Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

Chronic Activation of Liver X Receptor Sensitizes Mice to High Cholesterol Diet-Induced Gut Toxicity.

Cholesterol is essential for numerous biologic functions and processes, but an excess of intracellular cholesterol can be toxic. Intestinal cholesterol absorption is a major determinant of plasma cholesterol level. The liver X receptor (LXR) is a nuclear receptor known for its activity in cholesterol efflux and reverse cholesterol transport. In this study, we uncovered a surprising function of LXR in intestinal cholesterol absorption and toxicity. Genetic or pharmacologic activation of LXRα-sensitized mice to a high-cholesterol diet (HCD) induced intestinal toxicity and tissue damage, including the disruption of enterocyte tight junctions, whereas the same HCD caused little toxicity in the absence of LXR activation. The gut toxicity in HCD-fed LXR-KI mice may have been accounted for by the increased intestinal cholesterol absorption and elevation of enterocyte and systemic levels of free cholesterol. The increased intestinal cholesterol absorption preceded the gut toxicity, suggesting that the increased absorption was not secondary to tissue damage. The heightened sensitivity to HCD in the HCD-fed LXRα-activated mice appeared to be intestine-specific because the liver was not affected despite activation of the same receptor in this tissue. Moreover, heightened sensitivity to HCD cannot be reversed by ezetimibe, a Niemann-Pick C1-like 1 inhibitor that inhibits intestinal cholesterol absorption, suggesting that the increased cholesterol absorption in LXR-activated intestine is mediated by a mechanism that has yet to be defined.

Enzalutamide for the Treatment of Androgen Receptor-Expressing Triple-Negative Breast Cancer.

Purpose Studies suggest that a subset of patients with triple-negative breast cancer (TNBC) have tumors that express the androgen receptor (AR) and may benefit from an AR inhibitor. This phase II study evaluated the antitumor activity and safety of enzalutamide in patients with locally advanced or metastatic AR-positive TNBC. Patients and Methods Tumors were tested for AR with an immunohistochemistry assay optimized for breast cancer; nuclear AR staining > 0% was considered positive. Patients received enzalutamide 160 mg once per day until disease progression. The primary end point was clinical benefit rate (CBR) at 16 weeks. Secondary end points included CBR at 24 weeks, progression-free survival, and safety. End points were analyzed in all enrolled patients (the intent-to-treat [ITT] population) and in patients with one or more postbaseline assessment whose tumor expressed ≥ 10% nuclear AR (the evaluable subgroup). Results Of 118 patients enrolled, 78 were evaluable. CBR at 16 weeks was 25% (95% CI, 17% to 33%) in the ITT population and 33% (95% CI, 23% to 45%) in the evaluable subgroup. Median progression-free survival was 2.9 months (95% CI, 1.9 to 3.7 months) in the ITT population and 3.3 months (95% CI, 1.9 to 4.1 months) in the evaluable subgroup. Median overall survival was 12.7 months (95% CI, 8.5 months to not yet reached) in the ITT population and 17.6 months (95% CI, 11.6 months to not yet reached) in the evaluable subgroup. Fatigue was the only treatment-related grade 3 or higher adverse event with an incidence of > 2%. Conclusion Enzalutamide demonstrated clinical activity and was well tolerated in patients with advanced AR-positive TNBC. Adverse events related to enzalutamide were consistent with its known safety profile. This study supports additional development of enzalutamide in advanced TNBC.

Androgen Receptor Immunohistochemistry as a Companion Diagnostic Approach to Predict Clinical Response to Enzalutamide in Triple-Negative Breast Cancer.

