The Estrogen-Related Receptor Alpha Upregulates Secretin Expressions in Response to Hypertonicity and Angiotensin II Stimulation

Osmoregulation via maintenance of water and salt homeostasis is a vital process. In the brain, a functional secretin (SCT) and secretin receptor (SCTR) axis has recently been shown to mediate central actions of angiotensin II (ANGII), including initiation of water intake and stimulation of vasopressin (VP) expression and release. In this report, we provide evidence that estrogen-related receptor α (ERRα, NR3B1), a transcription factor mainly involved in metabolism, acts as an upstream activator of the SCT gene. In vitro studies using mouse hypothalamic cell line N-42 show that ERRα upregulates SCT promoter and gene expression. More importantly, knockdown of endogenous ERRα abolishes SCT promoter activation in response to hypertonic and ANGII stimulations. In mouse brain, ERRα coexpresses with SCT in various osmoregulatory brain regions, including the lamina terminalis and the paraventricular nucleus of the hypothalamus, and its expression is induced by hyperosmotic and ANGII treatments. Based on our data, we propose that both the upregulation of ERRα and/or the increased binding of ERRα to the mouse SCT promoter are two possible mechanisms for the elevated SCT expression upon hyperosmolality and central ANGII stimulation.


Introduction
Secretin (SCT) is a classical gastrointestinal hormone [1]. Beyond its best known actions on the regulation of bicarbonate, electrolytes and volume secretion from the pancreatic ductular epithelial cells, there is a growing body of evidence showing SCT as a neuropeptide in the CNS [2][3][4][5] and as an anti-diuretic hormone, SCT stimulate the process of renal water reabsorption in kidney by a VP-independent mechanisms [6]. SCT is found as a neurosecretory factor released from the neurohypophysis into circulation upon water deprivation or direct electrical stimulation of the paraventricular nuclei (PVN) [6,7] to control water reabsorption in the kidney [5]. Recently, the roles of SCT in the brain in water regulation were reported, and SCT was found to have similar functions as the osmoregulatory peptide, angiotensin II (ANGII) [8]. In these studies, with the aid of the SCTR 2/2 and SCT 2/2 mice, it was found that the presence of a functional SCT/ SCTR axis is a prerequisite for some actions of ANGII, including the upregulation of VP expression in the hypothalamic PVN and the release of VP protein from posterior pituitary into the circulation. In addition, SCT itself was found as a dipsogenic hormone that controls water intake behavior as well as mediates the dipsogenic effects of ANGII [8].
In specific brain regions, both plasma hypertonicity and circulating ANGII are able to stimulate the lamina terminalis, consisting of the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO), which are critical sites for regulating water homeostasis in rodents [9][10][11][12][13][14]. Not just that we found SCT and SCTR expression in these water regulation sites, we found also that SCT injected into the lateral ventricle of the brain could augment water drinking behavior, as well as VP expression and VP protein release. As ANGII via binding to AT 1 receptor could stimulate SCT expression in SFO and OVLT [7], it is therefore possible that SCT mediates central actions of ANGII partly by controlling the expression of SCT gene in these osmoregulatory brain regions.
The human and rat SCT promoter have previously been characterized [15][16][17], while more recently, small heterodimer partner (SHP), an orphan nuclear receptor, was identified as a repressor of the mouse SCT gene by blocking NeuroD-mediated gene activation in response to bile acids in the mouse duodenum [18]. Nuclear receptor superfamily is a large group of transcription factors that are involved in regulation of various physiological processes, including metabolism, cell growth and differentiation, development, and homeostasis [19]. A recent study on the genetically modified mouse model lacking estrogen-related receptor a (ERRa) revealed its functions in the renin-angiotensin system (RAS) to regulate renal sodium and potassium homeostasis [20], indicating potential roles of ERRa in osmoregulation. ERRa belongs to the NR3B1 subfamily of the orphan nuclear receptors and is apparently ligand-independent [21]. ERRa is expressed with the highest levels in tissues with high energy demands such as heart, kidney and adipose tissues [22,23]. ERRa recognizes the estrogen response element (ERE) in target genes as estrogen receptor (ER) does, but was found to have a higher preference for the ERE-half site or ERR response element, which is a single halfsite preceded by the three nucleotides TNA [24,25].
In the present study, we investigated a possible mechanism for the cross-interaction of SCT and ANGII central pathways in osmoregulation via ERRa. We found that ERRa co-expresses with SCT in various osmocenters in the CNS, and that its own expression is regulated by central ANGII and peripheral hyperosmotic stimulation. Using a hypothalamic cell model, ERRa was then found to interact specificity with an ERE-half site in the proximal region of the mouse SCT promoter to control SCT gene expression. After showing that SCT plays an indispensable role in mediating central effects of ANGII (7), the present study, thus, provide evidence to indicate that ERRa is one of the mechanisms that connects SCT with central effects of ANGII and/or plasma osmolality.

