Publications by authors named "Changlu Liu"

55 Publications

Molecular mechanism and structural basis of small-molecule modulation of the gating of acid-sensing ion channel 1.

Commun Biol 2021 Feb 9;4(1):174. Epub 2021 Feb 9.

Neuroscience Discovery, Janssen Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA, 92121, USA.

Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.
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http://dx.doi.org/10.1038/s42003-021-01678-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873226PMC
February 2021

IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells.

BMC Pulm Med 2020 Oct 23;20(1):278. Epub 2020 Oct 23.

The First Clinical Medical College, Jinan University, 601 W. Huangpu Avenue, Guangzhou, 510630, China.

Background: Epithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF.

Methods: Human lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting.

Results: Stimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, Vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA.

Conclusions: These results suggest that IL-32 induces EMT in A549 cells by triggering ER stress, and IL-32 may be a novel marker for IPF.
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http://dx.doi.org/10.1186/s12890-020-01319-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585222PMC
October 2020

Long Non-Coding RNA 691 Regulated PTEN/PI3K/AKT Signaling Pathway in Osteosarcoma Through miRNA-9-5p.

Onco Targets Ther 2020 22;13:4597-4606. Epub 2020 May 22.

Department of Joint Surgery, The Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot, Inner Mongolia Autonomous Region, People's Republic of China.

Background: Large amounts of researches indicate that non-coding RNAs play a crucial role in many malignancies. However, the potential mechanisms of non-coding RNAs involved in osteosarcoma tumorigenesis remain elusive.

Materials And Methods: The expression of long non-protein coding RNA 691 (lncRNA 691) in cell lines and paired osteosarcoma tissues was compared by qRT-PCR assay. Then, we explored the tumor suppressor function of lncRNA 691 with MTS and colony formation assay. Flow cytometry results showed lncRNA 691 can enhance cell apoptosis. Then, we predicted and verified the negative regulation relationship with miRNA and the miRNA's target gene. Lastly, we revealed the tumorigenesis function of lncRNA-691/miRNA/target gene axis in osteosarcoma.

Results: In our study, we disclosed that lncRNA 691 had low expression levels in osteosarcoma cell lines and tissues. Overexpression of lncRNA 691 could suppress the cell proliferation and induce cell apoptosis in MG-63 cell line. Then, bioinformatics analyses were performed and miR-9-5p was found to negatively regulate the lncRNA 691 expression and promote the osteosarcoma tumorigenesis in vitro. PTEN was predicted as the target gene of miR-9-5p. Luciferase reporter assay and RIP assay demonstrated the regulatory network of lncRNA 691/miR-9-5p/PTEN. We revealed that PTEN was downregulated by the overexpression of miR-9-5p and upregulated by the overexpression of lncRNA 691. At last, the apoptosis-associated protein of the lncRNA 691/miR-9-5p/PTEN/PI3K/AKT was further demonstrated.

Conclusion: LncRNA 691/miR-9-5p could regulate the tumorigenesis by regulating the PTEN/PI3K/AKT signal pathway in osteosarcoma.
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http://dx.doi.org/10.2147/OTT.S249827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250307PMC
May 2020

Putative role of GPR139 on sleep modulation using pharmacological and genetic rodent models.

Eur J Pharmacol 2020 Sep 9;882:173256. Epub 2020 Jun 9.

Department of Neuroscience, Janssen Research & Development, L.L.C, San Diego, CA, USA. Electronic address:

GPR139 is a G-protein coupled receptor expressed in circumventricular regions of the habenula and septum. Amino acids L-tryptophan and L-phenylalanine have been shown to activate GPR139 at physiologically relevant concentrations. The aim of the present study was to investigate the role of GPR139 on sleep modulation using pharmacological and genetic (GPR139 knockout mice, KO) rodent models. To evaluate the effects of GPR139 pharmacological activation on sleep, rats were orally dosed with the selective GPR139 agonist JNJ-63533054 (3-30 mg/kg). When acutely administered at the beginning of the light phase, the GPR139 agonist dose-dependently reduced non-rapid eye movement (NREM) latency and increased NREM sleep duration without altering rapid eye movement (REM) sleep. This effect progressively dissipated upon 7-day repeated dosing, suggesting functional desensitization. Under baseline conditions, GPR139 KO mice spent less time in REM sleep compared to their wild type littermates during the dark phase, whereas NREM sleep was not altered. Under conditions of pharmacologically enhanced monoamine endogenous tone, GPR139 KO mice showed a blunted response to citalopram or fluoxetine induced REM sleep suppression and an attenuated response to the wake promoting effect of amphetamine. These findings indicate an emerging role of GPR139 in the modulation of sleep states.
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http://dx.doi.org/10.1016/j.ejphar.2020.173256DOI Listing
September 2020

