Roland Reinehr

The G‐protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells

Sinusoidal endothelial cells (SEC) constitute a permeable barrier between hepatocytes and blood. SEC are exposed to high concentrations of bile salts from the enterohepatic circulation. Whether SEC are responsive to bile salts is unknown. TGR5, a G‐protein–coupled bile acid receptor, which triggers cAMP formation, has been discovered recently in macrophages. In this study, rat TGR5 was cloned and antibodies directed against the C‐terminus of rat TGR5 were developed, which detected TGR5 as a glycoprotein in transfected HepG2‐cells. Apart from Kupffer cells, TGR5 was detected in SEC of rat liver. SEC expressed TGR5 over the entire acinus, whereas endothelial cells of the portal or central veins were not immunoreactive toward TGR5 antibodies. In isolated SEC, TGR5 mRNA and protein were detected by reverse transcription (RT) PCR, immunofluorescence microscopy, and Western blot analysis. Bile salts increased cAMP in isolated SEC and induced mRNA expression of endothelial NO synthase (eNOS), a known cAMP‐dependent gene. In addition, bile acids activated eNOS by phosphorylation of eNOS at amino acid position 1177. In line with eNOS activation, bile acids induced NO production in liver slices. This is the first report on the expression of TGR5 in SEC. Conclusion: The data suggest that SEC are directly responsive toward specific bile salts. Regulation of eNOS in SEC by TGR5 connects bile salts with hepatic hemodynamics. This is of particular importance in cholestatic livers when bile salt concentrations are increased. (HEPATOLOGY 2007;45:695–704.)

Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices.

The role of NADPH oxidase (NOX) and the regulatory subunit p47phox for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47phox knock‐out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47phox, NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47phox and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47phox serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)ζ‐inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK‐801 and the intracellular Ca2+ chelator BAPTA‐AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47phox knock‐out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47phox‐dependent NADPH oxidase‐catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy.

Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation

The mechanisms underlying CD95 ligand (CD95L)‐ and hyperosmolarity‐induced activation of the CD95 system [Reinehr, R., Graf, D., Fischer, R., Schliess, F., and Haussinger, D. (2002) Hepatology 36, 602‐614] as initial steps of apoptosis were studied. Hyperosmotic exposure (405 mosmol/l) of rat hepatocytes induced within 1 min oxidative stress and antioxidant‐sensitive activation of the epidermal growth factor receptor (EGFR) and c‐Jun‐N‐terminal‐kinase (JNK). After 30 min of hyperosmotic exposure EGFR associated with CD95 and CD95 became tyrosine phosphorylated. Inhibition of JNK or protein kinase C (PKC) had no effect on EGFR phosphorylation but abolished CD95/EGFR association, CD95‐tyrosine phosphorylation, membrane targeting, and Fas‐associated death domain/caspase 8 recruitment to CD95 [death‐inducing signaling complex (DISC) formation]. Inhibition of EGFR tyrosine kinase activity prevented CD95 tyrosine phosphorylation and DISC formation but not hyperosmolarity‐induced EGFR phosphorylation and EGFR association with CD95. Tyrosine‐phosphorylated CD95 was enriched in the plasma membrane. All maneuvers preventing CD95 tyrosine phosphorylation inhibited CD95 membrane trafficking and DISC formation. Stimulation of EGFR by EGF induced EGFR phosphorylation but no association with CD95 or CD95 phosphorylation. Addition of CD95L also induced EGFR and JNK activation, EGFR/CD95 association, CD95 tyrosine phosphorylation, DISC formation, and CD95 membrane targeting with an inhibitor sensitivity profile similar to that of hyperosmotic CD95 activation, except that inhibition of PKC was ineffective. The data suggest that moderate hyperosmolarity or CD95L trigger oxidative stress and EGFR activation followed by a JNK‐dependent EGFR/CD95association and CD95 tyrosine phosphorylation, probably through EGFR tyrosine kinase activity. This provides a signal for CD95 membrane trafficking and DISC formation.

