Dirk Graf

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.

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis

BACKGROUND & AIMS Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.

Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver.

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI 3-kinase) in cultured hepatocytes. Ras activation was dependent on PI 3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI 3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI 3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.

Hydrophobic Bile Salts Induce Hepatocyte Shrinkage via NADPH Oxidase Activation

Hydrophobic bile salts activate NADPH oxidase through a ceramide and protein kinase Cζ-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. As shown in the present study, hydrophobic bile salts such as glycochenodeoxycholate, taurochenodeoxycholate or taurolithocholylsulfate (TLCS) also induce within 30min hepatocyte shrinkage in perfused rat liver. TLCS-induced hepatocyte shrinkage was strongly blunted in presence of desipramine, apocynin, bafilomycin and DIDS, i.e. maneuvres previously shown to inhibit TLCS-induced NADPH oxidase activation and the subsequent oxidative stress response. The antioxidant N-acetylcysteine inhibited TLCS-induced hepatocyte shrinkage. N-acetylcysteine by itself increased hepatocyte hydration, suggesting that a basal production of reactive oxygen intermediates is involved in the regulation of liver cell hydration. TLCS failed to induce shrinkage of hepatocytes from p47phox knock-out, but not control mice. Likewise, hepatocytes from p47phox knock-out mice were resistant towards TLCS-induced apoptosis and failed to activate the CD95 system. No cell shrinkage was observed in response to taurocholate and tauroursodesoxycholate, i.e. bile salts which do not induce an oxidative stress signal and apoptosis. NADPH oxidase activation also counteracts volume recovery in response to hyperosmotic hepatocyte shrinkage. The findings indicate that hydrophobic, proapoptotic bile salts induce hepatocyte shrinkage largely through NADPH oxidase-derived oxidative stress. Because cell shrinkage in turn activates NADPH oxidase, which blunts cell volume recovery, a vicious cycle ensues between oxidative stress and cell shrinkage, which propagates CD95 activation and may finally lead to apoptosis. In addition, cell shrinkage induced by proapoptotic bile salts may augment apoptosis by increasing protein breakdown and induction of cholestasis.

Bile acids PKA-dependently induce a switch of the IL-10/IL-12 ratio and reduce proinflammatory capability of human macrophages

That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS‐induced expression of proinflammatory cytokines without affecting the expression of the anti‐inflammatory cytokine IL‐10. This results in a macrophage phenotype that is characterized by an increased IL‐10/IL‐12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5‐dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF‐κB, and PKA‐dependent enhancement of LPS‐induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF‐κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti‐inflammatory phenotype characterized by an increased IL‐10/IL‐12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid‐induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti‐inflammatory and immunosuppressive effects of bile acids.

Role of p38(MAPK) in cell volume regulation of perfused rat liver.

In perfused rat liver, hypoosmotic exposure (225 mosmol/L) leads to a volume-regulatory decrease by release of K+, Cl- and HCO3- through Ba2+-, DIDS- and quinidine-sensitive ion channels. The underlying signal transduction mechanisms, however, are unknown. As hypoosmotic hepatocyte swelling leads to a rapid activation of extracellular signal regulated kinases (Erks) and of p38MAPK, the role of mitogen-activated protein kinases (MAPK) and PI-3-kinase in mediating the RVD in perfused rat liver was studied. The presence of the MEK inhibitor PD 098 059, which blocks the hypoosmotic activation of Erks, had no effect on the extent and time course of cell volume regulatory K+ efflux. However, inhibitors of p38MAPK such as SB 203 580 and PD 169 316, but not their inactive analogue SB 202 474, significantly delayed and diminished the volume-regulatory K+ efflux. Accordingly, in presence of these p38MAPK inhibitors, the hepatocytes remained in a more swollen state after completion of RVD. Inhibition of hypoosmotic Erk activation by pertussis or cholera toxin, erbstatin or genistein had no effect on RVD by hypoosmolarity. Likewise, neither inhibition of PI-3-kinase by wortmannin or LY 294 002 nor inhibition of S 6 phosphorylation by rapamycin nor protein kinase inhibition by H-7, H-89 or KT 5823 led to a significant change of RVD upon hypoosmolarity. The amount and time course of K+ release by oxidative stress upon addition of t-BOOH or H2O2 remained unaffected by inhibition of p38MAPK by SB 203 580, suggesting a specific inhibition of RVD-dependent K+ release by this inhibitor. The findings suggest that swelling-induced activation of p38MAPK, but not of Erks and PI-3-kinase, is involved in RVD in liver, whereas p38MAPK is apparently not involved in the net K+ release induced by oxidative stress.

