Publications by authors named "Bonne Thompson"

26 Publications

  • Page 1 of 1

Circulating 27-hydroxycholesterol and Risk of Colorectal Adenomas and Serrated Polyps.

Cancer Prev Res (Phila) 2021 Apr 6;14(4):479-488. Epub 2021 Jan 6.

Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.

The oxysterol 27-hydroxycholesterol (27-OHC) is an endogenous selective estrogen receptor modulator implicated in breast cancer etiology. It is unknown whether circulating 27-OHC is associated with colorectal neoplasia risk. Circulating 27-OHC was measured using LC/MS in fasting plasma collected at baseline from participants of the Vitamin D/Calcium Polyp Prevention Study, a completed randomized clinical trial. Participants were between 45 and 75 years old, recently diagnosed with ≥1 colorectal adenoma, and followed for new colorectal polyps during colonoscopic surveillance. Adjusted risk ratios (RR) with 95% confidence intervals (CI) of new colorectal polyps were estimated for quartiles of circulating 27-OHC using log-linear regression for repeated outcomes. Polyp phenotypes included any adenomas, advanced adenomas, hyperplastic polyps, and sessile serrated adenomas/polyps. Circulating 27-OHC was measured at baseline for 1,246 participants. Compared with participants with circulating 27-OHC below the first quartile (<138 ng/mL), those with circulating 27-OHC at or above the fourth quartile (≥201 ng/mL) had 24% higher risk of adenomas (RR, 1.24; 95% CI, 1.05-1.47) and 89% higher risk of advanced adenomas (RR, 1.89; 95% CI, 1.17-3.06). Stronger associations were observed among participants with advanced adenomas at baseline. Circulating 27-OHC was not associated with risk of hyperplastic polyps (RR, 0.90; 95% CI, 0.66-1.22) or sessile serrated adenomas/polyps (RR, 1.02; 95% CI, 0.50-2.07). Circulating 27-OHC may be a risk factor for colorectal adenomas but not serrated polyps. PREVENTION RELEVANCE: This study found that plasma concentration of 27-hydroxycholesterol, a metabolite of cholesterol that regulates lipid metabolism and acts as a selective estrogen receptor modulator, is associated with the risk of developing precursor lesions for colorectal cancer.
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http://dx.doi.org/10.1158/1940-6207.CAPR-20-0414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026496PMC
April 2021

GPR183-Oxysterol Axis in Spinal Cord Contributes to Neuropathic Pain.

J Pharmacol Exp Ther 2020 11 10;375(2):367-375. Epub 2020 Sep 10.

INRAe, UMR 1280, Physiopathologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France (F.K.-C., M.T., K.O.) and Physiogenex SAS, Prologue Biotech, Rue Pierre et Marie Curie, Laboratoryège-Innopole, France (F.B., T.S.)

Neuropathic pain is a debilitating public health concern for which novel non-narcotic therapeutic targets are desperately needed. Using unbiased transcriptomic screening of the dorsal horn spinal cord after nerve injury we have identified that (Epstein-Barr virus-induced gene 2) is upregulated after chronic constriction injury (CCI) in rats. GPR183 is a chemotactic receptor known for its role in the maturation of B cells, and the endogenous ligand is the oxysterol 7,25-dihydroxycholesterol (7,25-OHC). The role of GPR183 in the central nervous system is not well characterized, and its role in pain is unknown. The profile of commercially available probes for GPR183 limits their use as pharmacological tools to dissect the roles of this receptor in pathophysiological settings. Using modeling, we have screened a library of 5 million compounds to identify several novel small-molecule antagonists of GPR183 with nanomolar potency. These compounds are able to antagonize 7,25-OHC-induced calcium mobilization with IC values below 50 nM. intrathecal injections of these antagonists during peak pain after CCI surgery reversed allodynia in male and female mice. Acute intrathecal injection of the GPR183 ligand 7,25-OHC in naïve mice induced dose-dependent allodynia. Importantly, this effect was blocked using our novel GPR183 antagonists, suggesting spinal GPR183 activation as pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify this receptor as a potential target for therapeutic intervention. SIGNIFICANCE STATEMENT: We have identified several novel GPR183 antagonists with nanomolar potency. Using these antagonists, we have demonstrated that GPR183 signaling in the spinal cord is pronociceptive. These studies are the first to reveal a role for GPR183 in neuropathic pain and identify it as a potential target for therapeutic intervention.
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http://dx.doi.org/10.1124/jpet.120.000105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592849PMC
November 2020