The PAR2 signal peptide prevents premature receptor cleavage and activation.

PLoS One 2020 20;15(2):e0222685. Epub 2020 Feb 20.

Janssen Research & Development, LLC, San Diego, California, United States of America.

Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that there is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032737PMC
April 2020

GPR139 and Dopamine D2 Receptor Co-express in the Same Cells of the Brain and May Functionally Interact.

Front Neurosci 2019 26;13:281. Epub 2019 Mar 26.

Janssen Research and Development, LLC, San Diego, CA, United States.

GPR139, a G-coupled receptor that is activated by the essential amino acids L-tryptophan and L-phenylalanine, is predominantly expressed in the brain and pituitary. The physiological function of GPR139 remains elusive despite the availability of pharmacological tool agonist compounds and knock-out mice. Whole tissue RNA sequencing data from human, mouse and rat tissues revealed that GPR139 and the dopamine D receptor (DRD2) exhibited some similarities in their distribution patterns in the brain and pituitary gland. To determine if there was true co-expression of these two receptors, we applied double hybridization in mouse tissues using the RNAscope technique. GPR139 and DRD2 mRNA co-expressed in a majority of same cells within part of the dopaminergic mesolimbic pathways (ventral tegmental area and olfactory tubercle), the nigrostriatal pathway (compact part of substantia nigra and caudate putamen), and also the tuberoinfundibular pathway (arcuate hypothalamic nucleus and anterior lobe of pituitary). Both receptors mRNA also co-express in the same cells of the brain regions involved in responses to negative stimulus and stress, such as lateral habenula, lateral septum, interpeduncular nucleus, and medial raphe nuclei. GPR139 mRNA expression was detected in the dentate gyrus and the pyramidal cell layer of the hippocampus as well as the paraventricular hypothalamic nucleus. The functional interaction between GPR139 and DRD2 was studied using a calcium mobilization assay in cells co-transfected with both receptors from several species (human, rat, and mouse). The dopamine DRD2 agonist did not stimulate calcium response in cells expressing DRD2 alone consistent with the G signaling transduction pathway of this receptor. In cells co-transfected with DRD2 and GPR139 the DRD2 agonist was able to stimulate calcium response and its effect was blocked by either a DRD2 or a GPR139 antagonist supporting an interaction between GPR139 and DRD2. Taken together, these data showed that GPR139 and DRD2 are in position to functionally interact in native tissue.
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http://dx.doi.org/10.3389/fnins.2019.00281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443882PMC
March 2019

Characterization of a Selective, Orally Available, and Brain Penetrant Small Molecule GPR139 Agonist.

Front Pharmacol 2019 21;10:273. Epub 2019 Mar 21.

Janssen Research & Development, LLC, San Diego, CA, United States.

