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Nuclear receptor Pregnane X Receptor (PXR; NR1I2) has transcriptional regulation functions for energy homeostasis in the liver. Mouse PXR has a conserved phosphorylation motif at serine 347 (serine 350 in humans) within the ligand-binding domain. PXR phosphorylated at this motif is expressed in mouse livers in response to fasting. Mice with a PXR∗Ser347Ala knockin mutation (PXR KI) were generated to block phosphorylation, and utilized to investigate the role of Ser347 phosphorylation in vivo. PXR KI mice had decreased body weight at 8-weeks of age and had much greater weight loss after fasting compared with PXR WT mice. The cDNA microarray analysis of hepatic mRNAs showed that cell death or apoptotic signaling was induced in fasting PXR KI mice. Moreover, increasing hepatic lipids, triglycerides and the development of hypertriglyceridemia were observed in fasting PXR KI mice. These findings are indicative that blocking phosphorylation prevents mice from maintaining hepatic energy homeostasis. Thus, phosphorylated PXR may be an essential factor to prevent the liver from developing damage caused by fasting.

Visual Abstract

p38 phosphorylation levels and its involvement in the Cyp2b10 gene expression.

Characterization of interactions in CAR, GADD45B, MKK6, and p38 MAPK.

Expression of the GADD45 (A, B, and G) and Cyp2b10 genes following PB treatment.

Contact regions of CAR to GADD45B.

Phenobarbital, a nongenotoxic hepatocarcinogen, induces hepatic proliferation and promotes development of hepatocellular carcinoma (HCC) in rodents. Nuclear receptor constitutive active/androstane receptor (NR1I3/CAR) regulates the induction and promotion activities of phenobarbital. Here, it is demonstrated that phenobarbital treatment results in dephosphorylation of a tumor suppressor p38 MAPK in the liver of C57BL/6 and C3H/HeNCrlBR mice. The molecular mechanism entails CAR binding and inhibition of the growth arrest and DNA-damage-inducible 45 beta (GADD45B)-MAPK kinase 6 (MKK6) scaffold to repress phosphorylation of p38 MAPK. Phenobarbital-induced hepatocyte proliferation, as determined by BrdUrd incorporation, was significantly reduced in both male and female livers of GADD45B knockout (KO) mice compared with the wild-type mice. The phenobarbital-induced proliferation continued until 48 hours after phenobarbital injection in only the C57BL/6 males, but neither in males of GADD45B KO mice nor in females of C57BL/6 and GADD45B KO mice. Thus, these data reveal nuclear receptor CAR interacts with GADD45B to repress p38 MAPK signaling and elicit hepatocyte proliferation in male mice.Implications: This GADD45B-regulated male-predominant proliferation can be expanded as a phenobarbital promotion signal of HCC development in future studies.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/8/1309/F1.large.jpg. Mol Cancer Res; 16(8); 1309–18. ©2018 AACR.

Phosphorylation and interactions of p38 MAPK.

Structure modeling for CAR-GADD45G heterodimers.

Legends of Supplemental Figures

Androgen receptor, which regulates diverse biological processes for cell fate decisions, forms a homodimer in the cytoplasm and is monomerized by activation for nuclear translocation. Ser815 phosphorylated AR is expressed in mature prostates, with levels decreased by castration in mice or prostate cancer progression in humans. Here, we have examined the functional and biological roles of phosphorylation. AR phosphorylation at Ser815 stabilized homodimer formation in the cytoplasm, interrupting DHT-response nuclear translocation. cDNA microarray studies in castrated mouse prostates implied castration attenuates ER stress responses, suggesting AR phosphorylation acts on ER stress responses. In addition, AR Ser815Asp phospho-mimetic mutant expression augmented ER stress-induced death in PC-3 cells. These results suggested that phosphorylation at AR Ser815 modulates AR functions for maintaining the prostate.

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Human fetal, but not adult, livers express appreciable amounts of SULT1E1 protein, which is mimicked in human hepatoma-derived HepG2 cells cultured in high glucose (450 mg/dl) medium. Here, we have investigated this glucose signal that leads to phosphorylation of nuclear receptor RORα (NR1F1) at Ser100 and the transcription mechanism by which phosphorylated RORα transduces this signal to nuclear receptor HNF4α, activating the SULT1E1 promoter. The promoter is repressed by non-phosphorylated RORα which binds a distal enhancer (−943/−922 bp) and interacts with and represses HNF4α-mediated transcription. In response to high glucose, RORα becomes phosphorylated at Ser100 and reverses its repression of HNF4α promoter activation. Moreover, the casein kinase CK1α, which is identified in an enhancer-bound nuclear protein complex, phosphorylates Ser100 in in vitro kinase assays. During these dynamic processes, both RORα and HNF4α remain on the enhancer. Thus, RORα utilizes phosphorylation to integrate HNF4α and transduces the glucose signal to regulate the SULT1E1 gene in HepG2 cells and this phosphorylation-mediated mechanism may also regulate SULT1E1 expressions in the human liver.

