Annika Sommerfeld

TGR5 is essential for bile acid-dependent cholangiocyte proliferation in vivo and in vitro

Objective Cholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, elevation of BA levels enhances proliferation and invasiveness of cholangiocarcinoma (CCA) cells in animal models, thus promoting tumour progression. TGR5 is a G-protein coupled BA receptor, which is highly expressed in cholangiocytes and postulated to mediate the proliferative effects of BA. Design BA-dependent cholangiocyte proliferation was examined in TGR5-knockout and wild type mice following cholic acid (CA)-feeding and CBDL. TGR5-dependent proliferation and protection from apoptosis was studied in isolated cholangiocytes and CCA cell lines following stimulation with TGR5 ligands and kinase inhibitors. TGR5 expression was analysed in human CCA tissue. Results Cholangiocyte proliferation was significantly reduced in TGR5-knockout mice in response to CA-feeding and CBDL. Taurolithocholic acid and TGR5-selective agonists induced cholangiocyte proliferation through elevation of reactive oxygen species and cSrc mediated epidermal growth factor receptor transactivation and subsequent Erk1/2 phosphorylation only in wild type but not in TGR5-knockout-derived cells. In human CCA tissue TGR5 was overexpressed and the pathway of TGR5-dependent proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase (ERK)1/2 activation also translated to CCA cell lines. Furthermore, apoptosis was inhibited by TGR5-dependent CD95 receptor serine phosphorylation. Conclusions 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.

CD95 Ligand Is a Proliferative and Antiapoptotic Signal in Quiescent Hepatic Stellate Cells

BACKGROUND & AIMS Despite expression of CD95 (Fas) receptor, hepatic stellate cells (HSCs) are fairly resistant toward CD95 ligand (CD95L)-induced cell death. The underlying mechanisms and the function of the CD95 system in quiescent HSCs, however, are unknown. METHODS The effects of CD95L on quiescent, 1- to 2-day cultured rat HSCs were studied with regard to CD95 activation, signal transduction, proliferation, and apoptosis. RESULTS In quiescent HSCs, CD95L led to a rapid phosphorylation of the epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (Erk), and c-Src, but not of c-Jun-N-terminal kinase and p47(phox), an activating subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase. CD95L-induced EGFR and Erk phosphorylation were abolished after proteinase inhibition by GM6001 and in the presence of neutralizing epidermal growth factor antibodies, suggestive of a ligand-dependent EGFR phosphorylation in response to CD95L. In quiescent HSCs, CD95L did not induce apoptotic cell death but stimulated HSC proliferation and triggered a rapid inactivating CD95 tyrosine nitration that was not detected in activated HSCs (10-14 days of culture). EGFR phosphorylation, HSC proliferation, and CD95 tyrosine nitration were also triggered by tumor necrosis factor alpha and tumor necrosis factor-related apoptosis-inducing ligand. CONCLUSIONS In quiescent HSCs, CD95L and other death receptor ligands are mitogens through a ligand-dependent EGFR phosphorylation. Simultaneously, an antiapoptotic signaling is triggered by CD95L-induced CD95 tyrosine nitration. This unusual response to death receptor ligands may help quiescent HSCs to participate in liver regeneration following liver injury.

α5β1‐integrins are sensors for tauroursodeoxycholic acid in hepatocytes

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 α5β1 integrin‐mediated way. However, the molecular basis of TUDC‐sensing in the liver is unknown. We herein show that TUDC (20 μmol/L) induces in perfused rat liver and human HepG2 cells the rapid appearance of the active conformation of the β1 subunit of α5β1 integrins, followed by an activating phosphorylation of extracellular signal‐regulated kinases. TUDC‐induced kinase activation was no longer observed after β1 integrin knockdown in isolated rat hepatocytes or in the presence of an integrin‐antagonistic hexapeptide in perfused rat liver. TUDC‐induced β1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na+/taurocholate cotransporting peptide. Molecular dynamics simulations of a 3D model of α5β1 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 β1 integrins, which trigger signal transduction pathways toward choleresis. (HEPATOLOGY 2013)

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

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 p47phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Cζ, 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 (H2O2), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing 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.

Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

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.

Insulin Induces Swelling-dependent Activation of the Epidermal Growth Factor Receptor in Rat Liver

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 Tyr845 and Tyr1173, but not at Tyr1045, 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 β1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent 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 β1-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 β1-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.

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide- and protein kinase Cζ-dependent pathway, which leads to an activating phosphorylation of p47phox. The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl-]. Bafilomycin or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl-], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction.

Activation of integrins by urea in perfused rat liver

High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K+ channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552–560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of β1 integrins followed by an activation of focal adhesion kinase, c-Src, p38MAPK, extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α5β1 integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K+ efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38MAPK inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α5β1 integrin in water, urea, or thiourea solutions revealed significant conformational changes of α5β1 integrin in urea and thiourea solutions, in contrast to the simulation of α5β1 in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α5β1 integrin system.

Free Fatty Acids shift Insulin-induced Hepatocyte Proliferation towards CD95-dependent Apoptosis

Background: Hyperinsulinemia and increased blood levels of free fatty acids (FFA) trigger non-alcoholic steatohepatitis (NASH). Results: Insulin induces EGFR-activation and proliferation. In presence of FFA, however, insulin triggers apoptosis as EGFR triggers CD95-activation JNK-dependently. Conclusion: Insulin-induced EGFR-activation triggers proliferation, but shifts to CD95-dependent apoptosis when a JNK-signal is provided by FFA. Significance: The study provides new insights into the pathogenesis of NASH. Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2-terminal kinase (JNK)-dependent, but death receptor-independent way (2). As non-alcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFA-blood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR with CD95, subsequent CD95 tyrosine phosphorylation and formation of the death-inducing signaling complex (DISC). JNK inhibition restored the proliferative insulin effect in presence of FFAs and prevented EGFR/CD95 association, CD95 tyrosine phosphorylation and DISC formation. Likewise, in presence of FFAs insulin increased apoptosis in hepatocytes from wild type but not from Alb-Cre-FASfl/fl mice, which lack functional CD95. It is concluded that FFAs can shift insulin-induced hepatocyte proliferation toward hepatocyte apoptosis by triggering a JNK signal, which allows activated EGFR to associate with CD95 and to trigger CD95-dependent apoptosis. Such phenomena may contribute to the pathogenesis of NASH.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.