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. Author manuscript; available in PMC: 2023 Aug 23.
Published in final edited form as: FEBS Lett. 2018 Aug 10;592(16):2760–2768. doi: 10.1002/1873-3468.13199

Phenobarbital-induced phosphorylation converts nuclear receptor RORα from a repressor to an activator of the estrogen sulfotransferase gene Sult1e1 in mouse livers

Muluneh Fashe 1,*, Takuyu Hashiguchi 1,*, Myeongjin Yi 1, Rick Moore 1, Masahiko Negishi 1
PMCID: PMC10445657  NIHMSID: NIHMS982178  PMID: 30025153

Abstract

The estrogen sulfotransferase SULT1E1 sulfates and inactivates estrogen, which is reactivated via desulfation by steroid sulfatase, thus regulating estrogen homeostasis. Phenobarbital (PB), a clinical sedative, activates Sult1e1 gene transcription in mouse livers. Here, the molecular mechanism by which the nuclear receptors CAR, which is targeted by PB, and RORα communicate through phosphorylation to regulate Sult1e1 activation has been studied. RORα, a basal activity repressor of the Sult1e1 promoter, becomes phosphorylated at serine 100 and converts to an activator of the Sult1e1 promoter in response to PB. CAR regulates both the RORα phosphorylation and conversion. Our findings suggest that PB signals CAR to communicate with RORα via serine 100 phosphorylation, converting RORα from transcription repressor to activator of the Sult1e1 gene and inducing SULT1E1 expression in mouse livers.

Keywords: Phosphorylation, sulfotransferases, Nuclear receptors, RORα, CAR, phenobarbital, estrogen, liver

Introduction

Sulfotransferases (SULTs) are a family of cytosolic enzymes that transfer the sulfonate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to numerous endobiotics and xenobiotics (Negishi et al. 2001; Sato et al. 2009; Falany, Krasnykh, and Falany 1995; Gamage et al. 2006). Estrogen sulfotransferase (SULT1E1 or EST) is the only enzyme that sulfates estradiol with high affinity and efficacy at nM Km (Zhang et al. 1998; Falany, Krasnykh, and Falany 1995). Sulfated estradiol is an inactive hormone and can be reactivated through desulfation by steroid sulfatase (STS) ((Nakano et al. 2004; Lindsay et al. 2008; Cole et al. 2010; Purohit et al. 1994; Hobkirk 1993). SULT1E1 is a key enzyme that maintain the metabolic homeostasis of estrogen activity (Pasqualini et al. 1997; Falany, Krasnykh, and Falany 1995). Thus, any disruption of its expression can be detrimental in estrogen-dependent biological processes as well as disease developments (Tong et al. 2005; Chai et al. 2015).

Nuclear receptors are known to regulate hepatic SULT1E1 expression (Runge-Morris, Kocarek, and Falany 2013; Kodama and Negishi 2013), two of which are CAR and RORα. Whereas SULT1E1 is barely expressed in the liver of normal C57BL/6 mice under normal feeding conditions, it becomes highly expressed in C57BL/6-derived diabetogenic/insulin insensitive (db/db) or Staggerer mice (Song et al. 1995; Kang et al. 2007). CAR activators such as diallyl sulfide and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), induced SULT1E1 in mouse livers (Alnouti and Klaassen 2008; Sueyoshi et al. 2011). Insulin is known to repress CAR activation in mouse livers (Yasujima et al. 2016). Staggerer mouse bear the mutated RORα gene that expresses the non-functional truncated RORα. Therefore, both CAR and RORα appeared to regulate the Sult1e1 gene. However, whether and how do the two nuclear receptors communicate to regulate the gene remains unexplored.

Nuclear receptors possess highly conserved phosphorylation motif within their DNA binding domain (DBD) in both mice and humans (Negishi 2017; Hori, Moore, and Negishi 2016). CAR conserves this motif at threonine 38 and utilizes phosphorylation to regulate its activation (Shizu et al. 2017; Shizu et al. 2018; Mutoh et al. 2013; Mutoh et al. 2009). For example, phenobarbital, a widely used sedative drug, elicits an epidermal growth factor receptor-mediated cell signal that enables protein phosphatase 2A to dephosphorylate threonine 38 for CAR activation. Mouse estrogen receptor α (ERα) conserves the corresponding motif at serine 216, which was found to be phosphorylated in immune cells such as neutrophils (Shindo et al. 2013). Farnesoid X receptor (FXR) became phosphorylated at serine 154 in response to its agonist in mouse livers and this phosphorylation was linked to FXR activation, inactivation, and degradation (Hori, Moore, and Negishi 2016). Thus, this conserved phosphorylation initiated the integration of the nuclear receptors as a common regulatory signal. RORα also contains this motif at serine 100, although phosphorylation of this residue has not yet been investigated.

