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Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio)
- Source :
- Cell Biochemistry and Function. 32:675-682
- Publication Year :
- 2014
- Publisher :
- Wiley, 2014.
-
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. Copyright © 2014 John Wiley & Sons, Ltd.
- Subjects :
- Expression vector
fungi
Clinical Biochemistry
Danio
Cytochrome P450
Cell Biology
General Medicine
Biology
biology.organism_classification
Biochemistry
Molecular biology
Gene expression profiling
Open reading frame
CYP24A1
Complementary DNA
embryonic structures
polycyclic compounds
biology.protein
Zebrafish
Subjects
Details
- ISSN :
- 02636484
- Volume :
- 32
- Database :
- OpenAIRE
- Journal :
- Cell Biochemistry and Function
- Accession number :
- edsair.doi...........bbf4066b6dde7427c32347fb1bfb6bdd