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

Background The cytosolic calcium concentration is regulated by calcium-processing proteins such as transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca 2+ ATPase 1 (PMCA1). Those 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 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 were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486).Results 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 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 are regulated by E2, P4, and/or DEX and can be recovered by antagonist treatment.Conclusion These results indicate that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain. the distribution of calcium-processing proteins in rat and the regulation of calcium-processing proteins expression by steroid hormones. observed high expressions of calcium-processing proteins in the CT and HY and the regulation of expression of calcium-processing proteins by E2, P4, and DEX in immature rat brain. Our findings suggest calcium-processing by hormones, 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 via ER-, PR- and GR-dependent pathways in the CT and HY. Together, our data suggest that calcium-processing proteins in the brain are regulated by steroid hormones, which may serve as an important regulatory mechanism for cytosolic calcium absorption and release in the brain.

3 [2]. 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 integration [3,4]. Calcium homeostasis is the maintenance of (re)absorbed calcium amounts in the body and is important for essential body's functions, cell function, and cell survival [5]. To cope with large 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 several organs [2]. Calcium is processed by calcium transport proteins (channels, carriers, and pumps) and through interaction with cytosolic calcium buffers, which are regulated differentially at the plasma membrane and in calcium stores [6].
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 [8]. NCX1 is a counter-transporter membrane protein found in the plasma membrane, mitochondria, and endoplasmic reticulum of excitable cells [9].
PMCA1 is a transport protein in the plasma membrane of cells and is upregulated by E2 in the uterus [10]. When intracellular calcium concentrations increase, NCX1 and PMCA1 discharge calcium and vice versa [11,12].
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 spine in the hippocampus, protecting neurons. Neuroprotection of E2 is a calcium-dependent process and is related to the regulation of intracellular calcium levels [13]. P4 has an important neuroprotective effect and affects neuronal function by 4 regulating cellular activity and gene transcription through intracellular receptors that are abundant in the central nervous system (CNS) [14,15]. Glucocorticoids are a class of steroid hormones involved in calcium metabolism and can increase urinary calcium excretion and inhibit calcium absorption in the intestine [16]. They also have an essential role in the hypothalamus-pituitary-adrenal cortex axis [17]. Within the cell, DEX may protect the cell from calcium overload [18].
This study investigated the distribution of calcium-processing proteins in rat brain and the regulation of calcium-processing proteins expression by steroid hormones. We observed high expressions of calcium-processing proteins in the CT and HY and the regulation of expression of calcium-processing proteins by E2, P4, and DEX in immature rat brain. Our findings suggest that calcium-processing proteins are regulated by steroid hormones, which may serve as important regulators of cytosolic calcium absorption and release in the brain.

Animal treatment
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 housed in polycarbonate cages and were allowed to acclimate to their housing in an environmentally controlled room (temperature 23°C ± 2°C, relative humidity 50% ± 10%, frequent ventilation, and a 12 h light:dark cycle).
After approximately 1 week of acclimatization the rats were separated into seven groups (n = 7) and each group was subcutaneously (SC) injection with E2 (50 µg/kg body weight

RNA extraction and real-time PCR
Rats were euthanized by CO2 asphyxiation as described in previously [19]. The brains were removed and sagittally divided and the olfactory bulbs, cerebral cortex, hypothalamus, cerebellum, and brain stem were dissected from the right hemisphere.
Total RNA was extracted using the TRIzol (Ambion, Austine, TX, USA), according to the manufacturer's instructions. RNA purity was determined using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA). Next, first strand cDNA was prepared by subjecting 1 µg total RNA to reverse transcription using Moloney murine leukemia virus reverse transcriptase (ThermoFisher Scientific, Logen, UT, USA) with random primers (9-mers; TaKaRa Bio Inc., Kusatsu Japen). The cDNA template (2 µL) was added to 10 µL of 2× SYBR premix Ex Taq (TaKaRa Bio) and 10 pmol of each specific primer for ERa, PR, and GR genes. The primer sequences are showed in additional table 1.
By using an ABI 7300 Real-time PCR system (Applied Biosystems, Foster City, CA, USA), quantitative real-time PCR was conducted under the following conditions: 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s.
Fluorescence intensity was measured at the end of the extension phase of each reaction cycle, and the reaction cycle at which the PCR products exceeded the fluorescence intensity threshold in the exponential phase of PCR amplification was considered the threshold cycle (CT). Each data point was analyzed to the internal control (GAPDH) gene for determination of a normalized arbitrary value. Relative expression (R) was calculated by using the equation R = 2 -ΔΔCT and was normalized to the vehicle.

Expression of calcium-processing proteins in immature 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 Additional file 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 brain stem (BS). Significantly higher 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 significantly more highly expressed in CT and BS than in OB, HY, and CB (Fig. 1d), and the PMCA1 protein level was significantly higher in CT than in all other brain regions (Fig. 1e).

Localization of calcium-processing proteins in the immature rat brain regions by immunofluorescence staining
Distribution of calcium-processing proteins in the brain was examined by immunofluorescence staining. TRPV5-, TRPV6-, NCX1-, and PMCA1-positive cells were observed in OB, CT, dentate gyrus, thalamus, HY, ventral tegmental area (VTA), CB, and BS. We observed that calcium-processing proteins-positive cells were widely distributed in immature rat brain (Fig. 2).

