title: Hydrophobic bile acid apoptosis is regulated by sphingosine-1-phosphate receptor 2 in rat hepatocytes and human hepatocellular carcinoma cells.
abstract: The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.
authors.affiliation: Department of Clinical Science, Cummings School of Veterinary Medicine at Tufts University, Grafton, Massachusetts; and cynthia.leveille-webster@tufts.edu.
authors.forename: Cynthia R L
authors.initials: CR
authors.lastname: Webster
authors.affiliation: Department of Biomedical Science, Cummings School of Veterinary Medicine at Tufts University, Grafton, Massachusetts.
authors.forename: M Sawkat
authors.initials: MS
authors.lastname: Anwer
mesh_terms: D000818:Animals; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005999:Glycochenodeoxycholic Acid / Q000378:metabolism* / Q000633:toxicity; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008297:Male; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000081025:Sphingosine-1-Phosphate Receptors
chemical_list: C471998:JTE 013; D011720:Pyrazoles; D011725:Pyridines; D049349:Receptors, Lysosphingolipid; C477522:S1PR2 protein, human; D000081025:Sphingosine-1-Phosphate Receptors; C570053:sphingosine-1-phosphate receptor-2, rat; D005999:Glycochenodeoxycholic Acid; D051057:Proto-Oncogene Proteins c-akt; D048670:MAP Kinase Kinase 4
keywords: Akt; JNK; glycochenodeoxycholate; hepatocyte apoptosis; sphingosine-1-phosphate receptor