Niche-Specific Reprogramming of Epigenetic Landscapes Drives Myeloid Cell Diversity in Nonalcoholic Steatohepatitis.

Immunity 2020 06 1;52(6):1057-1074.e7. Epub 2020 May 1.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA; Department of Medicine, University of California, San Diego, La Jolla, CA, USA. Electronic address:

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.
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http://dx.doi.org/10.1016/j.immuni.2020.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305990PMC
June 2020

Cholesterol 25-hydroxylase promotes efferocytosis and resolution of lung inflammation.

JCI Insight 2020 06 4;5(11). Epub 2020 Jun 4.

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, North Carolina, USA.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.
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http://dx.doi.org/10.1172/jci.insight.137189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308063PMC
June 2020

Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol.

Nat Microbiol 2020 07 13;5(7):929-942. Epub 2020 Apr 13.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.
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http://dx.doi.org/10.1038/s41564-020-0701-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442315PMC
July 2020

A Medicinal Chemistry-Driven Approach Identified the Sterol Isomerase EBP as the Molecular Target of TASIN Colorectal Cancer Toxins.

J Am Chem Soc 2020 04 19;142(13):6128-6138. Epub 2020 Mar 19.

TASIN (Truncated APC-Selective Inhibitors) compounds are selectively toxic to colorectal cancer cells with mutations, although their mechanism of action remains unknown. Here, we found that TASINs inhibit three enzymes in the postsqualene cholesterol biosynthetic pathway including EBP, DHCR7, and DHCR24. Even though all three of these enzymes are required for cholesterol biosynthesis, only inhibition of the most upstream enzyme, EBP, led to cancer cell death via depletion of downstream sterols, an observation that was confirmed by genetic silencing of EBP. Pharmacologic inhibition or genetic silencing of either DHCR7 or DHCR24 had no impact on cell viability. By using photoaffinity probes to generate a relationship between chemical structure and probe competition, we identified compounds that selectively inhibit either EBP or DHCR7. These studies identify EBP, but not downstream enzymes in the cholesterol biosynthetic pathway, as a target in APC mutant colorectal cancer and also have implications for the clinical development of highly selective EBP inhibitors.
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http://dx.doi.org/10.1021/jacs.9b13407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236545PMC
April 2020

Biliopancreatic Diversion Induces Greater Metabolic Improvement Than Roux-en-Y Gastric Bypass.

Cell Metab 2019 11 3;30(5):855-864.e3. Epub 2019 Oct 3.

Center for Human Nutrition, Washington University School of Medicine, St. Louis, MO, USA; Departments of Medicine and Pharmacology, University of California, San Diego, San Diego, CA, USA. Electronic address:

Diabetes remission is greater after biliopancreatic diversion (BPD) than Roux-en-Y gastric bypass (RYGB) surgery. We used a mixed-meal test with ingested and infused glucose tracers and the hyperinsulinemic-euglycemic clamp procedure with glucose tracer infusion to assess the effect of 20% weight loss induced by either RYGB or BPD on glucoregulation in people with obesity (ClinicalTrials.gov number: NCT03111953). The rate of appearance of ingested glucose into the circulation was much slower, and the postprandial increases in plasma glucose and insulin concentrations were markedly blunted after BPD compared to after RYGB. Insulin sensitivity, assessed as glucose disposal rate during insulin infusion, was ∼45% greater after BPD than RYGB, whereas β cell function was not different between groups. These results demonstrate that compared with matched-percentage weight loss induced by RYGB, BPD has unique beneficial effects on glycemic control, manifested by slower postprandial glucose absorption, blunted postprandial plasma glucose and insulin excursions, and greater improvement in insulin sensitivity.
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http://dx.doi.org/10.1016/j.cmet.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876863PMC
November 2019

Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity.