Regulation of calcium-processing proteins in the CT and HY by steroid hormones
We confirmed relationships between the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins and the E2, P4, and DEX signal pathways in the immature rat brain. First, we observed double immunofluorescence staining to examine whether TRPV5-, TRPV6-, NCX1-, 9 and PMCA1-positive neurons are co-expressed with ERα, PR, and GR (Fig. 3a-d). There are no significant differences in expression of ERa, PR, and GR mRNA levels (Fig. 3e, f). We observed that ERα, PR, and GR receptors were expressed with TRPV5, TRPV6, NCX1, and PMCA1 in the neuronal cell.
Next, we examined whether E2, P4, and DEX influence the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins in the CT and HY (Fig. 4 and Additional file 1). In the E2-treated group, the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins in CT and HY were analyzed by examining western blot results (Fig. 4a-d). In the CT region, the levels of TRPV5, NCX1, and PMCA1 proteins were significantly decreased by E2, but TRPV6 protein expression did not change. TRPV5, NCX1, and PMCA1 protein levels in the CT region were recovered by ICI 182,780 (Fig. 4a, 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 recovered by ICI 182,780 in the HY region (Fig. 4c, d).
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 of TRPV6, NCX1, and PMCA1 proteins were recovered by RU486 in the CT region (Fig. 4e, f).
In the HY region, TRPV5 and TRPV6 levels were decreased by P4, but NCX1 and PMCA1 levels were increased. The levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were recovered by RU486 in the HY region (Fig. 4g, h).
The levels of NCX1 and PMCA1 proteins were increased by DEX in the CT and HY regions, and those levels were recovered by RU486 in both regions ( Fig. 4i-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 recovered 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 those effects were blocked by ICI 182,780 or RU486 treatments. The mRNA levels of ERa, PR, and GR showed no significant differences between the vehicle group and the E2-, P4-, and DEX-treated groups. Taken together, these results suggest that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may be a function of ER-, PR-and GR-dependent regulation in the CT and HY regions.

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 [11,16]. However, expression patterns and regulation of calciumprocessing proteins in brain regions have not been described. The present study demonstrated the expression patterns of TRPV5, TRPV6, NCX1, and PMCA1 proteins in a 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 in other brain regions [22][23][24].
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 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 [11,[25][26][27]. NCX1 is expressed in brain and is present in neurons, astrocytes, oligodendrocytes, and microglia, in which they have an important role in maintaining Na + and Ca 2+ homeostasis. In addition, NCX1 exerts a neuroprotective effect by promoting Ca 2+ influx and refilling in primary cortical neurons [28]. PMCA1 protein has an essential role in the regulation of intracellular calcium levels in neurons and in maintaining proper neuronal function and survival [3]. Therefore, this study was undertaken to demonstrate whether TRPV5, TRPV6, NCX1, and PMCA1 can maintain cytosolic calcium homeostasis in the immature rat brain.
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 [8], while NCX1 and PMCA1 are downregulated by E2 in rat esophagus [10] but upregulated by DEX in mouse lung [16].
However, the regulation of calcium-processing proteins in the brain by these steroid hormones has not been described. This study has successfully 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 E2, P4, and DEX treatment, and these proteins were co-expressed with the ERα, PR, and GR proteins. Intracellular calcium concentration is altered in neonatal astrocytes through ER by the action of E2 [29]. Moreover, E2 modulation of intracellular calcium concentration is dependent on ERα/β proteins associated with the plasma membrane [29].
E2 protects against mitochondrial oxidative stress in 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 hippocampus and dorsal root ganglion of rats [30]. Also, E2 regulates calcium levels through the L-type calcium channel Cav1.2 protein in rat primary cortical astrocytes [13]. E2 rapidly increases the free cytosolic calcium spike, mediating ERα-dependent pathway and intracellular calcium levels in hypothalamic astrocytes [31]. The TRPV6 protein expression is increased during the proestrus cycle in the mediobasal hypothalamus of mice, whereas TRPV5 protein expression is increased during metestrus and diestrus in several discrete nuclei and glial 12 cells; thus, TRPV5 and TRPV6 have emerged as potentially E2-regulated via ERα [26,27].
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 [23]. P4 has been shown to have a pleiotropic action in the brain and has neuroprotective effects in the brain [32]. PR has been found in several brain regions such as hippocampus, cortex, hypothalamus, and cerebellum. [23]. 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 signaling through inhibition of voltage-gated calcium channels has the neuroprotective effects [33].
Glucocorticoids can protect or support the normal functions of organs under stress from the injury of the CNS, and a high dose DEX treatment can decrease intracellular calcium in hypothalamic neurons [17]. DEX produces its 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 glutamate-induced cell death by decreasing calcium signaling [18].
The present study showed that the expressions of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and DEX via their mediation of the ER-, PR-, and GRdependent pathways in CT and HY, suggesting that the regulation of 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.

Conclusions
In summary, the results of this study have shown that TRPV5-, TRPV6-, NCX1-, and PMCA1-13 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 via ER-, PR-and GRdependent pathways in the CT and HY. Together, our data suggest that calcium-processing proteins in the brain are regulated by steroid hormones, which may serve as an important regulatory mechanism for cytosolic calcium absorption and release in the brain.

Consent to publish
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are included in this published article and are available from the corresponding author on reasonable request.  Figure 1 Expression levels of calcium-processing proteins in immature rat brain regions. Additioanl file_BMC neuro.pdf