Constructs and siRNA expressing vectors
The mouse SCT core promoter construct (mSCTP) (from 227 to 2399 bp relative to the start codon) was produced by 1) PCR amplification using mouse genomic DNA, 2) sequencing, and 3) subcloning into the pGL2-Basic vector. The mouse ERRa, ERRb and ERRc expression vectors were constructed using the pcDNA3 vector (Invitrogen, Carlsbad, CA). Site-directed mutants of the mSCTP promoter were constructed by a three-step mutagenesis method [26] using the mutagenic primers listed in Table 1. For gene silencing, two siRNAs specific for the mouse ERRa (siERRa-S1 and siERRa-S2) were constructed using oligos (pSi-ERRa S1 and pSi-ERRa S2; Table 1) and the pSilencer 4.1-CMV vector (Hind III/Bam HI sites; Ambion, Austin, TX). The siControl vector provided by the manufacturer was used as a negative control. Western blot analysis [18] was used to check the effectiveness of siRNA in silencing endogenous mouse ERRa. Briefly, N-42 cells were transfected with 2 mg of the siERRa-S1, siERRa-S2, siControl or pSilencer vector by GeneJuice (see later for transfection procedures). After 24 h, cells were lysed with RIPA buffer (50 mM Tris pH 7.4, 0.25% Na-deoxycholate, 1% NP-40, 150 mM NaCl and 1 mM EDTA), and cell lysates were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. After incubation with an anti-ERRa antibody (1:500 dilution; Millipore [27]) or GAPDH antibody (loading control, 1:1000; Cell signaling technology) at 4uC overnight, and subsequently by the horseradish peroxidase conjugated secondary antibody (1:1000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h, signals were detected by the Western Lightning plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer, Shelton, CT).

Primer
Sequence (59-30) siERRa-S1-S GATCCCTTTGCCTTTCCCGGGCCCTTCAAGAGAGGGCCCGGGAAAGGCAAAGGGA siERRa-S1-AS according to the manufacturer's instruction. For over-expression studies, the mouse SCT promoter (mSCTP) [15][16][17] luciferase construct (2 mg) was co-transfected with different amounts of the ERRa, ERRb, or ERRc expression vectors (0.5, 1.0, 2.0 mg), and pCMV-b-gal as an internal control (0.5 mg) per well. For gene silencing studies, cells were cotransfected with mSCTP luciferase construct (2 mg), pCMV-b-gal (0.5 mg) and various amounts of silencing or control vector (1 and 2 mg). Appropriate amount of the pcDNA3.1 was added so that the total amount of DNA used per well was the same. Cell extracts were harvested 48 h after transfection for luciferase and b-galactosidase assays. The promoter activities were measured in at least three independent experiments each in triplicate assays normalized with b-galactosidase activity. In saline treatment studies, 48 h after transfection, different concentrations of saline (25,50, and 100 mM) were added to the cells for 8 h at 37uC with 5% CO 2 for luciferase assay or RNA isolation.