Recently, our group along with another demonstrated that GPR139 can be activated by L-phenylalanine (L-Phe) and L-tryptophan (L-Trp) at physiologically relevant concentrations. GPR139 is discretely expressed in brain, with highest expression in medial habenula. Not only are the endogenous ligands catecholamine/serotonin precursors, but GPR139 expressing areas can directly/indirectly regulate the activity of catecholamine/serotonin neurons. Thus, GPR139 appears expressed in an interconnected circuit involved in mood, motivation, and anxiety. The aim of this study was to characterize a selective and brain penetrant GPR139 agonist (JNJ-63533054) in relevant models. JNJ-63533054 was tested for its effect on c-fos activation in the habenula and dorsal striatum. microdialysis experiments were performed in freely moving rats to measure basal levels of serotonin or dopamine (DA) in prefrontal cortex (mPFC) and nucleus accumbens (NAc). Finally, the compound was profiled in behavioral models of anxiety, despair, and anhedonia. The agonist (10-30 mg/kg, p.o.) did not alter c-fos expression in medial habenula or dorsal striatum nor neurotransmitter levels in mPFC or NAc. JNJ-63533054 (10 mg/kg p.o.) produced an anhedonic-like effect on urine sniffing, but had no significant effect in tail suspension, with no interaction with imipramine, no effect on naloxone place aversion, and no effect on learned helplessness. In the marble burying test, the agonist (10 mg/kg p.o.) produced a small anxiolytic-like effect, with no interaction with fluoxetine, and no effect in elevated plus maze (EPM). Despite GPR139 high expression in medial habenula, an area with connections to limbic and catecholaminergic/serotoninergic areas, the GPR139 agonist had no effect on c-fos in medial habenula. It did not alter catecholamine/serotonin levels and had a mostly silent signal in models commonly associated with these pathways. The physiological function of GPR139 remains elusive.
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http://dx.doi.org/10.3389/fphar.2019.00273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437111PMC
March 2019

Mutagenesis of GPR139 reveals ways to create gain or loss of function receptors.

Pharmacol Res Perspect 2019 02 7;7(1):e00466. Epub 2019 Feb 7.

Janssen Research & Development, LLC San Diego California.

GPR139 is a Gq-coupled receptor activated by the essential amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe). We carried out mutagenesis studies of the human GPR139 receptor to identify the critical structural motifs required for GPR139 activation. We applied site-directed and high throughput random mutagenesis approaches using a double addition normalization strategy to identify novel GPR139 sequences coding receptors that have altered sensitivity to endogenous ligands. This approach resulted in GPR139 clones with gain-of-function, reduction-of-function or loss-of-function mutations. The agonist pharmacology of these mutant receptors was characterized and compared to wild-type receptor using calcium mobilization, radioligand binding, and protein expression assays. The structure-activity data were incorporated into a homology model which highlights that many of the gain-of-function mutations are either in or immediately adjacent to the purported orthosteric ligand binding site, whereas the loss-of-function mutations were largely in the intracellular G-protein binding area or were disrupters of the helix integrity. There were also some reduction-of-function mutations in the orthosteric ligand binding site. These findings may not only facilitate the rational design of novel agonists and antagonists of GPR139, but also may guide the design of transgenic animal models to study the physiological function of GPR139.
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http://dx.doi.org/10.1002/prp2.466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367278PMC
February 2019

Re-evaluation of Adrenocorticotropic Hormone and Melanocyte Stimulating Hormone Activation of GPR139 .

Front Pharmacol 2018 2;9:157. Epub 2018 Mar 2.

Janssen Research and Development, LLC, San Diego, CA, United States.

It is now well established that GPR139, a G-protein coupled receptor exclusively expressed in the brain and pituitary, is activated by the essential amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) via G-coupling. The affinity and potency values of L-Trp and L-Phe are within the physiological concentration ranges of L-Trp and L-Phe. A recent paper suggests that adrenocorticotropic hormone (ACTH), α and β melanocyte stimulating hormones (α-MSH and β-MSH) and derivatives α-MSH/α-MSH can also activate GPR139 . We tested this hypothesis using guanosine 5'--(3-[S]thio)-triphosphate binding (GTPγS), calcium mobilization and [H]JNJ-63533054 radioligand binding assays. In the GTPγS binding assay, α-MSH, α-MSH/α-MSH, and β-MSH had no effect on [S]GTPγS incorporation in cell membranes expressing GPR139 up to 30 μM in contrast to the concentration dependent activation produced by L-Trp, JNJ-63533054, and TC-09311 (two small molecule GPR139 agonists). ACTH slightly decreased the basal level of [S]GTPγS incorporation at 30 μM. In the GPR139 radioligand binding assay, a moderate displacement of [H]JNJ-63533054 binding by ACTH and β-MSH was observed at 30 μM (40 and 30%, respectively); α-MSH, α-MSH/α-MSH did not displace any specific binding at 30 μM. In three different host cell lines stably expressing GPR139, α-MSH, and β-MSH did not stimulate calcium mobilization in contrast to L-Trp, JNJ-63533054, and TC-09311. ACTH, α-MSH/α-MSH only weakly stimulated calcium mobilization at 30 μM (<50% of EC). We then co-transfected GPR139 with the three melanocortin (MC) receptors (MC3R, MC4R, and MC5R) to test the hypothesis that ACTH, α-MSH, and β-MSH might stimulate calcium mobilization through a MCR/GPR139 interaction. All three MC peptides stimulated calcium response in cells co-transfected with GPR139 and MC3R, MC4R, or MC5R. The MC peptides did not stimulate calcium response in cells expressing MC3R or MC5R alone consistent with the G signaling transduction pathway of these receptors. In agreement with the previously reported multiple signaling pathways of MC4R, including G transduction pathway, the MC peptides produced a calcium response in cells expressing MC4R alone. Together, our findings do not support that GPR139 is activated by ACTH, α-MSH, and β-MSH at physiologically relevant concentration but we did unravel an interaction between GPR139 and the MCRs.
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http://dx.doi.org/10.3389/fphar.2018.00157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863515PMC
March 2018

