GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms.

mir-29 regulates Mcl-1 protein expression and apoptosis.

Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis.

Cathepsin B inactivation attenuates hepatocyte apoptosis and liver damage in steatotic livers after cold ischemia-warm reperfusion injury.

Hepatic steatosis predisposes the liver to cold ischemia-warm reperfusion (CI/WR) injury by unclear mechanisms. Because hepatic steatosis has recently been associated with a lysosomal pathway of apoptosis, our aim was to determine whether this cell-death pathway contributes to CI/WR injury of steatotic livers. Wild-type and cathepsin B-knockout (Ctsb(-/-)) mice were fed the methionine/choline-deficient (MCD) diet for 2 wk to induce hepatic steatosis. Mouse livers were stored in the University of Wisconsin solution for 24 h at 4 degrees C and reperfused for 1 h at 37 degrees C in vitro. Immunofluorescence analysis of the lysosomal enzymes cathepsin B and D showed a punctated intracellular pattern consistent with lysosomal localization in wild-type mice fed a standard diet after CI/WR injury. In contrast, cathepsin B and D fluorescence became diffuse in livers from wild-type mice fed MCD diet after CI/WR, indicating that lysosomal permeabilization had occurred. Hepatocyte apoptosis was rare in both normal and steatotic livers in the absence of CI/WR injury but increased in wild-type mice fed an MCD diet and subjected to CI/WR injury. In contrast, hepatocyte apoptosis and liver damage were reduced in Ctsb(-/-) and cathepsin B inhibitor-treated mice fed the MCD diet following CI/WR injury. In conclusion, these findings support a prominent role for the lysosomal pathway of apoptosis in steatotic livers following CI/WR injury.

Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.

Tumor necrosis factor-alpha (TNF-alpha) contributes to liver injury by inducing hepatocyte apoptosis. Recent evidence suggests that cathepsin B (cat B) contributes to TNF-alpha-induced apoptosis in vitro. The aim of the present study was to determine whether cat B contributes to TNF-alpha-induced hepatocyte apoptosis and liver injury in vivo. Cat B knockout (catB(-/-)) and wild-type (catB(+/+)) mice were first infected with the adenovirus Ad5I kappa B expressing the I kappa B superrepressor to inhibit nuclear factor-kappa B-induced survival signals and then treated with murine recombinant TNF-alpha. Massive hepatocyte apoptosis with mitochondrial release of cytochrome c and activation of caspases 9 and 3 was detected in catB(+/+) mice 2 hours after the injection of TNF-alpha. In contrast, significantly less hepatocyte apoptosis and no detectable release of cytochrome c or caspase activation occurred in the livers of catB(-/-) mice. By 4 hours after TNF-alpha injection, only 20% of the catB(+/+) mice were alive as compared to 85% of catB(-/-) mice. Pharmacological inhibition of cat B in catB(+/+) mice with L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline (CA-074 Me) also reduced TNF-alpha-induced liver damage. The present data demonstrate that a cat B-mitochondrial apoptotic pathway plays a pivotal role in TNF-alpha-induced hepatocyte apoptosis and liver injury.

Bid antisense attenuates bile acid-induced apoptosis and cholestatic liver injury.

Bile acids cause liver injury during cholestasis by inducing hepatocyte apoptosis by both Fas-dependent and -independent mechanisms. However, the Fas-independent apoptosis also appears to be death receptor-mediated. Because death receptor-mediated apoptosis in hepatocytes requires proapoptotic Bcl-2 BH3 domain only protein Bid, we postulated that Fas-independent but death receptor-mediated bile acid cytotoxicity would be Bid-dependent. We used Fas-deficient lymphoproliferative (lpr) mouse hepatocytes for these studies, and inhibited Bid expression using an antisense approach. Glychochenodeoxycholate (GCDC) was used to induce apoptosis. Bid cleavage and translocation to mitochondria was observed in GCDC-treated cells as assessed by immunoblot analysis and confocal imaging of Bid-green fluorescent protein, respectively. Bid translocation to mitochondria was associated with cytochrome c release. A Bid antisense 2'-MOE modified oligonucleotide inhibited Bid expression in hepatocytes and markedly attenuated hepatocytes apoptosis by GCDC. Treatment of lpr mice with Bid antisense also ameliorated liver injury following bile duct ligation of the mice, a model of extrahepatic cholestasis. These results suggest that bile acid cytotoxicity is Bid-dependent despite the absence of Fas. Bid antisense therapy is a promising approach for the treatment of cholestatic liver injury.

