Vitamin D signaling and the differentiation of developing dopamine systems

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VITAMIN D SIGNALING AND THE DIFFERENTIATION OF DEVELOPING DOPAMINE SYSTEMS

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RENATA A. N. PERTILE, ay XIAOYING CUI ay AND DARRYL W. EYLES a,b*

2

a

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Queensland Brain Institute, University of Queensland, Qld 4072, Australia

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b

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Queensland Centre for Mental Health Research, Wacol, Qld 4076, Australia

Abstract—Vitamin D regulates multiple factors including those involved in the ontogeny of dopaminergic systems. It has been shown that in neonatal rats maternally deprived of vitamin D, dopamine (DA) turnover is decreased with associated reductions in one catabolic enzyme, catechol-o-methyl transferase (COMT). To directly examine this signaling relationship, in the present study we have over-expressed the vitamin D receptor (VDR) in neuroblastoma SH-SY5Y cells in order to examine the mechanisms by which the active vitamin D hormone, 1,25(OH)2D3, via its receptor VDR, affects DA production and turnover. Our results show that VDR overexpression increases DA neuron differentiation by increasing tyrosine hydroxylase expression, DA production and decreasing the expression of NEUROG2 a marker of immature DA neurons. In the VDR-overexpressing cells, 1,25 (OH)2D3 further increased the levels of the DA-metabolites 3-MT and HVA and elevated COMT gene expression. Chromatin immunoprecipitation revealed that 1,25(OH)2D3 increased VDR binding in three regions of the COMT promoter, strongly suggesting direct regulation. In addition, 1,25(OH)2D3 treatment attenuated increased levels of MAOA, DRD2 and VMAT2 gene expression caused by the VDRoverexpression. Taken together, these results show VDR and 1,25(OH)2D3 are directly involved in regulating the expression of dopaminergic-associated genes and that this in vitro neuronal model is a useful tool for identifying the role of 1,25(OH)2D3 in DA neuronal development and maturation. Crown Copyright Ó 2016 Published by Elsevier Ltd on behalf of IBRO. All rights reserved.

Keywords: vitamin D receptor (VDR), vitamin D,1,25(OH)2D3, dopamine, COMT, SH-SY5Y, development. 11

*Correspondence to: D. W. Eyles, Neurobiology Laboratory, Queensland Brain Institute, University of Queensland, Qld 4072, Australia. E-mail address: [email protected] (D. W. Eyles). y These authors contributed equally to the work. Abbreviations: 3-MT, 3-methoxytyramine; ANOVA, analysis of variance; COMT, catechol-o-methyl transferase; DA, dopamine; DE, deoxypinephrine; DOPAC, 3,4-dihydroxyphenylacetic acid; DVD, vitamin D-deficient; HPLC, high pressure liquid chromatography; HVA, homovanillic acid; NA, noradrenaline; RA, retinoic acid; RXRs, retinoic acid receptors; TH, tyrosine hydroxylase; TR, thyroid hormone receptor; VDR, vitamin D receptor.

INTRODUCTION

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Among the neurotransmitter systems involved in the neurobiology of schizophrenia, abnormalities in dopamine (DA) regulation remain central in understanding the presentation of psychotic symptoms and their treatment (Carlsson and Lindqvist, 1963; Toda and Abi-Dargham, 2007). DA abnormalities also appear prior to the onset of psychosis, perhaps as a direct consequence of early adverse environmental factors. Early alterations in DA uptake and release may even represent a prodromal biomarker for this disease (Kuepper et al., 2012). Schizophrenia is a polygenetic group of diseases of neurodevelopment influenced by a variety of pre and post-natal environmental factors. Among such environmental risk factors, low maternal vitamin D has been implicated in this disease (McGrath et al., 2010; Eyles et al., 2012). The active vitamin D hormone, 1,25-dihydroxy vitamin D (1,25(OH)2D3) has been shown to influence calcium homeostasis, cell proliferation, differentiation, hormone secretion and also immune and neuronal functions (Matkovits and Christakos, 1995; Eyles et al., 2013). The receptor for vitamin D receptor (VDR) is widely present throughout the human brain being concentrated in the dopaminergically rich substantia nigra (Eyles et al., 2005). In rodents, the VDR 1st emerges in the developing mesencephalon at a time corresponding to the peak of dopaminergic cell birth (Cui et al., 2013). Recently we have also shown the VDR to be restricted to the nucleus of tyrosine hydroxylase- (TH) positive neurons in human and rodent brain (Eyles et al., 2012). Therefore, the VDR is appropriately positioned to regulate the ontogeny of DA neurons. Given that 1,25(OH)2D3 deficiency is associated with diseases such as schizophrenia and the ontogeny of DA neurons may represent a convergent pathway to this disease (Eyles et al., 2012) it has become crucial to understand the basic mechanisms behind how 1,25(OH)2D3, a steroid hormone who’s genomic actions are mediated by it’s receptor, the VDR, regulates DA neuron differentiation and function. Our work using the developmental vitamin D-deficient (DVD) animal model, has established 1,25(OH)2D3 as an essential factor in the ontogeny of DA systems (Eyles et al., 2012). We have shown DA turnover is altered in the brains of neonates from DVD-deficient dams. Specifically, the ratio of DA metabolites—3,4-dihydroxyphenyla cetic acid (DOPAC)/homovanillic acid (HVA) was shown to be reduced (Kesby et al., 2009; Eyles et al., 2012). Accordingly, this is accompanied by a reduction in the enzyme that catalyzes the conversion of DOPAC to

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HVA and DA to 3-methoxytyramine (3-MT) – catechol-omethyl transferase (COMT). Alterations in the COMT gene represent one of the most studied functional polymorphisms in schizophrenia (Tunbridge et al., 2006). However, the mechanism for how 1,25(OH)2D3 may regulate COMT expression remains unknown. Undifferentiated and differentiated neuroblastoma cells such as SH-SY5Y are widely used in studying DA neurons (Cheung et al., 2009; Lopes et al., 2010). The undifferentiated cells are highly proliferative and resemble an early neuronal progenitor while retinoic acid (RA) differentiated cells are considered post-mitotic differentiated cells, presenting a neuronal-like phenotype and mature neuronal markers (Lopes et al., 2010). RA activates neuronal differentiation genes thereby triggering a proneuronal differentiation process in these cells (Lopes et al., 2010; Korecka et al., 2013). However, the process of RA-mediated neuronal differentiation of these cells generally takes 5–7 days in order to achieve a mature neuron-like phenotype (Cheung et al., 2009). Previously we have studied the effects of 1,25(OH)2D3 on TH production in SH-SY5Y cells in which the VDR was over-expressed. In such cells we showed that 1,25 (OH)2D3 increased TH expression as well as the production of DA (Cui et al., 2015). However, this was in cells that were pre-treated with RA. We now wish to address whether initial vitamin D treatment in undifferentiated cells (no RA treatment) can also affect dopaminergic phenotype. Interpreting findings where RA differentiation involved is more complicated given the fact that the ligand bound retinoic acid receptors (RXRs) heterodimerise with the ligand bound VDR to exert many of their genomic actions. In order to address the role 1,25(OH)2D3 may play in the differentiation of DA neurons we have again employed the VDR-overexpression system in SH-SY5Y cells but in the absence of RA treatment. This system provides a simple and practical tool to further investigate the molecular mechanisms, mediated by 1,25(OH)2D3, that regulate DA neuron differentiation. Our findings here complement many of our previous studies implying that 1,25(OH)2D3 regulates many factors involved in the maturation and possible neuroprotection of developing DA neurons (Brown et al., 2003; Orme et al., 2013). This study also provides the 1st direct evidence for how this proven developmental epidemiological risk factor for schizophrenia can directly regulate the differentiation of DA neurons.

EXPERIMENTAL PROCEDURES

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Cell culture and transfection

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Human SH-SY5Y cells were cultured and transfected as previously described (Cui et al., 2015). Long-term maintenance of the stable transfected cells (SH-SY5Y/VDR+) was achieved in the presence of 0.6 mg/ml G418/geneticin (Life Technologies).

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1,25(OH)2D3 treatment

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SH-SY5Y wild-type and SH-SY5Y/VDR+ cells, seeded into 24-well plates, were cultured in standard media at a

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concentration of 10  104 cells per well. The cells were seeded in Hyclone FBS serum. Given the nature of these studies we selected this growth media as it does not contain RA. Cells were allowed to settle for 3 days and then the medium was replaced by complete DMEM/ F12 with 1% Charcoal Stripped-FBS (Gibco, Life technologies, Australia). The next day, cells were treated with 20 nM 1,25(OH)2D3 (Calcitriol, Calbiochem, Life Technologies) in serum-free medium supplemented with B27 (Invitrogen). After that, cells were cultured for another 7 days. 1,25(OH)2D3 was dissolved in DMSO and then diluted in ethanol. The vehicle control was 0.002% DMSO + 0.04% Ethanol. The medium was replaced every 3–4 days. For the Western Blot, flow cytometry, high pressure liquid chromatography (HPLC) and qPCR experiments, cells were cultured in 24 well plates in standard media at a concentration of 10  104 cells per well and treated with 1,25(OH)2D3 or vehicle control for 7 days. For the time-dependent expression of VDR and COMT the cells were plated as described above and samples were collected at 24 h, 48 h and 72 h for qPCR analysis. For the MTS experiment, SH-SY5Y and SH-SY5Y/VDR+ cells were seeded into 96- well plates in standard media at a concentration of 1  104 cells per well and treated with 1,25(OH)2D3 or vehicle for 7 days. For the ChIP-qPCR assays, SH-SY5Y and SHSY5Y/VDR+ cells were seeded into six well plates in standard media at a concentration of 50  104 cells per well and treated with 1,25(OH)2D3 or vehicle for 7 days.

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Western blot

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Western blot was performed as previously described (Cui et al., 2015). Briefly, cells were collected in lysis buffer and proteins were resolved by SDS–PAGE before being transferred to polyvinylidene fluoride membranes at 400 mA for two hours. The VDR and TH protein on the PVDF membrane were examined using anti-VDR (0.2 lg/mL) (Santa Cruz, USA) and anti-TH (0.1 lg/mL) (Merck Millipore, Australia) at 4 °C overnight, and then incubated with goat anti-mouse IRDye 680LT secondary antibody (1:100, Cell Signalling Technology, MA, USA) for one hour. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:50.000, Millipore, Germany) was used as a loading control. Fluorescent bands were visualized using an Odyssey Scanner. The intensity of the bands was evaluated using Image Studio Lite Version 4.0 software (LI-COR, USA).