LC/MS/MS Profiling of Tissue Oxysterols and its Application in Dextran Sodium Sulphate Induced Mouse Colitis Models.

Curr Top Med Chem 2017 ;17(24):2781-2790

Janssen R&D, LLC., 3210 Merryfield Row, San Diego, CA 92121. United States.

We have developed a workflow to extract, separate, and semi-quantify bioactive oxysterols from mouse colon tissues and fecal matters using solid- and liquid-phase extractions, enzymatic and chemical modifications, and stable-isotope dilution LC/MS/MS. The method was applied to a dextran sodium sulphate (DSS)-induced mouse colitis model, which revealed that one particular dihydroxycholesterol (diOHC), 7α,25-diOHC, was significantly elevated in both colon tissue and fecal matters of mice with colitis compared to that in naïve mice. The extent of 7α,25-diOHC elevation was positively correlated with colitis severity.
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http://dx.doi.org/10.2174/1568026617666170713165519DOI Listing
September 2017

High glucose-induced LIF suppresses osteoblast differentiation via regulating STAT3/SOCS3 signaling.

Cytokine 2017 03 5;91:132-139. Epub 2017 Jan 5.

Department of Orthopaedics, The First Affiliated Hospital of Chongqing Medical University, 400016 Chongqing, China. Electronic address:

High glucose (HG) is conceived to regulate bone metabolism in patients with diabetic mellitus (DM). In the present study, we examined the level of leukemia inhibitory factor (LIF), a pleiotropic cytokine in interleukin (IL)-6 family, in T2DM patients and investigated the regulation by HG on the induction of LIF/signal transducer and activator of transcription 3 (STAT3) signaling. Then we determined the regulation of HG and LIF on the osteoblast differentiation via measuring the ALP activity, matrix mineralization, and the expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), Osteocalcin (OCN) and osteopontin (OPN) in human osteoblast MG-63 cells. In addition, we evaluated the dependence of suppressor of cytokine signaling 3 (SOCS3)/STAT3 signaling in the progress. Results indicated significantly higher serum levels of high-sensitivity C-reactive protein (hsCRP), IL-1β, IL-6 and LIF in T2DM patients. HG induced markedly higher levels of these cytokines in vitro. Furthermore, either HG or LIF reduced the expression of ALP, OCN and RUNX2 in both mRNA and protein levels. In addition, LIF markedly promoted the expression of SOCS3, significantly upregulated the phosphorylation of STAT3 in MG-63 cells; and the downregulation of the four osteogenic differentiation-associated markers were restored by 50 or 100nM STAT3 inhibitor, JSI-124. In summary, this study has shown that LIF is implicated in the HG-mediated inhibition of osteoblast differentiation, via promoting STAT3/SOCS3 signaling. This study may provide insights into the signal pathway of HG-induced bone loss or delayed injured joint healing.
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http://dx.doi.org/10.1016/j.cyto.2016.12.016DOI Listing
March 2017

Fluoro analogs of bioactive oxy-sterols: Synthesis of an EBI2 agonist with enhanced metabolic stability.

