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Sphingolipids play vital roles in metabolism and regulation. Previously, the aryl hydrocarbon receptor (AHR), a ligand‐activated transcription factor, was reported to directly regulate ceramide synthesis genes by binding to their promoters. Herein, sphingosine kinase 2 (SPHK2), responsible for producing sphingosine‐1‐phosphate (S1P), was found to interact with AHR through LXXLL motifs, influencing AHR nuclear localization. Through mutagenesis and co‐transfection studies, AHR activation and subsequent nuclear translocation was hindered by SPHK2 LXXLL mutants or SPHK2 lacking a nuclear localization signal (NLS). Similarly, an NLS‐deficient AHR mutant impaired SPHK2 nuclear translocation. Silencing SPHK2 reduced AHR expression and its target gene CYP1A1, while SPHK2 overexpression enhanced AHR activity. SPHK2 was found enriched on the CYP1A1 promoter, underscoring its role in AHR target gene activation. Additionally, S1P rapidly increased AHR expression at both the mRNA and protein levels and promoted AHR recruitment to the CYP1A1 promoter. Using mouse models, AHR deficiency compromised SPHK2 nuclear translocation, illustrating a critical interaction where SPHK2 facilitates AHR nuclear localization and supports a positive feedback loop between AHR and sphingolipid enzyme activity in the nucleus. These findings highlight a novel function of SPHK2 in regulating AHR activity and gene expression. [ABSTRACT FROM AUTHOR]

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress (1,2). Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle (3), the immunological functions of the ApoMS1P complex are unknown. Here we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the [S1P.sub.1] receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM ([Apom.sup.-/-]) had increased proliferation of [Lin.sup.-] Sca-[1.sup.+] [cKit.sup.+] haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of [S1P.sub.1] suppressed LSK and CLP cell proliferation invivo. ApoM was stably associated with bone marrow CLPs, which showed active [S1P.sub.1] signalling in vivo (4). Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, [Apom.sup.-/-] mice developed more severe experimental autoimmune encephalomyelitis (5), characterized by increased lymphocytes in the central nervous system and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-[S1P.sub.1] signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities., S1P, a bioactive lysophospholipid mediator, interacts with vertebrate-specific S1P receptors to regulate various physiological functions6. Most cells express one or more of the G-protein-coupled S1P receptors ([S1P.sub.1-5]), which mediate cellular [...]

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1–5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P – S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed β-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal β-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination. [ABSTRACT FROM AUTHOR]