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Cell viability/proliferation assay

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Cell viability/proliferation assays were carried out using the MTS colorimetric assay (CellTiter 96Ò Proliferation Assay, Promega) following manufacturer’s instructions. In this test, metabolically active cells convert 3-(4,5-sulfo phenyl)-2H-tetrazolium (MTS) into formazan. In order to perform the test, the media of the wells was changed to 100 ll of standard culture media plus 20 ll of MTS solution and incubated for 1 h at 37 °C. After 1 h, 100 ll of the solution was transferred to a new 96 well plate and the absorbance was read at 485 nm using a plate reader.

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Flow cytometry to determine TH-positive cell number

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The TH-positive cell number was determined using flow cytometry, as previously described (Cui et al., 2015).

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Measurement of DA and metabolites

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Cells were collected in 200 lL of lysis buffer with 0.1 M perchloric acid and 50 ng/ml deoxypinephrine (DE). Samples were centrifuged for 10 min and 30 lL of supernatant was analyzed by HPLC as previously described (Kesby et al., 2009; Cui et al., 2015). Data were quantified by calculating peak-height ratios for each analyte relative to the internal standard DE. Noradrenaline (NA), DA and its metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT) were quantified. 3,4dihydroxyphenylacetic acid (DOPAC) and 5hydroxytryptamine (5-HT) were undetectable in these cells. Data are presented as nanogram per gram (ng/g) of cellular protein.

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Real time PCR

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Cells were collected with RLT buffer (Qiagen) and RNA was isolated with a RNAeasy Kit (Qiagen). cDNA synthesis was performed following manufacturer’s instructions (Life Technologies). The human primers used in this experiment are presented in Table 1. Quantitative PCR reactions were performed on a LightCycler 480 system (Roche Diagnostics, Australia). Thermal cycling conditions were as follows: a denaturation step at 95 °C for 10 min and then amplification for 45 cycles (95 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s). The relative expression of the genes examined was normalized to HPRT as housekeeping gene and the results were analyzed using the comparative threshold method.

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Chromatin immunoprecipitation (ChIP-qPCR assay)

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Cells were detached with 0.25% trypsin solution (Invitrogen), fixed with 1% formaldehyde for 3 min and then quenched with 0.125 M glycine. SDS lysis buffer (1%) was added and the samples were sonicated in a

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Table 1. qPCR primers sequences Gene

Primers

Forward 50 -GCAGTTCTCGCAGGACATTG-30 Reverse 50 -TGGATGCGTGAGGCATAGC-30 VDR Forward 50 -GTTCCAGGTGGGACTGAAGAAG-30 Reverse 50 -TCATCTTGGCATAGAGCAGGTG-30 COMT Forward 50 -TCCTAAATGCAAAGCACACC-30 Reverse 50 -CAATCCAGTGTTGCAGTTCAG-300 MAO-A Forward 50 -GTGTCAGCCAAAGCATGGAG-30 Reverse 50 -GCAGCAGATAGTCCTGAAATGC-30 GAPDH Forward 50 -TTTACATGTTTCCAATATGATTCCAC-30 Reverse 50 -TTGTCATACTTCTCATGGTTCACAC-30 HPRT Forward 50 -TGACCAGTCAACAGGGGACA-30 Reverse 50 -GCGACCTTGACCATCTTTGG-30 DRD2 Forward 50 -GGAAATTCAGCAGGATTCACTG-30 Reverse 50 -ATGCTGATGGCACACAAGTTC-30 VMAT2 Forward 50 -CCGTACATCCTCATTGCTGC-30 Reverse 50 -CCACCTCCCCATTTTGTGTG-30 TH

Covaris sonicator (Covaris, Australia) in order to shear the chromatin into 300–500 base pair fragments. Aliquots of chromatin were precleared for 2 h with protein A Dynabeads (Invitrogen). Genomic regions interacting with VDR were precipitated using an antiVDR rabbit antibody (5 lg, C-20, Santa Cruz Biotechnology, sc-1008x), and isolated with protein A Dynabeads. The positive control was anti-Histone H3K4me3 rabbit antibody (Active motif) and the negative control was normal rabbit IgG (Santa Cruz Biotechnology, sc-2027x). The samples were incubated overnight at 4 °C with the antibodies, and then washed with low- and high-salt buffers. Crosslinking was reversed by 4 h of incubation at 65 °C. Proteinase K was added to the samples followed by an incubation step of 1 h at 50 °C. ChIPed-DNA was purified using Qiagen PCR purification kit following manufacturer’s instructions. The purified products were used in qPCR reactions to amplify putative VDR-binding sites (VDREs) in the promoter region of COMT gene. The primers (Table 2) for putative VDREs, within 10 kb from the 50 upstream sequence in the COMT promoter were designed according to regions found using MAPPER search engine (Marinescu et al., 2005). The results are shown as fold change of DNA amount compared to IgG control.