Bioorg Med Chem Lett 2016 10 12;26(20):4888-4891. Epub 2016 Sep 12.

Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, United States. Electronic address:

Synthesis of several 7-hydroxy oxysterols and their potential roles as signaling molecules in the innate and adaptive immune responses is discussed. Discovery of a new, fluorinated, synthetic analog of the 7α,25-dihydroxycholesterol-the endogenous ligand of GPR 183 (EBI2), a G-protein coupled receptor highly expressed upon Epstein-Barr virus infection is described. Fluoro oxysterol 12 showed good metabolic stability while maintaining excellent EBI2 agonist activity.
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http://dx.doi.org/10.1016/j.bmcl.2016.09.029DOI Listing
October 2016

Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8.

J Pharmacol Exp Ther 2016 May 17;357(2):394-414. Epub 2016 Mar 17.

Janssen Research and Development, LLC, Neuroscience Therapeutic Area, San Diego, California (M.P.M., N.W., S.R., M.K.A., B.M.S., C.L., B.L., R.M.W., J.A.M., C.D., S.Y., A.D.W., N.I.C., T.W.L.); and Janssen Research and Development, a Division of Janssen Pharmaceutica NV, Neuroscience Therapeutic Area, Beerse, Belgium (L.V.D., T.S.).

Members of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediate the majority of fast synaptic transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic intervention. Here, we describe novel AMPA receptor modulators that require the presence of the accessory protein CACNG8, also known as transmembrane AMPA receptor regulatory protein γ8 (TARP-γ8). Using calcium flux, radioligand binding, and electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these compounds possess a novel mechanism of action consistent with a partial disruption of the interaction between the TARP and the pore-forming subunit of the channel. One of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. This molecule showed strong, dose-dependent inhibition of neurotransmission within the hippocampus, and a strong anticonvulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electroencephalogram, transient hyperlocomotion, no motor impairment on rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool for reversible AMPA receptor inhibition, particularly within the hippocampus, with potential therapeutic utility as an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism of action demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential number of druggable targets.
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http://dx.doi.org/10.1124/jpet.115.231712DOI Listing
May 2016

Editorial: Orphan GPCRs As Emerging Drug Targets.

Front Pharmacol 2015 15;6:295. Epub 2015 Dec 15.

Neuroscience Therapeutic Area, Janssen Global Services, LLC San Diego, CA, USA.

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http://dx.doi.org/10.3389/fphar.2015.00295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678218PMC
December 2015

A comprehensive investigation on adsorption of Ca (II), Cr (III) and Mg (II) ions by 3D porous nickel films.

J Colloid Interface Sci 2016 Feb 22;463:154-63. Epub 2015 Oct 22.

College of Science, Tsinghua University, Beijing 100084, PR China.

The present study reports the removal of Ca (II), Cr (III), Mg (II) ions from aqueous solution using 3D-porous nickel films (3DNFs) as a novel adsorbent material prepared by hydrogen bubble dynamic template (HBDT) method at room temperature. The structure morphology and the phase constitution of 3DNFs were characterized by FESEM, EDS and XRD. Adsorption process of Ca (II), Cr (III), Mg (II) ions was fast as the equilibrium was established within 30min, and the maximum adsorption at equilibrium was 44.1mg/g, 46.4mg/g and 32.7mg/g, respectively. The adsorption kinetics well fitted using a pseudo second-order kinetic model. The adsorption isotherm data of all the three metals fit well the Langmuir and Freundlich adsorption isotherm model. It was found out that kinetics of adsorption varies with initial concentration of metal ions. Thermodynamic parameters (i.e., the standard Gibbs free energies (ΔG), enthalpy change (ΔH), standard entropy change (ΔS)) were also evaluated. Thermodynamic analysis indicated that a high temperature is favored for the adsorption of metal ions by 3DNFs. These results suggest that 3DNFs have good potential application in effective adsorption of metal ions with satisfactory results.
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http://dx.doi.org/10.1016/j.jcis.2015.10.037DOI Listing
February 2016

Identification and SAR of Glycine Benzamides as Potent Agonists for the GPR139 Receptor.

