Publications by authors named "Marie-Gabrielle Ludwig"

22 Publications

  • Page 1 of 1

Targeting interleukin-4 to the arthritic joint.

J Control Release 2020 Oct 10;326:172-180. Epub 2020 Jul 10.

Institute of Pharmacy and Food Chemistry, University of Würzburg, DE-97074 Würzburg, Germany. Electronic address:

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.
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http://dx.doi.org/10.1016/j.jconrel.2020.07.005DOI Listing
October 2020

The proton-activated ovarian cancer G protein-coupled receptor 1 (OGR1) is responsible for renal calcium loss during acidosis.

Kidney Int 2020 05 25;97(5):920-933. Epub 2019 Dec 25.

Institute of Physiology, University of Zurich, Zürich, Switzerland; National Center for Competence in Research, NCCR Kidney.CH, Swiss National Science Foundation, Bern, Switzerland. Electronic address:

Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis. In the kidney, OGR1 mRNA was found in cells of the glomerulus, proximal tubule, and interstitium including endothelial cells. Wild type (OGR1) and OGR1 knockout (OGR1) mice were given standard chow without (control) or loaded with ammonium chloride for one or seven days to induce acute or chronic metabolic acidosis, respectively. No differences in responding to the acid load were observed in the knockout mice, except for higher plasma bicarbonate after one day. Bone mineral density, resorption activity of osteoclasts, and urinary deoxypyridinoline were similar between genotypes. During metabolic acidosis the expression levels of key proteins involved in calcium reabsorption, i.e. the sodium/proton exchanger (NHE3), the epithelial calcium-selective channel TRPV5, and the vitamin D-dependent calcium binding protein calbindin-D28k were all higher in the knockout mice compared to wild type mice. This is consistent with the previous demonstration that OGR1 reduces NHE3 activity in proximal tubules of mice. Wild-type mice displayed a non-linear positive association between urinary proton and calcium excretion which was lost in the knockout mice. Thus, OGR1 is a pH sensor involved in the hypercalciuria of metabolic acidosis by controlling NHE3 activity in the proximal tubule. Hence, novel drugs modulating OGR1 activity may improve renal calcium handling.
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http://dx.doi.org/10.1016/j.kint.2019.12.006DOI Listing
May 2020

Adhesion G protein-coupled receptors: opportunities for drug discovery.

Nat Rev Drug Discov 2019 11 28;18(11):869-884. Epub 2019 Aug 28.

Novartis Institute for Biomedical Research, Basel, Switzerland.

Adhesion G protein-coupled receptors (aGPCRs) - one of the five main families in the GPCR superfamily - have several atypical characteristics, including large, multi-domain N termini and a highly conserved region that can be autoproteolytically cleaved. Although GPCRs overall have well-established pharmacological tractability, currently no therapies that target any of the 33 members of the aGPCR family are either approved or in clinical trials. However, human genetics and preclinical research have strengthened the links between aGPCRs and disease in recent years. This, together with a greater understanding of their functional complexity, has led to growing interest in aGPCRs as drug targets. A framework for prioritizing aGPCR targets and supporting approaches to develop aGPCR modulators could therefore be valuable in harnessing the untapped therapeutic potential of this family. With this in mind, here we discuss the unique opportunities and challenges for drug discovery in modulating aGPCR functions, including target identification, target validation, assay development and safety considerations, using ADGRG1 as an illustrative example.
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http://dx.doi.org/10.1038/s41573-019-0039-yDOI Listing
November 2019

Glucocorticoid-loaded liposomes induce a pro-resolution phenotype in human primary macrophages to support chronic wound healing.

Biomaterials 2018 09 5;178:481-495. Epub 2018 Apr 5.

Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Basel, Switzerland. Electronic address:

Glucocorticoids are well established anti-inflammatory agents, however, their use to treat chronic inflammatory diseases is limited due to a number of serious side effects. For example, long-term local treatment of chronic wounds with glucocorticoids is prohibited by dysregulation of keratinocyte and fibroblast function, leading to skin thinning. Here, we developed and tested liposome formulations for local delivery of dexamethasone to primary human macrophages, to drive an anti-inflammatory/pro-resolution phenotype appropriate for tissue repair. The liposomes were loaded with the pro-drug dexamethasone-phosphate and surface-modified with either polyethylene glycol or phosphatidylserine. The latter was used to mimic phosphatidylserine-harboring apoptotic cells, which are substrates for efferocytosis, an essential pro-resolution function. Both formulations induced a dexamethasone-like gene expression signature in macrophages, decreased IL6 and TNFα release, increased secretion of thrombospondin 1 and increased efferocytosis activity. Phosphatidylserine-modified liposomes exhibited a faster uptake, a higher potency and a more robust phenotype induction than polyethylene glycol-modified liposomes. Fibroblast and keratinocyte cell cultures as well as a 3D skin equivalent model showed that liposomes applied locally to wounds are preferentially phagocytosed by macrophages. These findings indicate that liposomes, in particular upon shell modification with phosphatidylserine, promote dexamethasone delivery to macrophages and induce a phenotype suitable to support chronic wound healing.
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http://dx.doi.org/10.1016/j.biomaterials.2018.04.006DOI Listing
September 2018

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization.

