Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase

Abstract

Aryl hydrocarbon receptor (AhR), a cellular chemical sensor, controls cellular homeostasis, and sphingosine-1-phosphate (S1P), a bioactive intermediate of sphingolipid metabolism, is believed to have a role in immunity and inflammation, but their potential crosstalk is currently unknown. We aimed to determine whether there is a functional linkage between AhR signaling and sphingolipid metabolism. We showed that AhR ligands, including an environmental polycyclic aromatic hydrocarbon (PAH), induced S1P generation, and inhibited S1P lyase (S1PL) activity in resting cells, antigen/IgE-activated mast cells, and mouse lungs exposed to the AhR ligand alone or in combination with antigen challenge. The reduction of S1PL activity was due to AhR-mediated oxidation of S1PL at residue 317, which was reversible by the addition of an antioxidant or in cells with knockdown of the ORMDL3 gene encoding an ER transmembrane protein, whereas C317A S1PL mutant-transfected cells were resistant to the AhR-mediated effect. Furthermore, analysis of AhR ligand-treated cells showed a time-dependent increase of the ORMDL3-S1PL complex, which was confirmed by FRET analysis. This change increased the S1P levels, which in turn, induced mast cell degranulation via S1PR2 signaling. In addition, elevated levels of plasma S1P were found in children with asthma compared to non-asthmatic subjects. These results suggest a new regulatory pathway whereby the AhR-ligand axis induces ORMDL3-dependent S1P generation by inhibiting S1PL, which may contribute to the expression of allergic diseases.

Metformin inhibits IgE‐ and aryl hydrocarbon receptor‐mediated mast cell activation in vitro and in vivo

“…Consistent with our previous reports , an AhR ligand, FICZ, was shown to enhance the levels of mast cell degranulation and cytokine release following the cross linkage of Ag (OVA) and OVA‐specific IgE Abs (Fig ). To examine whether metformin was able to regulate IgE‐mediated mast cell activation and AhR ligand's impact, varying doses (1 μM–1 mM) of metformin was initially evaluated for its effect on mast cell degranulation.…”

“…Further, exposure of mast cells to FICZ increased the intracellular ROS levels, which were reversed following the addition of an antioxidant, N‐Acetyl‐Cysteine (NAC), and metformin (Fig A). Our previous report has shown that AhR negatively regulated the enzymatic activity of sphingolipid‐1‐phosphate lyase (S1PL) through increased ROS and oxidation of S1PL ; thus, we tested whether metformin could block the negative regulation of AhR on the enzymatic activity of S1PL through the inhibition of ROS production. As S1PL‐specific Abs were not available for immunoprecipitation assay and the low transfection efficiency of BMMCs, we employed an enforced overexpression of flag‐tagged‐S1PL in a human mast cell line, HMC‐1, instead.…”

“…Mouse bone marrow cells (BMMCs) and human mast cells 1 (HMC‐1) were cultured according to our previous publication and confirmed with FcεRI expression and leukotriene production .…”

“…The homogenates were then centrifuged for 15 min at 1500 × g to get the supernatant for S1P measurement. S1PL activity was determined according to our previous report . Briefly, the cells (∼2 × 10 6 cells) were lysed in 300 μL of potassium phosphate buffer (0.5 M, pH7.4), and the lysates were mixed with S1PL fluorogenic substrate (200 μM, Cayman Chemicals), Na 3 VO 4 (25 mM), NaF (25 mM), pyridoxal phosphate (0.25 mM), 5 μL of EDTA (0.5 mM),and 1 μL of DTT (1 mM).…”

“…Our recent reports showed that the AhR–ligand axis controls cellular Ca 2+ signaling, the endoplasmic reticulum, and mitochondrial stress response in mast cells, one of the major effector cell types in allergic and inflammatory responses . In addition, we discovered that AhR ligands inhibit the enzymatic activity of sphingosine‐1‐phosphate lyase (S1PL) through direct oxidation, leading to an increase in the level of sphingosine‐1‐phosphate (S1P) . S1P is a bioactive intermediate of sphingolipid metabolism that regulates a diverse range of cellular processes involved in immunity, inflammation and inflammatory disorders through S1P receptors (S1PRs) dependent and independent pathways .…”