PURPOSE: The androgen receptor (AR) is increasingly recognized as a potential biomarker for identifying a subset of patients with possible hormonally driven triple-negative breast cancer (TNBC). However, its performance as a companion diagnostic remains elusive. Thus, we evaluated AR expression by immunohistochemistry in patients with advanced TNBC before treatment with the AR inhibitor enzalutamide. METHODS: We optimized and validated immunohistochemistry assays in breast and prostate cancer cell lines and tissues using two commercial AR monoclonal antibodies (SP107 and AR441). AR expression was then examined in patients with advanced TNBC enrolled in a phase II study of enzalutamide (ClinicalTrials.gov identifier: NCT01889238) on archived or fresh tissue before treatment. Association with clinical response was assessed by sensitivity, specificity, positive predictive value (PPV), drop-out rate, and survival. RESULTS: AR expression was detected in 80% and 63% of breast cancer tissue using SP107 and AR441, respectively. SP107 was selected for additional analyses because of its higher sensitivity and robustness. Total AR nuclear staining demonstrated the best accuracy in predicting clinical response (area under receiver operating characteristic curve, 0.72; P = .0001). At a threshold of 10%, 74.6% of patients were AR positive, leading to 30% PPV, 90% sensitivity, and 30% specificity. These patients showed a significantly higher median progression-free survival (hazard ratio, 0.56; 95% CI, 0.36 to 0.88; P = .011) and overall survival (hazard ratio, 0.54; 95% CI, 0.32 to 0.91; P = .019) compared with those with AR-negative (< 10%) TNBC. CONCLUSION: At a threshold of ≥ 10% nuclear expression, the AR was associated with TNBC response to enzalutamide. However, the modest PPV may restrict its clinical application, and additional diagnostic tools may be helpful for improved patient selection.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1).

PURPOSE: Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia. EXPERIMENTAL DESIGN: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. RESULTS: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. CONCLUSIONS: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.

Retinal toxicity, in vivo and in vitro, associated with inhibition of nicotinamide phosphoribosyltransferase.

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein with intra- and extra-cellular functions as an enzyme, cytokine, growth factor, and hormone. NAMPT is of interest for oncology, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), which is considered a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. This manuscript describes NAMPT inhibitor-induced retinal toxicity that was identified in rodent safety studies. This toxicity had a rapid onset and progression and initially targeted the photoreceptor and outer nuclear layers. Using in vivo safety and efficacy rodent studies, human and mouse cell line potency data, human and rat retinal pigmented epithelial cell in vitro systems, and rat mRNA expression data of NAMPT, nicotinic acid phosphoribosyltransferase, and nicotinamide mononucleotide adenylyltransferease (NMNAT) in several tissues from rat including retina, we demonstrate that the retinal toxicity is on-target and likely human relevant. We demonstrate that this toxicity is not mitigated by coadministration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway. Further, modifying the physiochemical properties of NAMPT inhibitors could not sufficiently reduce retinal exposure. Our work highlights opportunities to leverage appropriately designed efficacy studies to identify known and measurable safety findings to screen compounds more rapidly and reduce animal use. It also demonstrates that in vitro systems with the appropriate cell composition and relevant biology and toxicity endpoints can provide tools to investigate mechanism of toxicity and the human translation of nonclinical safety concerns.

Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.

Direct reprogramming by overexpression of defined transcription factors is a promising new method of deriving useful but rare cell types from readily available ones. While the method presents numerous advantages over induced pluripotent stem (iPS) cell approaches, a focus on murine conversions and a reliance on retroviral vectors limit potential human applications. Here we address these concerns by demonstrating direct conversion of human fibroblasts to hepatocyte-like cells via repeated transfection with synthetic modified mRNAs. Hepatic induction was achieved with as little as three transcription factor mRNAs encoding HNF1A plus any two of the factors, FOXA1, FOXA3, or HNF4A in the presence of an optimized hepatic growth medium. We show that the absolute necessity of exogenous HNF1A mRNA delivery is explained both by the factor's inability to be activated by any other factors screened and its simultaneous ability to strongly induce expression of other master hepatic transcription factors. Further analysis of factor interaction showed that a series of robust cross-activations exist between factors that induce a hepatocyte-like state. Transcriptome and small RNA sequencing during conversion toward hepatocyte-like cells revealed global preferential activation of liver genes and miRNAs over those associated with other endodermal tissues, as well as downregulation of fibroblast-associated genes. Induced hepatocyte-like cells also exhibited hepatic morphology and protein expression. Our data provide insight into the process by which direct hepatic reprogramming occurs in human cells. More importantly, by demonstrating that it is possible to achieve direct reprogramming without the use of retroviral gene delivery, our results supply a crucial step toward realizing the potential of direct reprogramming in regenerative medicine.

