== A. hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knock down of the expression of S1P2by a recombinant lentivirus encoding S1P2shRNA, markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat main hepatocytes. Main hepatocytes prepared from S1P2knock out (S1P2/) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarilyviaS1P2in main rodent hepatocytes. Keywords:G protein coupled receptor, homology modeling, S1P receptor 2 knockout mice, cell signaling == Introduction == Over the past decade it has become obvious that bile acids are important regulatory molecules in the liver and gastrointestinal tract and function much like hormones. Bile acids have been shown to activate specific nuclear receptors [farnesoid receptor (FXR), pregnane receptor (PXR), and Vitamin D receptor] and cell signaling Spiramycin pathways [i.e., c-jun N-terminal kinase1/2 (JNK1/2), ERK1/2, AKT] at physiological concentrations (16). In 2002, a Gsprotein coupled receptor, TGR5/M-BAR, was reported to be activated by both conjugated and unconjugated bile acids (7,8). Subsequently, TGR5/M-BAR was shown to be involved in regulating energy expenditure by inducing the c-AMP-dependent thyroid hormone activating enzyme type 2 iodothyroxine deiodinase (D2). This enzyme converts metabolically inactive thyroxine (T4) into T3, a key hormone regulating energy metabolism in brown adipose tissue and muscle mass (9). TGR5/M-BAR also appears to play an important role in immune cells as bile acids are known to have immunoregulatory properties (10). TGR5/M-BAR is usually expressed in many cell types throughout the body including: neurons, astrocytes, cholangiocytes, macrophages, myocytes, and gallbladder epithelium (8,11). This receptor may play a protective role in hepatic sinusoidal endothelial cells in the liver (12). However, the expression of TGR5/M-BAR in main hepatocytes is very low. Our previous studies indicate that conjugated bile acids activated the ERK1/2 and AKT signaling pathwaysviaunidentified Giprotein coupled receptor(s) in main rodent hepatocytes andin vivo(13,14). Unconjugated bile acids can also activate the ERK1/2 and AKT pathways by at least two different mechanisms. We have previously reported evidence that deoxycholic acid (DCA) can activate the ERK1/2 and AKT pathways by stimulating the synthesis of superoxide ions, which was shown to inactivate phosphotyrosine phosphatase(s) resulting in the activation of the epidermal growth factor receptor (EGFR) (15). In addition, other laboratories have reported that DCA, chenodeoxycholic acid (CDCA) and taurochenodeoxycholic acid (TCDCA) can activate matrix metalloproteinase(s) that generate transforming growth factor (TGF), an EGFR ligand in cholangiocytes (16). Moreover, Raufman and co-workers reported that taurolithocholic acid (TLCA) and TDCA can activate the Gs-coupled M3 muscarinic receptor in gastric chief cells as well as human colon cancer cells (1719). The activation of the EGFR in colon cancer cells was by activation of matrix metalloproteinase gene expression resulting in the formation of heparin-binding EGF-like growth factor, also an EGFR ligand (20). Sphingosine 1-phosphate (S1P) is a membrane-derived lipid mediator involved in the regulation of Spiramycin fundamental cellular responses. Rabbit Polyclonal to TAF15 S1P is usually synthesized from sphingosine by either sphingosine kinase 1 (SphK1) or sphingosine kinase 2 (SphK2). SphK1 is located in the cytoplasm of mammalian cells and, following an external signal, translocates to the plasma membrane and converts sphingosine to S1P. S1P, a water soluble regulatory metabolite, is usually then actively transported by ATP-binding cassette transporter (ABC) C1 (ABCC1), and possibly ABCG2, in a regulated manner (21,22). Exogenous S1P has been shown to activate, in an autocrine/paracrine manner, at least 5 different GPCRs located on the surface of mammalian cells. The GPCRs activated by S1P have been linked to the activation of various cell signaling pathways, including ERK1/2 and AKT. SphK2 is usually primarily located in the nucleus and is activated by phosphorylation by pERK1/2 Spiramycin to produce S1P, a powerful inhibitor of histone deacetylase 1 and 2 (23). S1P2-mediated activation of ERK1/2 and AKT signaling cascades is usually linked.