Background Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia’s inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability. Methods Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability. Results Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests. Conclusions Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases.

Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.

Estrogen receptor α (ERα) conserves a phosphorylation motif at Serine 216. This site constitutes a protein kinase C phosphorylation motif located within the DNA binding domain (DBD) of ERα. The liver plays a critical role in the regulation of metabolism of various xenobiotics, fatty acids, and cholesterol or endogenous compounds. Moreover, numerous metabolizing enzymes are mainly expressed in the liver. In this chapter, we describe several practical experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in livers upon phenobarbital (PB) treatment. Also, this phosphorylation regulates the expression of estrogen sulfotransferase gene (SULT1E1) which has an important role to sulfate and inactivate estrogen. In response to PB, the conserved motif within the DBD activates the Sult1e1 gene. When this motif was mutated, the activation of Sult1e1 was suppressed significantly. This chapter also describes the use of a phospho-peptide antibody (αP-S216) in the chromatin immunoprecipitation (ChIP) assay, and the co-immunoprecipitation (Co-IP) assay visualized by Western blot analysis.

Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter, we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with αP-S216 or neutrophil marker Ly6G antibody) and double immunofluorescence staining of mouse uterine sections prepared from C3H/HeNCrIBR females revealed that phosphorylated ERα was expressed in all infiltrating neutrophils during hormonal cycles but not in any other of the other uterine cells. Neutrophils infiltrate the uterus from the bloodstream. White blood cells (WBC) were prepared from peripheral blood of C3H/HeNCrIBR females or males and double immunostained. Blood neutrophils also expressed phosphorylated ERα but in only about 20% of cells in both sexes. Only the neutrophils expressing phosphorylated ERα spontaneously migrated in in vitro Transwell migration assays and infiltrated the uterus in mice.

The androgen receptor (AR) regulates male sexual development. We have now investigated AR homodimerization, hormone-dependent monomerization and nuclear translocation in PC-3 and COS-1 cells, by utilizing mutations associated with the androgen insensitivity syndrome: Pro767Ala, Phe765Leu, Met743Val and Trp742Arg. AR wild type (WT) was expressed as a homodimer in the cytoplasm, while none of these mutants formed homodimers. Unlike AR WT which responded to 1 nM dihydrotestosterone (DHT) to dissociate and translocate into the nucleus, AR Pro767Ala and Phe765Leu mutants remain as the monomer in the cytoplasm. In the crystal structure of the AR LBD homodimer, Pro767 and Phe765 reside closely on a loop that constitutes the dimer interface; their sidechains interact with the Pro767 of the other monomer and with the DHT molecule in the ligand-binding pocket. These observations place Phe765 at a position to facilitate DHT binding to Pro767 and lead to dissociation of the AR homodimer in the cytoplasm. This Pro-Phe Met relay may constitute a structural switch that mediates androgen signaling and is conserved in other steroid hormone receptors.

Circulating estrogens levels significantly decrease in menopause and levels off in postmenopausal women. Accordingly, the liver represses levels of enzymes and membrane transporters, thereby decreasing capability of inactivating and excreting estrogens. Women increasingly develop type 2 diabetes during or after menopause. Estrogens are known to promote liver diseases in these women. Here, we have found that the estrogen inactivating sulfotransferase (SULT1E1) and an ATP-binding cassette subfamily G member 2 (ABCG2), a gene encoding breast cancer resistance protein that exports sulfated estrogens, increased their expression levels in diabetic women but not men. For the sulfotransferase gene, phosphorylated nuclear receptors ERα and RORα, at Ser212 and Ser100, respectively, bind their response elements to activate the SULT1E1 promoter in women. This coordinated increase in estrogen inactivation and excretion, and the phosphorylated nuclear receptor-mediated gene activation could be a defense mechanism against toxicities of estrogens through inactivation and excretion in the livers of women.

