
  • Phylogeny
  • Spleen/metabolism
  • Vitamin D/analogs & derivatives*
  • Vitamin D/metabolism
  • Vitamin D3 24-Hydroxylase/genetics*
  • Vitamin D3 24-Hydroxylase/metabolism
  • Zebrafish*
PubMed: 25290078 Full text @ Cell Biochem. Funct.
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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 (5-150 nM) or active 1,25D (0.1-10 nM) 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.
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