CAR requires Gadd45ß to promote phenobarbital-induced mouse liver tumors in early stage.

Phenobarbital (PB) is an archetypal substance used as a mouse hepatocellular carcinoma (HCC) promotor in established experimental protocols. Our previous results showed CAR is the essential factor for PB induced HCC promotion. Subsequent studies suggested Gadd45ß, which is induced by PB through CAR activation, is collaborating with CAR to repress TNF-α induced cell death. Here, we used Gadd45ß null mice (Gadd45ß KO) treated with N-diethylnitrosamine (DEN) at 5 weeks of age and kept the mice with PB supplemented drinking water from 7 to 57 weeks old. Compared with wild type mice, Gadd45ß KO mice developed no HCC in the PB treated group. Increases in liver weight were more prominent in wild type mice than KO mice. Microarray analysis of mRNA derived from mouse livers found multiple genes specifically up or down regulated in wild type mice but not null mice in DEN + PB groups. Further qPCR analysis confirmed two genes, Tgfbr2 and irisin/Fndc5, were up-regulated in PB treated wild type mice but no significant increase was observed in Gadd45ß KO mice. We focused on these two genes because previous reports showed that hepatic Irisin/Fndc5 expression was significantly higher in HCC patients and that irisin binds to TGF-ß receptor complex that includes TGFBR2 subunit. Our results revealed irisin peptide in cell culture media increased the growth rate of mouse hepatocyte-derived AML12 cells. Microarray analysis revealed that irisin-regulated genes in AML12 cells showed a significant association with the genes in the TGF-ß pathway. Expression of irisin/Fndc5 and Tgfbr2 induced growth of human HCC cell line HepG2. Thus, Gadd45ß plays an indispensable role in mouse HCC development regulating the irisin/Fndc5 and Tgfbr2 genes.

Phosphorylation of nuclear receptors: Novelty and therapeutic implications.

Nuclear receptors (NR) collectively regulate several biological functions in various organs. While NRs can be characterized by activation of the transcription of their signature genes, they also have other diverse roles. Although most NRs are directly activated by ligand binding, which induces cascades of events leading to gene transcription, some NRs are also phosphorylated. Despite extensive investigations, primarily focusing on unique phosphorylation of amino acid residues in different NRs, the role of phosphorylation in the biological activity of NRs in vivo has not been firmly established. Recent studies on the phosphorylation of conserved phosphorylation motifs within the DNA- and ligand-binding domains confirmed has indicated the physiologically relevance of NR phosphorylation. This review focuses on estrogen and androgen receptors, and highlights the concept of phosphorylation as a drug target.

Ser815 Phosphorylation stabilizes the androgen receptor homodimer and stimulates ER-stress induced cell death.

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.

Mice blocking Ser347 phosphorylation of pregnane x receptor develop hepatic fasting-induced steatosis and hypertriglyceridemia.

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.

Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling.

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.

Immunoprecipitation Analyses of Estrogen Receptor α Phosphorylated at Serine 216 in the Mouse Liver.

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.

Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils.

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.

Androgen receptor phosphorylated at Ser815: The expression and function in the prostate and tumor-derived cells.

Androgen is beneficial for the prostate with normal functions but creates a risk for prostate cancer progression. How androgen receptor (AR) mediates these various androgen actions 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 expression in association with prostatic development and castration decreases its expression levels in the mouse prostate. Functional analysis of AR in 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 a suppressive 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.

Sex-specific expression mechanism of hepatic estrogen inactivating enzyme and transporters in diabetic women.

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.

Estrogen Sulfotransferase (SULT1E1): Its Molecular Regulation, Polymorphisms, and Clinical Perspectives.

Estrogen sulfotransferase (SULT1E1) is a phase II enzyme that sulfates estrogens to inactivate them and regulate their homeostasis. This enzyme is also involved in the sulfation of thyroid hormones and several marketed medicines. Though the profound action of SULT1E1 in molecular/pathological biology has been extensively studied, its genetic variants and functional studies have been comparatively rarely studied. Genetic variants of this gene are associated with some diseases, especially sex-hormone-related cancers. Comprehending the role and polymorphisms of SULT1E1 is crucial to developing and integrating its clinical relevance; therefore, this study gathered and reviewed various literature studies to outline several aspects of the function, molecular regulation, and polymorphisms of SULT1E1.

Glucocorticoid receptor dimerization in the cytoplasm might be essential for nuclear localization.

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.

Nuclear receptor phosphorylation in xenobiotic signal transduction.

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.

PXR phosphorylated at Ser350 transduces a glucose signal to repress the estrogen sulfotransferase gene in human liver cells and fasting signal in mouse livers.

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.

RORα phosphorylation by casein kinase 1α as glucose signal to regulate estrogen sulfation in human liver cells.

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.

Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia.

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. Video abstract.

Phosphorylation of vaccinia-related kinase 1 at threonine 386 transduces glucose stress signal in human liver cells.

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 receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.

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.

Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

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.

Ligand induced dissociation of the AR homodimer precedes AR monomer translocation to the nucleus.

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.

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.

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 ß1 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.