Phosphatidylinositol-3-kinase p110γ contributes to bile salt-induced apoptosis in primary rat hepatocytes and human hepatoma cells.
OBJECTIVE
Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110α/β/γ), specific functions of which, in liver, are unclear. In other cell types, p110γ is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110γ mediates bile salt-induced hepatocyte cell death.


METHODS
Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110γ was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA.


RESULTS
All bile salts tested activated p110β, while p110α was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110γ. Inhibition of p110γ attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110γ markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor α- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation.


CONCLUSIONS
PI3K p110γ is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK.
Hohenester
Simon
S
Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, Amsterdam, The Netherlands. s.d.hohenester@amc.nl
Gates
Anna
A
Wimmer
Ralf
R
Beuers
Ulrich
U
Anwer
M Sawkat
MS
Rust
Christian
C
Webster
Cynthia R L
CR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D002478:Cells, Cultured; D058544:Class Ib Phosphatidylinositol 3-Kinase / Q000502:physiology*; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005999:Glycochenodeoxycholic Acid / Q000633:toxicity; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D008113:Liver Neoplasms / Q000473:pathology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D045162:Thiazolidinediones / Q000494:pharmacology
C505145:5-(2,2-difluorobenzo(1,3)dioxol-5-ylmethylene)thiazolidine-2,4-dione; D001647:Bile Acids and Salts; D004149:Dioxoles; D045162:Thiazolidinediones; D005999:Glycochenodeoxycholic Acid; D058544:Class Ib Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases