The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain

Journal Pre-proof The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain Seon Young Park (Conceptualization) (Methodology) (Visualization) (Writing - original draft), Yeong-Min Yoo (Validation)Data Curation), Eui-Man Jung (Conceptualization) (Methodology) (Visualization) (Writing - review and editing), Eui-Bae Jeung (Writing - review and editing) (Supervision)

PII:

S0891-0618(19)30237-6

DOI:

https://doi.org/10.1016/j.jchemneu.2020.101767

Reference:

CHENEU 101767

To appear in:

Journal of Chemical Neuroanatomy

Received Date:

18 December 2019

Revised Date:

11 February 2020

Accepted Date:

12 February 2020

Please cite this article as: Young Park S, Yoo Y-Min, Jung E-Man, Jeung E-Bae, The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain, Journal of Chemical Neuroanatomy (2020), doi: https://doi.org/10.1016/j.jchemneu.2020.101767

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The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain

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Seon Young Park, Yeong-Min Yoo, Eui-Man Jung* and Eui-Bae Jeung*

Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary

* Co-last and corresponding authors

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Medicine, Chungbuk National University, Cheongju, Chungbuk, 362-763 Republic of Korea

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Keywords: Calcium; TRPV5; TRPV6; NCX1; PMCA1; steroid hormones

* Corresponding author:

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Eui-Man Jung, Ph. D. Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28466

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Republic of Korea. E-mail: [email protected] Eui-Bae Jeung, D.V.M., Ph. D. Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28466 Republic of Korea. E-mail: [email protected] 1

Highlights 

TRPV5, TRPV6, NCX1 and PMCA1 are widely expressed in the immature rat brain.

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TRPV5, TRPV6, NCX1 and PMCA1 are regulated by ER-, PR- and GR-dependent pathways in cerebral cortex and hypothalamus.

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Calcium-processing proteins indicated that they may regulate by E2, P4, and/or DEX

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in cerebral cortex and hypothalamus.

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Abstract

The cytosolic calcium concentration is regulated by calcium-processing proteins such as

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transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodiumcalcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). Those

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calcium-processing proteins are important for physiological functions in the brain. The effects of steroid hormones on calcium-processing protein expressions in the brains are

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unknown. Thus, the effects of steroid hormones on the distribution, localization, and expressions of calcium-processing proteins in the brain were analyzed. Immature female rats

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were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486). We found that TRPV5 and TRPV6 proteins were highly expressed in the cerebral cortex (CT), hypothalamus (HY), and brain stem (BS) compared to that in the olfactory bulb (OB) and cerebellum (CB). Also, the NCX1 protein was highly expressed in CT and BS compared to that in OB, HY, and CB, and PMCA1 2

protein was highly expressed in CT compared to that in other brain regions. Furthermore, expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and/or DEX in the CT and HY. In summary, calcium-processing proteins are widely expressed in the immature rat brain, and expressions of calcium-processing proteins in CT and HY indicated that they may regulate by E2, P4, and/or DEX and can be attenuated by antagonist treatment. These results indicate that steroid hormone regulation of TRPV5,

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TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain.

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Abbreviations

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E2, estrogen; P4, progesterone; DEX, dexamethasone; RU486, mifepristone; VE, vehicle;

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OB, olfactory bulb; CT, cerebral cortex; HY, hypothalamus; CB, cerebellum; BS, brain stem

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1. Introduction Calcium concentration maintenance in nerve cells is needed for their proper development and function (Marcos et al., 2009), and is important in the temporal and spatial control of neuronal function (Hoenderop et al., 2005). Transient changes in the free calcium intracellular concentration have a role in the control of numerous neuronal processes ranging from electrical excitability and neurotransmitter release to gene expression and dendritic

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integration (Kenyon et al., 2010; Neher and Sakaba, 2008). Calcium homeostasis is the maintenance of (re)absorbed calcium amounts in the body and is important for essential body functions, cell function, and cell survival (Berliocchi et al., 2005). To cope with large

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variations in calcium input and output, organisms are equipped with regulatory systems to

control plasma calcium levels, and calcium fluxes between the extracellular compartment and

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several organs (Hoenderop et al., 2005). Calcium is processed by calcium transport proteins (channels, carriers, and pumps) and through interaction with cytosolic calcium buffers, which

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are regulated differentially at the plasma membrane and in calcium stores (Petersen et al., 1994).

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Transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1) have

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critical roles in calcium homeostasis and are regulated by steroid hormones in kidney (Jeon,

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2008). TRPV5 and TRPV6 are calcium channels involved in intracellular calcium influx. In addition, during the estrus cycle and pregnancy, TRPV6 is affected by steroid hormones involved in duodenal calcium absorption (Jung et al., 2011). NCX1 is a counter-transporter membrane protein found in the plasma membrane, mitochondria, and endoplasmic reticulum of excitable cells (Kiedrowski et al., 1994). PMCA1 is a transport protein in the plasma 4

membrane of cells and is upregulated by estrogen (E2) in the uterus (Kim et al., 2017). When intracellular calcium concentrations increase, NCX1 and PMCA1 discharge calcium and vice versa (Bindokas and Miller, 1995; Hwang et al., 2011). E2 exerts a variety of effects, including electrophysiological, metabolic, neurotrophic, and neuroprotective effects, on neurons in the brain. Also, E2 can induce or maintain the dendritic spines in the hippocampus, protecting neurons. Neuroprotection of E2 is a calcium-dependent

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process and is related to the regulation of intracellular calcium levels (He et al., 2016). Progesterone (P4) has an important neuroprotective effect and affects neuronal function by regulating cellular activity and gene transcription through intracellular receptors that are

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abundant in the central nervous system (CNS) (Mani and Oyola, 2012; Stein, 2008).

Glucocorticoids are a class of steroid hormones involved in calcium metabolism and can

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increase urinary calcium excretion and inhibit calcium absorption in the intestine (An et al., 2018). They also have an essential role in the hypothalamus-pituitary-adrenal cortex axis

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(Chen et al., 2011). Within the cell, dexamethasone (DEX) may protect the cell from calcium overload (Suwanjang et al., 2013).

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The distribution and expression of the calcium-processing proteins in the rat brain were identified by immunofluorescence and western blot, respectively. We observed high

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expressions of calcium-processing proteins in the CT and HY and the regulation of

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expression of calcium-processing proteins by E2, P4, and DEX in immature rat brain. Our findings suggest that calcium-processing proteins indicated that they may regulate by steroid hormones, which may serve as important regulators of cytosolic calcium absorption and release in the brain.

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2. Materials and methods 2.1. Reagents and chemicals The 17-estradiol (E2), dexamethasone (DEX), progesterone (P4), mifepristone (RU486), and corn oil were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA), and the ICI 182,780 was obtained from Tocris (Avonmouth, UK). Stock solutions were diluted with

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dimethyl sulfoxide (DMSO; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

2.2. Animal treatment

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Specific pathogen-free immature female Sprague-Dawley rats weighing 15–20 g at

postnatal day 10 were purchased from Samtako (Osan, Republic of Korea). The rats were

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housed in polycarbonate cages and were allowed to acclimate to their housing in an

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environmentally controlled room (temperature 23°C ± 2°C, relative humidity 50% ± 10%, frequent ventilation, and a 12 h light:dark cycle).

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After approximately 1 week of acclimatization the rats were separated into seven groups (n = 7) and each group was subcutaneously (SC) injected with E2 (50 µg/kg body weight

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[BW]), DEX (10 mg/kg BW), P4 (20 mg/kg), or vehicle (corn oil) once per day for 5 consecutive days. To evaluate the affected signal pathways, antagonist groups were injected

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with ICI 182,780 (10 mg/kg BW) and RU 486 (50 mg/kg BW) 30 min prior to hormone administration. At 12 h after final injection, all immature rats were euthanized with ether and tissue samples were collected. All experimental animal management and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University. 6

2.3. Immunofluorescence assay Immunofluorescence assay was performed as described previously (Jung et al., 2016; Jung et al., 2017). For examination of TRPV5, TRPV6, NCX1, and PMCA1 immunofluorescence, rat brain was fixed with 4% paraformaldehyde and serial 80 μm think coronal and sagittal sections were cut on a vibratome (Leica, VT1000S, Nussloch, Germany). To improve

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antibody penetration, brain sections were soaked in 0.5% Triton X-100 for 15min and incubated in blocking solution (5% normal goat serum, normal donkey serum and 0.3%

Triton X-100) for 1 h. Primary antibodies used were rabbit anti-TRPV5 antibody (Alomone

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Labs, Israel, ACC-035, 1:500), rabbit anti-TRPV6 (Alomone Labs, ACC-036, 1:500), mouse anti-NCX1 (Swant, Bellinzona, Switzerland, R3F1, 1:500), rabbit anti-PMCA1 (Swant,

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PMCA1, 1:500) for 24 h at 4°C. Following washing in PBS, appropriate secondary

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antibodies conjugated with Alexa Fluor dyes (Invitrogen, Carlsbad, CA, USA, 1:1000) were used to detect primary antibodies, and DAPI (ThermoFisher, D1306, 1:1000) was used to

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stain nuclei.

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2.4. Western blot assay

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Brain proteins were extracted with Pro-prep (iNtRON Bio, Sungnam, Kyeonggido, Republic of Korea), according to the manufacturer’s protocol. Protein concentration was determined by using the BCA assay (Sigma-Aldrich). Next, a 30-40 µg sample was resolved by performing tris-glycine SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Taunton, MA, USA). Next, the membrane was 7

blocked for 1 h with 5% skim milk dissolved in TBS-T. The membrane was incubated in primary antibodies diluted in 1% BSA for overnight at 4°C. Primary antibodies to rabbit antiTRPV5 (Alomone Labs, 1:1000), rabbit anti-TRPV6 (Alomone Labs, 1:1000), mouse antiNCX1 (Swant, 1:1000), rabbit anti-PMCA1 (Swant, 1:1000) and GAPDH (Santa Cruz, Biotechnology, 1:1000) were used. The membrane was washed four times for 10 min each with TBS-T. Horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling, Beverly,

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MA, USA, 1:3000) and anti-mouse IgG (Cell Signaling, 1:3000) were used as a secondary antibody with 2.5% skim milk dissolved in TBS-T for 60 min. The membrane was washed with TBS-T four times as previously mentioned, and immunoreactive proteins were

visualized by using the West-One Western Blot Detection System (iNtRON Bio), according

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to the manufacturer’s instructions. Signals were detected with the Chemi Doc EQ (Bio-Rad,

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Hercules, CA, USA) imaging system. Bands were normalized by GAPDH immunoreactive bands in the same membrane after stripping. Density measurements for each band were

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performed with NIH ImageJ. Background samples from an area near each lane were subtracted from each band to acquire mean band density. Raw blot images were shown in

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2.5. Data analysis

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Supplementary figure 1.

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Data were analyzed by applying a nonparametric one-way ANOVA followed by Tukey’s correction test for multiple comparisons. All experiments consisted of three separate trials and each used a minimum of four samples. Data were analyzed using GraphPad Prism (v.5.0; GraphPad Software, La Jolla, CA) and are presented as mean ± S.E.M values.

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3. Results 3.1. Expression of calcium-processing proteins in rat brain regions The expressions of calcium-processing proteins were examined in several regions of the immature rat brain by performing western blot assays (Fig. 1A and Supplementary figure 1). The results showed that calcium-processing proteins were expressed at comparable levels in the olfactory bulb (OB), cerebral cortex (CT), hypothalamus (HY), cerebellum (CB) and

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brain stem (BS). High expressions of TRPV5 and TRPV6 proteins were observed in the CT, HY, and BS, compared to those in the CB and OB (Fig. 1B, C). In addition, the NCX1 protein was more highly expressed in CT and BS than in OB, HY, and CB (Fig. 1D), and the PMCA1

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protein level was high in CT than in all other brain regions (Fig. 1E).

3.2. Localization of calcium-processing proteins in the rat brain regions by

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immunofluorescence staining

Distribution of calcium-processing proteins in the brain was examined by

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immunofluorescence staining. TRPV5-, TRPV6-, NCX1-, and PMCA1-positive cells were observed in the anterior olfactory nucleus of OB, the layer 4 of CT, the dentate gyrus of

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hippocampus, the mediodorsal thalamus, the paraventricular nucleus of HY, the ventral tegmental area (VTA), the declive of CB, and the dorsal motor nucleus of the vagus nerve of

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BS. We observed that calcium-processing proteins-positive cells were widely distributed in immature rat brain (Fig. 2).

