Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors.

Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.

Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion.

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

Hormonal Imbalances in Prader-Willi and Schaaf-Yang Syndromes Imply the Evolution of Specific Regulation of Hypothalamic Neuroendocrine Function in Mammals.

The hypothalamus regulates fundamental aspects of physiological homeostasis and behavior, including stress response, reproduction, growth, sleep, and feeding, several of which are affected in patients with Prader-Willi (PWS) and Schaaf-Yang syndrome (SYS). PWS is caused by paternal deletion, maternal uniparental disomy, or imprinting defects that lead to loss of expression of a maternally imprinted region of chromosome 15 encompassing non-coding RNAs and five protein-coding genes; SYS patients have a mutation in one of them, MAGEL2. Throughout life, PWS and SYS patients suffer from musculoskeletal deficiencies, intellectual disabilities, and hormonal abnormalities, which lead to compulsive behaviors like hyperphagia and temper outbursts. Management of PWS and SYS is mostly symptomatic and cures for these debilitating disorders do not exist, highlighting a clear, unmet medical need. Research over several decades into the molecular and cellular roles of PWS genes has uncovered that several impinge on the neuroendocrine system. In this review, we will discuss the expression and molecular functions of PWS genes, connecting them with hormonal imbalances in patients and animal models. Besides the observed hormonal imbalances, we will describe the recent findings about how the loss of individual genes, particularly MAGEL2, affects the molecular mechanisms of hormone secretion. These results suggest that MAGEL2 evolved as a mammalian-specific regulator of hypothalamic neuroendocrine function.

Emerging roles of the MAGE protein family in stress response pathways.

The melanoma antigen (MAGE) proteins all contain a MAGE homology domain. MAGE genes are conserved in all eukaryotes and have expanded from a single gene in lower eukaryotes to ∼40 genes in humans and mice. Whereas some MAGEs are ubiquitously expressed in tissues, others are expressed in only germ cells with aberrant reactivation in multiple cancers. Much of the initial research on MAGEs focused on exploiting their antigenicity and restricted expression pattern to target them with cancer immunotherapy. Beyond their potential clinical application and role in tumorigenesis, recent studies have shown that MAGE proteins regulate diverse cellular and developmental pathways, implicating them in many diseases besides cancer, including lung, renal, and neurodevelopmental disorders. At the molecular level, many MAGEs bind to E3 RING ubiquitin ligases and, thus, regulate their substrate specificity, ligase activity, and subcellular localization. On a broader scale, the MAGE genes likely expanded in eutherian mammals to protect the germline from environmental stress and aid in stress adaptation, and this stress tolerance may explain why many cancers aberrantly express MAGEs Here, we present an updated, comprehensive review on the MAGE family that highlights general characteristics, emphasizes recent comparative studies in mice, and describes the diverse functions exerted by individual MAGEs.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Tissue-Specific Regulation of the Wnt/ß-Catenin Pathway by PAGE4 Inhibition of Tankyrase.

Spatiotemporal control of Wnt/ß-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/ß-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the ß-catenin destruction complex. Although Wnt/ß-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/ß-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/ß-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/ß-catenin pathway.

A Novel Locus Predicts Spermatogenic Recovery among Childhood Cancer Survivors Exposed to Alkylating Agents.

Exposure to high doses of alkylating agents is associated with increased risk of impaired spermatogenesis among nonirradiated male survivors of childhood cancer, but there is substantial variation in this risk. Here we conducted a genetic study for impaired spermatogenesis utilizing whole-genome sequencing data from 167 nonirradiated male childhood cancer survivors of European ancestry from the St. Jude Lifetime Cohort treated with cyclophosphamide equivalent dose (CED) ≥4,000 mg/m2. Sperm concentration from semen analysis was assessed as the primary outcome. Common variants (MAF > 0.05) were adjusted for age at cancer diagnosis, CED, and top principal components. Rare/low-frequency variants (MAF ≤ 0.05) were evaluated jointly by various functional annotations and 4-kb sliding windows. A novel locus at 7q21.3 containing TAC1/ASNS was associated with decreased sperm concentration (rs7784118: P = 3.5 × 10-8). This association was replicated in two independent samples of SJLIFE survivors of European ancestry, including 34 nonirradiated male survivors treated with 0 < CED < 4,000 mg/m2 (P = 3.1 × 10-4) and 24 male survivors treated with CED ≥4,000 mg/m2 and radiotherapy <40 Gray (P = 0.012). No association was observed among survivors not exposed to alkylating agents included in the CED (P > 0.29). rs7784118 conferred 3.48- and 9.73-fold increases in risk for clinically defined oligospermia and azoospermia and improved prediction of normospermic, oligospermic, and azoospermic states by 13.7%, 5.3%, and 21.7%. rs7784118 was associated with decreased testosterone level, increased levels of follicle stimulating and luteinizing hormones, and 8.52-fold increased risk of Leydig cell failure. Additional research is warranted to determine how this SNP influences spermatogenesis and to assess its clinical utility in characterizing high-risk survivors and guiding intervention strategies. SIGNIFICANCE: The identified genetic markers harbor potential clinical utility in characterizing high-risk survivors and guiding intervention strategies including pretreatment patient counseling and use of fertility preservation services.