PLoS One 2017 24;12(8):e0183679. Epub 2017 Aug 24.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183679PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570424PMC
October 2017

Design and synthesis of potent and orally active GPR4 antagonists with modulatory effects on nociception, inflammation, and angiogenesis.

Bioorg Med Chem 2017 08 29;25(16):4512-4525. Epub 2017 Jun 29.

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.

GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screening hit 1. The compound shows potent cellular activity and is efficacious in animal models of angiogenesis, inflammation and pain.
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http://dx.doi.org/10.1016/j.bmc.2017.06.050DOI Listing
August 2017

Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling.

JCI Insight 2017 Jun 2;2(11). Epub 2017 Jun 2.

Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.
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http://dx.doi.org/10.1172/jci.insight.93700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453702PMC
June 2017

Exploring Glucocorticoid Receptor Agonists Mechanism of Action Through Mass Cytometry and Radial Visualizations.

Cytometry B Clin Cytom 2017 01;92(1):42-56

Developmental and Molecular Pathway, Computational Biology, Novartis Institute for Biomedical Research, Novartis Campus, CH-4056 Basel, Switzerland.

Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data. © 2016 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21499DOI Listing
January 2017

Adhesion GPCR Function in Pulmonary Development and Disease.

Handb Exp Pharmacol 2016 ;234:309-327

Department of Pediatrics, Section of Pulmonary Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA.

Classic G-protein-coupled receptors (GPCRs) control multiple aspects of pulmonary physiology as demonstrated by loss-of-function experiments in mice and pharmacologic targeting of GPCRs for treatment of several pulmonary diseases. Emerging data demonstrate critical roles for members of the adhesion GPCR (aGPCR) family in pulmonary development, homeostasis, and disease. Although this field is still in its infancy, this chapter will review all available data regarding aGPCRs in pulmonary biology, with a particular focus on the aGPCR for which the most substantial data to date exist: Adgrf5.
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http://dx.doi.org/10.1007/978-3-319-41523-9_14DOI Listing
June 2017

Interleukin-4-Clicked Surfaces Drive M2 Macrophage Polarization.

Chembiochem 2016 Nov 19;17(22):2123-2128. Epub 2016 Oct 19.

Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.

Driving macrophage (Mϕ) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin-4 (IL-4) promotes polarization into the M2-Mϕ phenotype, but its systemic use is constrained by dose-limiting toxicity. Consequently, we developed IL-4-decorated surfaces aiming at sustained and localized activity. IL-4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell-stimulation ability and binding affinity to IL4Rα similar to those of wt-IL-4. Copper-catalyzed (CuAAC) and copper-free strain-promoted (SPAAC) 1,3-dipolar azide-alkyne cycloadditions were used to site-selectively anchor IL-4 to agarose surfaces. These surfaces had sustained IL-4 activity, as demonstrated by TF-1 cell proliferation and M2, but not M1, polarization of M-CSF-generated human Mϕ. The approach provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity.
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http://dx.doi.org/10.1002/cbic.201600480DOI Listing
November 2016

Proton detection and breathing regulation by the retrotrapezoid nucleus.

J Physiol 2016 Mar 19;594(6):1529-51. Epub 2016 Feb 19.

Department of Pharmacology, University of Virginia, Charlottesville, VA, 22908, USA.

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H(+) ]. RTN neurons are glutamatergic. In vitro, their activation by [H(+) ] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome.
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http://dx.doi.org/10.1113/JP271480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799966PMC
March 2016

The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

Am J Physiol Gastrointest Liver Physiol 2015 Sep 23;309(6):G475-90. Epub 2015 Jul 23.

Novartis Institutes for Biomedical Research, Basel, Switzerland;

The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.
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http://dx.doi.org/10.1152/ajpgi.00408.2014DOI Listing
September 2015

PHYSIOLOGY. Regulation of breathing by CO₂ requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons.

Science 2015 Jun 11;348(6240):1255-60. Epub 2015 Jun 11.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.

Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H(+) and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency, and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2 (K(2P)5), a pH-sensitive K(+) channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel, and essential central mediators of respiratory chemosensitivity.
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http://dx.doi.org/10.1126/science.aaa0922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5171229PMC
June 2015

G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation.