Immunity 2019 10 3;51(4):655-670.e8. Epub 2019 Oct 3.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.
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http://dx.doi.org/10.1016/j.immuni.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800814PMC
October 2019

Uptake of HDL-cholesterol contributes to lipid accumulation in clear cell renal cell carcinoma.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 12 9;1864(12):158525. Epub 2019 Sep 9.

Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Electronic address:

Clear cell renal cell carcinoma (ccRCC), which accounts for the majority of kidney cancer, is known to accumulate excess cholesterol. However, the mechanism and functional significance of the lipid accumulation for development of the cancer remains obscure. In this study, we analyzed 42 primary ccRCC samples, and determined that cholesterol levels of ~ 70% of the tumors were at least two-fold higher than that of benign kidney tissues. Compared to tumors without cholesterol accumulation, those containing excess cholesterol expressed higher levels of scavenger receptor BI (SR-B1), a receptor for uptake of HDL-associated cholesterol, but not genes involved in cholesterol synthesis and uptake of LDL-associated cholesterol. To further determine the roles of sterol accumulation for cancer development, we implanted ccRCC from patients into mouse kidneys using a mouse ccRCC xenograft model. Feeding mice with probucol, a compound lowing HDL-cholesterol, markedly reduced levels of cholesterol in tumors containing excess cholesterol. This treatment, however, did not affect growth of these tumors. Our study suggests that cholesterol overaccumulation in ccRCC is the consequence of increased uptake of HDL-cholesterol as a result of SR-B1 overexpression, but the lipid accumulation by itself may not play a significant role in progression of the cancer.
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http://dx.doi.org/10.1016/j.bbalip.2019.158525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832851PMC
December 2019

Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric G.

Nature 2019 07 5;571(7764):279-283. Epub 2019 Jun 5.

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric G protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a G-coupled activation mechanism of human SMO. Notably, the G protein presents a different arrangement from that of class-A GPCR-G complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.
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http://dx.doi.org/10.1038/s41586-019-1286-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777001PMC
July 2019

Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition.

Nat Commun 2019 06 5;10(1):2452. Epub 2019 Jun 5.

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

3-β-hydroxysteroid-Δ, Δ-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.
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http://dx.doi.org/10.1038/s41467-019-10279-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549186PMC
June 2019

Target identification reveals lanosterol synthase as a vulnerability in glioma.

Proc Natl Acad Sci U S A 2019 04 28;116(16):7957-7962. Epub 2019 Mar 28.

Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065;

Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.
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http://dx.doi.org/10.1073/pnas.1820989116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475387PMC
April 2019

Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice.

Elife 2019 02 20;8. Epub 2019 Feb 20.

Departments of Molecular Genetics, Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States.

Autosomal-dominant Schnyder corneal dystrophy (SCD) is characterized by corneal opacification owing to overaccumulation of cholesterol. SCD is caused by mutations in UBIAD1, which utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K. Using cultured cells, we previously showed that sterols trigger binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase (HMGCR), thereby inhibiting its endoplasmic reticulum (ER)-associated degradation (ERAD) (Schumacher et al. 2015). GGpp triggers release of UBIAD1 from HMGCR, allowing maximal ERAD and ER-to-Golgi transport of UBIAD1. SCD-associated UBIAD1 resists GGpp-induced release and is sequestered in ER to inhibit ERAD. We now report knockin mice expressing SCD-associated UBIAD1 accumulate HMGCR in several tissues resulting from ER sequestration of mutant UBIAD1 and inhibition of HMGCR ERAD. Corneas from aged knockin mice exhibit signs of opacification and sterol overaccumulation. These results establish the physiological significance of UBIAD1 in cholesterol homeostasis and indicate inhibition of HMGCR ERAD contributes to SCD pathogenesis.
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http://dx.doi.org/10.7554/eLife.44396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402834PMC
February 2019

Three-phase liquid extraction: a simple and fast method for lipidomic workflows.