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Statistical analysis

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The statistical analysis of the effects of VDR and 1,25 (OH)2D3 on DA production and DA-associated gene and protein expression were conducted with a two-way analysis of variance (ANOVA) followed by post hoc Bonferroni’s test. For time-dependent expression and ChIP-qPCR experiment differences between groups were tested for significance using a One-way ANOVA. A statistically significant result was recorded when p < 0.05.

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RESULTS

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VDR overexpression increases TH cell number, gene and protein expression

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Obviously the over-expression of VDR in SH-SY5Y cells led to a dramatic elevation in VDR mRNA and protein. 1,25 (OH)2D3 treatment had no effect on wt cells but significantly further increased VDR mRNA (cell line  treatment F(1, 24) = 17.33, p = 0.0003) and protein expression (cell line  treatment F(1, 36) = 5.118, p = 0.03) in SH-SY5Y/ VDR+ cells when compared to vehicle at 7 days (Fig. 1A, B). VDR over-expression also induced TH mRNA (F(1, 24) = 7.9239, p = 0.009) and protein expression (F(1, 32) = 15.889, p = 0.0001) when compared to wt cells but the addition of 1,25(OH)2D3 had no additional effect (Fig. 1C, D). The effect of over expressing the VDR on increasing TH mRNA and protein appeared to be directly due to an increase in the percentage of TH-expressing cells. Over expressing VDR virtually doubled the number of THpositive cells in culture (F(1, 12) = 8.576, p = 0.01) (Fig. 1E). We interpret this finding as a direct effect on

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Table 2. ChIP-qPCR primers sequences and their location within the human COMT promoter

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Region

Location

Primers

1

9804 to 9790

2

8970 to 8956

3

7919 to 7905

4

7458 to 7444

5

6203 to 6189

6

5923 to 5909

7

4808 to 4794

8

3162 to 3148

9

2267 to 2253

10

1721 to 1708

11

1124 to 1110

Forward 50 -GCCAGAGTTGTTTTCGATAATGG-30 Reverse 50 -CTTCTCCCCAAGGTAGGAG-30 Forward 50 -GAAACCATTAGCCAGGACCG-30 Reverse 50 -CTACAGGCTTGCACCACCA-30 Forward 50 -GAGCTTAGCAGGTGGAGGAT-30 Reverse 50 -GAGGGTAAATCTGGGCCCTG-30 Forward 50 -TCTGAGAGATCTGATCGGCG-30 Reverse 50 -TCTCAACTACTGGGGCATGA-30 Forward 50 -GAGAGATCTGATCGGCGCAT-30 Reverse 50 -CCCAACGCCCCAGGAATTAT-30 Forward 50 -TGACCCACCTTCTCCAAACC-30 Reverse 50 -GAGCCAGGACAGGTCGAG-30 Forward 50 -ACATAGCCACATCTCCCGC-30 Reverse 50 -TGGGTCTACGGGTATGATGG-300 Forward 50 -CAAGACCAGCCTCAACATGGA-30 Reverse 50 -CCTGGCCTCTTCGCATTTTCT-30 Forward 50 -GGGTACAGGGTATTTTGGGGA-30 Reverse 50 -GTCTCGAACTCCTGACCTCA-30 Forward 50 -GGCAACATAGCGAAACCCACCT-30 Reverse 50 -AAATGGTGCAATCTCGGCTC-30 Forward 50 -ATACCACACGATCCAGTTGG-30 Reverse 50 -GTACGTGTTCCCCTGTAGCT-30

TH neuron differentiation as overall viability of cultures was unaffected by either VDR over-expression or the addition of ligand (F(1, 19) = 1.402, p = 0.25; F(1, 19) = 0.01990, p = 0.89) (Fig. 1F). We also examined the expression of Neurogenin 2 (NEUROG2), a marker that is expressed in very early DA neurons immediately, i.e. before specification factors such as Nurr1 are expressed (Smidt and Burbach, 2007). Over-expression of the VDR reduced this marker (F(1, 20) = 7.4921, p = 0.013) again consistent with the apparent enhanced differentiation and maturation of DA neurons in these cells (Fig. 1G). On all these measures the 1,25(OH)2D3 ligand had no effect after 7 days of culture. VDR overexpression increases TH products and 1,25 (OH)2D3 increases DA turnover The TH enzyme is rate limiting in DA and noradrenalin (NA) synthesis. Over-expression of the VDR in SHSY5Y cells increased DA (F(1, 40) = 19.301, p = 0.0001), NA (F(1, 40) = 9.4380, p = 0.004) and the DA metabolites 3-MT (F(1, 40) = 69.104, p = 0.0001) and HVA (F(1, 40) = 34.842, p = 0.0001) relative to wt. While there was no effect of the 1,25(OH)2D3 ligand on DA or NA synthesis its addition led to a further increase in DA turnover with enhanced production of 3-MT (cell line  treatment F(1, 40) = 15.295, p = 0.0003) and HVA (F(1, 40) = 8.2323, p = 0.006) while DOPAC was not able to be detected (Fig. 2). VDR overexpression alters the expression of dopaminergic genes associated with terminal differentiation and 1,25(OH)2D3 affects the expression of enzymes involved in DA turnover When we examined other factors consistent with a terminally differentiated DA neuron we showed the overexpression of VDR increased vesicular