The SCT expression in N42 cells after saline and ANGII treatment
Cell was seeded in 6-well plates at a density 2.0610 5 cells/well. After 2 d, at which the cell confluence was about 80%, saline treatment was carried out. Different concentrations of saline were added to the cells so that the final concentrations of the medium were 0, 25, 50, and 100 mM. The plate was then incubated at 37uC with 5% CO2 for various time periods (2 h, 4 h or, 8 h) before harvest. For ANGII treatments, the culture mediums were replaced by the medium with ANGII in various final concentration (ranged from 10 27 to 10 211 M). The cells were collected for 8 h before the RNA isolation and protein extraction. Total RNA were isolated by TRIzol reagent (Invitrogen). The isolated RNA was reverse transcribed with oligo-dT primer and Superscript III reverse-transcriptase (Invitrogen). One-tenth of the first strand cDNAs was then used as the template in qRT-PCR for measuring mouse GAPDH, SCT, ERRa, and S14 mRNA levels by the SYBR Green PCR Master Mix kit (Applied Biosystems, Foster city, CA) with specific primers ( Table 1). The fluorescence signals were measured in real time during the extension step by the 7300 Real Time PCR System (Applied Biosystems). The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence signal reached 10-fold standard deviation of the baseline (from cycles 2 to 10). The ratio change in the target gene relative to the GAPDH control gene was determined by the 2 2DD Ct method [28]. For western blot analysis, cells were lysed with RIPA and western blot analysis of ERRawere performed as described above

MTS Assay
The saline treatments for N-42 cells was performed based on previous studies [29,30], Cell proliferation assays were performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, MI) according to the manufacturer's instructions. N-42 cells were seeded at an optimized density of 2000 cells per well in a 96-well plate and maintained at 37uC with 5% CO 2 . After 48 h when cell confluence was about 70%, different concentrations of saline (0, 50, 100, 150, 200, 300, 400 mM) were added for 8 h. After salt treatment, CellTiter 96 Aqueous One Solution Reagent was added for 2 h and absorbance at 490 nm, which is directly proportional to the number of living cells in the culture, was measured. Wells with plain medium with no cells were included as the blank and each sample was repeated at least 5 times in this study.

Chromatin Immunoprecipitation (ChIP) Assay
ChIP assays were performed as described previously [33]. The mouse hypothalamus was isolated and nuclear extracts were prepared as described in [31]. The DNA-protein complexes in N-42 cells and mouse hypothalamus were cross-linked with 1.42% formaldehyde and harvested in IP buffer (150 mM NaCl, 50 mM Tris-HCL (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride and 10 mg/ml leupeptin). To shear the chromatin into DNA fragments, samples were sonicated (Sonifier 450; Branson Ultrasonics, Danbury, CT) by 1s-long pulses for 15 times at 50% maximum power output for 2 rounds. The DNA-ERRa complex was immunoprecipitated with 2 mg rabbit anti-ERRa IgG (Upstate, Millipore, Billerica, MA) in ice bath followed by the addition of protein A-agarose (Cruz Biotechnology, Santa Cruz, CA). DNA was isolated in Chelex-100 slurry (BioRad, Hercules, CA) and the protein was removed by proteinase K digestion (0.2 mg/ml). The purified DNA was used for quantitative real-time PCR (qRT-PCR) with specific primers (SCTP-F and SCTP-R; Table 1), targeting the mSCTP. The relative occupancy of the immunoprecipitation factor at the locus was estimated by 2 (Ct mock -Ct specific ) and normalized with input DNA, where Ct mock and Ct specific represent the mean threshold cycles (Ct) of qRT-PCR (in triplicates) on purified DNA samples from mock and specific ERRa immunoprecipitation.