In the present study, we have examined whether RORα regulates the Sult1e1 promoter through phosphorylation at its serine 100 and, if so, what is the role of CAR in this RORα-mediated regulation. Experiments utilized both Staggerer and CAR KO mice for in vivo studies including chromatin immunoprecipitation assays as well as phosphomimetic and non-phosphomimetic mutants of RORα (RORα S100D and RORα S100A, respectively) for Huh-7 cell-based reporter assays. A phospho-S100 peptide antibody was used to detect phosphorylation of RORα at serine 100. Experimental findings will be presented to characterize RORα as a transcriptional repressor and CAR as the master regulating the function of RORα through serine 100 phosphorylation to activate the Sult1e1 promoter in response to phenobarbital.

Methods and Materials

Materials:

Antibodies against RORα: #sc-6062 and #PP-H3910-00 were from Santa Cruz Biotechnology, Inc. (TX, USA) and Perseus Proteomics, Inc (Tokyo, Japan), respectively; TOPO TA cloning kit with pCR2.1 TOPO and Power SYBR Green Master Mix from Invitrogen (NY, USA); an antibody to GFP-HRP (#ab6663) was from Abcam; HRP-conjugated anti-FLAG M2 (S8592) was from Sigma-Aldrich. Anti-P-Ser100 peptide antibody (αP-Ser 100) against hRORα was generated by Genescript (NJ, USA). Max DNA Polymerase was from Takara (CA, USA); FuGENE 6 from Roche Diagnostics (IN, USA); Bio-Rad Protein Assay reagent was from Bio-Rad (NC, USA); WesternBright ECL and Sirius HRP substrate was from Advansta (CA, USA); Protein Kinase C and Dual Luciferase Assay System were from Promega (WI, USA); phosphatase inhibitor cocktail-2 and its-3 from Sigma-Aldrich (MO, USA); DNA polymerase Klenow fragment and restriction enzymes were from New England Biolabs (MA, USA); ChIP IT Express kit was from Active Motif (CA, USA).

Plasmids:

pGL3 vectors harboring mouse Sult1e1 promoter were constructed in our previous study (Kodama et al. 2011). Human RORα was sub-cloned into pCR2.1 vector from hRORαWT/pcDNA3.1, hRORα S100A/pcDNA3.1, and hRORα S100D/pcDNA3.1. RORE/p-tk-luc (Giguere et al. 1994) was generously obtained from Anton Jetten, NIEHS. All constructs were verified by nucleotide sequencing. Luciferase reporter plasmid harboring the 237 bp (−167/+87) of the mouse Sult1e1 promoter., called this −167bp was constructed with pGL3 plasmid. Thirteen bps (−112/−99) were internally deleted within the context of the −167 bp promoter to produce −167 bp ∆DR1 promoter. In pGL3

Animals:

Mice were maintained under the standard condition at the NIEHS. Animal experiments were conducted per protocols approved by the animal ethics committee at NIEHS/NIH. Both Car+/+and Car−/− mice in C57BL/6 background were produced in house (Yamamoto et al. 2004). Staggerer mice (RORα sg/sg) were obtained from the Immunity, Inflammation, and Disease Laboratory, NIEHS. Phenobarbital (100 mg/kg body weight) in phosphate-buffered saline (PBS) or PBS was intraperitoneally injected into 10- to 15-week-old male mice for 6 or 24 hrs.

Cell culture and transfection.

The protocol used was detailed in our previous report (Hashiguchi et al. 2016). Briefly, COS-1 cells grown in DMEM supplemented with 10% FBS, penicillin (100U/ml) and streptomycin (100μg/ml) were transfected with given plasmids using FuGENE 6® per manufacturer’s instructions (Roche Diagnostics).