Bile salt—induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation

BACKGROUND & AIMS Hydrophobic bile acids induce CD95-dependent hepatocyte apoptosis. METHODS The mechanisms of bile acid-induced CD95 activation were studied in 24-hour cultured rat hepatocytes, in situ-perfused rat livers, and livers from bile duct-ligated rats. RESULTS Within 1 minute, the proapoptotic bile salts taurolithocholate-3-sulfate and glycochenodeoxycholate induced oxidative stress and EGF receptor (EGF-R) tyrosine phosphorylation followed by rapid c-Jun-N-terminal kinase (JNK) activation. Thereafter, EGF-R associated with CD95 with subsequent CD95 tyrosine phosphorylation, CD95 membrane targeting, and death-inducing signal complex (DISC) formation. All of these responses were also triggered by taurochenodeoxycholate except that DISC formation only occurred in the presence of phosphatidylinositol 3-kinase inhibitors. No activation of EGF-R or CD95 was observed with tauroursodeoxycholate or taurocholate. Taurolithocholate-3-sulfate-induced EGF-R phosphorylation was sensitive to N-acetylcysteine (NAC) and genistein, whereas CD95/EGF-R association was inhibited by NAC, JNK, or protein kinase C inhibition but not by AG1478. However, the latter compound as well as NAC, genistein, inhibition of JNK, or protein kinase C inhibited CD95 tyrosine phosphorylation, membrane trafficking, and DISC formation. CONCLUSIONS Induction of apoptosis by hydrophobic bile salts involves EGF-R activation and EGF-R-dependent CD95 tyrosine phosphorylation, which triggers CD95 membrane targeting and Fas-associated death domain/caspase-8 recruitment. The latter step is apparently also controlled by phosphatidylinositol 3-kinase.

Selenoprotein P expression is controlled through interaction of the coactivator PGC‐1α with FoxO1a and hepatocyte nuclear factor 4α transcription factors

Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine‐tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor‐γ coactivator 1α (PGC‐1α) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)‐4α, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO‐responsive element daf16‐binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC‐1α. Moreover, the PGC‐1α‐inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC‐1α and SeP caused by dexamethasone treatment. In a brain‐derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC‐1α together with FoxO1a, and overexpression of HNF‐4α potentiated this effect. Conclusion: High‐level expression of SeP in liver is ensured by concerted action of the coactivator PGC‐1α and the transcription factors FoxO1a and HNF‐4α. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose‐6‐phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC‐1α as a key regulator of selenium homeostasis. (HEPATOLOGY 2008;48:1998‐2006.)

Chronic liver disease is triggered by taurine transporter knockout in the mouse

Taurine is an abundant organic osmolyte with antioxidant and immunomodulatory properties. Its role in the pathogenesis of chronic liver disease is unknown. The liver phenotype was studied in taurine transporter knockout (taut?/?) mice. Hepatic taurine levels were ~21, 15 and 6 ?mol/g liver wet weight in adult wild‐type, heterozygous (taut+/?) and homozygous (taut?/?) mice, respectively. Immunoelectronmicroscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut?/?) mice. Compared with wild‐type mice, (taut?/?) and (taut+/?) mice developed moderate unspecific hepatitis and liver fibrosis with increased frequency of neoplastic lesions beyond 1 year of age. Liver disease in (taut?/?) mice was characterized by hepatocyte apoptosis, activation of the CD95 system, elevated plasma TNF‐? levels, hepatic stellate cell and oval cell proliferation, and severe mitochondrial abnormalities in liver parenchymal cells. Mitochondrial dysfunction was suggested by a significantly lower respiratory control ratio in isolated mitochondria from (taut?/?) mice. Taut knockout had no effect on taurine‐conjugated bile acids in bile; however, the relative amount of cholate‐conjugates acid was decreased at the expense of 7‐keto‐cholateconjugates. In conclusion, taurine deficiency due to defective taurine transport triggers chronic liver disease, which may involve mitochondrial dysfunction.

