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  • br Discovery of S PR as a CBA

    2019-05-22


    Discovery of S1PR2 as a CBA sensor in the liver Early studies reported that activation of the ERK1/2 and AKT signaling pathways by CBAs was PTX-sensitive, suggesting a role for a Gαi-linked GPCR. To identify which GPCR activates the ERK1/2 and AKT signaling pathways, the cDNAs encoding more than a dozen lipid-activated GPCRs were expressed individually in HEK293 cells and screened for activation of these cell signaling pathways by TCA. It was discovered that, if TCA was added to HEK293 cells over-expressing S1PR2, the ERK1/2 and AKT signaling pathways were activated significantly in a concentration-dependent manner. These results led to discovery of a link between CBAs and sphingosine-1-phosphate (S1P) purchase GSK256066 signaling. S1P is synthesized from sphingosine by sphingosine kinase (SphK)1 or SphK2. SphK1 is located primarily in the cytoplasm of mammalian cells, whereas SphK2 is located in the nucleus and mitochondria. S1P is secreted from mammalian purchase GSK256066 by the transporters ABCC1 and ABCG2 as well as a facilitated diffusion transporter (spinster homolog 2) probably in a regulated manner. Secreted S1P can act in an autocrine or paracrine manner to activate five GPCRs (Fig. 2), but the expression pattern of S1PRs is specific according to cell type. For example, hepatocytes express only S1PRs 1, 2 and 3, but immune cells express all S1PRs. Additional studies showed that S1PR2 was the most highly expressed S1PR in primary rodent hepatocytes and human primary hepatocytes. Activation of the ERK1/2 and AKT signaling pathways by TCA was blocked significantly by an S1PR2 antagonist (JTE-013) or knockdown of S1PR2 mRNA by specific shRNA in primary hepatocytes. Moreover, JTE-013 was shown to inhibit AKT activation significantly and to decrease the induction of expression of SHP mRNA by TCA using a model of chronic bile fistulae in rats. This study demonstrated a link between the insulin-signaling pathway and FXR activation in vivo. Finally, molecular modeling of the five known S1PRs showed that only S1PR2 could accommodate TCA binding in its putative active site. What is the physiologic significance of S1PR2 activation in the liver? It was observed that feeding of a high-fat diet (HFD) to mice up-regulated expression of SphK2. Moreover, intestinal infusion of TCA into chronic bile fistulae in rats or over-expression of the gene encoding S1PR2 up-regulated hepatic expression of SphK2 significantly, but did not increase the levels of mRNA, protein or activity of SphK1. In contrast, primary hepatocytes isolated from S1PR2−/− mice had significantly lower levels of nuclear SphK2 protein and activity. Measurement of S1P in nuclei isolated from the hepatocytes of wild-type and S1PR2−/− mice showed lower levels of S1P in the nuclei from S1PR2−/− mice. In total, these results showed an important link between CBAs, S1PR2, as well as up-regulation of nuclear levels of SphK2 and S1P. It has been reported that S1P strongly inhibits expression of histone deacetylases 1/2 (HDAC1/2) in cancer cells. Histone acetylation is a key regulator of gene expression in mammalian cells. When the levels of histone acetylation (H3K9, H4K5, and H2BK12) were measured in the nuclei of hepatocytes isolated from S1PR2−/−or SphK2−/− mice, acetylation was decreased markedly. Moreover, there was a differential down-regulation of expression of genes encoding the enzymes involved in nutrient metabolism in hepatocytes isolated from S1PR2−/− and SphK2−/− mice. Over-expression of the gene encoding S1PR2 in hepatocytes from wild-type mice up-regulated expression of SphK2 markedly but not of SphK1. Expression of several genes involved in the metabolism of sterols and lipids was also up-regulated significantly, especially genes encoding the enzymes involved in fatty-acid oxidation and cholesterol transport (Table 1). As a control, the cDNA encoding mouse S1PR2 was over-expressed in mouse hepatocytes isolated from SphK2−/− mice. The results showed no significant increase in expression of genes encoding the enzymes involved in lipid metabolism, suggesting that SphK2 is required for the up-regulation of expression of these genes.