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. 2011;6(8):e23689.
doi: 10.1371/journal.pone.0023689. Epub 2011 Aug 24.

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism

Chia-Hao Lin  1 , I-Lun Tsai, Che-Hsien Su, Deng-Yu Tseng, Pung-Pung Hwang
Affiliations

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism

Chia-Hao Lin et al. PLoS One. 2011.

Abstract

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Zebrafish mr and gr expression profiles.
Determined by RT-PCR, mr and gr mRNA in various tissues of adults (A), and during developmental stages of embryos (B). β-actin was used as the internal control.
Figure 2
Figure 2. Ca2+ influx and gene expressions of Ca2+ regulation-related genes.
Ca2+ influx (A) and mRNA expression (B) of 3-dpf zebrafish embryos acclimated to low- (0.02 mM Ca2+) or high-Ca2+ (2.00 mM Ca2+) artificial fresh water. mRNA expression analyzed by qPCR and values were normalized to β-actin. Values are the mean ± SEM (n = 4∼6). *Significant difference (Student's t-test, p<0.05).
Figure 3
Figure 3. Effects of exogenous cortisol in 3-dpf zebrafish embryos.
Ca2+ content (A), Ca2+ influx (B) and mRNA expressions (C). mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7).
Figure 4
Figure 4. Effects of exogenous cortisol on ecac-expressing cells in 3-dpf zebrafish embryos.
In situ hybridization analysis indicated ecac signals (A) and density of ecac-expressing cells (B). abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7). Scale bar 100 µm.
Figure 5
Figure 5. Specificity and effectiveness of MR MO and GR MO.
MR and GR cRNA (with GFP fusion) were injected into embryos respectively (A, B), and embryos coinjection of MR/GR MO with cRNA (C, D). Western blot were used to detect GR and MR protein expressions in wild type (WT) and the MO-injected embryos at 3 dpf (E).
Figure 6
Figure 6. Effects of MR MO and GR MO in 3-dpf zebrafish embryos.
Ca2+content (A), Ca2+ influx (B), and mRNA expressions (C). mRNA expressions were analyzed by qPCR and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6 or 7).
Figure 7
Figure 7. Effects of MR MO and GR MO on ecac-expressing cells in 3-dpf zebrafish embryos.
In situ hybridization analysis indicated ecac signals (A) and density of ecac-expressing cells (B). abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7). Scale bar 100 µm.
Figure 8
Figure 8. Effects of MR MO and GR MO on zebrafish embryos with cortisol treatment.
Ca2+ influx (A) and ecac mRNA expression (B) were analyzed in 3-dpf zebrafish embryos injected with GR MO or MR MO with cortisol treatment. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 9
Figure 9. Effects of GR cRNA on GR-SB MO-injected zebrafish embryos.
Ca2+ influx (A) and ecac mRNA expression (B) were also analyzed in 3-dpf zebrafish embryos injected with GR-SB MO or GR-SB MO with GR cRNA. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 10
Figure 10. Effect of MR MO and GR MO on ecac mRNA expression with low Ca2+ treatment.
ecac mRNA expression were analyzed in 3-dpf zebrafish embryos injected with GR MO or MR MO with low Ca2+ (0.02 mM; LCa) treatment. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 11
Figure 11. Effects of exogenous cortisol on mRNA expressions of the vitamin D3-related genes.
qPCR was used to analyze mRNA expression and values were normalized to β-actin. (A) mRNA expressions in 1-dpf zebrafish embryos. (B) mRNA expressions in 3-dpf zebrafish embryos. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6).
Figure 12
Figure 12. Effects of MR MO and GR MO on mRNA expression of the vitamin D3-related genes.
(A) mRNA expressions in 1-dpf zebrafish embryos. (B) mRNA expressions in 3-dpf zebrafish embryos. mRNA expression was analyze by qPCR and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6).
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