ACS Med Chem Lett 2015 Sep 20;6(9):1015-8. Epub 2015 Jul 20.

Janssen Research & Development, LLC , San Diego, California 92121, United States.

A focused high throughput screening for GPR139 was completed for a select 100K compounds, and new agonist leads were identified. Subsequent analysis and structure-activity relationship studies identified (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl)benzamide 7c as a potent and selective agonist of hGPR139 with an EC50 = 16 nM. The compound was found to cross the blood-brain barrier and have good drug-like properties amenable for oral dosing in rat.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569879PMC
September 2015

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

Mol Pharmacol 2015 Nov 8;88(5):911-25. Epub 2015 Sep 8.

Janssen Research & Development LLC, San Diego, California.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.
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http://dx.doi.org/10.1124/mol.115.100412DOI Listing
November 2015

A clinical study of the rotational alignment of the femoral component in total knee arthroplasty.

J Phys Ther Sci 2015 Jul 22;27(7):2077-81. Epub 2015 Jul 22.

Operation Room, The Second Affiliated Hospital of Inner Mongolia Medical University, China.

[Purpose] The reasons for femorotibial rotational malalignment after total knee arthroplasty (TKA) were analyzed to provide evidence for clinical knee joint surgery and to reduce complications. [Subjects and Methods] Ninety knees of 60 patients were selected and randomly divided into two groups (n=30). For one group, rotational alignment of the femoral component was determined by the transepicondylar axis and TKA was performed. For the other group, rotational alignment of the femoral component was conducted through 3° external rotation of the posterior femoral condyles. Knee joint specimens were operated with TKA and various biomechanical indices were measured. [Results] The femoral epicondylar axis was a constant, reliable reference for femoral component rotational alignment. When the femoral component was rotated by 0° versus the epicondylar axis, the peak contact pressure on the patellofemoral joint was optimal. When the femoral component was arranged in parallel with Whiteside's line, the peak contact pressure on the patellofemoral joint varied largely. The patellofemoral contact areas of the two groups were similar. [Conclusion] Axial rotational alignment of the femoral component influenced the contact pressure of patellofemoral joints in TKA more significantly than external rotation of the femoral condyles. It is more reliable to use the femoral epicondylar axis as the reference for the rotational alignment of the femoral component.
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http://dx.doi.org/10.1589/jpts.27.2077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540821PMC
July 2015

7α, 25-dihydroxycholesterol-mediated activation of EBI2 in immune regulation and diseases.

Front Pharmacol 2015 24;6:60. Epub 2015 Mar 24.

Neuroscience Therapeutic Area, Janssen Pharmaceutical Research & Development, LLC, San Diego CA, USA.

EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.
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http://dx.doi.org/10.3389/fphar.2015.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371701PMC
April 2015

Bayesian variable selection for hierarchical gene-environment and gene-gene interactions.

Hum Genet 2015 Jan 26;134(1):23-36. Epub 2014 Aug 26.

Biomathematics and Biostatistics Program, Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.

We propose a Bayesian hierarchical mixture model framework that allows us to investigate the genetic and environmental effects, gene by gene interactions and gene by environment interactions in the same model. Our approach incorporates the natural hierarchical structure between the main effects and interaction effects into a mixture model, such that our methods tend to remove the irrelevant interaction effects more effectively, resulting in more robust and parsimonious models. We consider both strong and weak hierarchical models. For a strong hierarchical model, both the main effects between interacting factors must be present for the interactions to be considered in the model development, while for a weak hierarchical model, only one of the two main effects is required to be present for the interaction to be evaluated. Our simulation results show that the proposed strong and weak hierarchical mixture models work well in controlling false-positive rates and provide a powerful approach for identifying the predisposing effects and interactions in gene-environment interaction studies, in comparison with the naive model that does not impose this hierarchical constraint in most of the scenarios simulated. We illustrate our approach using data for lung cancer and cutaneous melanoma.
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http://dx.doi.org/10.1007/s00439-014-1478-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282989PMC
January 2015

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

Proc Natl Acad Sci U S A 2014 Aug 4;111(33):12163-8. Epub 2014 Aug 4.