monoamine transporter (VMAT2) (cell line  treatment F(1, 20) = 53.381, p = 0.0001) and decreased dopamine receptor D2 (DRD2) transcripts (cell line  treatment F(1, 20) = 24.844, p = 0.0001). Interestingly the addition of 1,25(OH)2D3 appeared to specifically reduce these two factors in the SH-SY5Y cells in which the VDR was over-expressed (VMAT2, F(1, 20) = 36.588, p = 0.000; DRD2, F(1, 20) = 6.8163, p = 0.02). Also given 1,25 (OH)2D3 treatment increased 3-MT and HVA, we analyzed the expression of genes responsible for their formation from DA. Over-expression of the VDR had no effect on expression of these genes, however the addition of 1,25(OH)2D3 increased COMT (cell line  treatment F(1, 20) = 16.996, p = 0.0001) and decreased MAO-A (cell line  treatment F(1, 20) = 14.457, p = 0.001) (Fig. 3). Vitamin D directly regulates COMT expression Our previous studies have alerted us to the possibility that 1,25(OH)2D3 may regulate COMT expression in vivo (Kesby et al., 2009). Here we show the increase in COMT expression by 1,25(OH)2D3 as well as its immediate product 3-MT in vitro. Given the broader interest in this enzyme in psychiatric disease we elected to examine whether it was a direct gene target of 1,25(OH)2D3. We identified 16 putative 1,25(OH)2D3-responsive elements (VDREs) within 10 kb in the promoter region of this gene making it an excellent candidate. We 1st re-examined the time profile for 1,25(OH)2D3-mediated increase in this gene. We show that COMT is rapidly upregulated by 1,25(OH)2D3 within 24 h (24 h F(1, 14) = 29.120, p = 0.000; 48 h F(1, 14) = 147.12, p = 0.000; 72 h F(1, 14) = 152.36, p = 0.000) (Fig. 4A). To our surprise this was even faster than the well-known upregulation of the VDR by its ligand which we have used here as a positive control (24 h F(1, 14) = .40648, p = 0.53; 48 h F(1,

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E

25

15 10 5 0.01 0.00

SH-SY5Y

0.3

**

0.1

SH-SY5Y

% of TH positive cells relative to SH-SY5Y

*

-D +D

300 200 100

SH-SY5Y

5 4

F

-D +D

**

0.1

SH-SY5Y

0.3

SH-SY5Y/VDR+

***

-D +D

0.2

0.1

SH-SY5Y

SH-SY5Y/VDR+

2.0

-D +D

1.5 1.0 0.5 0.0

SH-SY5Y/VDR+

*

0.2

0.0

SH-SY5Y/VDR+

400

0

Relative mRNA expression NEUROG2

D -D +D

*

0.3

0.0

SH-SY5Y/VDR+

0.2

0.0

B VDR protein relative to GAPDH

***

20

500

G

-D +D

TH protein relative to GAPDH

Relative mRNA expression TH

C

***

30

Live cells (ABS490nm)

Relative mRNA expression VDR

A

SH-SY5Y

SH-SY5Y/VDR+

-D +D

3 2 1 0

SH-SY5Y

SH-SY5Y/VDR+

Fig. 1. Increasing expression of the vitamin D receptor (VDR) (SH-SY5Y/VDR+) increases DA neuron differentiation independent of the vitamin D hormone 1,25(OH)2D3 in SH-SY5Y cells and treated with vehicle (D) or 1,25(OH)2D3 (+D) for 7 days. (A, B) 1,25(OH)2D3 (+D), further increases VDR mRNA and protein in VDR overexpressing (SH-SY5Y/VDR+) cells; (C, D) VDR over-expression increase tyrosine hydroxylase (TH) mRNA and protein independent of 1,25(OH)2D3; (E, F) VDR-overexpression more than doubled the number of TH+ cells in culture assessed by flow cytometry without affecting total survival; (G) VDR-overexpression decreased a marker of immature DA neurons NEUROG2. Data = mean ± SEM (n = 5–7, *p < 0.05, **p < 0.01; ***p < 0.001).

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= 17.392, p = 0.001; 72 h F(1, 14) = 37.285, p = 0.0001) (Fig. 4B). Primers were designed against 11 putative VDREs spanning the 10 kb prior to the transcript start site (TSS) of the COMT gene (Fig. 5A). ChIP assays revealed 1,25 (OH)2D3 treatment increased VDR association at all 11 sites along the COMT promoter (Fig. 5B) when compared to vehicle control. We chose to repeatedly examine three of these sites: primers 5 and 6 were shown to present slightly higher signal levels compared to the other regions and considering that they are next 14)

to each other they are likely to represent a cluster; Primer 11 was chosen as it is closest to the TSS. Clustering of VDREs and proximity to the TSS have been shown to be a key criteria for functional VDREs in other gene promoters (Heikkinen et al., 2011). These three regions are located at 6203, 5923 and 1124 kb from the transcription start site, respectively. We show that 1,25(OH)2D3 significantly enhanced the association of the VDR to sites 5 (F(1, 18) = 6.5699, p = 0.03), 6 (F(1, 18) = 5.1142, p = 0.05) and 11 (F(1, 18) = 6.7895, p = 0.03) (Fig. 5C).