J Lipid Res 2019 03 4;60(3):694-706. Epub 2019 Jan 4.

Center for Human Nutrition University of Texas Southwestern Medical Center, Dallas, TX 75390

An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. Here we present a novel three-phase lipid extraction (3PLE) technique using a single-step liquid-liquid extraction (LLE) that allows both extraction and fractionation of lipids by polarity. 3PLE is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesteryl esters), while the middle organic phase contains the major glycerophospholipids. Thin-layer chromatography, radioactive labeling, and MS were used to confirm lipid partitioning. 3PLE efficiency was demonstrated for bovine liver, human pooled plasma, mouse liver, mouse brain, and mouse white adipose tissue. Compared with the gold-standard Bligh/Dyer LLE, 3PLE showed significant advantages. For direct-infusion workflows, there was a decrease in ion suppression with a corresponding increased number of lipid species identified. For LC/MS workflows, increased signal intensities were observed for lower-abundance lipid species such as phosphatidic acid and phosphatidylserine. 3PLE also proved to be a valuable tool for fatty acid profiling by GC/MS, allowing for the separate identification of neutral and polar fatty acids.
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http://dx.doi.org/10.1194/jlr.D090795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399505PMC
March 2019

Cilia-Associated Oxysterols Activate Smoothened.

Mol Cell 2018 10;72(2):316-327.e5

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.
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http://dx.doi.org/10.1016/j.molcel.2018.08.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503851PMC
October 2018

27-Hydroxycholesterol, an Endogenous SERM, and Risk of Fracture in Postmenopausal Women: A Nested Case-Cohort Study in the Women's Health Initiative.

J Bone Miner Res 2019 01 7;34(1):59-66. Epub 2018 Dec 7.

School of Medicine, Stanford University, Stanford, CA, USA.

27-Hydroxycholesterol (27HC) is a purported, novel endogenous SERM. In animal models, 27HC has an anti-estrogen effect in bone, and 17β-estradiol mitigates this effect. 27HC in relation to fracture risk has not been investigated in humans. Depending on the level of bioavailable 17β-estradiol (bioE ), 27HC may increase fracture risk in postmenopausal women and modify the fracture risk reduction from menopausal hormone therapy (MHT). To test these a priori hypotheses, we conducted a nested case-cohort study of 868 postmenopausal women within the Women's Health Initiative Hormone Therapy (WHI-HT) trials. The WHI-HT tested conjugated equine estrogens versus placebo and separately conjugated equine estrogens plus progestin versus placebo. Fracture cases were 442 women who had an adjudicated incident hip or clinical vertebral fracture during the WHI-HT follow-up. The subcohort included 430 women randomly selected at WHI-HT baseline, four of whom had a subsequent fracture. Of the 868 women, 266 cases and 219 non-cases were assigned to the placebo arms. Cox models estimated hazard ratios for incident fracture in relation to pre-randomization circulating levels of 27HC and 27HC/bioE molar ratio. Models adjusted for age, race/ethnicity, total cholesterol, bioE , sex hormone-binding globulin, 25-hydroxyvitamin D, diabetes, osteoporosis, prior MHT use, BMI, falls history, and prior fracture. In women assigned to placebo arms, those in the middle and the highest tertiles of 27HC/bioE had an up to 1.9-fold (95% confidence intervals, 1.25 to 2.99) greater risk of fracture than women in the lowest tertile. In women assigned to MHT arms, fracture risk increased with continuous 27HC/bioE levels but not with categorical levels. 27HC levels alone were not associated with fracture risk. 27HC and 27HC/bioE did not modify the fracture risk reduction from MHT. In postmenopausal women, circulating levels of 27HC relative to bioE may identify those at increased risk of fracture. © 2018 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478389PMC
January 2019

Cell-specific discrimination of desmosterol and desmosterol mimetics confers selective regulation of LXR and SREBP in macrophages.