Real-time PCR (qPCR):

Total RNAs prepared from mouse livers was reverse transcribed using a High Capacity cDNA Archive kit (Life Technologies). Taqman probes used were Mm00499178_m1 and Mm99999915_g1 for SULT1E1 and GAPDH, respectively. qPCR was performed with CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

ChIP assays:

About 100 mg liver samples were sliced and incubated in a 1% formaldehyde for 10 min followed by a 5-minute incubation in glycine at room temperature before homogenization. Nuclear suspensions were obtained by filtering homogenates through a Cell Strainer (Falcon), incubated in the lysis buffer and sonicated per a manufacturer’s guideline (Activ motif). Immunoprecipitations were performed overnight with an RORα (#PP-H3910-00) antibody or a p-Ser100 using protein G conjugated magnetic beads. The beads were washed with ChIP buffers, chromatins were eluted and the cross-linking was reversed prior to PCR (36 cycles) amplification using primers targeting the proximal region (−168/+68) of mouse Sult1e1 promoter.

Extraction of nuclear proteins:

Huh-7 cells in a 10cm dish were transfected with human RORα expression vectors (RORα WT/pcDNA3.1, RORα S100A/pcDNA3.1 or RORαS100D pcDNA3.1) without or with mouse CAR. The cells were homogenized with a buffer consisting of 10 mM HEPES, pH 7.6, 10 mM KCl and 1.5 mM MgCl2 in the presence of 0.3% NP-40 and centrifuged at 2000 g for 5 min. The pellet was suspended in a lysis buffer composed of 10 mM HEPES, 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA and 1 mM Na3VO4 in 20% glycerol and incubated at 4 °C for 30 min after addition of 400 mM NaCl. The nuclear proteins were obtained by centrifuging the lysate at 38000 rpm for 30 min.

Gel shift assays:

Gel mobility shift assays were performed as described in our previous study (Mutoh et al. 2009). Briefly, a 5’-overhanging double stranded oligonucleotides were filled in with DNA polymerase Klenow fragment in the presence of [α−32P]dATP or with T4 polynucleotide kinase in the presence of [γ−32P] ATP to generate radioactive probes. For super-shifts, mixtures were incubated with 1 µg of anti-RORα antibody at room temperature for 10 min. Nucleotide sequences used as probes are described in each figure legends where appropriate.

In vitro phosphorylation assays:

GST-hRORα WT and its alanine mutant (RORα S100A) were bacterially expressed and purified for substrates through glutathione sepharose 4B (GE Healthcare). These purified proteins (5 μg) were incubated with 50 mU of pure PKC in 30 μl of TBS buffer containing 2 mM CaCl2, 2 mM DTT, 17 mM MgCl2, 0.6mg/ml phosphatidyl serine and 170 μM ATP in the presence or absence of 0.5 μl (50 mU) of PKC at 30°C for 1 h for subsequent Western blot analysis using αP-Ser100 antibody or anti-GST antibody in 5% BSA solution (1:500 dilution).

Western blot:

Five μg of RORα proteins for the in vitro phosphorylation assay or 5 μg for the Huh-7 cells nuclear contents were electrophoresed on a 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes at 10 V or 15 V for 1 h. The membranes were probed with a given antibody overnight at 4°C, reacted with HRP-conjugated goat anti-rabbit (#Sc-2004), monkey anti-mouse (#Sc-2314) or donkey anti-goat IgG secondary antibodies using WesternBright ECL or Sirius kit, visualized by a C-DiGit Chemiluminescent Western Blot Scanner (LI-COR, Inc).

Co-IP assays:

COS-1 cells co-transfected with given plasmids for 24 h were lysed in Immunoprecipitation (IP) buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, protease inhibitors, and phosphatase inhibitor cocktails. Lysate was collected by centrifugation, sonicated, and resulted supernatants (500 µg protein) were incubated with anti-GFP antibody-conjugated agarose at 4°C for 4 h. Resins were washed with the IP buffer, subjected to SDS-PAGE for Western blot analysis.

Reporter assays:

COS-1 cells were co-transfected with a given RORα expression vector and p-tk-luc plasmids harboring RORE (Giguere et al. 1994) and control Renilla luciferase plasmids for 24 hrs. Cell lysates were prepared to measure firefly luciferase activities using Dual Luciferase Assay System and normalized with Renilla luciferase activities.

Statistical analysis.

The statistical significance was analyzed by multiple t-test (Holm-Sidak method) with alpha = 0.05 on GraphPad prism software suite.