3.3. Regulation of calcium-processing proteins in the CT and HY by steroid hormones We confirmed relationships between the expressions of TRPV5, TRPV6, NCX1, and 9

PMCA1 proteins and the E2, P4, and DEX signal pathways in the immature rat brain. We examined whether E2, P4, and DEX influence the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins in the CT and HY (Fig. 3 and Supplementary figure 1). In the E2treated group, the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins in CT and HY were analyzed by examining western blot results (Fig. 3A-D). In the CT region, the levels of TRPV5, NCX1, and PMCA1 proteins were significantly decreased by E2, but TRPV6

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protein expression did not change. TRPV5, NCX1, and PMCA1 protein levels in the CT region were attenuated by ICI 182,780 (Fig. 3A, B). In the HY region, the levels of TRPV6, NCX1, and PMCA1 proteins were increased by E2, but TRPV5 protein expression showed

no significant change. The levels of TRPV6, NCX1, and PMCA1 proteins were attenuated by

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ICI 182,780 in the HY region (Fig. 3C, D).

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The expressions of TRPV6, NCX1, and PMCA1 proteins were significantly increased by P4, but TRPV5 protein expression showed no significant change in the CT region. The levels

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of TRPV6, NCX1, and PMCA1 proteins were attenuated by RU486 in the CT region (Fig. 3E, F). In the HY region, TRPV5 and TRPV6 levels were decreased by P4, but NCX1 and

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PMCA1 levels were increased. The levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were attenuated by RU486 in the HY region (Fig. 3G, H).

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The levels of NCX1 and PMCA1 proteins were increased by DEX in the CT and HY

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regions, and those levels were attenuated by RU486 in both regions (Fig. 3I-L). The levels of TRPV5 and TRPV6 proteins did not significantly change in the CT region. However, TRPV5 protein expression was decreased by DEX and was attenuated by RU486 in the HY region. TRPV6 protein expression did not significantly change in the HY region. Overall, calcium-processing proteins were affected by E2, P4, and DEX treatments, and 10

those effects were blocked by ICI 182,780 or RU486 treatments. These results suggest that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may be a function of estrogen receptor (ER)-, progesterone receptor (PR)- and glucocorticoid receptor

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(GR)-dependent regulation in the CT and HY regions.

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4. Discussion Calcium-processing proteins, such as TRPV5, TRPV6, NCX1, and PMCA1, regulate intracellular calcium homeostasis in epithelial cells. Previous reports have also shown a role of calcium-processing proteins in calcium homeostasis in duodenum, heart, kidney, lung, and uterus (An et al., 2018; Hwang et al., 2011). However, expression patterns and regulation of calcium-processing proteins in brain regions have not been described. The present study demonstrated the expression patterns of TRPV5, TRPV6, NCX1, and PMCA1 proteins in a

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variety of brain regions such as OB, CT, HY, CB, and BS. The TRPV5, TRPV6, NCX1, and PMCA1 proteins were detected in all brain regions. We focused on the CT and HY brain

regions because many proteins and receptors are expressed more highly in those regions than

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in other brain regions (Brinton et al., 2008; Morimoto et al., 1996; Perez et al., 2003).

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In the brain, TRPV5 and TRPV6 have important roles in the activity of calcium ion channels in neurons, with calcium regulation signaling increasing or decreasing the

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intracellular calcium level. TRPV5 and TRPV6 are expressed in several discrete nuclei and cells within the brain, and they have a role in neuroendocrine regulation (Hwang et al., 2011;

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Kumar et al., 2017a; Kumar et al., 2017b; van Abel et al., 2005). NCX1 is expressed in brain and is present in neurons, astrocytes, oligodendrocytes, and microglia, in which they have an

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important role in maintaining Na+ and Ca2+ homeostasis. In addition, NCX1 exerts a neuroprotective effect by promoting Ca2+ influx and refilling in primary cortical neurons

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(Molinaro et al., 2016). PMCA1 protein has an essential role in the regulation of intracellular calcium levels in neurons and in maintaining proper neuronal function and survival (Kenyon et al., 2010). Therefore, this study was undertaken to demonstrate whether TRPV5, TRPV6, NCX1, and PMCA1 can maintain cytosolic calcium homeostasis in the immature rat brain.