Improving risk assessment.

Widespread sharing and analysis of clinical trial data and a U.S. government initiative to engineer nonclinical cell-based models that mimic human biological processes have the potential to improve predictions of drug-related adverse events.

Characterization of rat liver proteins adducted by reactive metabolites of menthofuran.

Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.

Activation of liver X receptor increases acetaminophen clearance and prevents its toxicity in mice.

UNLABELLED: Overdose of acetaminophen (APAP), the active ingredient of Tylenol, is the leading cause of drug-induced acute liver failure in the United States. As such, it is necessary to develop novel strategies to prevent or manage APAP toxicity. In this report, we reveal a novel function of the liver X receptor (LXR) in preventing APAP-induced hepatotoxicity. Activation of LXR in transgenic (Tg) mice or by an LXR agonist conferred resistance to the hepatotoxicity of APAP, whereas the effect of LXR agonist on APAP toxicity was abolished in LXR-deficient mice. The increased APAP resistance in LXR Tg mice was associated with increased APAP clearance, increased APAP sulfation, and decreased formation of toxic APAP metabolites. The hepatoprotective effect of LXR may have resulted from the induction of antitoxic phase II conjugating enzymes, such as Gst and Sult2a1, as well as the suppression of protoxic phase I P450 enzymes, such as Cyp3a11 and Cyp2e1. Promoter analysis suggested the mouse Gst isoforms as novel transcriptional targets of LXR. The suppression of Cyp3a11 may be accounted for by the inhibitory effect of LXR on the PXR-responsive transactivation of Cyp3a11. The protective effect of LXR in preventing APAP toxicity is opposite to the sensitizing effect of pregnane X receptor, constitutive androstane receptor, and retinoid X receptor alpha. CONCLUSION: We conclude that LXR represents a potential therapeutic target for the prevention and treatment of Tylenol toxicity.

Mechanism of subendocardial cell proliferation in the rat and relevance for understanding drug-induced valvular heart disease in humans.

A number of drugs and drug candidates, including fenfluramine and ergot derivatives, are associated with valvulopathy in humans; however, these responses are poorly predicted from animal studies. In vitro and in vivo evidence suggests that these compounds exert their pathological effect through activation of serotonin 2B receptor (5HT2BR) signaling. However, the variable effect of fenfluramine and other 5HT2BR agonists in rodents has cast doubt on the relevance of animal findings to predicting human risk. Herein, a candidate compound, RO3013, induced subendocardial cell proliferation in the mitral and tricuspid valves in rats after only 3 days of daily dosing. Additionally, there was a treatment-related increase in immunostaining of the proliferation marker Ki67, and phosphorylated Smad3 in the heart indicative of TGFß signaling co-localized with 5HT2BR expression. To substantiate the hypothesis that RO3013-induced valvular proliferation is secondary to 5HT2BR activation, the compound was evaluated in vitro and found to bind to the human 5HT2BR with a K(i) of 3.8µM; however, it was virtually devoid of agonist activity in a functional assay in human cells. By contrast, RO3013 bound to the rat 5HT2BR with a K(i) of 1.2µM and activated the receptor with an EC50 of 0.5µM. This agonist potency estimate is in good agreement with the free plasma concentrations of RO3013 at which valvular proliferation was observed. These results suggest that the rat may be susceptible to 5HT2BR-mediated valvular proliferation similar to humans; yet, the significant differences between binding and functional activities can be a possible explanation for the observed species-selective receptor responses.

Identification of a kinase profile that predicts chromosome damage induced by small molecule kinase inhibitors.

Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs) are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases), yielding a 113x290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT). Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM) for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity). This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing compounds, thereby providing a basis for rational drug design away from genotoxicity.

In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates.