The bile acid glycochenodeoxycholate induces trail-receptor 2/DR5 expression and apoptosis.

Toxic bile salts induce hepatocyte apoptosis by both Fas-dependent and -independent mechanisms. In this study, we examined the cellular mechanisms responsible for Fas-independent, bile acid-mediated apoptosis. HuH-7 cells, which are known to be Fas deficient, were stably transfected with the sodium-dependent bile acid transporting polypeptide. The toxic bile acid glycochenodeoxycholate (GCDC)-induced apoptosis in these cells in a time- and concentration-dependent manner. Apoptosis and mitochondrial cytochrome c release were inhibited by transfection with dominant negative FADD, CrmA transfection, or treatment with the selective caspase 8 inhibitor IETD-CHO. These observations suggested the Fas-independent apoptosis was also death receptor mediated. Reverse transcriptase-polymerase chain reaction demonstrated tumor necrosis factor-R1, tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, -R2/DR5, and TRAIL, but not tumor necrosis factor-alpha expression by these cells. GCDC treatment increased expression of TRAIL-R2/DR5 mRNA and protein 10-fold while expression of TRAIL-R1 was unchanged. Furthermore, aggregation of TRAIL-R2/DR5, but not TRAIL-R1/DR4 was observed following GCDC treatment of the cells. Induction of TRAIL-R2/DR5 expression and apoptosis by bile acids provides new insights into the mechanisms of hepatocyte apoptosis and the regulation of TRAIL-R2/DR5 expression.

Viral fusogenic membrane glycoprotein expression causes syncytia formation with bioenergetic cell death: implications for gene therapy.

Viral fusogenic membrane glycoproteins (FMGs) are candidates for gene therapy of solid tumors because they cause cell fusion, leading to formation of lethal multinucleated syncytia. However, the cellular mechanisms mediating cell death after FMG-induced cell fusion remain unclear. The present study was designed to examine the mechanisms by which FMG expression in hepatocellular carcinoma cells lead to cell death. Transfection of Hep3B cells with the Gibbon Ape leukemia virus hyperfusogenic envelope protein (GALV-FMG) resulted in the formation of multinucleated syncytia that reached a maximum 5 days after transfection (100 nuclei/syncytia). The syncytia were viable for a period of 2 days and then rapidly lost viability by day 5. Mitochondrial dysfunction occurred in GALV-FMG-induced syncytia prior to loss of viability with loss of the mitochondrial membrane potential, cellular ATP depletion, and release of mitochondrial cytochrome c-GFP into the cytosol. The pan-caspase inhibitor, Z-VAD-fmk, did not prevent cell death. However, glycolytic generation of ATP with fructose effectively increased cellular ATP and preserved syncytial viability. These data suggest that expression of FMG in hepatoma cells results in the formation of multinucleated syncytia, causing mitochondrial failure with ATP depletion, a bioenergetic form of cell death with necrosis. This form of cell death should be effective in vivo and enhance the bystander effect, suggesting that FMG-based gene therapy deserves further study for the treatment of hepatocellular and other cancers.

Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c.

TNF-alpha-induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-alpha-induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-alpha in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-alpha/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis of isolated hepatocytes was markedly diminished in cells from CatB(-/-) mice. Deletion of the CatB gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-alpha and an adenovirus construct expressing the IkappaB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-alpha-treated hepatocytes.

Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas.

Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.

The transforming growth factor beta(1)-inducible transcription factor TIEG1, mediates apoptosis through oxidative stress.

Transforming growth factor beta(1) (TGF-beta(1))-inducible transcription factors have recently elicited interest because of their critical role in the regulation of cell proliferation, differentiation, and apoptosis. We have previously reported that the TGF-beta(1)-inducible transcription factor, TIEG1, induces apoptosis in a pancreas-derived cell line. However, the mechanisms underlying the apoptotic effects of this transcription factor remain to be defined. In this study, using the TGF-beta(1)-sensitive Hep 3B cell line, we have defined the mechanistic sequence of events that characterize TIEG1-mediated apoptosis and compared these events with the changes observed during TGF-beta(1)-induced apoptosis. Both TGF-beta(1)- and TIEG1-induced cell death were accompanied by an increase in the generation of reactive oxygen species and a loss of the mitochondrial membrane potential preceding the morphological changes of apoptosis. In contrast, increases in caspase 3-like activity and glutathione (GSH) depletion occurred later in the apoptotic process, concurrent with the morphological features of apoptosis. The antioxidant, trolox, decreased the formation of reactive oxygen species and apoptosis. These results demonstrate that similar to TGF-beta(1), TIEG1 induces apoptosis by a mechanism involving the formation of reactive oxygen species.

Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas.

Cholestatic liver injury appears to result from the induction of hepatocyte apoptosis by toxic bile salts such as glycochenodeoxycholate (GCDC). Previous studies from this laboratory indicate that cathepsin B is a downstream effector protease during the hepatocyte apoptotic process. Because caspases can initiate apoptosis, the present studies were undertaken to determine the role of caspases in cathepsin B activation. Immunoblotting of GCDC-treated McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polymerase and lamin B1 to fragments that indicate activation of effector caspases. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-induced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Examination of the mechanism of GCDC-induced caspase 8 activation revealed that dominant-negative FADD inhibited apoptosis and that hepatocytes isolated from Fas-deficient lymphoproliferative mice were resistant to GCDC-induced apoptosis. After GCDC treatment, immunoprecipitation experiments demonstrated Fas oligomerization, and confocal microscopy demonstrated DeltaFADD-GFP (Fas-associated death domain-green fluorescent protein, aggregation in the absence of detectable Fas ligand mRNA. Collectively, these data suggest that GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B.

Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases.

As part of this thematic series on mitochondria in cell death, we would like to review our data on: (1) the role of the mitochondrial permeability transition (MPT) in hepatocyte necrosis during cholestasis; and (2) the concept that endogenous mitochondrial protease activity may lead to the MPT. Many chronic human liver diseases are characterized by cholestasis, an impairment in bile flow. During cholestasis an accumulation of toxic hydrophobic bile salts in the hepatocyte causes necrosis. We tested the hypothesis that toxic hydrophobic bile salt, glycochenodeoxycholate (GCDC), causes hepatocyte necrosis by inducing the MPT. GCDC induces a rapid, cyclosporin A-sensitive MPT. The hydrophilic bile salt, ursodeoxycholate (UDCA), prevents the GCDC-induced MPT and hepatocyte necrosis providing an explanation for its beneficial effect in human liver disease. We have also demonstrated that the calcium-dependent MPT is associated with an increase in calpain-like protease activity and inhibited by calpain inhibitors. In an experimental model of cholestasis, mitochondrial calpain-like protease activity increases 1.6-fold. We propose for the first time that activation of mitochondrial proteases may initiate the MPT and cell necrosis during cholestasis.

Cholestasis confers resistance to the rat liver mitochondrial permeability transition.

BACKGROUND & AIMS: Bile salts can cause hepatocyte death by inducing the mitochondrial permeability transition (MPT). However, the slow progression of human cholestatic liver diseases suggests that hepatocytes adapt to resist the MPT. Bcl-x, a protein, and increased mitochondrial cardiolipin, a membrane lipid, elevate the threshold for the MPT. Our aims were to determine if liver mitochondria become resistant to the MPT during cholestasis and, if so, if the resistance is mediated by Bcl-x and/or increased cardiolipin. METHODS: Hepatocytes and liver mitochondria were obtained from bile duct-ligated (BDL) rats and sham-operated rats (control). RESULTS: After addition of glycochenodeoxycholate (GCDC), the magnitude of the MPT was reduced in mitochondria from BDL rats vs. controls. Although Bcl-xL was not increased, mitochondrial cardiolipin content was significantly greater in BDL rats vs. controls. Cell viability was also increased in hepatocytes from BDL rats vs. controls after treatment with GCDC. Feeding BDL rats a fatty acid-deficient diet prevented the increase in mitochondrial cardiolipin content; mitochondria and hepatocytes from these rats were susceptible to the MPT and hepatocellular death by GCDC. CONCLUSIONS: These data suggest that an increase in mitochondria cardiolipin content occurs during cholestasis as an adaptive phenomenon to resist cell death by the MPT.

Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes.

BACKGROUND & AIMS: Bile salt-induced apoptosis is mediated by a trypsin-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced hepatocyte apoptosis. METHODS: Rats, isolated rat hepatocytes, and a rat hepatoma cell line stably transfected with a bile salt transporter (McNtcp.24) were used for this study. RESULTS: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in trypsin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as cathepsin B. Specific, structurally dissimilar cathepsin B inhibitors blocked glycochenodeoxycholate (GCDC)-induced apoptosis in cultured rat hepatocytes. Furthermore, stable transfection of McNtcp.24 cells with the complementary DNA for cathepsin B in the antisense orientation reduced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, cathepsin B cellular localization during apoptosis was determined by immunoblot analysis of nuclear cell fractions, immunocytochemistry, and by determining the compartmentation of expressed cathepsin B fused to green fluorescent protein. All three approaches showed translocation of cathepsin B from the cytoplasm to the nucleus during GCDC-induced apoptosis. CONCLUSIONS: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bile salt-induced apoptosis of hepatocytes.

