Action of vitamin D and the receptor, VDRa, in calcium handling in zebrafish (Danio rerio) - PubMed (original) (raw)

Action of vitamin D and the receptor, VDRa, in calcium handling in zebrafish (Danio rerio)

Chia-Hao Lin et al. PLoS One. 2012.

Abstract

The purpose of the present study was to use zebrafish as a model to investigate how vitamin D and its receptors interact to control Ca(2+) uptake function. Low-Ca(2+) fresh water stimulated Ca(2+) influx and expressions of epithelial calcium channel (ecac), vitamin D-25-hydroxylase (cyp2r1), vitamin D receptor a (vdra), and vdrb in zebrafish. Exogenous vitamin D increased Ca(2+) influx and expressions of ecac and 25-hydroxyvitamin D(3)-24-hydroxylase (cyp24a1), but downregulated 1α-OHase (cyp27b1) with no effects on other Ca(2+) transporters. Morpholino oligonucleotide knockdown of VDRa, but not VDRb, was found as a consequence of calcium uptake inhibition by knockdown of ecac, and ossification of vertebrae is impaired. Taken together, vitamin D-VDRa signaling may stimulate Ca(2+) uptake by upregulating ECaC in zebrafish, thereby clarifying the Ca(2+)-handling function of only a VDR in teleosts. Zebrafish may be useful as a model to explore the function of vitamin D-VDR signaling in Ca(2+) homeostasis and the related physiological processes in vertebrates.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Effect of different Ca2+ concentrations on mRNA expressions of Ca2+-related genes.

(A) mRNA expressions in 3-d post-fertilization (dpf) zebrafish embryos. (B) mRNA expressions in 5-dpf zebrafish embryos. (C) mRNA expressions in gills of adult zebrafish. mRNA expressions were analyzed by a qPCR, and values were normalized to β-actin. Values are the mean ± SD (n = 4∼6). Student’s t-test, *p<0.05; **p<0.05; ***p<0.001.

Figure 2

Figure 2. Effects of exogenous 1α,25(OH)2D3 (20 µg/l) in 3-d post-fertilization (dpf) zebrafish embryos.

Ca2+ influx (A), Ca2+ content (B), mRNA expressions in 3-dpf zebrafish embryos (C). mRNA expressions were analyzed by a qPCR, and values were normalized to β-actin. Values are the mean ± SD (n = 7∼10). Student’s t-test, *p<0.05; **p<0.01, ***p<0.001.

Figure 3

Figure 3. Alignment and phylogenetic analysis of vitamin D receptors (VDRs).

(A) Alignment of amino-acid sequences of zebrafish (z)VDRa and VDRb. (B) Phylogenetic analysis of vertebrate VDRs. The consensus line denotes a consensus (asterisk), similarity (: or.), or difference (-) between zVDRa and zVDRb. Bold letters indicate the DNA-binding domain, and underlined letters indicate the ligand-binding domain. Phylogenetic analyses were conducted using MEGA5. The phylogenetic analyses were inferred using Neighbor-joining trees and were bootstrapped (600 pseudosamples) to assess the robustness. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The unit of the scale bar is the number of amino-acid substitutions per site.

Figure 4

Figure 4. Zebrafish vdra and vdrb expression profiles.

mRNA expression patterns in developing stages (A) and various tissues (B) were determined by an RT-PCR using β-actin as the internal control. (C) vdra and vdrb mRNA expressions in different tissues of adult zebrafish were analyzed by a qPCR using β-actin as the internal control. Values are the mean ± SD (n = 3). E, eye; K, kidney; Sk, skin; I, intestine; G, gill; H, heart; M, muscle; B, brain; L, liver; Ov, ovary; Te, testis.

Figure 5

Figure 5. Effects of vitamin D receptor (VDR)a and VDRb morpholino oligonucleotides (MOs) in 3-d post-fertilization (dpf) zebrafish embryos.

Ca2+ influx (A), Ca2+ content (B), mRNA expression (C), density of _ecac_-expressing cells (D), and ecac signals (E). mRNA expressions were analyzed by a qPCR using β-actin as the internal control. Different letters indicate a significant difference (p<0.05) using a one-way ANOVA followed by Tukey’s multiple-comparison test. Values are the mean ± SD (n = 6 or 7). Scale bar:100 µm.

Figure 6

Figure 6. Effects of vitamin D receptor (VDR)a and VDRb morpholino oligonucleotides (MOs) on zebrafish embryos with 1α,25(OH)2D3 (20 µg/l) or low Ca2+ (0.02 mM; LCa) treatment.

Ca2+ influx (A, C) and ecac mRNA expression (B, D). mRNA expression of ecac was analyzed by a qPCR using β-actin as the internal control. Different letters indicate a significant difference (p<0.05) using one-way ANOVA followed by Tukey’s multiple-comparison test. Values are the mean ± SD. (n = 6∼8). High Ca2+ (HCa): 2.00 mM.

Figure 7

Figure 7. Effects of vitamin D receptor (VDR)a and VDRb morpholino oligonucleotides (MOs) on cyp27b1 and cyp24a1 mRNA expressions in 3-d post-fertilization (dpf) zebrafish embryos treated with 1α,25(OH)2D3 (20 µg/l).

(A) cyp27b1 mRNA expression. (B) cyp24a1 mRNA expression. mRNA expressions were analyzed by a qPCR using β-actin as the internal control. Different letters indicate a significant difference (p<0.05) using one-way ANOVA followed by Tukey’s multiple-comparison test. Values are the mean ± SD. (n = 5–6).

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This study was financially supported by the grant to P.P.H. from the National Science Council, Taiwan, R.O.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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