The glucocorticoid receptor (GR) plays an important role in steroid-dependent regulation of metabolism, development, and the immune response in humans. Although GR is known to be activated by the binding of glucocorticoid, the mechanism of action is poorly understood. We investigated dimerization of GR in the cytoplasm and nuclear trans-localization in response to treatment with the ligand dexamethasone. GFP-tagged GR and FLAG-tagged GR were co-expressed in COS-1 cells, and cell lysates were subjected to co-immunoprecipitation assay with anti-GFP antibody to determine their dimerization. FLAG-GR was co-precipitated with GFP-GR in the cytoplasmic fraction of COS-1 cells. Treatment with the GR agonist dexamethasone significantly decreased the cytoplasmic interaction between FLAG- and GFP-GR, and significantly increased interaction of the GRs in the nuclear fraction. The two amino acids, Pro625 and Ile628 known to be located in GR-GR dimer interface, were mutated to alanine and the influence of the mutation on dimerization, ligand-dependent nuclear localization, and transcriptional activities were determined. Mutant GR showed a dramatic decrease in interaction in the cytoplasmic fraction and no detectable nuclear translocation in the presence or absence of dexamethasone. Furthermore, luciferase assays showed that mutant GR showed no detectable transcriptional activation via the GR-responsive DNA element (GRE) compared to the wild-type. Our results suggest that GR exists as a dimer in the cytoplasm and this dimerization may be essential for GRE-mediated transcriptional activation following ligand binding.

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

Androgen receptor (AR) regulates male sexual development and maintenance. AR forms a homodimer in the cytoplasm and monomerizes following hormonal activation, translocating to the nucleus in Cos-1 cells (Shizu et al. Scientific reports. 2019). Utilizing Ser815 of AR, the conserved phosphorylation residue within the ligand binding domains of steroid hormone receptors (NR3C), whether and how this phosphorylation regulates AR functions was investigated. While, like AR WT, a phosphomimic AR S815D mutant formed a homodimer in the cytoplasm, unlike the WT, this mutant remained as a homodimer in the cytoplasm even after hormone treatment. Apparently, Ser815 phosphorylation disabled AR’s capability to monomerize and nuclear translocate in Cos-1 cells. A phospho-Ser815 peptide antibody was used to detect phosphorylation of endogenous AR in mouse as well as human prostates. Immunohistochemistry showed phosphorylation present in both the cytoplasm and nucleus. Mouse prostates were cell fractionated in cell membrane, mitochondria, endoplasmic reticulum (ER) and cytosolic fractions for subsequent Western blot analysis. While AR was found in all of these fractions, phosphorylated AR was only detected in the ER and cytosolic fractions. A cDNA microarray analysis of PC-3 cells with ectopic expression of AR S815D suggested that phosphorylated AR may regulate ER stress.

Vaccinia-related kinase 1 (VRK1) is a chromatin-associated Ser-Thr kinase that regulates numerous downstream factors including DNA repair as well as stress factors c-Jun and p53. Both c-Jun and p53 are phosphorylated at Ser63 and Thr18, respectively, in response to low glucose (40 mg/dl of medium) but not high glucose (140 mg/dl of medium) in human hepatoma-derived Huh-7 cells. Here, we have determined the molecular mechanism by which VRK1 phosphorylates these residues in response to glucose in Huh-7 cells. Human VRK1 auto-phosphorylates Ser376 and Thr386 in in vitro kinase assays. In Huh-7 cells, this auto-phosphorylation activity is regulated by glucose signaling; Thr386 is auto-phosphorylated only in low glucose medium, while Ser376 is not phosphorylated in either medium. A correlation of this low glucose response phosphorylation of Thr386 with the phosphorylation of c-Jun and p53 suggests that VRK1 phosphorylated at Thr386 catalyzes this phosphorylation. In fact, VRK1 knockdown by siRNA decreases and over-expression of VRK1 T386D increases phosphorylated c-Jun and p53 in Huh-7 cells. Phosphorylation by VRK1 of c-Jun but not p53 is regulated by cadherin Plakophilin-2 (PKP2). The PKP2 is purified from whole extracts of Huh-7 cells cultured in low glucose medium and is characterized to bind a C-terminal peptide of the VRK1 molecules to regulate its substrate specificity toward c-Jun. siRNA knockdowns show that PKP2 transduces low glucose signaling to VRK1 only to phosphorylate c-Jun, establishing the low glucose-PKP2-VRK1-c-Jun pathway as a glucose stress signaling pathway.

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.

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.