Intracerebroventricular (ICV) incannulation and drug administration
Mice were anesthetized and surgery was conducted in aseptic conditions. Procedures for ICV were performed as described [34,35]. For injections into the lateral ventricle, the coordinates of cannula implantation were determined according to the mouse brain atlas [36]. The cannula (11-mm-long, 21-gauge stainless steel tubing) was stereotaxically placed such that the tip was 0.5 mm caudal to bregma, 1.0 mm lateral to midline, and 2.0 mm below the dura. The placement was confirmed by injection of a dye. Three days were allowed for recovery before ICV injection. Central injections were done using PE-10 tubing attached to an injector and a 10-ml Hamilton syringe. Artificial cerebrospinal fluid (ACSF; according to Alzet protocol, 2 or 5 ml), ANGII (100 ng/2 ml; 002-12; Phoenix Pharmaceuticals, Belmont, CA), or saline (0.25 or 0.50 M/5 ml) were injected into the lateral ventricle.
Water deprivation and saline drinking followed by Lasercapture microdissection (LCM) and qRT-PCR To induce hypertonic serum in mice, normal drinking water was either removed for 1 d or replaced with 2% saline for 5 d.
Treated and control mice were then sacrificed and their brains were collected. For ICV studies, 1 h after the ICV injection, similarly, brains of treated and control mice were isolated. For whole brain studies, total RNA was extracted by the TriPure isolation reagent (Roche Molecular Biochemicals, Basel, Switzerland). For study of the specific brain areas, the brains were embedded in OCT compound (Sarura Finetek, Torrance, CA) and stored at 220uC. The frozen brain was then sectioned (8-mm thick) using a cryostat microtome (Jung CM3000, Lieca Microsystems, Leitz, Germany) and mounted on plain slices. Brain sections at the level of the lamina terminalis and PVN were stained with hematoxylin and eosin and imaged on the PixCell IIe Laser Capture Microdissection System (Arcturus, Sunnyvale, CA). Target cells were located according to the mouse brain atlas and were captured on CapSure HS LCM Caps (Arcturus). RNA of captured cells was prepared using the PicoPure RNA Isolation Kit (Arcturus). The isolated RNA was reverse transcribed and realtime PCR were performed as described above.

Immunohistochemical (IHC) staining
IHC staining was performed as described [6,35]. Brains isolated from mice were fixed in 3.7% formalin, embedded in paraffin, and sectioned (7 mm). Paraffin sections were dewaxed and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol. Microwave antigen retrieval was performed with citric acid buffer at pH 6.0 for 10 min, followed by blocking of non-immunological binding with 5% normal goat serum for 2 h. Sections were then incubated with rabbit anti-ERRa IgG (1:500 dilution; Millipore) overnight at 4uC.  Data was calculated using the equation: 2 (Ct mock -Ct specific ) and normalized with Input, which was defined as 1.00. A mock immunoprecipitation using rabbit anti-IgG and without using antibody were carried out as negative control. Data represent the mean 6 SEM of three experiments.*p,0.05; **p,0.001, compared with the control. (B) EMSA using the ERE-half site as the oligo. Left: Nuclear extract of mouse hypothalamus (10 mg), pcDNA (control), and in vitro translated ERRa protein were pre-incubated with the oligo for 15 minutes at room temperature. Arrows indicate specific protein-DNA complexes that contain ERRa (Complex I) and unknown protein (complex II). Middle: Hypothalamic nuclear extract of control or water deprived or saline-drank mice were used (n = 4/group) in EMSA. Right: Supershift assay of ERRa proteins. Antibodies (2 mg) specific for ERRa and AP1 (Santa Cruz) proteins were preincubated with the nuclear extract (10 mg) for 20 min at room temperature. Arrows indicate specific protein-DNA complexes that contain ERRa (Complex I), non-specific complex (Complexes II) and ERRa supershift (SS). (C) ChIP assay showing the in vivo binding of ERRa on mouse SCT promoter in mouse hypothalamus after water deprivation and saline treatment (left) and ICV-ANGII injection in mice (1 and 4 h) (right). doi:10.1371/journal.pone.0039913.g005

Statistical analysis
All data are shown as means 6 standard error SEM. The deviations between groups were analyzed using the computer software PRISM (version 3.0, GraphPad Software, La Jolla, CA). Unpaired t-test was performed when only two groups were under consideration, whereas data from more than two groups were analyzed by one-way ANOVA, followed by Dunnett's test.

