TGR5 is an important mediator of BA-induced cholangiocyte proliferation in vivo and in vitro. Furthermore, TGR5 protects cholangiocytes from death receptor-mediated apoptosis. These mechanisms may protect cholangiocytes from BA toxicity under cholestatic conditions, however, they may trigger proliferation and apoptosis resistance in malignantly transformed cholangiocytes, thus promoting CCA progression.

Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in the perfused rat liver in an a 5 b 1 integrin-mediated way. However, the molecular basis of TUDC-sensing in the liver is unknown. We herein show that TUDC (20 lmol/L) induces in perfused rat liver and human HepG2 cells the rapid appearance of the active conformation of the b 1 subunit of a 5 b 1 integrins, followed by an activating phosphorylation of extracellular signal-regulated kinases. TUDCinduced kinase activation was no longer observed after b 1 integrin knockdown in isolated rat hepatocytes or in the presence of an integrin-antagonistic hexapeptide in perfused rat liver. TUDC-induced b 1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na 1 /taurocholate cotransporting peptide. Molecular dynamics simulations of a 3D model of a 5 b 1 integrin with TUDC bound revealed significant conformational changes within the head region that have been linked to integrin activation before. Conclusions: TUDC can directly activate intrahepatocytic b 1 integrins, which trigger signal transduction pathways toward choleresis.

No abstract

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47 phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase C, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H 2 O 2 ), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the deathinducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95 2 death receptor (1-5). The underlying molecular mechanisms are complex and involve a Yes-dependent, but ligand-independent activation of the epidermal growth factor receptor (EGFR), which catalyzes CD95-tyrosine phosphorylation as a prerequisite for CD95 oligomerization, formation of the death-inducing signaling complex (DISC), and apoptosis induction (6, 7). Bile acids also activate EGFR in cholangiocytes (8) and activated hepatic stellate cells (HSC) (9), however, the mechanisms underlying bile acid-induced EGFR activation in HSC remained unclear (9). Surprisingly, bile acid-induced EGFR activation in HSC does not trigger apoptosis but results in a stimulation of cell proliferation (9). The behavior of quiescent HSC toward CD95 ligand (CD95L) is also unusual. CD95L, which is a potent inducer of hepatocyte apoptosis (10 -12), triggers activation of the EGFR in quiescent HSC, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death signaling through CD95-tyrosine nitration (13). Similar observations were made with other death receptor ligands, i.e. tumor necrosis factor-␣ (TNF-␣) and TNF-rela...

Background: Hyperosmotically induced hepatic cholestasis involves Mrp2 and Bsep retrieval from canalicular membrane. Results: Hyperosmolarity induces NADPH oxidase isoform (NOX)-driven ROS-formation triggering Fyn-dependent bile acid transporter retrieval from canalicular membrane, which may be mediated by increased cortactin phosphorylation. Conclusion: NOX, Fyn, and cortactin are critical players in hyperosmolarity-induced cholestasis. Significance: New molecular insights in short term regulation of canalicular bile acid excretion are given.

Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development.

Background:Little is known about the effect of hyperosmotic hepatocyte shrinkage on Na ϩ -taurocholate cotransporting

The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr 845 and Tyr 1173 , but not at Tyr 1045 , suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin-and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin ␤1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrindependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294 -21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin ␤1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin ␤1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin-or hypoosmolarity-induced hepatocyte swelling triggers an integrin-and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.Apart from its metabolic effects, insulin exerts proliferative effects in the liver and other organs (1-5). Much effort has been devoted to the understanding of insulin signaling and its complexity (6 -11), which involves tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1) 2 and activation of a variety of protein kinases such as mitogen-activated protein (MAP) kinases ERK1/2 and p38 MAPK (3,12). In rat liver, insulin stimulates Na ϩ /H ϩ antiport and K ϩ /Na ϩ /2Cl Ϫ co-transport, thereby inducing hepatocyte swelling (13). Evidence has been presented that insulin-induced hepatocyte swelling is an integral part of insulin signaling and mediates proteolysis inhibition by the hormone through a swelling-induced p38 MAPK activation (14). Like hypoosmotic hepatocyte swelling, insulin-induced hepatocyte swelling is sensed by the integrin system with subsequent activation of c-Src kinase and downstream MAP kinases ERK1/2 and p38 MAPK (14 -16). In line with this, the insulin-or hypoosmolarity-induced inhibition of autophagic proteolysis is largely abolished in presence of a...