Inflamm Bowel Dis 2015 Jun;21(6):1269-81

*Division of Gastroenterology and Hepatology, University Hospital Zürich, Zürich, Switzerland; †Institute of Physiology, University of Zürich, Zürich, Switzerland; ‡Department of Clinical Pharmacology and Toxicology, University Hospital Zürich, Zürich, Switzerland; and §Novartis Institutes for Biomedical Research, Basel, Switzerland.

Background: A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa.

Mtehods: OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) residential peritoneal macrophages.

Results: Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-κB inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways.

Conclusions: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-κB inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.
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http://dx.doi.org/10.1097/MIB.0000000000000375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450952PMC
June 2015

Transcriptional regulation and functional characterization of the oxysterol/EBI2 system in primary human macrophages.

Biochem Biophys Res Commun 2014 Apr 27;446(3):663-8. Epub 2014 Jan 27.

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland. Electronic address:

Oxysterols such as 7 alpha, 25-dihydroxycholesterol (7α,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein-coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses. Activation of EBI2 by specific oxysterols leads to chemotaxis of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adaptive immune response is established by a stromal cells subset here we investigate the involvement of the oxysterol/EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes needed for ligand production such as cholesterol 25-hydroxylase (CH25H), sterol 27-hydroxylase (CYP27A1), and oxysterol 7α-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharides (LPS) triggers a strong up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Stimulation of EBI2 expressed on macrophages leads to calcium mobilization and to directed cell migration. Supernatants of LPS-stimulated macrophages are able to stimulate EBI2 signaling indicating that an induction of CH25H, CYP27A1, and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is a study characterizing the oxysterol/EBI2 pathway in primary monocyte-derived macrophages. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity.
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http://dx.doi.org/10.1016/j.bbrc.2014.01.069DOI Listing
April 2014

The proton-activated receptor GPR4 modulates glucose homeostasis by increasing insulin sensitivity.

Cell Physiol Biochem 2013 ;32(5):1403-16

Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP.

Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed.

Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARa, IL-6, IL-10, TNFa, and TGF-1b in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes.

Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues.
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http://dx.doi.org/10.1159/000356578DOI Listing
August 2014

Orphan G protein-coupled receptor GPR116 regulates pulmonary surfactant pool size.

Am J Respir Cell Mol Biol 2013 Sep;49(3):348-57

Perinatal Institute, Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116(Δexon17), were generated. Gpr116(Δexon17) mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116(Δexon17) mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116(Δexon17) type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.
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http://dx.doi.org/10.1165/rcmb.2012-0439OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824053PMC
September 2013

The proton-activated G protein coupled receptor OGR1 acutely regulates the activity of epithelial proton transport proteins.

Cell Physiol Biochem 2012 3;29(3-4):313-24. Epub 2012 Apr 3.

Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.
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http://dx.doi.org/10.1159/000338486DOI Listing
August 2012

Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor.

Angiogenesis 2011 Dec 2;14(4):533-44. Epub 2011 Nov 2.

Novartis Institutes for Biomedical Research, Forum 1 Novartis Campus, CH-4056, Basel, Switzerland.

The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.
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http://dx.doi.org/10.1007/s10456-011-9238-9DOI Listing
December 2011

Receptors for protons or lipid messengers or both?

J Recept Signal Transduct Res 2006 ;26(5-6):599-610

Novartis Institutes for Biomedical Research, Basel, Switzerland.

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.
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http://dx.doi.org/10.1080/10799890600932220DOI Listing
March 2007

Proton-sensing G-protein-coupled receptors.

Nature 2003 Sep;425(6953):93-8

Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.
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http://dx.doi.org/10.1038/nature01905DOI Listing
September 2003

Characterization of the human adenylyl cyclase gene family: cDNA, gene structure, and tissue distribution of the nine isoforms.

J Recept Signal Transduct Res 2002 Feb-Nov;22(1-4):79-110

Department of Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.

The membrane-bound adenylyl cyclases (ACs) represent one of the major families of effector enzymes for G protein-coupled receptors. Eight human AC isoforms, encoded by separate genes, have been identified up to now. However, in several cases only partial cDNA sequences are available (ADCY1,2,5). A ninth expected isoform, the human ortholog of rat ADCY4, has not been described yet. Using the high inter-species homology of mammalian AC isoforms, we searched the human genome and we succeeded to identify full-length coding sequences for all enzymes. Where required, missing sequence information was provided experimentally. Analysis of genomic sequences from the Celera database also allowed us to determine the exon-intron boundaries for ADCY1-9 and to establish the gene structures. We found that human AC genes comprise 11 to 26 exons, which are distributed over 16 to 430kb. We further report expression profiles for the nine ACs in a panel of 16 human tissues and in human embryonic kidney (HEK) cells.
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http://dx.doi.org/10.1081/rrs-120014589DOI Listing
June 2003