ERRa upregulates SCT gene in the hypothalamic N-42 cell line
In view of a recent study revealing a potential function of ERRa in the renin-angiotensin system (RAS) for osmoregulation [9] and the presence of an ERE-half site in the proximal region of the mouse (from 2149 to 2144 bp, relative to the start codon), rat and human SCT promoters (Fig. 1A), we initially investigated the in vitro function of ERRs to control the mouse SCT gene in a mouse hypothalamic N-42 cell-line. In this study, each of the three isoforms of ERR (a, b and c) was overexpressed with the mouse SCT promoter-luciferase construct. As shown in Fig. 1B, the SCT promoter was significantly (1.92-fold and 2.18-fold for 1.0 and 2.0 mg of ERRa, respectively) activated only by ERRa, which is a constitutive activator [37,38], whereas overexpression of neither ERRb nor ERRc showed an activation effect on mouse SCT promoter. Consistent with this observation, endogenous SCT mRNA levels were significantly elevated (1.63-fold and 2.15-fold for 1.0 and 2.0 mg of ERRa, respectively) when ERRa was overexpressed in N-42 cells (Fig. 1C). In order to confirm further a functional role of ERRa, two siRNA vectors specific for targeting the mouse ERRa were designed. Silencing of the endogenous ERRa in N-42 cells, by using either of the silencing vectors (S1 or S2), resulted in considerable drops in the mouse SCT promoter activities (S1: 39.7% and 63.2% decrease; S2: 26.4% and 51.6% decrease) (Fig. 1D), and also in the endogenous SCT transcript levels (S1: 38.9% and 57.2% decrease; S2: 42.4% decrease for 2 mg) (Fig. 1E). The siRNA were functional in lowering endogenous ERRa expression as indicated by Western blotting (Fig. 1F), in which ERRa protein levels were substantially reduced. These findings suggested that ERRa is responsible for the upregulation of mouse SCT expression in the hypothalamic cells. The direct relationship between the ERE-half site and the ERRa mediated activation of mSCTP were confirmed by mutation analysis (Fig. 1G). The promoter activities were increased with ERRa cotransfection in the mutants that not related to ERE-half site (M1 1.8 fold and M4 1.9 fold when compare to pKS+ control), whereas the mutation of ERE-half site can severely decrease ERRa-mediated activation effects (only 1.08 fold for M2 and 1.13 fold for M3). The data clearly suggested that the ERE-half site is the critical motif for the ERRa responsiveness in mSCTP.

ERRa is a mediator of the hypertonicity-and ANGIIinduced upregulation of SCT
After showing ERRa as an activator of the SCT gene, our next task was to study if ERRa was involved also in the osmoregulatory pathway. We have previously revealed that SCT expression was upregulated in the mouse hypothalamus in response to water deprivation and saline drinking [8]. To examine the effect of hypertonic shock on the mouse SCT gene, N-42 cells were treated with different concentrations of hypertonic salt treatment (25,50 and 100 mM) for 2, 4 and 8 h (Fig. 2C) as in a previous study [29]. Before the treatment, the toxicity of hypertonic saline to the N42 cells was tested by MTS assay. The corresponding osmolality in different concentration of NaCl was shown in Fig. 2A. The MTS assay (Fig. 2B) suggested that the lethal concentration 50 of NaCl is around 150 mM. Therefore, the maximum concentration of NaCl that used in the N42 cells treatment is 100 mM. Hypertonic treatment of cells led to time-and dose-dependent increases in SCT promoter activities (Fig. 2C), in which the 8-h treatment with 100 mM saline resulted in the strongest induction (2.45-fold). Likewise, hypertonic saline treatment could stimulate endogenous SCT expression in a similar pattern (Fig. 2D). In this study, the mouse house-keeping gene S14 was used as a control which expression was not affected by hypertonic saline under all the conditions tested (Fig. 2E). Consistently, ANGII peptide dosedependently (10 211 to 10 27 M, 8 h treatment) activated mouse SCT promoter (Fig. 2C) as well as endogenous SCT mRNA levels (Fig. 2D). ANGII at the concentration 10 210 M was found to produce the greatest stimulation (2.89 folds). Again, ANGII treatment was unable to change the expression of the mouse S14 housekeeping gene, as a control.
Similar to their effects on SCT, both hypertonic shock and ANGII could also stimulate mouse ERRa mRNA expression (Fig. 3A) as well as the ERRa protein level (Fig. 3B) in this hypothalamic cell model. When siERRa construct (pSi-mERRa-S1) was employed to silence endogenous ERRa, consistent with findings in Fig. 2D, the effects of both hypertonic saline and ANGII treatment were almost completely abolished ( Figure 3D). This upregulation of mouse SCT expression is dependent on the ERE-half site. As shown in Fig. 3C, the mutation of this motif (M2 and M3) cause a large and significant reduction in the response to the saline and ANGII treatment. Taken together, our data show the potential function of ERRa in mediating stimulatory effects of hypertonic saline and ANGII on SCT expression.

In vivo functions of ERRa in osmoregulation in the mouse brains
To study the in vivo functions of ERRa to upregulate SCT in response to hypertonic treatment and ANGII, we initially investigated expression levels of ERRa in mouse brain in response to hyperosmotic stimuli and central ANGII administration. We found that ERRa transcript levels in the mouse cerebrum were significantly elevated after water deprivation, saline drinking or ICV-ANGII in mice (Fig. 4A). We have previously shown the localization of SCT transcript and protein in various osmoregulatory sites, including the SFO, OVLT and MnPO, and that SCT transcript levels were significantly enhanced upon water deprivation and saline drinking [8]. By IHC stainings, ERRa proteins were also found in sites collectively known as lamina terminalis and PVN of the mouse brain (Fig. 4B). By LCM-coupled with qRT-PCR, expression changes of ERRa in these brain regions were studied (Fig. 4C). ERRa transcript levels in the PVN, SFO, MnPO and OVLT were significantly augmented after water deprivation and saline drinking (Fig. 4C). One hour after ICV-ANGII injection, ERRa levels in the PVN and OVLT were elevated while its levels in the SFO and MnPO were unchanged (Fig. 4C) The expression changes of ERRa could therefore activate SCT expression in these areas as shown in our in vitro studies in N42 cells.
In vitro and in vivo binding of ERRa to the ERE-half site of the mouse SCT promoter Despite the presence of an ERE-half site in the mouse SCT promoter (Fig. 1A), there was no previous report showing the functional interaction of ERRa with this motif. In the present study, using EMSA and ChIP assay, we seek to investigate the binding of ERRa to the SCT promoter ERE-half site in N-42 cells and mouse hypothalamus (Fig. 5). In the ChIP assay (Fig. 5A), we observed no PCR signal from the negative controls including the no antibody, and anti-rabbit IgG control. The controls show neither nonspecific precipitations nor PCR contamination in this assay. Positive PCR signals are found in the samples treated with ERRa antibody. This suggested the in vivo binding of ERRa with the mouse SCT promoter. The ChIP assay coupled to qRT-PCR, more importantly, showed that both saline and ANGII treatments were able to increase the ERRa binding. EMSA using mouse hypothalamic nuclear extract showed the presence of two DNAprotein complexes (I and II) (Fig. 5B left). To confirm the identity of these complexes, we performed EMSA by incubating the same probe with in vitro translated mouse ERRa proteins, which was found to form a complex with a size similar to complex I. In the ERRa supershift assay, the intensity of complex I was decreased and a supershift band was observed (Fig. 5B right). No supershift band can be observed in the control (AP1 antibody). This supershift assays suggests that complex I contains ERRa protein.
To show changes in binding of ERRa, we have used nuclear extracts prepared from the hypothalamus of mice under water deprivation and saline drinking, and we found that the intensities of complex I were increased after these treatments (Fig. 5B  middle).
Consistent with results in these in vitro studies (Fig. 5A), ChIP assay showed the in vivo occupancy of the SCT ERE-half site with ERRa was greatly induced by water deprivation, saline drinking, and 4 h after the ICV-ANGII administration (5.83-fold) in the mouse hypothalamus (Fig. 5C). In summary, the above findings indicate the increased expression and binding of ERRa with the SCT ERE-half site in the mouse hypothalamus is a mechanism for the elevated SCT expressions in response to hyperosmolality and central ANGII stimulation.

