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Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-? that regulates neoangiogenesis.

ABSTRACT: Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)?. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPAR?-specific gene reporter by ?12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPAR? ligand binding domain is important for binding to S1P. PPAR? functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-? coactivator 1 (PGC1)? binds to PPAR? in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPAR?:PGC1? complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPAR? target genes with PGC1? and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPAR? antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPAR? by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPAR?, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPAR?:PGC1? complex may be a useful target to manipulate neovascularization.

SUBMITTER: Parham KA

PROVIDER: S-EPMC6137673 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis.

Parham Kate A KA Zebol Julia R JR Tooley Katie L KL Sun Wai Y WY Moldenhauer Lachlan M LM Cockshell Michaelia P MP Gliddon Briony L BL Moretti Paul A PA Tigyi Gabor G Pitson Stuart M SM Bonder Claudine S CS

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20150518 9

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of t ...[more]

PMID: 25985799

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Project description:BackgroundHumans are exposed to a complex mixture of environmental chemicals that impact bone and metabolic health, and traditional exposure assessments struggle to capture these exposure scenarios. Peroxisome proliferator activated receptor-gamma (PPARγ) is an essential regulator of metabolic and bone homeostasis, and its inappropriate activation by environmental chemicals can set the stage for adverse health effects. Here, we present the development of the Serum PPARγ Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum.MethodsFirst, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPARγ and RXRα, the obligate DNA-binding partner of PPARγ, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPARγ ligand and/or LG100268, a synthetic RXR ligand, to characterize the dose response and determine the simplest and most efficacious format. Following optimization of the bioassay, we assessed the cumulative activation of PPARγ by ligands in serum from mice treated with a PPARγ ligand and commercial human serum samples.ResultsCos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPARγ. Co-transfection of an RXR expression vector with the PPARγ expression vector did not increase PPRE transcriptional activity induced by rosiglitazone. Treatment with an RXR ligand marginally increased PPRE transcriptional activity in the presence of transfected PPARγ, and co-treatment with an RXR ligand reduced rosiglitazone-induced PPRE transcriptional activity. Therefore, the final bioassay protocol consists of transfecting Cos-7 cells with a PPARγ expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10 μL of serum, and measuring luminescence and fluorescence after a 24 h incubation. Sera from mice dosed with rosiglitazone induced PPRE transcriptional activity in the SPAA in a dose-dependent and PPARγ-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPARγ-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands.ConclusionsThe SPAA can reliably measure total PPRE transcriptional activity in small volumes of serum. This system provides a sensitive, straightforward assay that can be reproduced in any cell culture laboratory.

Dataset Information

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-? that regulates neoangiogenesis.

Publications

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis.

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