title: Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio).
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.
authors.affiliation: Department of Orthopaedic Surgery, UCLA-Orthopaedic Hospital, Los Angeles, CA, 90095, United States.
authors.forename: Rene F
authors.initials: RF
authors.lastname: Chun
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Blatter
authors.forename: Stephanie
authors.initials: S
authors.lastname: Elliott
authors.forename: Sorel
authors.initials: S
authors.lastname: Fitz-Gibbon
authors.forename: Sandra
authors.initials: S
authors.lastname: Rieger
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sagasti
authors.forename: John S
authors.initials: JS
authors.lastname: Adams
authors.forename: Martin
authors.initials: M
authors.lastname: Hewison
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D006801:Humans; D007668:Kidney / Q000378:metabolism; D023421:Models, Animal; D009206:Myocardium / Q000378:metabolism; D010802:Phylogeny; D013154:Spleen / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish
chemical_list: D014807:Vitamin D; C104450:25-hydroxyvitamin D; D065668:Vitamin D3 24-Hydroxylase
keywords: CYP24A1; CYP27B1; cytochrome P450; metabolism; vitamin D