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.

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.

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.

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.

Mechanical Strength of the Proximal Tibia Following Total Knee Arthroplasty: A Cadaveric Study of Resection Depth and Bone Density.

BACKGROUND: Tibial component failure has been a problem in total knee arthroplasty, it is still undetermined how tibial resection depth affects the strength to support a tibial component. This study examined the relationship between the resection depth and the bone density and the mechanical strength to support the tibial component. MATERIALS AND METHODS: Eight matched pairs of fresh, frozen cadaver lower legs were imaged with computed tomography to assess the bone density. A right tibia was resected at minimum resection level and a left tibia was resected at deep resection level. After the tibial component was implanted with cement on each tibia, it was loaded on a materials testing load frame to measure the stiffness and the load to failure. RESULTS: The average bone density at the minimum resection level of the tibia was significantly higher than at deep level (p=0.0003). The average stiffness and load to failure of the proximal tibia were 1105 N/mm (range 889 to 1303 N/mm) and 5626 N (range 3360 to 9098 N). There was no statistical correlation between tibial resection depth and the axial stiffness (p=0.4107) or the load to failure (p=0.1487). CONCLUSIONS: Although the bone density at a minimum resection level was higher than that at a deep level, the strength to support the tibial component was not statistically higher at a minimum cutting level than at a deeper cutting level proportionally. Surgeons may not need to minimize a proximal tibial bone resection to maintain a stronger support for a tibial component.

Twenty-Five-Years and Greater, Results After Nonmodular Cemented Total Knee Arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) has been shown to be very successful with long-term follow-ups. But there are no reports showing prosthesis survival at 25-30 years. Here, we report the outcomes for 25-30 years using the Anatomic Graduated Component (Biomet, Warsaw, IN) TKA and elucidate the etiology and cause of failure of the components. METHODS: We reviewed the outcomes of 5649 primary total knee arthroplasties for 25-30 years using the Anatomic Graduated Component. Statistical analysis was performed by the Kaplan-Meier survival analysis. Clinical outcomes included the Knee Society Score and standardized radiographs to check for loosening of the implants. The reason for revision surgery was reviewed retrospectively. We compared our results with those at another institution with similar long-term follow-up. RESULTS: There were 112 failures, 48 with aseptic loosening and 25 with instability for an overall prosthesis survival rate of 94.2% at 25 years and 92.4% at 30 years follow-up. In the third decade after TKA, patients are substantially more likely to experience death than experience a failing prosthesis, with a 3811% greater risk of dying relative to failing (Risk ratio = 38.1, Odds ratio = 56.7, P < .0001). CONCLUSION: There was a greater risk of dying than failing over time. The primary reason for revision knee surgery was due to aseptic loosening of the prosthesis followed by instability.

SLC13A5 is a novel transcriptional target of the pregnane X receptor and sensitizes drug-induced steatosis in human liver.

The solute carrier family 13 member 5 (SLC13A5) is a sodium-coupled transporter that mediates cellular uptake of citrate, which plays important roles in the synthesis of fatty acids and cholesterol. Recently, the pregnane X receptor (PXR, NR1I2), initially characterized as a xenobiotic sensor, has been functionally linked to the regulation of various physiologic processes that are associated with lipid metabolism and energy homeostasis. Here, we show that the SLC13A5 gene is a novel transcriptional target of PXR, and altered expression of SLC13A5 affects lipid accumulation in human liver cells. The prototypical PXR activator rifampicin markedly induced the mRNA and protein expression of SLC13A5 in human primary hepatocytes. Utilizing cell-based luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays, we identified and functionally characterized two enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction. Functional analysis further revealed that rifampicin can enhance lipid accumulation in human primary hepatocytes, and knockdown of SLC13A5 expression alone leads to a significant decrease of the lipid content in HepG2 cells. Overall, our results uncover SLC13A5 as a novel target gene of PXR and may contribute to drug-induced steatosis and metabolic disorders in humans.

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.

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.

Relationship between early physical activity after total knee arthroplasty and postoperative physical function: are these related?

BACKGROUND: Physical activity is associated with physical function; however, the relationship between early physical activity after total knee arthroplasty (TKA) and postoperative physical function remains unclear. The purpose of this study was to evaluate the association of early physical activity after TKA with postoperative physical function. METHODS: Timed Up and Go test (TUG) of 47 patients was assessed preoperatively and at 10 days, 3 months, and 6 months postoperatively. Physical activity from the second to the ninth day after TKA was measured with accelerometer, and the correlation with pre- and postoperative physical function was evaluated . A multiple linear regression was used to predict TUG at 6 months after TKA. RESULTS: Postoperative physical activity correlated with preoperative TUG (ρ = -0.485, p < 0.001), TUG at 10 days (ρ = -0.675, p < 0.001), 3 months (ρ = -0.441, p < 0.01), and 6 months (ρ = -0.368, p < 0.05) after surgery. Multiple linear regression indicated that only the preoperative TUG was associated with TUG at 6 months. Postoperative physical activity was not an independent factor predicting TUG at 6 months after TKA. CONCLUSION: Our study demonstrated that patients with better physical function have higher physical activity in the early postoperative period, whereas it does not affect physical function at 6 months after TKA. In the early postoperative period, increasing physical activity may not always be necessary to improve postoperative physical function. We also confirmed that preoperative physical function affects postoperative physical function. These findings may be beneficial in improving rehabilitation programs in the early postoperative period.