Proc Natl Acad Sci U S A 2018 05 9;115(20):E4680-E4689. Epub 2018 Apr 9.

Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093;

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.
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http://dx.doi.org/10.1073/pnas.1714518115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960280PMC
May 2018

Genetic, anatomic, and clinical determinants of human serum sterol and vitamin D levels.

Proc Natl Acad Sci U S A 2014 Sep 8;111(38):E4006-14. Epub 2014 Sep 8.

Departments of Molecular Genetics,

An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10(-7)). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism.
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http://dx.doi.org/10.1073/pnas.1413561111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183318PMC
September 2014

Bazedoxifene and conjugated estrogen prevent diet-induced obesity, hepatic steatosis, and type 2 diabetes in mice without impacting the reproductive tract.

Am J Physiol Endocrinol Metab 2014 Aug 17;307(3):E345-54. Epub 2014 Jun 17.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas; and

Despite the capacity of estrogens to favorably regulate body composition and glucose homeostasis, their use to combat obesity and type 2 diabetes is not feasible, because they promote sex steroid-responsive cancers. The novel selective estrogen receptor modulator (SERM) bazedoxifene acetate (BZA) uniquely antagonizes both breast cancer development and estrogen-related changes in the female reproductive tract. How BZA administered with conjugated estrogen (CE) or alone impacts metabolism is unknown. The effects of BZA or CE + BZA on body composition and glucose homeostasis were determined in ovariectomized female mice fed a Western diet for 10-12 wk. In contrast to vehicle, estradiol (E₂), CE, BZA, and CE + BZA equally prevented body weight gain by 50%. In parallel, all treatments caused equal attenuation of the increase in body fat mass invoked by the diet as well as the increases in subcutaneous and visceral white adipose tissue. Diet-induced hepatic steatosis was attenuated by E₂ or CE, and BZA alone or with CE provided even greater steatosis prevention; all interventions improved pyruvate tolerance tests. Glucose tolerance tests and HOMA-IR were improved by E₂, CE, and CE + BZA. Whereas E₂ or CE alone invoked a uterotrophic response, BZA alone or CE + BZA had negligible impact on the uterus. Thus, CE + BZA affords protection from diet-induced adiposity, hepatic steatosis, and insulin resistance with minimal impact on the female reproductive tract in mice. These combined agents may provide a valuable new means to favorably regulate body composition and glucose homeostasis and combat fatty liver.
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http://dx.doi.org/10.1152/ajpendo.00653.2013DOI Listing
August 2014

27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth.

Cell Rep 2013 Nov 7;5(3):637-45. Epub 2013 Nov 7.

Department of Pediatrics, Division of Pulmonary and Vascular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

To date, estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells, and it stimulates cell-autonomous, ER-dependent, and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally modulated, nonaromatized ER ligand that promotes ER+ breast tumor growth.
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http://dx.doi.org/10.1016/j.celrep.2013.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950897PMC
November 2013

A mouse macrophage lipidome.

J Biol Chem 2010 Dec 5;285(51):39976-85. Epub 2010 Oct 5.

Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.
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http://dx.doi.org/10.1074/jbc.M110.182915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3000979PMC
December 2010

Subcellular organelle lipidomics in TLR-4-activated macrophages.

J Lipid Res 2010 Sep 23;51(9):2785-97. Epub 2010 Jun 23.

Department of Chemistry and Biochemistry and Department of Pharmacology, University of California, San Diego, CA 92093, USA.

Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo(2)-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.
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http://dx.doi.org/10.1194/jlr.M008748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2918461PMC
September 2010

25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production.

Proc Natl Acad Sci U S A 2009 Sep 15;106(39):16764-9. Epub 2009 Sep 15.

Department of Molecular Genetics and The Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.
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http://dx.doi.org/10.1073/pnas.0909142106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757821PMC
September 2009

Identification and quantitation of sorbitol-based nuclear clarifying agents extracted from common laboratory and consumer plasticware made of polypropylene.

Anal Chem 2008 Jul 6;80(14):5532-41. Epub 2008 Jun 6.

Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.

Reported here is the mass spectral identification of sorbitol-based nuclear clarifying agents (NCAs) and the quantitative description of their extractability from common laboratory and household plasticware made of polypropylene. NCAs are frequently added to polypropylene to improve optical clarity, increase performance properties, and aid in the manufacturing process of this plastic. NCA addition makes polypropylene plasticware more aesthetically pleasing to the user and makes the product competitive with other plastic formulations. We show here that several NCAs are readily extracted with either ethanol or water from plastic labware during typical laboratory procedures. Observed levels ranged from a nanogram to micrograms of NCA. NCAs were also detected in extracts from plastic food storage containers; levels ranged from 1 to 10 microg in two of the three brands tested. The electron ionization mass spectra for three sorbitol-based nuclear clarifying agents (1,3:2,4-bis-O-(benzylidene)sorbitol, 1,3:2,4-bis-O-(p-methylbenzylidene)sorbitol, 1,3:2,4-bis-O-(3,4-dimethylbenzylidene)sorbitol) are presented for the native and trimethylsilyl-derivatized compounds together with the collision-induced dissociation mass spectra; gas and liquid chromatographic data are also reported. These NCAs now join other well-known plasticizers such as phthalate esters and bisphenol A as common laboratory contaminants. While the potential toxicity of NCAs in mammalian systems is unknown, the current data provide scientists and consumers the opportunity to make more informed decisions regarding the use of polypropylene plastics.
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http://dx.doi.org/10.1021/ac8005632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727714PMC
July 2008

Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.

Methods Enzymol 2007 ;432:145-70

Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

We describe the development of a high performance liquid chromatography mass spectrometry (HPLC-MS) method that allows the identification and quantitation of sterols in mammalian cells and tissues. Bulk lipids are extracted from biological samples by a modified Bligh/Dyer procedure in the presence of eight deuterated sterol standards to allow subsequent quantitation and determination of extraction efficiency. Sterols and other lipids are resolved by HPLC on a reverse-phase C18 column using a binary gradient of methanol and water, both containing 5mM ammonium acetate. Sterol identification is performed using an Applied Biosystems (Foster City, CA) 4000 QTRAP mass spectrometer equipped with a TurboV electrospray ionization source and operated in the positive (+) selected reaction monitoring (SRM) mode. The total run time of the analysis is 30 min. Sterols are quantitated by comparison of the areas under the elution curves derived from the detection of endogenous compounds and isotopically labeled standards. The sensitivity of the method for sterol detection ranges between 10 and 2000 fmol on-column. Cultured RAW 264.7 mouse macrophages contain many different sterols, including the liver X receptor (LXR) ligand 24,25-epoxycholesterol. Tissues such as mouse brain also contain large numbers of sterols, including 24(s)-hydroxycholesterol, which is involved in cholesterol turnover in the brain. The extraction procedure described is flexible and can be tailored to sample type or information sought. The instrumental analysis method is similarly adaptable and offers high selectivity and sensitivity.
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http://dx.doi.org/10.1016/S0076-6879(07)32006-5DOI Listing
March 2008

Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.

J Clin Invest 2006 Nov;116(11):2985-94

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher's form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease.
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http://dx.doi.org/10.1172/JCI29224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1626112PMC
November 2006