MAGE cancer-testis antigens protect the mammalian germline under environmental stress.

Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.

Correction: γKlotho is a novel marker and cell survival factor in a subset of triple negative breast cancers.

[This corrects the article DOI: 10.18632/oncotarget.6006.].

Store-Operated Calcium Channel Complex in Postsynaptic Spines: A New Therapeutic Target for Alzheimer's Disease Treatment.

Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in aging and Alzheimer's disease (AD). The stability of mushroom spines depends on stromal interaction molecule 2 (STIM2)-mediated neuronal-store-operated Ca2+ influx (nSOC) pathway, which is compromised in AD mouse models, in aging neurons, and in sporadic AD patients. Here, we demonstrate that the Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 channels form a STIM2-regulated nSOC Ca2+ channel complex in hippocampal mushroom spines. We further demonstrate that a known TRPC6 activator, hyperforin, and a novel nSOC positive modulator, NSN21778 (NSN), can stimulate activity of nSOC pathway in the spines and rescue mushroom spine loss in both presenilin and APP knock-in mouse models of AD. We further show that NSN rescues hippocampal long-term potentiation impairment in APP knock-in mouse model. We conclude that the STIM2-regulated TRPC6/Orai2 nSOC channel complex in dendritic mushroom spines is a new therapeutic target for the treatment of memory loss in aging and AD and that NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD. SIGNIFICANCE STATEMENT: Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in Alzheimer's disease (AD). This study demonstrated that Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 form stromal interaction molecule 2 (STIM2)-regulated neuronal-store-operated Ca2+ influx (nSOC) channel complex in hippocampal synapse and the resulting Ca2+ influx is critical for long-term maintenance of mushroom spines in hippocampal neurons. A novel nSOC-positive modulator, NSN21778 (NSN), rescues mushroom spine loss and synaptic plasticity impairment in AD mice models. The TRPC6/Orai2 nSOC channel complex is a new therapeutic target and NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD.

γKlotho is a novel marker and cell survival factor in a subset of triple negative breast cancers.

Over the last decade, breast cancer mortality has declined. However, triple negative breast cancer (TNBC) remains a challenging problem mostly due to early recurrence and lack of molecularly driven treatments. There is a critical need to identify subgroups of TNBC with common molecular features that can be therapeutically targeted. Here we show that in contrast to Klotho and ßKlotho, the third member of the Klotho protein family, γKlotho, is overexpressed in more than 60% of TNBCs and correlates with poorer disease progression. Furthermore, we find that γKlotho is expressed in a subset of TNBC cell lines promoting cell growth. Importantly, we demonstrate that in these cells γKlotho is necessary for cell survival and that its depletion leads to constitutive ERK activation, cell cycle arrest and apoptosis. Interestingly, we observe increased oxidative stress in γKlotho-depleted cells suggesting that γKlotho enables cancer cells to cope with an oxidative environment and that cells become dependent on its expression to maintain this survival advantage. These findings indicate that γKlotho might be a potential marker for patients that would benefit from treatments that alter oxidative stress and constitutes a novel drug target for a subset of TN breast cancers.

USP7 Acts as a Molecular Rheostat to Promote WASH-Dependent Endosomal Protein Recycling and Is Mutated in a Human Neurodevelopmental Disorder.

Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels by counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insights into endosomal trafficking, illuminate the cooperativity between an ubiquitin ligase and a deubiquitinating enzyme, and establish a role for USP7 in human neurodevelopmental disease.

Degradation of AMPK by a cancer-specific ubiquitin ligase.

AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth-restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and are sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.

Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression.

1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug-drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and ß-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug-drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.

Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist.

In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

Cholesterogenic genes expression in brain and liver of ganglioside-deficient mice.