Results

SULT1E1 expression in mouse livers:

Expression of mouse SULT1E1 mRNA was studied in mouse livers before or after phenobarbital treatment. First, CAR KO mice were utilized to examine how CAR regulated these expressions. The basal expression was increased over 2-fold in the liver of CAR KO mice. Phenobarbital treatment increased SULT1E1 mRNA levels about 20-fold in the liver of CAR WT mice, while the mRNA was not increased in that of CAR KO mice (Fig. 1A). Subsequently, C57BL/6-derived RORαsg/sg mice, which lack functional RORα, and C57BL/6 (hereafter, just called RORα KO and WT for practical reason) were utilized. Levels of basal SULT1E1 mRNA expression were about 130-fold higher in the liver of RORα KO mice over the RORα WT mice (Fig. 1B). Phenobarbital treatment increased SULT1E1 mRNA about 10-fold in in the liver of RORα WT mice. About 4-fold increase was observed in RORα KO mice but was not statistically significant. Thus, the basal expression was repressed by both CAR and RORα whereas the phenobarbital-induced expression depended on CAR and was significantly attenuated in the absence of RORα (Figure 1A&B).

Figure 1. Hepatic PB-induced expression of the mouse Sult1e1 gene in CAR KO or RORα KO mice.

Figure 1.

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A, 10–15 weeks old Car+/+and Car−/− mice (n=3) and B, 10 to15 weeks old RORα +/+ and Staggerer (RORα sg/sg) mice (n = 3) were injected with PBS or 100 mg/kg PB for 24 hrs. The livers were collected and the total RNA was isolated for the cDNA synthesis. qPCR was performed for determination of Sult1e1 mRNA levels using GAPDH mRNA as endogenous control. The asterisk (*) between the groups represents statistically significant difference (p<0.05) and ns stands for statistically insignificant differences (p>0.05).

Phosphorylation of serine 100:

Since phenobarbital is known to regulate CAR through phosphorylation, we examined the hypothesis that RORα might also be regulated via phenobarbital-induced phosphorylation, by focusing on the conserved phosphorylation motif within the DBD. RORα conserves this motif at serine 100 within its DBD (Figure 2A). Bacterially expressed recombinant RORα proteins were incubated with protein kinase C in in vitro kinase assays and were subjected to Western blot analysis with αP-Ser100 antibody specific to RORα (Fig. 2B). This antibody detected RORα WT but not RORα S100A, indicating that serine 100 was phosphorylated. Gel shift assays with in vitro translated RORα proteins showed that, consistent with phosphorylation (Fig. 2B), RORα S100D was unable to bind a known response DNA element (RORE and HRE) for RORα binding (Fig. 1C). Thus, RORα can be phosphorylated at serine 100, disabling its DNA binding ability.

Figure 2. Characterization of phosphorylated RORα at S100.

Figure 2.

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A, The sequence of DNA binding domain in RORα. The amino acid sequences including a phosphorylation site between RORα, CAR, HNF 4α and FXR was aligned below as shown in blue. B, In vitro phosphorylation of the serine 100 for RORα by recombinant PKCs. GST-RORα was purified and used as the substrate. After phosphorylation, the proteins were subjected to Western blotting using αP-Ser100 peptide antibody. C, Gel shift assays. In vitro transcribed/translated proteins of hRORα was incubated with 32P-labeled probe, electrophoresed on a polyacrylamide gel, and detected by autoradiography using X-ray film. Probes used: 5’-GATCTCGACTCGTATATCAAGGTCATGCTG for RORE or 5’-GATCTCGACAGTGACCCTTTTAACCAGGTCAGTGAG

RORα regulation of the Sult1e1 promoter:

Having these observations, the −167 bp Sult1e1 promoter was examined. This promoter was placed in front of luciferase reporter gene and co-expressed with RORα WT, non-phosphomimetic RORα S100A or phosphomimetic RORα S100D in COS-1 cells for subsequent luciferase assays (Fig. 3A). Whereas this promoter was highly active, it was effectively repressed by RORα WT and RORα S100A, but not RORα S100D (Fig. 3A). Thus, RORα functioned as a repressor for the −167 bp Sult1e1 promoter in cell-based reporter assays. ChIP assays were employed to examine RORα binding to the Sult1e1 promoter in mouse livers before or after phenobarbital treatment. ChIP with an anti-RORα antibody showed that RORα bound the promoter before treatment and continued to bind it even after treatment (Fig. 3B). On the other hand, αP-Ser100 antibody detected RORα only after phenobarbital treatment (Fig. 3B). The binding pattern before treatment was consistent with RORα that repressed the basal SULT1E1 mRNA expression in mouse livers. RORα appeared to become phosphorylated at serine 100 in response to phenobarbital; this phosphorylated RORα was retained on the promoter.