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B

A 75

75

-D +D

***

NA ng/g of protein

DA ng/g of protein

30 15

30 15 0

SH-SY5Y

SH-SY5Y

SH-SY5Y/VDR+

D

75

-D +D

***

***

45

75

SH-SY5Y/VDR+

30 15

-D +D

***

60

60

HVA ng/g of protein

3-MT ng/g of protein

+D

45

0

C

-D

60

60 45

**

**

45 30 15 0

0 SH-SY5Y

SH-SY5Y

SH-SY5Y/VDR+

SH-SY5Y/VDR+

Fig. 2. Production and turnover of catecholamines in SH-SY5Y and SH-SY5Y/VDR+ cells. Cells were cultured for 7 days in vehicle (D) or 1,25 (OH)2D3 (+D). (A) Dopamine (DA), (B) Noradrenaline (NA); (C) 3-methoxytyramine (3-MT) and (D) homovanillic acid (HVA). VDR overexpression increased DA and NA production when compared to SH-SY5Y cells. 1,25(OH)2D3 increased DA turnover by increasing the conversion to 3-MT and HVA. DOPAC was not detected in these cells. Data = mean ± SEM (n = 9, **p < 0.01; ***p < 0.001).

***

0.4

-D +D

0.3 0.2 0.1 0.0

SH-SY5Y

***

0.5

SH-SY5Y/VDR+

C

-D +D

0.4 0.3

***

0.2 0.1 0.0

SH-SY5Y

SH-SY5Y/VDR+

D 1.5

Relative mRNA expression COMT

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Fig. 3. Effect of 1,25(OH)2D3 on dopamine-associated gene expression in SH-SY5Y and SH-SY5Y/VDR+ cells. (A) Vesicular monoamine transporter 2 (VMAT2), and (B) Dopamine receptor D2 (DRD2), (C) Catechol-o-methyl transferase (COMT), (D) Monoamine oxidase A (MAO-A) mRNA expression after exposure to vehicle (D) and 1,25(OH)2D3 (+D). Overexpression of VDR caused an increase in VMAT2 gene expression and a decrease in DRD2 when compared to SH-SY5Y cells. 1,25(OH)2D3 specifically upregulated COMT mRNA and downregulated VMAT2 and DRD2 genes after 7 days of treatment. Data = mean ± SEM (n = 6, ***p < 0.001). 361

DISCUSSION

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There are a number of main findings from this study. 1st we show that overexpression of the VDR in a neural cell

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line is capable of driving these cells down a dopaminergic lineage in the absence of any other known dopaminergic differentiation agents such as RA. 2nd 1,25(OH)2D3 further enhances VDR’s genomic

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3.0 2.5 2.0 1.5

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Relative mRNA expression VDR

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Fig. 4. Time-dependent actions of 1,25(OH)2D3 on the expression of dopamine-associated genes in SH-SY5Y/VDR+ cells. (A) COMT and (B) VDR mRNA expression after exposure to vehicle (D) and 1,25(OH)2D3 (+D). Data = mean ± SEM (n = 8, *p < 0.05; ***p < 0.001).

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Fig. 5. 1,25(OH)2D3 increases VDR binding to the COMT promoter. (A) Location of the designed VDR primers within 10 kb from the COMT transcription start site. (B) qPCR screening showing increased VDR binding to the COMT promoter after 48 h of exposure to vehicle (D) or 1,25 (OH)2D3 (+D) in SH-SY5Y/VDR+ cells. (C) Quantitation of VDR binding to three pre-defined sites (arrows) showing a significant doubling of VDR binding to these three sites in the presence of 1,25(OH)2D3. Fold enrichment was calculated relative to IgG antibody. Data = mean ± SEM (n = 5, * p < 0.05).

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actions and regulates the expression of several dopaminergic-associated genes. 3rd 1,25(OH)2D3 upregulates COMT expression as well as its direct product the DA metabolite 3-MT. 4th 1,25(OH)2D3 enhances VDR binding to VDREs within the COMT promoter strongly suggesting direct regulation. SH-SY5Y neuroblastoma cells are commonly used in studies focusing on the differentiation and function of DA

neurons. Before differentiation these cells resemble cells in an early stage in neuronal development (Voigt and Zintl, 2003), providing a means by which to compare the influence of different treatments on neuronal progenitors and neuronal differentiation. The VDR has a vast list of cell-specific target genes (Heikkinen et al., 2011). Here we transfected the recombinant VDR into SH-SY5Y. The level of the VDR expressed in these SH-SY5Y cells