BCL-2 Family Inhibitors Enhance Histone Deacetylase Inhibitor and Sorafenib Lethality via Autophagy and Overcome Blockade of the Extrinsic Pathway to Facilitate Killing

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway.

Endosomal Acidification and Activation of NADPH Oxidase Isoforms Are Upstream Events in Hyperosmolarity-induced Hepatocyte Apoptosis

Hyperosmotic exposure of rat hepatocytes induced a rapid oxidative-stress(ROS) response as an upstream signal for proapoptotic CD95 activation. This study shows that hyperosmotic ROS formation involves a rapid ceramide- and protein kinase Cζ (PKCζ)-dependent serine phosphorylation of p47phox and subsequent activation of NADPH oxidase isoforms. Hyperosmotic p47phox phosphorylation and ROS formation were sensitive to inhibition of sphingomyelinases and were strongly blunted after knockdown of acidic sphingomyelinase (ASM) or of p47phox protein. Hyperosmolarity induced a rapid bafilomycin- and 4,4 ′-diisothiocyanostilbene-2,2 ′-disulfonic acid disodium salt (DIDS)-sensitive acidification of a vesicular compartment, which was accessible to endocytosed fluorescein isothiocyanate-dextran and colocalized with ASM, PKCζ, and the NADPH oxidase isoform Nox 2 (gp91phox). Bafilomycin and DIDS prevented the hyperosmolarity-induced increase in ceramide formation, p47phox phosphorylation, and ROS formation. As shown recently (Reinehr, R., Becker, S., Höngen, A., and Häussinger, D. (2004) J. Biol. Chem. 279, 23977-23987), hyperosmolarity induced a Yes-dependent activation of JNK and the epidermal growth factor receptor (EGFR), followed by EGFR-CD95 association, EGFR-catalyzed CD95-tyrosine phosphorylation, and translocation of the EGFR-CD95 complex to the plasma membrane, where formation of the deathinducing signaling complex occurs. These proapoptotic responses were not only sensitive to inhibitors of sphingomyelinase, PKCζ, or NADPH oxidases but also to ASM knockdown, bafilomycin, and DIDS, i.e. maneuvers largely preventing hyperosmolarity-induced endosomal acidification and/or ceramide formation. In hepatocytes from p47phox knock-out mice, hyperosmolarity failed to activate the CD95 system. The data suggest that hyperosmolarity induces endosomal acidification as an important upstream event for CD95 activation through stimulation of ASM-dependent ceramide formation and activation of NADPH oxidase isoforms.

Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca(2+) levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS; however, combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca(2+). Quenching of ROS did not affect drug-induced ceramide/dihydro-ceramide levels, whereas quenching of Ca(2+) reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knockdown of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings show that induction of cytosolic Ca(2+) by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.

Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes

Astrocyte swelling is observed in different types of brain injury. We studied a potential contribution of swelling to protein tyrosine nitration (PTN) by using cultured rat astrocytes exposed to hypoosmotic (205 mosmol/L) medium. Hypoosmolarity (2 h) increases total PTN by about 2‐fold in 2 h. The hypoosmotic PTN is significantly inhibited by the NMDA receptor antagonist MK‐801, the nitric oxide synthase (NOS) inhibitor L‐NMMA, the extracellular Ca2+ chelator EGTA and the calmodulin antagonist W13, suggesting the involvement of NMDA receptor activation, influx of extracellular Ca2+ and Ca2+/calmodulin‐dependent NO synthesis. Further, superoxide dismutase plus catalase and uric acid strongly inhibit hypoosmotic PTN, suggesting the involvement of the toxic metabolite peroxynitrite (ONOO−) as a nitrating agent. Hypoosmotic astrocyte swelling rapidly stimulates generation of reactive oxygen intermediates; this process is prevented by MK‐801 and EGTA. In addition, MK‐801 inhibits the hypoosmotic elevation of [Ca2+]i. The findings support the view that astrocyte swelling as induced, for example, by toxins relevant for hepatic encephalopathy is sufficient to produce oxidative stress and PTN and thus contributes to altered astroglial and neuronal function.