In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Background: S1P 3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtypeselective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist. Methodology/Principal Findings: Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts. Conclusions/Significance: We have developed the first-reported monoclonal antibody that selectively recognizes a lipidactivated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsi

7H9 prevents lethality caused by LPS administration.

<p>Survival curve of mice treated with LPS (8 mg/kg, i.p.) on day 0. Mice were pre-treated with vehicle (PBS), IgG (normal mouse IgG), or 7H9.</p

S1P signaling.

<p>S1P is an extracellular signaling molecule, generated by the phosphorylation of sphingosine, that exerts a variety of effects on a family of 5 cognate GPCRs.</p

7H9 blocks activation of S1P₃.

<p>(A-D) 7H9 blocks S1P₃-mediated arrestin translocation. β-arrestin (green) is cytosolic in quiescent cells and appears as diffuse labeling (A). Following stimulation with 1 µM S1P (B), arrestin translocates to the plasma membrane and is rapidly internalized into intracellular vesicles (arrows). In contrast, when cells are pre-treated with 1 µg/ml 7H9 arrestin localization is diffuse and cytoplasmic in both the absence (C) and presence (D) of 1 µM S1P. (E-H) 7H9 blocks S1P-dependent internalization of S1P₃. Epitope-tagged S1P₃ is normally abundant on the plasma membrane (E, arrows), but is internalized into intracellular vesicles (arrowheads) upon stimulation with 1 µM S1P (F). Following pre-treatment of cells with 7H9, S1P₃ remains localized to the plasma membrane in the absence (G) or presence (H) of 1 µM S1P. (I) 7H9 blocks S1P₃-dependent calcium mobilization. S1P₃-expressing cells exhibited increased intracellular [Ca²⁺] upon stimulation with 100 nM S1P (blue, antibody control). Cells within the same culture that did not express S1P₃-EGFP (green, receptor control) showed no change in [Ca_i²⁺]. Similarly, cells expressing S1P₃ that were pre-treated with 7H9 (red, 7H9) also showed no response to 100 nM and 1 µM S1P, but gave a partial response at 5 µM. (J) 7H9 blocks S1P₃-dependent inhibition of AC. cAMP was measured in S1P₃-expressing cells by ELISA and normalized to controls. The graph represents the change in cAMP content from unstimulated cells, relative to the change observed in stimulated cells with no 7H9 pre-treatment. *p<0.05, **p<0.01. Error bars  =  S.E.M.</p

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite, Caloric Intake and Fat Storage in <i>Drosophila</i>

<div><p>Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use <i>Drosophila</i> genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor <i>dNepYr</i>, resulting in caloric intake-dependent obesity.</p></div

Sphingolipidomic profiles of SL mutants.

<p><b>Sph = Sphingosine; S1P = Sphingosine 1-Phosphate; C = ceramide;</b> Dihydroceramide (C_14:0), ceramide (C_14:1) and ceramide diene (C_14:2) subspecies are shown and represent the degree of saturation on the sphingoid backbone. C_20:0, C_22:0 and C_24:0 denote the length and saturation of the second fatty acid chain connected to the sphingoid backbone in these ceramides.</p>*<p>denotes p-value<0.05.</p

Sphingolipid regulation of caloric intake and fat mobilization.

<p>Both saturated and unsaturated fats act as precursor “input” molecules in the production of “output” sphingolipid intermediates, specifically ceramide and S1P, which act to transduce a physiological response. (Left) Sphingolipid metabolism regulates Akh cell viability and function (Right). After a meal, S1P accumulates downstream of elevated ceramide. S1P, either directly or indirectly (through dRYamide), induces dNepY receptor signaling in the hindgut, inducing appetite suppression, reduced caloric intake and downregulation of <i>dNepYr</i> mRNA expression (negative feedback).</p

The ceramide:S1P rheostat's role in regulating <i>dNepYr</i> expression and caloric intake.

<p>(A) <i>dNepYr</i> expression is upregulated in <i>Sk2^KG6050894</i> and downregulated in <i>Sply⁰⁵⁹⁰¹</i> mutants, with concurrent changes in overlapping cis-NAT's of pancreatic TAG lipase genes. Two day old <i>Sk2^KG6050894</i> flies and wt flies were administered 0 uM, 1 uM, 10 uM and 100 uM dRYamide (1∶1 dRYamide 1∶2) in solid food for 6 days, after which (B) TG levels, (C) rate of dose dependent TG decline, and (D) <i>dNepYr</i> mRNA levels were measured. Three to five day old <i>Sk2^KG6050894</i> flies were also administered S1P analogue FTY720P in the CAFE, after which (E) caloric intake (F) and <i>dNepYr</i> mRNA levels were measured. Error bars are represented by the S.E.M. p-values<*0.05, **<0.01, ***p<0.001.</p

Differential expression of Lipid Metabolic and Apoptotic genes in SL mutants.

<p>DNA microarray analysis in conjunction with DAVID bioinformatics analysis was used to identify distinct subsets of genes mapped to elucidated pathways. Downregulation (Fold change <1.5) is shown in green while upregulation (Fold change >1.5) is shown in red, up to a maximum of 3-fold or greater difference. All changes >3 fold are represented by the brightest color. No change is displayed as black. (A)These data show that diene-accumulating, Akh cell-ablating <i>ifc⁴</i> mutants exhibit upregulation of proapoptotic genes and downregulation of anti-apoptotic genes, while diene-depleting, Akh cell-expanding <i>lace^k05305/2</i> mutants' exhibit downregulation of proapoptotic genes and upregulation of anti-apoptotic genes. (B) These data show that appetite suppressed S1P accumulating <i>Sply⁰⁵⁹⁰¹</i> mutants' downregulate lipogenic pathways (FA biosynthesis) and upregulate lipid utilizing pathways (Fatty acid Oxidation and Oxidative Phosphorylation). Conversely, high appetite, S1P depleting <i>Sk2^KG050894</i> mutants upregulate lipogenic pathways (specifically FA synthase) and downregulate lipid utilizing pathways.</p

Classic hallmarks of obesity.

<p>Obesity in flies is characterized by classic hallmarks of obesity observed in higher organisms. (A) Whole fly triglyceride (TAG) and (B) hemolymph TAG levels were measured in µg of TAG per mg flies and normalized to wt flies. (C) Resistance to starvation-induced death was measured as the mean % of the population that survives over time of 3 independent experiments. (n = 100 flies) (D) Mean body weight (mg) was measured in six sets of n = 100 flies. (E–K) One-Day old larval origin fat body cells stained with lipid positive Nile Red (Red) and a nuclear stain DAPI (blue). (L) Distribution of fat body cell size (µm) from 50 randomly selected fat bodies from n = 4 flies used to calculate (M) mean fat body cell size. Error bars are represented by the S.E.M. p-values *<0.05, **<0.01, ***<0.001.</p