Figure 3. RORα binds the Sult1e1 promoter and suppresses the gene expression.

Figure 3.

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A, a 167 bp (−167/+87) DNA fragment of the mouse Sult1e1 promoter was placed in front of luciferase reporter gene and co-expressed with ectopic RORα WT, RORα S100A, RORα S100D, CAR WT, CAR T38A or CAR T38D in COS-1 cells. The cells were lysed and the firefly luciferase activities were measured by using the Dual Luciferase Assay System and normalized with Renilla luciferase activities. B, Mouse liver chromatins for ChIP assays were prepared per the Activmotif’s guidelines and the RORα binding onto the proximal Sult1e1 promoter was examined. DNA fragment amplification primers were: 5’-ACCCAAAGGGGAGAAACAGC-3’ and 5’-TCGAATGGCAGCACGATTCT-3’ (−168/+68). Two mice were used in each group and each column represent an individual mouse.

Our ChIP assays amplified the 236 bp (−168/+68) within which the −118/−94 bp region contains nested a DR1 motif overlapped with an A/T-rich sequence, both of which can be RORα binding elements (Fig. 4A). This DR-sequence was deleted within the context of the −167 bp promoter to examine whether RORα regulated the −167 bp Sult1e1 promoter activities through the motif in cell-based reporter assays. As expected, deletion disabled RORα WT and RORα S100A promoter regulation (Fig. 4B). On the other hand, to our surprise, the RORα S100D mutant was found to activate this deletion promoter (Fig. 4B). Apparently, this deletion may have created a promoter mimicking chromatin structure through which phosphorylated RORα activated the Sult1e1 promoter in mouse livers. This observation also revealed that the DR1 motif overlapped by A/T rich sequence was essential for the basal activity of the promoter. In addition to RORα, other nuclear factors may also bind this motif. For instance, (Gong et al. 2007) identified a DR4 motif overlapping the DR1 motif that can possibly bind CAR.

Figure 4. RORα binds to the proximal region of the mSULT1E1 promoter.

Figure 4.

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A, DNA sequences and motif map of proximal Sult1e1 promoter. B, The −167 bp promoter or-Luc or the −167 bp DR1promoter-Luc was co-expressed with expression hRORα WT, RORα S100A or hRORα S100D in COS-1 cells. Luciferase activities were normalized with Renilla luciferase activities. C, RORα WT, RORα S100A or RORα S100D was co-expressed in the absence or presence of ectopic CAR in Huh-7 cells for 48 hrs. from which nuclear extracts were prepared and used in gel shift assays using oligonucleotide (−118/−94) as prob. D, Nuclear extracts were subjected to Western blot analysis to confirm expression levels of RORα proteins. E, Chromatins were prepared from livers of CAR WT and CAR KO mice treated with PB for 6 hrs. and subjected to ChIP assays as described in the Methods section and in the legend of Figure 3. In the graph on the right side, the band intensities of PCR amplified DNAs for each group from two different samples immunoprecipitated by p-RORα were quantified by using gel imaging software (Image J/NIH).

CAR regulation of RORα activities:

Given the fact that RORα underwent phosphorylation to regulate the Sult1e1 promoter in response to phenobarbital, CAR KO mice were used to examine whether CAR was involved in this. First, gel shift assays were employed, for which nuclear extracts were prepared from Huh-7 cells transfected with RORα in the presence or absence of CAR. Nuclear extracts containing RORα S100A were most effectively shifted in the absence of CAR but were barely shifted in the presence of CAR (Fig. 4C). Conversely, those containing the RORα S100D mutant greatly increased the shifted band in the presence of CAR (the right-side lane of Fig. 4C). Although confirmation with super-shift was not possible, a weak gel-shift band appeared with CAR-transfected extracts could be an indication of CAR binding to DR4. Western blots showed the expression levels of these proteins (Fig. 4D). Since nuclear expression levels of RORα proteins were nearly identical in the presence or absence of CAR, these results suggested that CAR repressed RORα binding to the promoter while stimulated the binding of RORα S100D. Subsequently, ChIP assays were performed to examine RORα binding to Sult1e1 promoter in livers of CAR WT and CAR KO mice before or after phenobarbital treatment (Fig. 4E). Confirming the observation in Fig. 3B, in CAR WT mice, an RORα antibody detected RORα bound the promoter in both before and after phenobarbital treatment. Moreover, αP-Ser100 peptide antibody detected RORα only after phenobarbital treatment. With CAR KO mice, it was found that a P-S100 peptide antibody, as well as a RORα antibody, detected RORα on the promoter before but not after treatment (Fig. 4E). These observations suggested that CAR regulated phosphorylation and retained phosphorylated RORα on the promoter in response to phenobarbital.