The development of predictive toxicology assays is necessary to optimize the drug candidate selection process. The colony forming assay (CFA) is used routinely to assess bone marrow toxicity and represents a viable tool for the discovery toxicologist, but the assay is not widely accepted as a standard screening tool due to technical challenges. A higher throughput and standardized version of the assay recently was developed such that the proliferative capacity of a cell lineage is measured indirectly via ATP levels, replacing the cumbersome identification and enumeration of specific colonies. In this study, a high-throughput assay of bone marrow toxicity prediction using the granulocyte, erythrocyte, monocyte, and macrophage (GEMM) progenitor cell lineage was evaluated using a training set of 56 structurally diverse compounds with known in vivo bone marrow effects. In general, compounds identified as toxic in vivo had lower IC(50) values, whereas those identified as non-toxic had higher IC(50) values. Concordance (i.e., predictive accuracy) to in vivo bone marrow toxicity results was 82% when an in vitro toxicity threshold of 20 microM was used. Additional experiments in other hematopoietic lineages were conducted to determine if predictivity of several false positive and negative compounds in the GEMM lineage could be improved; however an increase in sensitivity or specificity was not observed. The high-throughput GEMM assay has good concordance to in vivo bone marrow toxicity results and, with the high-throughput and standardized format, can be incorporated readily into the pharmaceutical toxicological screening paradigm, aiding in the early identification of compounds that eventually may fail due to bone marrow toxicity.

Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds.

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.

Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.

UNLABELLED: Gallstone disease is a hepatobiliary disorder due to biochemical imbalances in the gallbladder bile. In this report, we show that activation of nuclear receptor liver X receptor (LXR) sensitized mice to lithogenic diet-induced gallbladder cholesterol crystallization, which was associated with dysregulation of several hepatic transporters that efflux cholesterol, phospholipids, and bile salts. The combined effect of increased biliary concentrations of cholesterol and phospholipids and decreased biliary concentrations of bile salts in LXR-activated mice led to an increased cholesterol saturation index and the formation of cholesterol crystals. Interestingly, the lithogenic effect of LXR was completely abolished in the low-density lipoprotein receptor (Ldlr) null background or when the mice were treated with Ezetimibe, a cholesterol-lowering drug that blocks intestinal dietary cholesterol absorption. These results suggest that LDLR-mediated hepatic cholesterol uptake and intestinal cholesterol absorption play an essential role in LXR-promoted lithogenesis. CONCLUSION: The current study has revealed a novel lithogenic role of LXR as well as a functional interplay between LXR and LDLR in gallbladder cholesterol crystallization and possibly cholesterol gallstone disease (CGD). We propose that LXR is a lithogenic factor and that the LXR transgenic mice may offer a convenient CGD model to develop therapeutic interventions for this disease.

Estrogen deprivation and inhibition of breast cancer growth in vivo through activation of the orphan nuclear receptor liver X receptor.

Estrogen plays an important role in normal physiology. It is also a risk factor for breast cancer, and antiestrogen therapies have been shown to be effective in the treatment and prevention of breast cancers. The liver is important for estrogen metabolism, and a compromised liver function has been linked to hyperestrogenism in patients. In this report, we showed that the liver X receptor (LXR) controls estrogen homeostasis by regulating the basal and inducible hepatic expression of estrogen sulfotransferase (Est, or Sult1e1), an enzyme critical for metabolic estrogen deactivation. Genetic or pharmacological activation of LXR resulted in Est induction, which in turn inhibited estrogen-dependent uterine epithelial cell proliferation and gene expression, as well as breast cancer growth in a nude mouse model of tumorigenicity. We further established that Est is a transcriptional target of LXR, and deletion of the Est gene in mice abolished the LXR effect on estrogen deprivation. Interestingly, Est regulation by LXR appeared to be liver specific, further underscoring the role of liver in estrogen metabolism. Activation of LXR failed to induce other major estrogen-metabolizing enzymes, suggesting that the LXR effect on estrogen metabolism is Est specific. In summary, our results have revealed a novel mechanism controlling estrogen homeostasis in vivo and may have implications for drug development in the treatment of breast cancer and other estrogen-related cancerous endocrine disorders.

Activation of LXRs prevents bile acid toxicity and cholestasis in female mice.

UNLABELLED: Liver X receptors (LXRs) have been identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. In this study, we found that LXRs also affect sensitivity to bile acid toxicity and cholestasis. Activation of LXRalpha in transgenic mice confers a female-specific resistance to lithocholic acid (LCA)-induced hepatotoxicity and bile duct ligation (BDL)-induced cholestasis. This resistance was also seen in wild-type female mice treated with the synthetic LXR ligand TO1317. In contrast, LXR double knockout (DKO) mice deficient in both the alpha and beta isoforms exhibited heightened cholestatic sensitivity. LCA and BDL resistance in transgenic mice was associated with increased expression of bile acid-detoxifying sulfotransferase 2A (Sult2a) and selected bile acid transporters, whereas basal expression of these gene products was reduced in the LXR DKO mice. Promoter analysis showed that the mouse Sult2a9 gene is a transcriptional target of LXRs. Activation of LXRs a l so suppresses expression of oxysterol 7alpha-hydroxylase (Cyp7b1), which may lead to increased levels of LXR-activating oxysterols. CONCLUSION: We propose that LXRs have evolved to have the dual functions of maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism and, at the same time, preventing toxicity from bile acid accumulation.

A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.

The pregnane X receptor (PXR) was isolated as a xenosensor regulating xenobiotic responses. In this study, we show that PXR plays an endobiotic role by impacting lipid homeostasis. Expression of an activated PXR in the livers of transgenic mice resulted in an increased hepatic deposit of triglycerides. This PXR-mediated lipid accumulation was independent of the activation of the lipogenic transcriptional factor SREBP-1c (sterol regulatory element-binding protein 1c) and its primary lipogenic target enzymes, including fatty-acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC-1). Instead, the lipid accumulation in transgenic mice was associated with an increased expression of the free fatty acid transporter CD36 and several accessory lipogenic enzymes, such as stearoyl-CoA desaturase-1 (SCD-1) and long chain free fatty acid elongase. Studies using transgenic and knock-out mice showed that PXR is both necessary and sufficient for Cd36 activation. Promoter analyses revealed a DR-3-type of PXR-response element in the mouse Cd36 promoter, establishing Cd36 as a direct transcriptional target of PXR. The hepatic lipid accumulation and Cd36 induction were also seen in the hPXR "humanized" mice treated with the hPXR agonist rifampicin. The activation of PXR was also associated with an inhibition of pro-beta-oxidative genes, such as peroxisome proliferator-activated receptor alpha (PPARalpha) and thiolase, and an up-regulation of PPARgamma, a positive regulator of CD36. The cross-regulation of CD36 by PXR and PPARgamma suggests that this fatty acid transporter may function as a common target of orphan nuclear receptors in their regulation of lipid homeostasis.

Dual role of orphan nuclear receptor pregnane X receptor in bilirubin detoxification in mice.

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are implicated in xenobiotic and endobiotic detoxification, including the clearance of toxic bilirubin. Previous studies have suggested both overlapping and preferential regulation of target genes by these receptors, but the mechanism of cross-talk remains elusive. Here we reveal a dual role of PXR in bilirubin detoxification in that both the loss and activation of PXR led to protection from hyperbilirubinemia induced by bilirubin infusion or hemolysis. The increased bilirubin clearance in PXR-null mice was associated with selective upregulation of detoxifying enzymes and transporters, and the pattern of regulation is remarkably similar to that of transgenic mice expressing the activated CAR. Interestingly, the increased bilirubin clearance and associated gene regulation were absent in the CAR-null or double-knockout mice. In cell cultures, ligand-free PXR specifically suppressed the ability of CAR to induce the multidrug resistance associated protein 2 (MRP2), a bilirubin-detoxifying transporter. This suppression was, at least in part, the result of the disruption of ligand-independent recruitment of coactivator by CAR. In conclusion, PXR plays both positive and negative roles in regulating bilirubin homeostasis, and this provides a novel mechanism that may govern receptor cross-talk and the hierarchy of xenobiotic and endobiotic regulation. PXR is a potential therapeutic target for clinical treatment of jaundice. (HEPATOLOGY 2005;41:497-505.).