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Many studies have suggested that calcium-processing proteins, depending on tissue specificity, are regulated by E2, P4, and DEX steroid hormones. For example, TRPV6 is upregulated by E2 and P4 in mouse duodenum (Jung et al., 2011), while NCX1 and PMCA1 are downregulated by E2 in rat esophagus (Kim et al., 2017) but upregulated by DEX in mouse lung (An et al., 2018). However, the regulation of calcium-processing proteins in the brain by these steroid hormones has not been described. This study has successfully

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demonstrated the regulation of calcium-processing proteins by E2, P4, and DEX in the CT and HY.

There were significant changes in the expressions of calcium-processing proteins following

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E2, P4, and DEX treatment. Intracellular calcium concentration is altered in neonatal

astrocytes through ER by the action of E2 (Chaban et al., 2004). Moreover, E2 modulation of

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intracellular calcium concentration is dependent on ERα/β proteins associated with the plasma membrane (Chaban et al., 2004). E2 protects against mitochondrial oxidative stress in

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the hippocampus, cerebral cortex, and hypothalamus in ovariectomized female rats. In addition, E2 is involved in the regulation of calcium concentration via TRPV1 in the

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hippocampus and dorsal root ganglion of rats (Yazgan and Naziroglu, 2017). Also, E2 regulates calcium levels through the L-type calcium channel Cav1.2 protein in rat primary

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cortical astrocytes (He et al., 2016). E2 rapidly increases the free cytosolic calcium spike,

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mediating ERα-dependent pathway and intracellular calcium levels in hypothalamic astrocytes (Micevych et al., 2007). The TRPV6 protein expression is increased during the proestrus phase of the cycle in the mediobasal hypothalamus of mice, whereas TRPV5 protein expression is increased during metestrus and diestrus in several discrete nuclei and glial cells; thus, TRPV5 and TRPV6 have emerged as potentially E2-regulated via ERα 13

(Kumar et al., 2017a; Kumar et al., 2017b). Therefore, the roles of calcium-processing proteins following E2, P4, and DEX treatment are tissue-specific and are associated with ERα, PR, and GR, respectively. E2 acts in concert with P4 to control several non-reproductive brain functions, such as cognition and neuroprotection (Brinton et al., 2008). P4 has been shown to have a pleiotropic action in the brain and has neuroprotective effects in the brain (Meffre et al., 2013). PR has

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been found in several brain regions such as hippocampus, CT, HY, and CB.(Brinton et al., 2008). The wide distribution of PR in the brain suggests that this receptor may regulate neuroprotection, cognition, and motor and sensory functions. P4 regulation of calcium

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signaling through inhibition of voltage-gated calcium channels has the neuroprotective effects (Luoma et al., 2012). Glucocorticoids can protect or support the normal functions of organs

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under stress from the injury of the CNS, and a high dose DEX treatment can decrease intracellular calcium in hypothalamic neurons (Chen et al., 2011). DEX produces its

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biochemical function mainly by binding to the GR, which is expressed in nearly all cell types but has varying effects in different cell types. In cortical neurons, DEX can protect against

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glutamate-induced cell death by decreasing calcium signaling (Suwanjang et al., 2013). The present study showed that the expressions of TRPV5, TRPV6, NCX1, and PMCA1

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proteins were regulated by E2, P4, and DEX in CT and HY, suggesting that the regulation of

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intracellular calcium concentration via TRPV5, TRPV6, NCX1, and PMCA1 proteins by E2, P4, and DEX may provide important neuroprotective effects in the brain. In the future, we will address the concentration of cytosolic calcium or calcium signaling pathway after treated with steroid hormones in the brain.