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is similar to that found in adult rat kidney cells (data not shown). Our previous study using SH-SY5Y cells in which the VDR was over-expressed showed 1,25(OH)2D3 was capable of upregulating TH and DA in pre-differentiated cells (Cui et al., 2015). In this study, we have explored whether the over-expression of the VDR itself is capable of enhancing dopaminergic differentiation independently of RA. We ensure the absence of small differentiation factors in our culture system such as RA by the use of HycloneTM-FBS and charcoal stripped-FBS. In our previous study using SH-SY5Y cells in which the VDR was over-expressed we showed the addition of RA for 7 days induced approximately 12% of cells to express TH. Here we show the over-expression of the VDR was sufficient to produce a similar percentage of TH-expressing cells (approximately 11%) in the absence of RA after 7 days in culture. 1,25(OH)2D3-independent actions of VDR on the expression of DA-associated genes Though nuclear receptors are transcription factors activated by their specific steroid hormone ligands, some nuclear receptors like the estrogen-related receptor (ERR), retinoid organ receptor (ROR), VDR and thyroid hormone receptor (TR) can affect transcription in the absence of ligand (Matkovits and Christakos, 1995; Sato et al., 2007; Sever and Glass, 2013). Interactions of nuclear receptors with coactivators, corepressors and post-translational modifications such as phosphorylation regulate the transcriptional activity of nuclear receptors in the absence of ligand (Sever and Glass, 2013). The unliganded VDR was found to interact with lymphoid enhancer factor (Lef1) on regulatory regions of the canonical Wnt and hedgehog target genes and is essential for the induction of these pathways during the postmorphogenic hair cycle (Lisse et al., 2014). The occupancy of the VDR on these regulatory sites was not enriched by ligand treatment. It is well-known that Wnt and hedgehog pathways play a critical role in dopaminergic differentiation (Alves dos Santos and Smidt, 2011). Wnt/Lef1 signaling is also present in SHSY-5Y cells (Vieira et al., 2015). Therefore, in our study, it is plausible that the unliganded VDR might interact with Lef1 to modulate dopaminergic differentiation. Further experiments examining this possibility are now warranted. The ligand-independent action of VDR has been demonstrated in CV-1 and COS-7 cells (monkey kidney tissue) (Matkovits and Christakos, 1995; Skorija et al., 2005). There are also pharmacological agents that can phosphorylate VDR and RXRs to activate transcription in CV-1 cells without the presence of 1,25(OH)2D3 (Matkovits and Christakos, 1995). Furthermore, DA itself has been shown to activate VDR-mediated transcription in a dose-dependent manner (Matkovits and Christakos, 1995). Given the increase in DA shown here in the absence of 1,25(OH)2D3 this may represent a possible mechanism for VDR-mediated transcription in our model system. Some other dramatic phenotypes mediated by the VDR have also been shown to be ligand independent such as alopecia (Skorija et al., 2005). Regarding ligandindependent signaling of other nuclear receptors, the

estrogen receptor (ERa) can initiate transcription in the absence of estrogen via the human X box-binding protein 1 (XBP-1) (Ding et al., 2003). Ligand-independent repression of some nuclear receptors such as the TR mediated by the Nuclear Receptor Co-Repressor (N-CoR) have been also reported (Horlein et al., 1995). The thyroid hormone receptor perhaps best represents a model of ligand-independent suppression and ligand-dependent activation of the transcription of the same genes (Sato et al., 2007). Differentiation to a more mature dopaminergic phenotype in VDR over-expressing cells was also marked by a corresponding reduction in early postmitotic factors involved in DA differentiation such as NEUROG2 (Kele et al., 2006; Korecka et al., 2013). Previous studies have shown differentiating SH-SY5Y cells with RA also downregulates NEUROG2 over a similar time-frame (Korecka et al., 2013). NEUROG2 is an early essential gene for midbrain DA neuron development. In NEUROG2 null mice, around 86% of TH+ neurons fail to develop in the ventral midbrain region at the end of the neurogenic period E14.5 (Kele et al., 2006). Furthermore, NEUROG2 is expressed in post-mitotic Nurr1+ precursors, but not in fully differentiated TH+ neurons. We consider this further evidence that vitamin D signaling is capable of directly differentiating DA neurons.

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1,25(OH)2D3-dependent actions of VDR on the expression of DA-associated genes

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1,25(OH)2D3 is well known to enhance the expression of it’s own receptor the VDR. We verified this at both the protein and transcript level thus ensuring both the 1,25 (OH)2D3 concentration chosen, and our model system, were appropriate to examine the genomic actions of vitamin D. VMAT2 expression was increased in SHSY5Y cells in which the VDR was over-expressed when compared to wt cells. This expression was significantly diminished in these cells when the 1,25(OH)2D3 ligand was added. Interestingly there was no effect of the ligand in wt cells. VMAT2 transports DA into the synaptic vesicle and it is important for the maintenance of presynaptic DA homeostasis in dopaminergic neurons (Ham et al., 2012). The packaging of DA into vesicles is important to protect DA neurons against oxidative stress induced by free cytosolic DA (Guillot and Miller, 2009). It may be that the VDR-mediated increase in VMAT2 seen here was in response to this increased DA synthesis. This finding is in accordance with other studies using SH-SY5Y cells where treatment of SH-SY5Y cells with DA (600 lM) has been shown to downregulate VMAT2 mRNA (Ham et al., 2012). After 7 days in culture, VDR overexpression decreased DRD2 transcripts compared to wt cells. 1,25 (OH)2D3 further decreased DRD2 levels in SH-SY5Y/ VDR+ cells but similar to the situation with VMAT2 had no effect on wt cells. DRD2 receptors have been previously shown to be inversely related to DA and NA concentrations in SH-SY5Y cells (Deslauriers et al., 2011). The increase in DA produced by VDR overexpression therefore potentially explains this finding. The fact that 1,25(OH)2D3 itself did not appear to further

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Fig. 6. Model showing the overall effects of VDR over-expression (SH-SY5Y/VDR+) on dopaminergic elements in the presence and absence of 1,25(OH)2D3. (A) VDR-overexpression increases TH and as a result DA production. SH-SY5Y/VDR+ cells deal with this increase in DA by increasing VMAT2 production in order to sequester potentially oxidatively reactive DA. (B) Treatment of VDR-overexpressing cells (which have the same high level of TH and DA production) with 1,25(OH)2D3 leads to an alternative neuroprotective action of a direct upregulation of COMT expression with a subsequent increase in inactive DA metabolites 3-MT and HVA. (Filled circles: DA); (Gray cylinders: VMAT2); single arrow up to twofold alteration; two arrows >twofold alteration.