Janssen Research and Development, LLC, San Diego, CA 92121; and

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.
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http://dx.doi.org/10.1073/pnas.1322807111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143045PMC
August 2014

Involvement of β-arrestin-2 and clathrin in agonist-mediated internalization of the human cannabinoid CB2 receptor.

Curr Mol Pharmacol 2014 ;7(1):67-80

College of Life Sciences, Zhejiang University, Zijingang Campus, 388 Yuhang Tang Rd., Hangzhou, 310058, China.

The CB2 cannabinoid receptor is a promising therapeutic target for the treatment of inflammatory diseases, neuropathic pain, liver diseases, cancer and cardiovascular diseases. Obtaining detailed information on the internalization and trafficking of the human CB2 receptor in response to agonist will have a significant impact on drug discovery. Visualization and quantitative detection of EGFP-tagged CB2 receptor showed that, upon WIN55,212-2 stimulation, the CB2 receptor was rapidly internalized in a dose- and time-dependent manner from the cell membrane into the cytoplasm. Pretreatment with hypertonic sucrose, MDC clathrin inhibitor, or siRNA-mediated knock-down of clathrin heavy chain led to significant inhibition of agonist-induced CB2 internalization. Using the RNA interference method, we showed that knockdown of β-arrestin2 expression significantly impaired receptor internalisation. Further investigation demonstrated that the internalized CB2 receptors were co-localized with the early endosome probe and were recycled to the cell surface after the removal of agonist, but treatment with specific cell-permeable proteasome inhibitor MG132 a inhibited the recycling of internalized CB2 receptor, suggesting that the proteasome-mediated degradation pathway may be involved in CB2 internalization. Moreover, the single residue Ser(352) and residue cluster S(335)S(336)T(338)T(340) at the C-terminal tail are shown to be essential for receptor phosphorylation and β-arrestin2 association. These data provide new insights into the mechanisms regulating agonist-mediated internalization and trafficking of the human CB2 receptor.
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http://dx.doi.org/10.2174/1874467207666140714115824DOI Listing
September 2015

A novel method to access chiral nonnatural 2,4-disubstituted pyrrolidines from aldehydes and nitroolefins only with an α-substituent.

Chem Commun (Camb) 2013 May 8;49(40):4561-3. Epub 2013 Apr 8.

School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.

A series of α-substituted nitroolefins were employed in organocatalytic asymmetric Michael reactions with aldehydes. γ-Nitro carbonyl products were achieved in good yields (up to 86%) with good stereoselectivities (up to 96% ee and 24 : 1 dr). Reduction of the nitro group followed by intramolecular reductive amination successfully afforded various novel optically active 2,4-disubstituted pyrrolidine compounds.
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http://dx.doi.org/10.1039/c3cc40583dDOI Listing
May 2013

Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.

Mol Pharmacol 2012 Dec 28;82(6):1094-103. Epub 2012 Aug 28.

Janssen Pharmaceutical Research and Development, San Diego, California, USA.

Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid β(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.
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http://dx.doi.org/10.1124/mol.112.080275DOI Listing
December 2012

Natural and orthogonal interaction framework for modeling gene-environment interactions with application to lung cancer.

Hum Hered 2012 9;73(4):185-94. Epub 2012 Aug 9.

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Objectives: We aimed at extending the Natural and Orthogonal Interaction (NOIA) framework, developed for modeling gene-gene interactions in the analysis of quantitative traits, to allow for reduced genetic models, dichotomous traits, and gene-environment interactions. We evaluate the performance of the NOIA statistical models using simulated data and lung cancer data.

Methods: The NOIA statistical models are developed for additive, dominant, and recessive genetic models as well as for a binary environmental exposure. Using the Kronecker product rule, a NOIA statistical model is built to model gene-environment interactions. By treating the genotypic values as the logarithm of odds, the NOIA statistical models are extended to the analysis of case-control data.

Results: Our simulations showed that power for testing associations while allowing for interaction using the NOIA statistical model is much higher than using functional models for most of the scenarios we simulated. When applied to lung cancer data, much smaller p values were obtained using the NOIA statistical model for either the main effects or the SNP-smoking interactions for some of the SNPs tested.

