First we tested the ability of the natural ligand of S1P2 (S1P) to activate ERK and AKT in primary rat hepatocytes. S1P showed a concentration-dependent increase of p-AKT GSK1120212 research buy and p-ERK at nM levels. Both p-ERK and p-AKT levels were saturated at 50-100 nM S1P (Supporting Fig. 1). We further determined whether TCA had a similar effect on ERK and AKT activation as S1P in primary rat hepatocytes. TCA also dose-dependently induced activation of ERK1/2 and AKT, but at μM levels (Fig. 2). Both p-ERK1/2 and p-AKT levels were saturated at 50-100 μM TCA (Fig. 2B). Several articles have reported that S1P receptors are expressed and functional

in the liver.33, 34 The expression of S1P1 and S1P2 in mouse, rat, and human primary hepatocytes was identified by RT-PCR (Supporting Fig. 2) and further confirmed by DNA sequencing. The addition of S1P (100 nM) to primary rat hepatocytes significantly activated both the ERK1/2 and AKT signaling pathways (Supporting Fig. 3). Moreover, JTE-013, a S1P2 chemical antagonist, as well as PTX, blocked the activation of both ERK1/2 and AKT by S1P (Supporting Fig. 3). We have

reported before that TCA-induced ERK1/2 and AKT activation was PTX sensitive in primary rat hepatocytes.13 In the present study, we determined that TCA-induced ERK1/2 and AKT activation was also significantly blocked by JTE-013 (10 μM) in primary rat hepatocytes (Fig. 3). We further examined whether other SCH772984 manufacturer conjugated bile acids have similar effects on ERK1/2 and AKT activation. In addition to TCA, TDCA, TUDCA, GCA, and GDCA also induced activation of ERK1/2 and AKT in primary rat hepatocytes, and their effects MCE公司 were blocked by JTE-013 to different degrees (Fig. 4). These results indicate that S1P2 may be a major GPCR in the activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids and S1P in primary hepatocytes. In order to further identify the role of S1P2 in TCA-mediated activation of ERK and AKT, lentiviral shRNA specifically targeting rat S1P2 was constructed. The knockdown efficiency was determined by real-time RT-PCR in

primary rat hepatocytes. The mRNA level of S1P2 was reduced by 50% after 48-hour transduction of lentiviral shRNA (Supporting Fig. 4). Transduction of primary rat hepatocytes with S1P2 shRNA lentivirus markedly inhibited the activation of ERK1/2 (60%) and AKT (70%) by TCA compared with a control lentivirus. Moreover, shRNA against S1P2 also inhibited S1P-mediated activation of ERK1/2 (51%) and AKT (72%) (Fig. 5). Primary hepatocytes were prepared from S1P2−/− mice and matched with wild-type control mice. It was observed that male, but not female, S1P2−/− mice livers were difficult to infuse with collagenase solution, and the hepatocyte cell yields from male mice livers were quite low. Therefore, all experiments were run with hepatocytes prepared from female S1P2−/− mice.