Discussion

SULT1E1 mRNA augmented its basal expression and attenuated the rate of phenobarbital-induced increase in the liver of RORα KO mice. Consistent with its role as a transcription repressor, RORα was found to suppress the Sult1e1 promoter in cell-based reporter assays and was detected on the promoter in ChIP assays with mouse livers. RORα became phosphorylated at serine 100 and was retained on the activated Sult1e1 promoter in response to phenobarbital treatment, indicating the role of phosphorylated RORα as a transcription activator which was further confirmed by the finding that the phosphomimetic RORα S100D mutant activated the Sult1e1 promoter in cell-based reporter assays. Thus, phenobarbital-induced phosphorylation converted RORα repressing the Sult1e1 gene to activating it. Observations obtained with CAR KO mice demonstrated that CAR, acting as a master regulator, is essential for this conversion of RORα through serine 100 phosphorylation.

Serine 100 and threonine 38 are the conserved phosphorylation motifs within the DBDs of RORα and CAR, respectively. CAR inactivates its constitutive activation activity by phosphorylation at threonine 38. In response to phenobarbital, threonine 38 is dephosphorylated by protein phosphatase 2A to activate CAR (Mutoh et al. 2013; Mutoh et al. 2009). Conversely, it has now been determined that RORα becomes phosphorylated at serine 100 in response to phenobarbital. Therefore, RORα reciprocates serine 100 phosphorylation with CAR dephosphorylation at threonine 38 (Fig. 5). In the absence of phenobarbital, CAR represses phosphorylation of serine 100 since this residue is phosphorylated in CAR KO mice before phenobarbital treatment. In the presence of CAR, serine 100 can only be phosphorylated in response to phenobarbital. Taken these observations in consideration, CAR phosphorylated at threonine 38 represses RORα phosphorylation at serine 100, enabling non-phosphorylated RORα to bind and repress the Sult1e1 promoter. Conversely, once threonine 38 dephosphorylated, CAR is no longer able to retain RORα in its non-phosphorylated form, disabling RORα’s ability to repress the Sult1e1 promoter (Fig. 5). The phosphorylated RORα remained on the promoter to activate it, by mostly likely acting as a co-activator, since phosphorylation inhibited direct DNA binding of RORα. Consistent with these observations, RORα S100D mutant activated the deletion promoter in reporter assays, which may have mimicked a state of the endogenous promoter. Both CAR and RORα are constitutive active nuclear receptors, for which the so-called “endogenous ligands” equivalent to steroid hormones for nuclear hormone receptors may not be present. Instead, CAR and RORα utilize the common phosphorylation motif for their communication to activate the Sult1e1 promoter in response to phenobarbital. This concept of phosphorylation-mediated mechanism can be applied to many nuclear receptors which are constitutively activated.

Figure 5.

Figure 5.

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Proposed mechanism for regulation of SULT1E1 expression by CAR and RORα. In this, the LBD of nuclear receptors are depicted in asymmetric shape. CAR utilizes different surfaces to form a homodimer and a heterodimer with RXR (Shizu et al. 2017; Shizu et al. 2018). Non-phosphorylated RORα binds and suppress the Sult1e1 promoter before PB treatment, although the possibility remains that this is an indirect binding through an additional protein. After PB, CAR become dephosphorylated at threonine 37 and retains RORα phosphorylated at serine 100 on the promoter as a co-activator. Whether phosphorylated RORα directly interacts with and co-activates CAR remains not yet proved. However, if it does, the CAR indicated as a PXR heterodimer in the hatched box may utilizes an opposite surface to interact with phosphorylated RORα. Our observations indicated phosphorylated CAR may prevent RORα from being phosphorylated. It remains elusive whether this prevention takes place either in the cytoplasm or in the nucleus, since CAR is phosphorylated in the cytoplasm as well as can be re-phosphorylated in the nucleus (Hori et al., 2016). Our ChIP assays also suggested that PB altered the Sult1e1 promoter in a CAR-independent manner. Our previous work demonstrated PB-induced but CAR-independent chromatin modification of the Cyp2b10 promoter (Ohno et al. 2014). Hatched arrow indicates such modification that may occur with the Sult1e1 promoter.