Conclusion: The NOIA statistical models are usually more powerful than the functional models in detecting main effects and interaction effects for both quantitative traits and binary traits.
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http://dx.doi.org/10.1159/000339906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534768PMC
January 2013

Identification of Hydroxybenzoic Acids as Selective Lactate Receptor (GPR81) Agonists with Antilipolytic Effects.

ACS Med Chem Lett 2012 Aug 11;3(8):637-9. Epub 2012 Jun 11.

Janssen Research & Development, LLC , San Diego, California 92121, United States.

Following the characterization of the lactate receptor (GPR81), a focused screening effort afforded 3-hydroxybenzoic acid 1 as a weak agonist of both GPR81 and GPR109a (niacin receptor). An examination of structurally similar arylhydroxy acids led to the identification of 3-chloro-5-hydroxybenzoic acid 2, a selective GPR81 agonist that exhibited favorable in vivo effects on lipolysis in a mouse model of obesity.
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http://dx.doi.org/10.1021/ml3000676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025785PMC
August 2012

3,5-Dihydroxybenzoic acid, a specific agonist for hydroxycarboxylic acid 1, inhibits lipolysis in adipocytes.

J Pharmacol Exp Ther 2012 Jun 20;341(3):794-801. Epub 2012 Mar 20.

Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA.

Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the β-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) ∼150 μM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.
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http://dx.doi.org/10.1124/jpet.112.192799DOI Listing
June 2012

2-[(3S)-5-Oxooxolan-3-yl]isoindoline-1,3-dione.

Acta Crystallogr Sect E Struct Rep Online 2012 Jan 3;68(Pt 1):o24. Epub 2011 Dec 3.

Department of Chemistry and Chemical Engineering, Sichuan University of Arts and Science, Sichuan Key Laboratory of Characteristic Plant Development and Research, Sichuan Dazhou 635000, People's Republic of China.

The oxolan-2-one ring in the title compound, C(12)H(9)NO(4), has an envelope conformation with the atom linking the two five-membered rings being the flap atom.
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http://dx.doi.org/10.1107/S160053681105135XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3254364PMC
January 2012

Study of GPR81, the lactate receptor, from distant species identifies residues and motifs critical for GPR81 functions.

Mol Pharmacol 2011 Nov 23;80(5):848-58. Epub 2011 Aug 23.

Johnson and Johnson Pharmaceutical Research and Development, San Diego, CA 92121, USA.

Receptors from distant species may have conserved functions despite significant differences in protein sequences. Whereas the noncritical residues are often changed in distant species, the amino acids critical in receptor functions are often conserved. Studying the conserved residues between receptors from distant species offers valuable information to probe the roles of residues in receptor function. We identified two zebrafish receptors (zGPR81-1 and zGPR81-2) that show approximately 60% identity to human GPR81, GPR109a, and GPR109b but respond only to l-lactate and not to the GPR109a ligands. Protein sequence comparison among zebrafish GPR81s, mammalian GPR81s, GPR109a, and GPR109b identified a common structure (six Cys residues at the extracellular domains that potentially form three disulfide bonds) in this subfamily of receptors. In addition, a number of residues conserved in all GPR81s but not in GPR109s have been identified. Furthermore, we identified a conserved motif, C165-E166-S167-F168, at the second extracellular loop of GPR81. Using site-directed mutagenesis, we showed that Arg71 at the transmembrane domain 2 is very critical for GPR81 function. In addition, we demonstrated that the C165-E166-S167-F168 motif at the second extracellular loop is critical for GPR81 function, and the conserved six Cys residues at the extracellular regions are necessary for GPR81 function. It is important to mention that for those residues important for GPR81 function, the corresponding residues or motifs in GPR109a are also critical for GPR109a function. These findings help us better understand the interaction between lactate and GPR81 and provide useful information for GPR81 ligand design.
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http://dx.doi.org/10.1124/mol.111.074500DOI Listing
November 2011

Oxysterols direct B-cell migration through EBI2.

Nature 2011 Jul 27;475(7357):519-23. Epub 2011 Jul 27.

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, California 92121, USA.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.
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http://dx.doi.org/10.1038/nature10226DOI Listing
July 2011