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increase DA yet it did decrease DRD2 expression is likely confounded by the increased turnover of DA or suggests some alternative mechanism for ligand-dependent regulation of DRD2.

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1,25(OH)2D3 directly regulates COMT expression

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COMT gene polymorphisms are associated with alterations in COMT protein activity and have long been associated with psychiatric disorders such as schizophrenia (Strous et al., 1997). Developmental Vitamin D deficiency is a known developmental risk factor for schizophrenia (McGrath et al., 2010). We have previously shown COMT expression and subsequent DA turnover to be reduced in neonatal forebrains from developmentally vitamin D deficient rat dams (Kesby et al., 2009). Our findings here complement these data in that the addition of 1,25(OH)2D3 in this in vitro system increases COMT expression. Collectively these data strongly suggest 1,25(OH)2D3 directly regulates COMT transcription. A rapid in silico analysis using the Mapper Engine Software (Marinescu et al., 2005) predicted numerous VDR-binding sites within the promoter of the COMT gene again suggesting direct regulation. We therefore elected to conduct ChIP-qPCR in the VDR overexpressing SH-SY5Y cells in order to verify whether the ligand bound VDR directly associated with the COMT promoter. The regulation of gene transcription through VDR occurs with its heterodimerization with retinoid X receptors (RXR) and the high-affinity binding of this complex to vitamin D response elements (VDREs) on the promoter region of target genes (Prufer et al., 2000). Although both VDR and RXR can bind to DNA in the absence of 1,25(OH)2D3, the addition of the 1,25 (OH)2D3 ligand is believed to further stabilize this heterodimer promoting its binding to the VDREs (Haussler et al., 1998; Prufer et al., 2000). The co-repressors are then released and co-activators are recruited to promote gene expression (Smith and O’Malley, 2004).

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The presence of several VDREs in the promoter of a gene suggests that they may act synergistically (Sinkkonen et al., 2005; Heikkinen et al., 2011). Several studies show that multiple VDREs – commonly 3–4 – are found in the promoter region of 1,25(OH)2D3-responsive genes (Sinkkonen et al., 2005; Saramaki et al., 2006; Heikkinen et al., 2011). Here we reveal that the ligand bound VDR was enriched by about twofold in three VDRE-containing regions within the promoter of the COMT gene. This level of enrichment compares favourably with other genes known to be directly regulated by ligand-bound VDR such as the cell cycle regulators Cyclin C (Sinkkonen et al., 2005) and p21kip2 (Saramaki et al., 2006). Collectively our previous published data considered in the light of these new findings strongly indicate COMT is under the direct control of vitamin D.

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CONCLUSIONS

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Taken together our findings again confirm that vitamin Drelated signaling is an important differentiation factor for DA neurons. Increasing VDR signaling within SH-SY5Y neurons increases the production of DA via an increase in the number of newly differentiated TH-positive neurons. In order to deal with this increase in catecholamines which are potentially a source of oxidative damage these cells upregulate the packaging mechanism for DA, VMAT2 (Fig 6A). In the presence of the active vitamin D hormone, 1,25(OH)2D3, these cells would appear to promote an alternative mechanism to protect themselves from subsequent oxidative damage by degrading DA to inactive metabolites (Fig 6B). These findings extend the roles vitamin D signaling may play in the ontogeny of dopaminergic systems. Here we show vitamin D signaling can affect the regulation of numerous genes important in the differentiation of DA neurons such as TH, COMT, MAOA, VMAT2, DRD2 and NEUROG2. We have examined the ligand-dependent regulation of one of these genes COMT in greater detail. Our ChIP studies strongly

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suggest COMT expression is under the direct regulation of the ligand bound VDR. These data continue to suggest 1,25(OH)2D3 is not only an important regulator of DA neuron function but may directly affect the early differentiation and subsequent ontogeny of developing DA neurons in the embryonic brain. Such findings may prove informative for the emerging concept that factors proposed as environmental risk factors for serious psychiatric disease such as Developmental Vitamin D deficiency may have early adverse consequences for the ontogeny of DA systems (Eyles et al., 2012).

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COMPETING INTERESTS

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The authors declare that they have no conflicts of interest with the contents of this article.

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AUTHOR CONTRIBUTIONS

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R.A.N.P designed, performed and analyzed most of the experiments; X.C. also designed, assisted and helped analyzed most of the experiments; D.E. designed experiments and analyzed data. R.A.N.P and D.E wrote the manuscript.

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Acknowledgments—The authors thank Dr. James Kesby and Tim Reeks for their technical support in the HPLC experiments. Funding for this study was provided by the National Health and Medical Research Council of Australia (APP1024239). R.A.N. Pertile had a scholarship from CNPq – Brazil.

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(Accepted 13 July 2016) (Available online xxxx)

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