Although the molecular mechanism utilized by CAR to regulate phosphorylation of RORα remains unexplored, the scenario of which may be speculated based on our previous findings. This conserved motif was first characterized as a site phosphorylated by protein kinase C (PKC) but can be phosphorylated by additional kinases. For example, p38 MAPK formed a complex with CAR and phosphorylated threonine 38 in the nucleus, suggesting that threonine 38 dephosphorylated in the cytoplasm can scaffold p38 MAPK in the nucleus (Hori, Moore, and Negishi 2016). p38 MAPK appeared also to phosphorylate RORα at serine 100 in in vitro kinase assays (Supplemental Fig.1). Moreover, CAR and RORα interacted as shown by co-immunoprecipitation assays (Supplemental Fig. 2). Thus, the scenario may be hypothesized that non-phosphorylated CAR scaffolds p38 MAPK to phosphorylate RORα at serine 100. To repress RORα phosphorylation, phosphorylated CAR may stimulate this phosphorylation or prevent it, the molecular mechanism for which remains beyond our speculation. Nevertheless, both CAR and RORα are co-localized in both phosphorylated and non-phosphorylated forms in the nucleus, possibly allowing them to communicate to regulate a gene. One outstanding question raised is how can CAR, RORα and likely protein kinase and phosphatase be compartmentalized on the promoter during these sequential processes.

In conclusion, the molecular mechanism by which phenobarbital utilizes two nuclear receptors to activate the Sult1e1 gene in mouse livers has now been described. In response to PB, CAR and RORα communicated through phosphorylation at the conserved site within their DBDs in this activation mechanism. Forty-one nuclear receptors conserve this phosphorylation motif and they may integrate this conserved phosphorylation as a communication language to form signal networks, regulating tremendously diversified biological functions.

Supplementary Material

Supp FigS1

Supplemental figure 1. p38 mediated phosphorylation of RORα at serine 100. COS-1 cells were transfected with GFP-RORα WT, GFP-RORα S100A or mock vector along with or without FLAG-human p38 for 24 h. The transfected cells were lysed and subjected to immunoprecipitation as described in Materials and Methods section. Western blot analysis was performed with αP-Ser100 peptide antibody and anti-RORα antibody for IP samples. For inputs, 54 μg of cell lysates was used for electrophoresis and detected with anti-GFP antibody, anti-FLAG antibody and β-actin.

Supp FigS2

Supplemental figure 2. Co-immunoprecipitation of CAR with RORα. COS-1 cells were transfected with GFP-CAR and FLAG-RORα for 24 h, from which whole extracts were incubated with a GFP antibody-conjugated agarose resin to precipitate CAR, for subsequent Western blot analysis with a FLAG antibody to detect RORα. For input, 5 μg of the total protein was applied. The β-actin level was also evaluated as a positive control.

Acknowledgements

We thank DNA sequence core at NIEHS for their excellent assistance. Human RORα1/pCMX and RORE/p-tk-luc were kindly provided from Anton Jetten (NIEHS). This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences: Z01ES1005-01

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigS1

Supplemental figure 1. p38 mediated phosphorylation of RORα at serine 100. COS-1 cells were transfected with GFP-RORα WT, GFP-RORα S100A or mock vector along with or without FLAG-human p38 for 24 h. The transfected cells were lysed and subjected to immunoprecipitation as described in Materials and Methods section. Western blot analysis was performed with αP-Ser100 peptide antibody and anti-RORα antibody for IP samples. For inputs, 54 μg of cell lysates was used for electrophoresis and detected with anti-GFP antibody, anti-FLAG antibody and β-actin.

NIHMS982178-supplement-Supp_FigS1.pptx (203.2KB, pptx)
Supp FigS2

Supplemental figure 2. Co-immunoprecipitation of CAR with RORα. COS-1 cells were transfected with GFP-CAR and FLAG-RORα for 24 h, from which whole extracts were incubated with a GFP antibody-conjugated agarose resin to precipitate CAR, for subsequent Western blot analysis with a FLAG antibody to detect RORα. For input, 5 μg of the total protein was applied. The β-actin level was also evaluated as a positive control.

NIHMS982178-supplement-Supp_FigS2.pptx (154.8KB, pptx)

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