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5. Conclusions In summary, the results of this study have shown that TRPV5-, TRPV6-, NCX1-, and PMCA1-positive cells were highly expressed and widely distributed in the rat brain. The expressions of these proteins were observed in the OB, CT, dentate gyrus, thalamus, HY, VTA, CB, and BS. In addition, TRPV5, TRPV6, NCX1, and PMCA1 were expressed in the brain, and their expressions were regulated by E2, P4, and DEX in the CT and HY. Together, our data suggest that calcium-processing proteins in the brain indicated that they may

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regulate by steroid hormones, which may serve as an important regulatory mechanism for

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cytosolic calcium absorption and release in the brain.

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Ethical statement All animal experiments were carried out in accordance with the Standard Operation Procedures of Laboratory Animal Center, Chungbuk National University (CBNU), Korea. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of CBNU.

Author Statement

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Seon Young Park: Conceptualization, Methodology, Visualization, Writing - Original Draft; Yeong-Min Yoo: Validation, Data Curation; Eui-Man Jung: Conceptualization,

Methodology, Visualization, Writing - Review & Editing; Eui-Bae Jeung: Writing - Review

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Competing interests

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& Editing, Supervision

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The authors declare that they have no competing interests. Funding

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This work was supported by a Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of

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Education, Science and Technology (2017K1A4A3014959 and NRF-2017R1A2B2005031).

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and TRPV6: regulation and implications for disease. Naunyn Schmiedebergs Arch Pharmacol

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Yazgan, Y., Naziroglu, M., 2017. Ovariectomy-Induced Mitochondrial Oxidative Stress,

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Apoptosis, and Calcium Ion Influx Through TRPA1, TRPM2, and TRPV1 Are Prevented by 17beta-Estradiol, Tamoxifen, and Raloxifene in the Hippocampus and Dorsal Root Ganglion

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Figure legends Fig 1. Expression levels of calcium-processing proteins in rat brain regions. (A) Samples from different regions of the olfactory bulb (OB), cerebral cortex (CT), hypothalamus (HY), cerebellum (CB), and brain stem (BS) in the immature rat brain were analyzed by western blot method. (B) TRPV5, (C) TRPV6, (D) NCX1, and (E) PMCA1 results are presented as bar graphs. Expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins were normalized to that of the internal control gene (GAPDH). Data expressed as means ± S.E.M. Statistical

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significance was determined by using one-way ANOVA with Tukey’s correction test. *P < 0.05 versus OB, **p < 0.01 versus OB, ***p < 0.001 versus OB.

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Fig. 2. Distribution of calcium-processing proteins in rat brain regions. TRPV5, TRPV6,

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NCX1, and PMCA1 proteins were identified in the immature rat brain by immunofluorescence assays. Localization was performed with the indicated antibodies in the

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anterior olfactory nucleus of OB, the layer 4 of CT, the dentate gyrus of hippocampus, the mediodorsal thalamus, the paraventricular nucleus of HY, the ventral tegmental area (VTA),

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the declive of CB, and the dorsal motor nucleus of the vagus nerve of BS. Scale bar, 200 m.

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Fig. 3. Effect of steroid hormones on expressions of calcium-processing proteins in the rat brain. The expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by

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E2 (A, B) in CT and (C, D) HY, regulated by P4 (E, F) in CT and (G, H) HY, and regulated by DEX (I, J) in CT and (K, L) HY. The expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by steroid hormone in the CT and HY. The expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins were normalized to that of the internal control gene (GAPDH). Data expressed as means ± S.E.M. Statistical significance was determined 21

by one-way ANOVA with Tukey’s correction test. *P < 0.05 versus vehicle (VE), **p < 0.01 versus VE, ***p < 0.001 versus VE, #p < 0.05 versus agonist, ##p < 0.01 versus agonist, and p < 0.001 versus agonist.