Ceramide induces hepatocyte cell death through disruption of mitochondrial function in the rat.

Although ceramide signaling pathways have been implicated in cell death, neither their role in hepatocellular death nor the cellular mechanisms mediating ceramide-induced cell death are known. The mitochondrial membrane permeability transition (MMPT) has been proposed as a common final pathway in cell death. Thus the aims of our study were to determine if ceramides cause hepatocellular death by necrosis and not apoptosis as confirmed by morphology and the absence of internucleosomal DNA cleavage. Ceramide-mediated hepatocyte necrosis was acyl chain-length, concentration, and time-dependent. Ceramides induced cell necrosis was associated with adenosine triphosphate (ATP) depletion and mitochondrial depolarization suggesting that ceramides caused mitochondrial dysfunction. In isolated mitochondria, ceramides induced the cyclosporine A-sensitive MMPT in an acyl chain-length and concentration dependent manner. Ceramide toxicity was specific as the less potent dihydro form did not induce cell necrosis, significant ATP depletion, mitochondrial depolarization nor the MMPT. In conclusion, ceramide induced cell death is acyl-chain length dependent and mediated by the MMPT. These data show for the first time that ceramide acts as a mediator of hepatocyte necrosis by causing mitochondrial failure.

Cytoprotection by fructose and other ketohexoses during bile salt-induced apoptosis of hepatocytes.

Toxic bile salts cause hepatocyte necrosis at high concentrations and apoptosis at lower concentrations. Although fructose prevents bile salt-induced necrosis, the effect of fructose on bile salt-induced apoptosis is unclear. Our aim was to determine if fructose also protects against bile salt-induced apoptosis. Fructose inhibited glycochenodeoxycholate (GCDC)-induced apoptosis in a concentration-dependent manner with a maximum inhibition of 72% +/- 10% at 10 mmol/L. First, we determined if fructose inhibited apoptosis by decreasing adenosine triphosphate (ATP) and intracellular pH (pHi). Although fructose decreased ATP to <25% of basal values, oligomycin (an ATP synthase inhibitor) did not inhibit apoptosis despite decreasing ATP to similar values. Fructose (10 mmol/L) decreased intracellular pH (pHi) by 0.2 U. However, extracellular acidification (pH 6.8), which decreased hepatocyte pHi 0.35 U and is known to inhibit necrosis, actually potentiated apoptosis 1.6-fold. Fructose cytoprotection also could not be explained by induction of bcl-2 transcription or metal chelation. Because we could not attribute fructose cytoprotection to metabolic effects, alterations in the expression of bcl-2, or metal chelation, we next determined if the poorly metabolized ketohexoses, tagatose and sorbose, also inhibited apoptosis; unexpectedly, both ketohexoses inhibited apoptosis. Because bile salt-induced apoptosis and necrosis are inhibited by fructose, these data suggest that similar processes initiate bile salt-induced hepatocyte necrosis and apoptosis. In contrast, acidosis, which inhibits necrosis, potentiates apoptosis. Thus, ketohexose-sensitive pathways appear to initiate both bile salt-induced cell apoptosis and necrosis, whereas dissimilar, pH-sensitive, effector mechanisms execute these two different cell death processes.

Induction of the mitochondrial permeability transition by protease activity in rats: a mechanism of hepatocyte necrosis.

BACKGROUND & AIMS: The mitochondrial membrane permeability transition (MMPT) has been proposed as a mechanism of cell necrosis. In contrast, it has been suggested that enhanced activity of calpain-like proteases causes cell necrosis. To integrate these concepts, the hypothesis that stimulation of mitochondrial calpain-like protease activity induces the MMPT was developed. METHODS: Calpain-like protease activity and the MMPT were measured in rat liver mitochondria. The mitochondrial membrane potential and cell necrosis were measured in rat hepatocytes. RESULTS: The protease inhibitor Cbz-Leu-Leu-Tyr-CHN2 inhibited both calpain-like protease activity and induction of the MMPT by Ca2+ and tert-butyl hydroperoxide. This effect of Cbz-Leu-Leu-Tyr-CHN2 was specific because serine, aspartate, and metalloprotease inhibitors did not inhibit the MMPT. The protease inhibitor Cbz-Leu-Leu-Tyr-CHN2 also delayed the onset of mitochondrial depolarization and cell necrosis during treatment of rat hepatocytes with tert-butyl hydroperoxide, a model of oxidative stress relevant to human disease. CONCLUSIONS: These data suggest a unifying hypothesis linking calpain-like protease activity to the MMPT in cell necrosis. We propose for the first time that activation of mitochondrial calpain-like protease activity can function as a cytolytic trigger initiating the MMPT in cell necrosis.