Multimodal treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the most common liver cancer with an increasing incidence and it accounts for the third most common cause of cancer-related death worldwide. Even though the clinical diagnosis and management of HCC improved significantly in the last decades, this malignant disease is still associated with a poor prognosis. It has to be distinguished between patients with HCCs, which developed from liver cirrhosis, and patients without underlying liver cirrhosis as classification systems, prognosis estimation and therapy recommendations differ in-between. In case of HCC in patients with liver cirrhosis in Europe, treatment allocation and prognosis estimation are mainly based on the Barcelona-Clinic Liver Cancer (BCLC) staging system. Based on this staging system different surgical, interventional radiological/sonographical and non-interventional procedures have been established for the multimodal treatment of HCC. The BCLC classification system represents a decision guidance; however because of its limitations in selected patients treatment allocation should be determined on an individualized rather than a guideline-based medicine by a multidisciplinary board in order to offer the best treatment option for each patient. This review summarizes the current management of HCC and illustrates controversial areas of therapeutic strategies.

Enhanced insulin sensitivity of gene targeted mice lacking functional KCNQ1

The pore-forming K+-channel alpha-subunit KCNQ1 is expressed in a wide variety of tissues including heart, skeletal muscle, liver, and epithelia. Most recent evidence revealed an association of the KCNQ1 gene with the susceptibility to type 2 diabetes. KCNQ1 participates in the regulation of cell volume, which is, in turn, critically important for the regulation of metabolism by insulin. The present study explored the influence of KCNQ1 on insulin-induced cellular K+ uptake and glucose metabolism. Insulin (100 nM)-induced K+ uptake was determined in isolated perfused livers from KCNQ1-deficient mice (kcnq1(-/-)) and their wild-type littermates (kcnq1(+/+)). Moreover, plasma glucose and insulin levels, intraperitoneal glucose (3 g/kg) tolerance, insulin (0.15 U/kg)-induced hypoglycemia, and peripheral uptake of radiolabeled 3H-deoxy-glucose were determined in both genotypes. Insulin-stimulated hepatocellular K+ uptake was significantly more sustained in isolated perfused livers from kcnq1(-/-) mice than from kcnq1(+/+)mice. The decline of plasma glucose concentration following an intraperitoneal injection of insulin was again significantly more sustained in kcnq1(-/-) than in kcnq1(+/+) mice. Both fasted and nonfasted plasma glucose and insulin concentrations were significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Following an intraperitoneal glucose injection, the peak plasma glucose concentration was significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Uptake of 3H-deoxy-glucose into skeletal muscle, liver, kidney and lung tissue was significantly higher in kcnq1(-/-) than in kcnq1(+/+)mice. In conclusion, KCNQ1 counteracts the stimulation of cellular K+ uptake by insulin and thereby influences K+-dependent insulin signaling on glucose metabolism. The observations indicate that KCNQ1 is a novel molecule affecting insulin sensitivity of glucose metabolism.

Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms.

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 micromol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38(MAPK), and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.

Osmotic regulation of insulin-induced mitogen-activated protein kinase phosphatase (MKP-1) expression in H4IIE rat hepatoma cells.

A contribution of intracellular dehydration to insulin resistance has been established in human subjects and in different experimental systems. Here the effect of hyperosmolarity (405 mosmol/l) on insulin-induced mitogen-activated protein (MAP) kinase phosphatase (MKP)-1 expression was studied in H4IIE rat hepatoma cells. Insulin induces robust MKP-1 expression which correlates with a vanadate-sensitive decay of extracellular-signal-regulated kinase (Erk-1/Erk-2) activity. Hyperosmolarity delays MKP-1 accumulation by insulin and this corresponds to impaired MKP-1 synthesis, whereas MKP-1 degradation remains unaffected by hyperosmolarity. Rapamycin, which inhibits signalling downstream from the mammalian target of rapamycin (mTOR) and a peptide inhibiting protein kinase C (PKC) zeta/lambda abolish insulin-induced MKP-1 protein but not mRNA expression, suggesting the involvement of the p70 ribosomal S6 protein kinase (p70S6-kinase) and/or the eukaryotic initiation factor 4E-binding proteins (4E-BPs) as well as atypical PKCs in MKP-1 translation. Hyperosmolarity induces sustained suppression of p70S6-kinase and 4E-BP1 hyperphosphorylation by insulin, whereas insulin-induced tyrosine phosphorylation of the insulin receptor (IR) beta subunit and the IR substrates IRS1 and IRS2, recruitment of the phosphoinositide 3-kinase (PI 3-kinase) regulatory subunit p85 to the receptor substrates as well as PI 3-kinase activation, and Ser-473 phosphorylation of protein kinase B and Thr-410/403 phosphorylation of PKC zeta/lambda are largely unaffected under hyperosmotic conditions. The hyperosmotic impairment of both, MKP-1 expression and p70S6-kinase hyperphosphorylation by insulin is insensitive to K(2)CrO(4), calyculin A and vanadate, and inhibition of the Erk-1/Erk-2 and p38 pathways. The suppression of MKP-1 may further contribute to insulin resistance under dehydrating conditions by allowing unbalanced MAP kinase activation.