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###

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Figure 1

A

OB

CT

HY

CB

BS TRPV5 TRPV6 NCX1 PMCA1

Olfactory bulb (OB) Cerebral cortex (CT) Hypothalamus (HY) Cerebellum (CB) Brain stem (BS)

GAPDH

***

250 200 150

***

**

100 50

D

100 50 0

**

E ***

250 200

*

150

lP

100 50 0

300 250

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300

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NCX1/GAPDH related expression (%)

150

-p

0

200

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300

TRPV6/GAPDH related expression (%)

C

PMCA1/GAPDH related expression (%)

TRPV5/GAPDH related expression (%)

B

23

200 150 100 50 0

***

***

**

Brain stem VTA

Thalamus

Dentate gyrus

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-p

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Hypothalamus

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na

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Cerebellum

Jo Cerebral cortex

Olfactory bulb

Figure 2 TRPV5 TRPV6

24

NCX1 PMCA1

Figure 3-1

A

B

Vehicle E2 ICI 182,780

160 ###

E2

E2+ICI

Relative expression (%)

VE

TRPV5 TRPV6 NCX1 PMCA1

120

80

#

**

***

** 40

GAPDH 0

E2

E2+ICI

Relative expression (%)

VE

TRPV5 TRPV6 NCX1 PMCA1 GAPDH

TRPV5 250

TRPV6

NCX1

***

***

200

#

150

##

100 50

0

TRPV6

NCX1

re Relative expression (%)

P4+RU

TRPV5

G

Jo

VE

ur

na

TRPV6

P4

NCX1 PMCA1

Vehicle P4 RU 486

250

***

***

200

PMCA1

***

150 ###

100

###

###

50

GAPDH 0 TRPV5

H P4+RU TRPV5 TRPV6 NCX1 PMCA1

Relative expression (%)

P4

lP

F

VE

**

##

TRPV5

E

PMCA1

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D

-p

C

TRPV6

180

PMCA1

** ###

150

*** ##

120 90

NCX1

## ###

*

**

TRPV5

TRPV6

60 30

GAPDH 0

25

NCX1

PMCA1

Figure 3-2 Vehicle DEX RU 486

J DEX

DEX+RU

Relative expression (%)

VE

TRPV5 TRPV6 NCX1 PMCA1

500

***

400 300

**

200 100

GAPDH 0

VE

DEX

DEX+RU TRPV5 TRPV6 NCX1

GAPDH

400 300

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NCX1

100

PMCA1

##

***

200

###

###

***

0

TRPV5

26

TRPV6

***

500

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PMCA1

TRPV5 600

-p

L Relative expression (%)

K

##

###

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I

TRPV6

NCX1

PMCA1

Supplementary figure 1-1

Figure 1A OB

CT

HY

CB

BS 100 kDa 70 kDa

TRPV5

NCX1

130 kDa 100 kDa

PMCA1

180 kDa 130 kDa

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TRPV6

100 kDa 70 kDa

40 kDa 35 kDa

-p

GAPDH

Figure 3A

Figure 3C E2

E2+ICI

100 kDa 70 kDa

TRPV5

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TRPV5

100 kDa 70 kDa

na

TRPV6

130 kDa 100 kDa

NCX1

180 kDa 130 kDa 40 kDa 35 kDa

27

VE

E2

E2+ICI 130 kDa 100 kDa

TRPV6

100 kDa 70 kDa

NCX1

130 kDa 100 kDa

PMCA1

180 kDa 130 kDa

GAPDH

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PMCA1

GAPDH

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VE

40 kDa 35 kDa

Supplementary figure 1-2

Figure 3E

Figure 3G VE

P4

P4+RU

TRPV5 TRPV6

VE

PMCA1

TRPV5

100 kDa 70 kDa

100 kDa 70 kDa

TRPV6

100 kDa 70 kDa

NCX1

130 kDa 100 kDa

PMCA1

180 kDa 130 kDa

40 kDa 35 kDa

40 kDa 35 kDa

-p

GAPDH

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130 kDa

GAPDH

Figure 3I

Figure 3K DEX DEX+RU

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VE

100 kDa 70 kDa

TRPV5

lP

TRPV5

TRPV6

100 kDa 70 kDa 100 kDa 70 kDa

130 kDa 100 kDa

NCX1

130 kDa 100 kDa

PMCA1

180 kDa 130 kDa

GAPDH

40 kDa 35 kDa

40 kDa 35 kDa

Jo

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DEX DEX+RU

TRPV6

180 kDa 130 kDa

PMCA1

VE

100 kDa 70 kDa

na

NCX1

GAPDH

P4+RU

100 kDa 70 kDa

130 kDa 100 kDa

NCX1

P4

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