Discussion
By reviewing the 59 sequence (1800 bp upstream of the transcriptional start site) of the mouse SCT gene, an ERE-half site, a putative binding site for the orphan nuclear receptor ERR, is present at 2149 bp (Fig. 1A). In the past, there were several studies revealing essential regulatory sequence motifs within the human and rat SCT promoter [15,16,39], and recently, the mouse SCT gene has been shown to be regulated by Sp1/Sp3 interacting with two GC-boxes (at 2125 and 2158) and NeuroD binding with an E-box motif (at 2167), [17,18]. In the present study, we provided evidences that ERRa interacting with the ERE-half site in the mouse SCT promoter is implicated for mediating central effects of ANGII.
There have been studies suggesting estrogen plays functional roles in fluid regulation in rodent and human [40][41][42]. ERRs are closely related to ERs, with which they share almost identical DBD (68% amino acid homology) [43][44][45]. In view of its close relationship with the ER, and that ERRa is often shown to act synergistically with ERa [25], ERRa is hypothesized as a possible candidate that participates in osmoregulation. In the present study, ERRa expression was revealed to be responsive to ANGII stimulation in the hypothalamic cells, and in the lamina terminalis and PVN of mouse brain. This is similar to previous study on ERa that water deprivation led to increased ERa expression in the SFO [46]. ERa is localized extensively in the osmosensitive neurons in the lamina terminalis and the SON, whereas ERb is prominently expressed in the VP magnocellular neurons of rat hypothalamus [47,48]. Likewise, using immunohistochemical staining and realtime PCR, the present study showed that ERRa proteins and transcripts were localized in the lamina terminalis and PVN, which are important brain regions for the control of VP secretion and drinking behavior.
In agreement with its expression patterns in high energy demand tissues, ERRa is generally recognized as a transcription factor that regulates expression of genes involved in bioenergetics [49,50], as a biomarker for several human cancers [51][52][53], bone homeostasis [54], as well as in cell proliferation and migration [55,56]. The ERRa knockout mice are viable, fertile and display no gross anatomical alterations, but have reduced body weight with less peripheral fat mass [57]. More recently, a physiological study of ERRa deficient (ERRa 2/2 ) mice have identified the potential of ERRa in regulating blood pressure and water homeostasis, by controlling renal sodium and potassium homeostasis and the RAS [20]. In summary, phenotypic analysis revealed that the ERRa 2/2 mice have reduced water intake, urine volume, and increased Na + in blood. These indicate that ERRa is dipsogenic and diuretic. Also, ERRa knockout mice are deficient in renal Na + /K + handling with a mechanism that favor Na + retention, leading to the increased blood Na + and thus increased water reabsorption and decreased urine volume. Using genomewide ChIP-on-ChIP analysis and expression profile comparison of ERRa 2/2 and WT mice, the study also identified a number of target genes regulated by ERRa in kidney. Some of these are genes involved in water regulation, including the RAS genes (Ren1, Agt, Ace2), VP receptor (Avpr1a, Avpr2), and tonicity-responsive element (TonE) binding protein (TonEBP). Our findings show that ANGII upregulates ERRa in the lamina terminalis and PVN in the brain; while Tremblay et al. suggested that ERRa could downregulate RAS genes in kidney, which might work as a negative feedback control [20]. Compatible with our study that shows ANGII can stimulate ERRa, and thus SCT and VP expression; VP receptor genes in kidney were found to be downregulated in the ERRa 2/2 mice [20], indicating that ERRa is responsible for intensifying the renal actions of VP. In summary, this report suggests new functions of ERRa in osmoregulation by regulating SCT's action in the brain. In view of an indispensable role of SCT and SCTR axis in mediating functions of ANGII in the central nervous system, this study not only confirmed, but also filled in the missing gap between ANGII and SCT in this newly discovered osmoregulatory pathway.