Phenobarbital Induces SLC13A5 Expression through Activation of PXR but Not CAR in Human Primary Hepatocytes.

Phenobarbital (PB), a widely used antiepileptic drug, is known to upregulate the expression of numerous drug-metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)-dependent but CAR-independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type and CAR-knockout HepaRG cells, whereas such induction was fully abolished in the PXR-knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream from the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR-mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB-mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.

Bone turnover markers in the early stage of rapidly progressive osteoarthritis of the hip.

OBJECTIVE: Previous reports have demonstrated that patients with end-stage rapidly progressive osteoarthritis of the hip (RPOH) show significantly higher serum levels of bone turnover markers than those with osteoarthritis (OA). However, the characteristics of bone turnover markers in the early stage of RPOH remain unclear. This study aimed to elucidate the association of bone turnover markers with disease progression in the early stage of RPOH. METHODS: This study included 29 postmenopausal female patients with joint space narrowing >2 mm demonstrated on a series of radiographs and computed tomography within 1 year following the onset of hip pain. The study also included 9 postmenopausal female patients with hip OA secondary to developmental dysplasia showing femoral head destruction. Cortical thickness index (CTI) associated with bone mineral density of the hip was analyzed. Serum concentrations of tartrate-resistant acid phosphatase-5b (TRACP-5b) and bone alkaline phosphatase (BAP) were evaluated. RESULTS: RPOH was classified into two types on the basis of the absence (type 1, n=13) or presence (type 2, n=16) of subsequent destruction of the femoral head within 1 year following disease onset. TRACP-5b and BAP significantly increased in RPOH type 2 compared with type 1 and OA. Receiver operating characteristic curve analyses indicated that TRACP-5b and BAP could differentiate RPOH type 2 from type 1 within 1 year following the onset. CTI showed no difference among the RPOH types 1 and 2 and OA. CONCLUSION: High serum levels of bone turnover markers may be associated with destruction of the femoral head in the early stage of RPOH.

Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the alpha-helix spanning from residues 29-42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.

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.

Characterization of rapidly progressive osteoarthritis of the hip in its early stage.

OBJECTIVE: This study aimed to characterize the process of disease progression in the early stage of rapidly progressive osteoarthritis of the hip (RPOH) and clarify its association with potential pathological factors of RPOH. METHODS: This monocentric retrospective study included 41 female patients who met the criteria for RPOH, chondrolysis >2 mm during 12 months from the onset of hip pain based on a series of radiographs and computed tomography. This study also included 9 female patients with osteoarthritis secondary to developmental dysplasia of the hip (DDH) who demonstrated chondrolysis >2 mm during 12 months from the onset of hip pain. Cortical thickness index (CTI) correlated with bone mineral density of the hip, pelvic tilt, and serum concentrations of matrix metalloproteinase (MMP)-3 were analyzed. RESULTS: RPOH was classified into two types based on the absence (type 1, n=17) and presence (type 2, n=24) of subsequent femoral head destruction within 12 months after the onset of hip pain. MMP-3 significantly increased in RPOH type 2 compared with type 1 and DDH. Increased posterior pelvic tilt was found in RPOH type 2 compared with DDH. Logistic regression and receiver operating characteristic curve analyses indicated that MMP-3 may be associated with differentiation between RPOH types 1 and 2. No difference was found in the CTI between the RPOH types and DDH. CONCLUSION: Disease progression of RPOH during 12 months after the onset of hip pain could be classified into two distinct types based on the absence (type 1) and presence (type 2) of femoral head destruction in association with MMP-3 and pelvic tilt as biological and mechanical factors, respectively. MMP-3 may be helpful to differentiate these two types in the early stage of RPOH.

Vertebroplasty Using Allograft Bone Chips with Posterior Instrumented Fusion in the Treatment of Osteoporotic Vertebral Fractures with Neurological Deficits.

INTRODUCTION: In general, osteoporotic vertebral fractures with neurological deficits require surgery. However, the ideal surgical method remains controversial. We evaluated the efficacy of combining posterior instrumented fusion and vertebroplasty using allograft bone chips. METHODS: Twelve patients (five men, seven women; age 68-84 years, mean age 75.9 years) with osteoporotic vertebral fractures with neurological deficits were reviewed retrospectively. They underwent posterior instrumented fusion and vertebroplasty, using allograft bone, at our institution between January 2007 and June 2016. We assessed the surgical results, radiologically and neurologically, after a mean follow-up of 37.3 months. RESULTS: The mean local kyphosis angle was 10° before surgery, -3.3° immediately after surgery, and 4.4° at follow-up. The average spinal canal compromise was 26.9% before surgery and 19.5% at follow-up. All patients achieved bony fusion and none needed additional surgery. All patients improved by at least one grade on the modified Frankel grading system. CONCLUSIONS: Combining vertebroplasty, using allograft bone chips, and posterior instrumented fusion appears to be an effective option for osteoporotic vertebral fractures with neurological deficits.

PPP1R16A, the membrane subunit of protein phosphatase 1beta, signals nuclear translocation of the nuclear receptor constitutive active/androstane receptor.

Constitutive active/androstane receptor (CAR), a member of the nuclear steroid/thyroid hormone receptor family, activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. We have now identified a membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A) as a novel CAR-binding protein. When CAR and R16A are coexpressed in mouse liver, CAR translocates into the nucleus. Close association of R16A and CAR molecule on liver membrane was shown by fluorescence resonance energy transfer (FRET) analysis using expressed yellow fluorescent protein (YFP)-CAR and CFP-R16A fusion proteins. R16A can form dimer through its middle region, where protein kinase A phosphorylation sites are recently identified. Translocation of CAR by R16A correlates with the ability of R16A to form an intermolecular interaction via the middle region. Moreover, this interaction is enhanced by PB treatment in mouse liver. R16A specifically interacted with PP1beta in HepG2 cells despite the highly conserved structure of PP1 family molecules. PP1beta activity was inhibited by R16A in vitro and coexpression of PP1beta in liver can prevent YFP-CAR translocation into mouse liver. Taken together, R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1beta activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial.

The peripheral benzodiazepine receptor ligand 1-(2-chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide is a novel antagonist of human constitutive androstane receptor.

As a promiscuous xenobiotic sensor, the constitutive androstane receptor (CAR; NR1I3) regulates the expression of multiple drug-metabolizing enzymes and transporters in liver. The constitutively activated nature of CAR in the cell-based transfection assays has hindered its use as a predictor of metabolism-based drug-drug interactions. Here, we have identified 1-(2-chlorophenylmethylpropyl)-3-isoquinoline-carboxamide (PK11195), a typical peripheral benzodiazepine receptor (PBR) ligand, as a selective and potent inhibitor of human (h) CAR. In cell-based transfection assays, PK11195 inhibited the constitutive activity of hCAR more than 80% at the concentration of 10 microM, and the PK11195-inhibited activity was efficiently reactivated by the direct CAR activator, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime, but not by the indirect hCAR activator, phenobarbital. Mammalian two-hybrid and GST pull-down assays showed that PK11195 repressed the interactions of hCAR with the coactivators steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein 1 to inhibit hCAR activity. The inhibition by PK11195 specifically occurred to the hCAR: PK1195 strongly activated human pregnane X receptor (PXR), whereas it did not alter the activity of the mouse CAR and mouse PXR. In addition, PBR played no role in the PK11195 inhibition of hCAR because the inhibition fully occurred in the HeLa cells in which the PBR was knocked down by small interfering RNA. In the Car(-/-) mouse liver, PK11195 translocated enhanced yellow fluorescent protein-hCAR into the nucleus. These results are consistent with the conclusion that PK11195 is a novel hCAR-specific antagonist that represses the CAR-coactivator interactions to inhibit the receptor activity inside the nucleus. Thus, PK11195 can be used as a chemical tool for studying the molecular basis of CAR function.

Identification of Ginkgo biloba as a novel activator of pregnane X receptor.

Pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that plays a role not only in drug metabolism and transport but also in various other biological processes. Ginkgo biloba is a herbal medicine commonly used to manage memory impairment. Treatment of primary cultures of rat hepatocytes with G. biloba extract increases the mRNA expression of CYP3A23, which is a target gene for rat PXR. The present study was conducted to test the hypothesis that G. biloba extract activates PXR. Treatment of mouse PXR (mPXR) or human PXR (hPXR)-transfected HepG2 cells with G. biloba extract at 200 microg/ml increased mPXR and hPXR activation by 3.2- and 9.5-fold, respectively. Dose-response analysis showed a log-linear increase in hPXR activation by the extract over the range of 200 to 800 microg/ml. To determine whether G. biloba extract induces hPXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. G. biloba extract at 200, 400, and 800 microg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The same concentrations of the extract increased CYP3A5 (1.3-3.6-fold) and P-glycoprotein (ABCB) 1 (2.7-6.4-fold) mRNA expression. At concentrations (5 and 10 microM) that did not down-regulate PXR gene expression and were not cytotoxic, L-sulforaphane (an hPXR antagonist) decreased CYP3A4, CYP3A5, and ABCB1 gene expression in cells treated with G. biloba extract. In summary, G. biloba extract activated mPXR and hPXR in a cell-based reporter gene assay and induced CYP3A4, CYP3A5, and ABCB1 gene expression in hPXR-expressing LS180 cells.

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation.

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.