The aim of this study was to determine the effect of changed ganglioside profile on transcription of selected genes involved in cholesterol homeostasis. For that purpose, the expression of 11 genes related to cholesterol synthesis, regulation, and cholesterol transport was investigated in selected brain regions (frontal cortex, hippocampus, brain stem, cerebellum) and liver of St8sia1 knockout (KO) mice characterized by deficient synthesis of b- and c-series gangliosides and accumulation of a-series gangliosides. The expression of majority of the analyzed genes, as determined using quantitative real time PCR, was slightly higher in St8sia1 KO compared to wild-type (wt) controls. More prominent changes were observed in Hmgr, Cyp51, and Cyp46 expression in brain (hippocampus and brain stem) and Srebp1a, Insig2a, and Ldlr in liver. In addition, the expression of master transcriptional regulators, Srebp1a, Srebp1c, and Insig2a, as well as transporters Ldlr and Vldlr differed between liver and brain, and within brain regions in wt animals. Cyp46 expression was expectedly brain-specific, with brain region difference in both wt and St8sia1 KO. The established change in transcriptome of cholesterogenic genes is associated to specific alteration of ganglioside composition which indicates relationship between gangliosides and regulation of cholesterol metabolism.

Nonalcoholic Fatty liver disease: focus on lipoprotein and lipid deregulation.

Obesity with associated comorbidities is currently a worldwide epidemic and among the most challenging health conditions in the 21st century. A major metabolic consequence of obesity is insulin resistance which underlies the pathogenesis of the metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. It comprises a disease spectrum ranging from simple steatosis (fatty liver), through nonalcoholic steatohepatitis (NASH) to fibrosis, and ultimately liver cirrhosis. Abnormality in lipid and lipoprotein metabolism accompanied by chronic inflammation is the central pathway for the development of metabolic syndrome-related diseases, such as atherosclerosis, cardiovascular disease (CVD), and NAFLD. This paper focuses on pathogenic aspect of lipid and lipoprotein metabolism in NAFLD and the relevant mouse models of this complex multifactorial disease.

Research resource: Comprehensive expression atlas of the fibroblast growth factor system in adult mouse.

Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals.

Adaptation of cholesterol synthesis to fasting and TNF-alpha: profiling cholesterol intermediates in the liver, brain, and testis.

Key players in pathogenesis of metabolic disorders are disturbed cholesterol balance and inflammation. In addition to cholesterol also sterol intermediates are biologically active, however, surprisingly little is known about their synthesis and roles. The aim of our study was to assess the interplay between the inflammatory cytokine TNF-alpha and cholesterol synthesis by measuring cholesterol and its intermediates in the liver, brain, and testis. Liquid chromatography-mass spectrometry has been applied to profile sterols of normally fed mice, during fasting and after TNF-alpha administration. In mice on normal chow diet, sterols other than cholesterol represent 0.5% in the liver, 1% in brain and 5% in testis. In the liver only 7-dehydrocholesterol, lanosterol and desmosterol were detected. Major sterol intermediates of the brain are desmosterol, testis meiosis activating sterol (T-MAS), and 7-dehydrocholesterol while in testis T-MAS predominates (4%), followed by desmosterol, lanosterol, 7-dehydrocholesterol and others. In 20h fasting there is no significant change in cholesterol of the three tissues, and no significant change in intermediates of the liver. In the brain sterol intermediates are lowered (significant for zymosterol) while in the testis the trend is opposite. TNF-alpha provokes a significant raise of some intermediates whereas the level of cholesterol is again unchanged. The proportion of sterols in the liver rises from 0.5% in controls to 1.2% in TNF-alpha-treated mice, which is in accordance with published expression profiling data. In conclusion, our data provide novel insights into the interaction between the inflammatory cytokine TNF-alpha and the tissue-specific cholesterol biosynthesis of the liver, brain and testis.

Regulation of bile acid synthesis by fat-soluble vitamins A and D.

Bile acids are required for proper absorption of dietary lipids, including fat-soluble vitamins. Here, we show that the dietary vitamins A and D inhibit bile acid synthesis by repressing hepatic expression of the rate-limiting enzyme CYP7A1. Receptors for vitamin A and D induced expression of Fgf15, an intestine-derived hormone that acts on liver to inhibit Cyp7a1. These effects were mediated through distinct cis-acting response elements in the promoter and intron of Fgf15. Interestingly, transactivation of both response elements appears to be required to maintain basal Fgf15 expression levels in vivo. Furthermore, whereas induction of Fgf15 by vitamin D is mediated through its receptor, the induction of Fgf15 by vitamin A is mediated through the retinoid X receptor/farnesoid X receptor heterodimer and is independent of bile acids, suggesting that this heterodimer functions as a distinct dietary vitamin A sensor. Notably, vitamin A treatment reversed the effects of the bile acid sequestrant cholestyramine on Fgf15, Shp, and Cyp7a1 expression, suggesting a potential therapeutic benefit of vitamin A under conditions of bile acid malabsorption. These results reveal an unexpected link between the intake of fat-soluble vitamins A and D and bile acid metabolism, which may have evolved as a means for these dietary vitamins to regulate their own absorption.