Nuclear serine protease activity contributes to bile acid-induced apoptosis in hepatocytes.

Glycodeoxycholate (GDC) induces apoptosis in hepatocytes by a mechanism associated with DNA cleavage by endonucleases. In many models of apoptosis, proteolysis is required prior to DNA cleavage. Our aims were to determine if enhanced proteolysis is a mechanism causing GDC-mediated apoptosis. In cultured rat hepatocytes exposed to 50 microM GDC for 4 h, nonlysosomal proteolysis increased by 65% compared with controls. The serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK; 100 microM) reduced cell death from apoptosis by 75% after 4 h of treatment with GDC. TLCK also inhibited DNA fragmentation. There was a twofold increase in nuclear serinelike protease activity during GDC-induced apoptosis accompanied by a 2.5-fold reduction in nonnuclear serine protease activity, suggesting translocation of the protease from the cytosol to the nucleus. Zn2+, an inhibitor of apoptosis, also inhibited nonlysosomal proteolysis and nuclear serinelike protease activity. These novel data suggest that nonlysosomal serinelike protease activity contributes to hepatocyte apoptosis. These data may be important in understanding apoptosis in other cell types and in providing insight into the mechanisms of liver injury during cholestasis.

Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection.

Ursodeoxycholate (UDCA), a hydrophilic bile salt, ameliorates hepatocellular injury by toxic bile salts and is used to treat cholestatic liver disease. However, the mechanisms of bile salt-mediated hepatocyte necrosis and UDCA cytoprotection remain unclear. Hepatocyte necrosis is thought to be caused by the mitochondrial membrane permeability transition (MMPT). Thus, the aims of our study were to determine if a toxic bile salt, glycochenodeoxycholate (GCDC) induces the MMPT and if so, whether UDCA prevents the bile salt-induced MMPT. The MMPT was assessed in isolated rat liver mitochondria. Cell viability was measured in isolated rat hepatocytes. GCDC induced the MMPT in a dose-dependent manner. The GCDC-induced MMPT was partially blocked by cyclosporin A plus trifluoperazine, known inhibitors of the MMPT. UDCA also inhibited the GCDC-induced MMPT, and partially blocked the MMPT by phenylarsene oxide, an established mediator of the MMPT. UDCA or cyclosporin A plus trifluoperazine protected against loss of hepatocyte viability during treatment with GCDC. In conclusion, GCDC induces a MMPT; a finding providing a physicochemical explanation for the bioenergetic form of cell necrosis caused by toxic bile salts. UDCA cytoprotection may, in part, be due to inhibition of the bile salt-induced MMPT.

Increases of intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes.

Retention of bile salts by the hepatocyte contributes to liver injury during cholestasis. Although cell injury can occur by one of two mechanisms, necrosis versus apoptosis, information is lacking regarding apoptosis as a mechanism of cell death by bile salts. Our aim was to determine if the bile salt glycodeoxycholate (GDC) induces apoptosis in rat hepatocytes. Morphologic assessment included electron microscopy and quantitation of nuclear fragmentation by fluorescent microscopy. Biochemical studies included measurements of DNA fragmentation, in vitro endonuclease activity, cytosolic free Ca2+ (Cai2+), and cytosolic free Mg2+ (Mgi2+). Morphologic studies demonstrated typical features of apoptosis in GDC (50 microM) treated cells. The "ladder pattern" of DNA fragmentation was also present in DNA obtained from GDC-treated cells. In vitro endonuclease activity was 2.5-fold greater with Mg2+ than Ca2+. Although basal Cai2+ values did not change after addition of GDC, Mgi2+ increased twofold. Incubation of cells in an Mg(2+)-free medium prevented the rise in Mgi2+ and reduced nuclear and DNA fragmentation. In conclusion, GDC induces apoptosis in hepatocytes by a mechanism promoted by increases of Mgi2+ with stimulation of Mg(2+)-dependent endonucleases. These data suggest for the first time that changes of Mgi2+ may participate in the program of cellular events culminating in apoptosis.