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.

Keywords: CYP24A1; CYP27B1; cytochrome P450; metabolism; vitamin D.

Figure 1. Induction of cyp24a1 mRNA expression by 25D and 1,25D identifies a functional 25-hydroxyvitamin D-1-hydroxylase in zebrafish

Zebrafish larvae (day 5) were treated with increasing doses of: A) 25D (5-150 nM), B) 1,25D (0.1-10 nM) or vehicle (0.1% ethanol) for six hours, and expression of cyp24a1 mRNA determined by qRT-PCR. Data are expressed as ΔΔCt = ΔCt (cyp24a1) - ΔCt(elfa). C) Zebrafish larvae (day 5) were treated with increasing doses of itraconazole (ITRA, 0, 0.1, and 1.0 μM) one hour prior to six hour incubation with vehicle, 5 nM 25D or 0.1 nM 1,25D. Expression of cyp24a1 was then determined by qRT-PCR and expressed relative to vehicle ITRA set to 100%. Fold = 2^ΔΔCt. Data are shown as n=3 replicates ± SD. * P < 0.05.

Figure 2. Amino acid sequence comparison between zebrafish Cyp27b1 and human CYP27B1

The amino acid sequence for Cyp27b1 was compared with human CYP27B1 using NCBI BLAST. Heme binding domain was marked in bold. Ferredoxin binding domain was marked with underline. Q66 and T408 (bold and asterik) have been identified as residues found mutated in human type I rickets suggesting functionally significant amino acids. The cDNA sequence has been deposited to Genbank (KM262796).

Figure 3. Expression of zebrafish cyp27b1 in kidney, spleen and heart

Quantitative real-time PCR was conducted on cDNA prepared from heart (H1, H2) spleen (S1, S2) and kidney (K1, K2). The graphs display the data from triplicate qPCR assessments of two independent preparations of the respective organs. The data is presented as relative fold expression with H2 (lowest expression sample) set to one-fold and error bars ± SD.

Figure 4. Zebrafish cyp27b1 cDNA encodes a functional 25-hydroxyvitamin D-1α-hydroxylase activity

A) HKC-8, a human proximal tubule kidney cell line, was transfected with indicated amounts of pcDNA3.1-zcyp27b1 expression plasmid or empty vector. The resulting cells were incubated with vehicle (0.1% ethanol) or 100 nM 25D for six hours. qRT-PCR analysis was used to quantify expression of mRNA for the 1,25D-VDR-induced catabolic enzyme 24-hydroxylase (CYP24A1) in human HKC-8 cells. Data are expressed as fold-induction of CYP24A1 expression relative to 100 nM 25D incubated empty vector control cells set to a value of one. Data are shown as mean ± SE for n=3 replicates; * P < 0.05. B) HKC-8 transfected with plasmid expressing zebrafish cyp27b1 or empty vector was analyzed by HPLC for 1,25D₃ synthesis after two-hour incubation with 300,000 cpm ³H-25D₃. ³H-1,25D₃ production was calculated and normalized to mg protein per sample and expressed as fmol/mg/hr. The graph displays the average of N=3 (empty vector) and N=4 (cyp27b1) HPLC runs. Representative traces are shown of analysis of HKC-8 transfected with C) pcDNA3.1 empty vector or D) pcDNA3.1-zcyp27b1.

Figure. 5. Phylogenetic analysis of CYP27B1 found in various species

CYP27B1 evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood (-8036.2717) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 487 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. Zebrafish Cyp27a1 and Cyp27c1 were included as outgroups. CYP27A1 and CYP27C1 from chicken were included since no CYP27B1 was found for that species.