Phenobarbital, a nongenotoxic hepatocarcinogen, induces hepatic proliferation and promotes development of hepatocellular carcinoma (HCC) in rodents. Nuclear receptor constitutive active/androstane receptor (NR1I3/CAR) regulates the induction and promotion activities of phenobarbital. Here, it is demonstrated that phenobarbital treatment results in dephosphorylation of a tumor suppressor p38 MAPK in the liver of C57BL/6 and C3H/HeNCrlBR mice. The molecular mechanism entails CAR binding and inhibition of the growth arrest and DNA-damage-inducible 45 beta (GADD45B)-MAPK kinase 6 (MKK6) scaffold to repress phosphorylation of p38 MAPK. Phenobarbital-induced hepatocyte proliferation, as determined by BrdUrd incorporation, was significantly reduced in both male and female livers of GADD45B knockout (KO) mice compared with the wild-type mice. The phenobarbital-induced proliferation continued until 48 hours after phenobarbital injection in only the C57BL/6 males, but neither in males of GADD45B KO mice nor in females of C57BL/6 and GADD45B KO mice. Thus, these data reveal nuclear receptor CAR interacts with GADD45B to repress p38 MAPK signaling and elicit hepatocyte proliferation in male mice. Implications: This GADD45B-regulated male-predominant proliferation can be expanded as a phenobarbital promotion signal of HCC development in future studies. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/8/1309/F1.large.jpg. Mol Cancer Res; 16(8); 1309–18. ©2018 AACR.

Androgen is beneficial for the prostate with normal functions but creates a risk for prostate cancer progression. How androgen receptor (AR) mediates these various actions of androgen remains elusive. AR conserves a phosphorylation motif within its ligand-binding domain throughout species. Here, we have found AR phosphorylated at Ser815 (P-AR) is expressed in normal tissues of both human and mouse prostates. P-AR begins being expressed in association with prostatic development and decreases its expression levels by castration in the mouse prostate. Functional analysis of AR in the prostate cancer PC-3 cells showed ligand-induced AR nuclear translocation and transactivation were disturbed by its phosphorylation at Ser815. Moreover, P-AR suppressed oncogenic AKT signaling suggesting AR has a suppression function for prostate cancer development. In fact, AR phosphorylation levels progressively decrease in human prostates as cancer worsens. These findings showed androgen might utilize P-AR to self-antagonize oncogenic signals and cancer progression believed to be regulated by non-phosphorylated AR (NonP-AR). By differing its target genes and signal regulations from those of NonP-AR, P-AR co-expression with NonP-AR may be the molecular basis for androgen to balance its actions and to control disease developments.

Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us.

Comparison of 11 human nuclear receptor amino acid sequences revealed a conserved phosphorylation motif within their DNA-binding domains as an intramolecular signal that regulates proteolytic degradation. Nuclear receptors use this signal to either degrade or proscribe degradation through either the proteasome or nonproteasome pathways. A phosphomimetic farnesoid X receptor (FXR) S154D mutant neither bound to nor trans-activated an FXR-response element-driven reporter gene and was rapidly degraded in COS-1 cells. Ectopically expressed FXR had increased Ser154 phosphorylation in COS-1 cells after ligand treatment, and knock-down of the nuclear vaccinia-related kinase 1 (VRK1) greatly reduced this phosphorylation. FXR was phosphorylated at Ser154 in the nucleus of centrilobular hepatocytes only in ligand-treated mice. Thus, FXR Ser154 phosphorylation is a rheostat for activation and subsequent degradation that controls receptor levels and activity.

Hepatic estrogen sulfotransferase (SULT1E1), the enzyme that inactivates estrogen, regulates metabolic estrogen homeostasis. Here, we have demonstrated how nuclear receptor PXR regulated the SULT1E1 gene in response to glucose in human hepatoma-derived cells and in response to fasting in mouse livers. The SULT1E1 gene was activated by a nuclear receptor HNF4α-RORα complex binding on an upstream enhancer of the SULT1E1 promoter in cells cultured in high glucose medium (Hu and Negishi, 2020). The SULT1E1 gene was repressed in cells cultured in low glucose medium, in which PXR was phosphorylated at Ser350 by vaccinia virus-related kinase 1. Phosphorylated PXR interacted with this complex, retaining HNF4α on and dissociating RORα from the enhancer as a phosphorylated PXR complex. Therefore, in response to low glucose, phosphorylated PXR transduced a low glucose signal to repress the SULT1E1 gene in cells. Hepatic Sult1e1 mRNA was induced in PXR wild type (WT) male mice in response to fasting, whereas this induction was synergistically increased in phosphorylation-blocking PXR Ser347Ala (Ser350 in human) KI males over that observed in PXR WT males. As phosphorylated PXR repressed the Sult1e1 gene, it increased its binding to the Sult1e1 promoter in WT males. The absence of phosphorylated PXR resulted in the synergistic activation of the Sult1e1 gene in PXR KI males. Apparently, phosphorylated PXR functioned as a transcriptional repressor to the SULT1E1/Sult1e1 gene in human liver cells and mouse livers.

Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA-binding domain. Here we have generated RXRα knock-in mice (RxrαT167A) bearing a single mutation of Thr 167 to alanine and examined the roles of Thr 167 in the regulation of energy metabolism within adipose, muscle, and liver tissues. RxrαT167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in the white adipose tissue (WAT) and ATP citrate lyase in the muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor β1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in RxrαT167A during feeding, as observed with RXRα wild-type mice. However, RxrαT167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr 167. Thus, Thr 167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintenance of glucose homeostasis.

Co-chaperone cytoplasmic constitutive active/androstane receptor retention protein (CCRP), a member of heat shock protein (HSP) 40, was first characterized to retain a nuclear-destined protein in the cytoplasm. Here we have used CCRP KO mice and demonstrated that CCRP suppresses lipopolysaccharide (LPS)-induced cardiac toxicity in mice. LPS treatment decreased heart rates in CCRP KO mice, but not in wild-type (WT) mice. In addition, LPS-treated KO mice showed reduced fraction shortening, an indicator of ventricular contractile function, to a greater degree than WT mice did. Rat cardiomyocyte-derived H9c2 cells, in which CCRP is not expressed, were used to examine a cell signal through which CCRP suppressed LPS-induced cardiac toxicity. Overexpression of CCRP prevented p65, a nuclear factor κB (NFκB) subunit, from accumulating in the nucleus after LPS treatment. As observed with H9c2 cells, nuclear accumulation of p65 was found to be higher in the hearts of KO mice than WT mice after LPS treatment. Furthermore, induction of TNFα by LPS was markedly suppressed by CCRP in H9c2 cells as well as in LPS-treated mouse serum. In supporting the notion that CCRP repressed the LPS-induced NFκB signaling, pretreatment with pyrrolidinedithiocarbamate, an NFκB signaling inhibitor, or anti-TNF-α antibody before LPS treatment restored heart rates decreased in KO mice after LPS treatment in a dose-dependent manner. Our present study characterized a novel physiological role of CCRP in protecting cardiac functions through the inhibition of NFκB signaling.

The constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is a well-known xenosensor that regulates hepatic drug metabolism and detoxification. CAR activation can be elicited by a large variety of xenobiotics, including phenobarbital (PB) which is not a directly binding CAR ligand. The mechanism of CAR activation is complex and involves translocation from the cytoplasm into the nucleus, followed by further activation steps in the nucleus. Recently, epidermal growth factor receptor (EGFR) has been identified as a PB-responsive receptor, and PB activates CAR by inhibiting the EGFR signaling. In addition to regulation of drug metabolism, activation of CAR has multiple biological end points such as modulation of xenobiotic-elicited liver injury, and the role of CAR in endobiotic functions such as glucose metabolism and cholesterol homeostasis is increasingly recognized. Thus, investigations on the molecular mechanism of CAR activation are critical for the real understanding of CAR-mediated processes. Here, we summarize the current understanding of mechanisms by which CAR activators regulate gene expression through cellular signaling pathways and the roles of CAR on xenobiotic-elicited hepatocellular carcinoma, liver injury, glucose metabolism and cholesterol homeostasis.

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer–heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD–LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D–LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD–LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

Low glucose stimulated phosphorylation of pregnane X receptor (PXR) at Ser350 in correlation with an increased gluconeogenesis in human hepatoma-derived HepG2 cells. Only glucose, but neither insulin nor glucagon, stimulated this phosphorylation. Here, serine/threonine kinase, vaccinia related kinase 1 (VRK1)-mediated phosphorylation of PXR is now defined as this glucose-elicited novel signal. In low glucose conditions, VRK1 directly phosphorylates PXR at Ser350, enabling PO3-PXR to scaffold protein phosphatase PP2Cα. This PP2Cα dephosphorylates serine/threonine kinase 2 (SGK2) at Thr193. This dephosphorylation dissociates SGK2 from and actives the phosphoenolpyruvate carboxykinase 1 (PCK1) gene as phosphorylated SGK2 binds and represses the gene. Conversely, VRK1 self-represses its activity to phosphorylate PXR through cyclin-dependent kinase 2 (CDK2) in high glucose conditions, resulting in the repression of the PCK1 gene. This PXR phosphorylation was also observed in fasting mouse livers. Thus, the VRK1-CDK2-PXR-PP2Cα-SGK2 pathway can be a novel physiological cell signaling that regulates gluconeogenesis in response to glucose.

The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR.

Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs-null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs-null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice.