- Email: [email protected]
NCX-1OOO, an no-derivative of ursodeoxycholic acid protects against thi-mediated liver damage in mice
April 2000
is mediated through the Bile acid-responsive element (IR-1) via FXRlRXR subsequent to their binding by bile acids.
3 ATYPICAL PROTEIN KINASE C ISOFORM A (PKC-Z), BUT NOT AKT KINASE MEDIATES TAUROCHENODEOXYCHOLATE-ACTIVATED SURVIVAL SIGNALS. Christian Rust, Carlos V. Paya, Jorge Moscat, Larry M. Karnitz, Gregory J. Gores, Mayo Clin, Rochester, MN; Ctr de Biologia Molecular, Madrid, Spain. Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile salts. We have recently demonstrated that the hydrophobic yet nontoxic bile salt taurochenodeoxycholate (TCDC) activates a PI 3-kinaseINF-KB dependent survival signal, preventing its inherent toxicity. PI 3-kinase activates NF-KB indirectly either through Akt kinase or atypical protein kinase c isoforms such as ( (PKC-0. Thus, the AIMS of our study were to test the HYPOTHESIS that TCDC-induced PI 3-kinase-dependent activation of NF-KB is mediated by Akt and/or PKC-{ METHODS: We employed the McNtcp.24 rat hepatoma cell line which is stably transfected with a bile salt transporter and undergoes bile saltinduced apoptosis. Apoptosis was quantitated using the nueleic acid binding dye DAPI and fluorescent microscopy. Akt activation was assessed using a specific antibody against the phosphorylated form of Akt and immunoblot analysis. PKC-( was immunoprecipitated from cell Iysates and its activity measured using the substrate myelin basic protein and [y_32p]_ATP. Transfections with dominant negative and wildtype PKC-( were used to alter PKC-( activity. NF-KB transcriptional activity was assessed by a luciferase reporter gene assay. RESULTS: Akt was not activated by TCDC. In contrast, PKC-( activity was increased by more than 100% after stimulation with 200 pM TCDC. Furthermore, PKC-( activation was blocked by the PI 3-kinase inhibitor wortmannin (250 nM), suggesting that PKC-( is downstream of PI 3-kinase. In cells transfected with wildtype PKC-(, TCDC-induced NF-KB transcriptional activity was more than doubled as compared to unstimulated controls. This TCDCinduced activation was blocked in cells transfected with the dominant negative PKC-(. These data suggest that PKC-( can activate NF-KB in hepatocytes. Although transfection of McNtcp.24 cells with dominant negative PKC-( did not induce significant apoptosis by itself, TCDC (50 f.LM) increased apoptosis 4-fold above control in cells expressing this plasmid. These data demonstrate the importance of PKC-( in TCDCmediated survival signals. CONCLUSIONS: Collectively, these results indicate that PKC-(, but not Akt, is mediating TCDC-induced survival signaling downstream of PI 3-kinase and upstream of NF-KB. PKC-( appears to be a key kinase mediating survival signals in hepatocytes. Activation of this signaling cascade could prove useful in decreasing the proapoptotic effects of toxic bile salts in cholestasis.
4 PROTECTIVE PATHWAYS IN TNFA- AND FAS-MEDIATED APOPTOSIS IN HEPATOCYTES. Etsuro Hatano, Cynthia A. Bradham, Alexandre Mignon, David A. Brenner, Univ of North Carolina, Chapel Hill, NC; Univ, Paris, France. NFKB-mediated upregulation of iNOS and nitric oxide protect mouse hepatocytes from TNFa- and Fas-mediated apoptosis. Because iNOS is not sufficient for complete protection from TNFa- and Fas-mediated apoptosis, additional NFKB-responsive genes are required. Recent reports have shown that phosphorylation of Bad by PI-3KJAkt abolishes its binding to Bcl-xl and results in Bcl-xl, homodimer formation and anti-apoptotic activity. Our AIMS are to elarify the effect of Bcl-xl, and to investigate NFKB-independent pathways in TNFa- and Fas-mediated apoptosis. METHODS: Primary hepatocytes were isolated from wild type (wt)-mice, Bcl-xl., or Bel-2 transgenic mice. Hepatocytes were treated with TNFaor agonistic anti-Fas antibody (102) with actinomycinD (ActD) or after infection with an adenoviral IKB superrepressor. To block PI3K, p38, or JNK, hepatocytes were treated with LY294002, SB203580, or dominant-negative TAK adenovirus, respectively. Apoptosis was assessed by propidium iodide and trypan blue staining. JNK activity was determined by kinase assay. RESULTS: J02 or TNFainduced massive apoptosis in ActD-sensitized or IKB super-repressor expressing hepatocytes (12± I % of control viability after 17 h). Overexpression of Bcl-xl, suppressed Pas-mediated apoptosis in ActD-sensitized hepatocytes (34±4%) and hepatocytes expressing IKB super-repressor (53 ±6%). In contrast, Bel-xL failed to protect from TNFamediated apoptosis in ActD-sensitized (l.l±l.l%) or Ad5IKB-infected (l.7±0.7%) hepatocytes. Bel-2 overexpression also protected AcID-sensitized hepatocytes from Fas, but not TNFa-, mediated cytotoxicity. Inhibition of PI3K by LY294002 sensitized wt hepatocytes to J02 (27.1 ±2.5%) and, to lesser extent, to TNFa(62.2±4.4%). TNFatreatment induced JNK activation, whereas Fas-induced JNK activation was minimal. However, blockage of JNK by dnTAK increased both TNFa- (27.4%) and Fas(36.8%) mediated cytotoxicity. SB203580 treatment promoted TNFa- and Fas-mediated cytotoxicity (65.3±3.3%, 41.2±4.5%, respectively). CONCLUSION: Bcl-xl, or Bel-2 protects hepatocytes from Fas-, but not TNFa-, mediated apoptosis, suggesting that TNFa- and Fas-signaling pathways are differentially regulated by anti-apoptotic Bel-2 family members in hepatocytes. Thus the Bel-2 family must have other functions than inhibiting the MPT. The PI3K1Akt pathway, an activator of Bel-xL, has a protective role in both TNFa- and Fas-mediated apoptosis. Furthermore, JNK and p38 activation are anti-apoptotic in hepatocytes.
AASLDA895
5 NCX-IOOO, AN NO·DERIVATIVE OF URSODEOXYCHOLIC ACID PROTECTS AGAINST THI-MEDIATED LIVER DAMAGE IN MICE. Fiorucci Stefano, Santucci Luca, Mencarelli Andrea, Oliviai Morell. Piero Del Soldato, Antonio Morelli, Univ of Perugia, Perugia, Italy. Background: Concanavalin A (Con A)-induced hepatitis is an immunomediated disease in which assembly of CD4+ T cells and Thl-like cytokines causes a Fas-mediated liver cell death. The Con A-induced hepatitis is an useful model to test irnmuno-modulatory drugs. Nitric oxide (NO) modulates Thl response in vitro. NCX-looo is an NO derivative of ursodeoxycholic acid (URSO)generated by adding an NO-releasing mojety to the steroid molecule. Aims: to investigate whether NCX-l 000 modulates Thl-like response in Con A-treated mice and protects from acute liver injury induced by this lectin. Methods: Balb/c mice were injected with 0.3 mg/mouse Con A alone or in combination with NCX-looo (5, 15 or 30 mg/kg) or equimolar doses of URSO. To assess whether protection was maintained in established disease both drugs (15 mg/kg) were administered 2,4 and 8 hours after Con A. Results. NCX-looo, but not URSO, caused a dose-dependent protection against liver damage induced by Con A. At the dose of 30 and 15/mg. NCX-looo caused a =80% reduction of aminotransferase plasma levels (AST levels were: 121.4 ± 11 in control mice; 2456.3 ± 234 in mice treated with Con A and 78.4 ± 21 UIIL in mice treated with Con a in combination with NCX-IOOO 15/mg/kg. P>O.OOI versus Con A). and 40-805 reduction of IL-lf3. IL-12. IL-18, IFNy and TNFa plasma concentrations without affecting liver cytokine mRNA expression. NCX-lOOO administration completely protected from liver damage induced by Con A as assessed by liver histology and prevented Fas, Fas ligand (L) and IL-2 receptor upregulation on spleen lymphocytes and Fas L expression on hepatocyte. NCX-lOOO but not URSO inhibited IL-lf3, IL-12. IL-18 and IFNyrelease from Con A-stimulated spleen lymphocytes. Con A administration resulted in a time-dependent increase in caspase 3, 8 and 9 activity in liver tissues. NCX-looo, but not URSO, prevented caspase 3, 8 and 9 activation induced by Con A. The inhibition of caspase-3, 8 and 9 by NCX-looo was reversible by the addition of DTT. which is consistent with S-nitrosylation as the mechanism of caspase inhibition. In contrast to URSO. NCX-lOOO administered 2 hours after Con A was still able to protect against liver damage induced by Con A. Conclusions. Thl-like response induced by Con A results in activates a caspase cascade in the liver. NCX-looo an NO derivative of ursodeoxycholic acid causes the S-nitrosylationlinhibition of caspases involved in apoptosis and inhibit Thl-like response induced by Con A injection.
6 HETEROZYGOSITY FOR MITOCHONDRIAL TRIFUNCTIONAL PROTEIN DEFECT CAUSES HEPATIC STEATOSIS AND ELEVATED LIVER ENZYMES IN MICE. Jamal A. Ibdah, Hyacinth Paul. Yiwen Zhao, Scott Binford, Ken Salleng, Mark Cline. Wake Forest Univ Sch of Medicine. Winston-Salem. NC. Background: Little is known about the potential role of recessively inherited fatty acid oxidation disorders in development of fatty liver disease in the adult carriers. Affected children develop micro- and macro-vesicular steatosis associated with hepatic. cardiac, and muscular abnormalities often leading to death if untreated. Mitochondrial trifunctional protein (TFP) is a hetero-octamer of 4 a and 4 f3 subunits that catalyze the final 3 steps of long chain fatty acid oxidation. Heterozygosity for a TFP mutation is :f 1% in the general population. Hypothesis: Heterozygosity for TFP predisposes to development of fatty liver disease. Methods: We used gene targeting replacement strategy to generate a-subunit null allele. Routine histology and Oil 0 Red stain were used to assess hepatic changes in 6-month old +/- and +1+ littermates fed regular chow or 3-week 35% calorie fat diet with high content of long chain saturated fatty acids. Histological changes were correlated with serum ALT levels. Results: -/- mice were TFP deficient by enzymatic assays and lack both a and f3 subunits by Western blot analysis. All -/- mice suffered an early sudden death within 6-36 hours after birth with significant micro- and macro-vesicular fatty infiltration of the liver and acute degenerative changes in the muscle and heart. Table I lists the extent of hepatic steatosis and ALT levels in +/- and +1+ mice fed regular chow or high fat diet. +/- mice developed mild hepatic steatosis and elevated ALT on regular chow diet. High fat diet caused extensive hepatic steatosis without necrosis and significantly higher ALT levels in +/- mice compared to +/+ mice. Conelusions: Heterozygosity for TFP mutations can be important in development of fatty liver disease particularly under oxidative stress. Table 1 Diet
Genotype
Regular Chow
+/+(n:6) +/-(n=61 +/+ (n=6) +/-(n=6)
High Fat
a:0= nolipidosis, 4= extensive lipidosis b:P= 0.052 c: P= 0.016
AlTlUlL) b
51±22 79±27b 107±37' 225+93'
Fat Stain Score'
0 1 2 3.5
is mediated through the Bile acid-responsive element (IR-1) via FXRlRXR subsequent to their binding by bile acids.
3 ATYPICAL PROTEIN KINASE C ISOFORM A (PKC-Z), BUT NOT AKT KINASE MEDIATES TAUROCHENODEOXYCHOLATE-ACTIVATED SURVIVAL SIGNALS. Christian Rust, Carlos V. Paya, Jorge Moscat, Larry M. Karnitz, Gregory J. Gores, Mayo Clin, Rochester, MN; Ctr de Biologia Molecular, Madrid, Spain. Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile salts. We have recently demonstrated that the hydrophobic yet nontoxic bile salt taurochenodeoxycholate (TCDC) activates a PI 3-kinaseINF-KB dependent survival signal, preventing its inherent toxicity. PI 3-kinase activates NF-KB indirectly either through Akt kinase or atypical protein kinase c isoforms such as ( (PKC-0. Thus, the AIMS of our study were to test the HYPOTHESIS that TCDC-induced PI 3-kinase-dependent activation of NF-KB is mediated by Akt and/or PKC-{ METHODS: We employed the McNtcp.24 rat hepatoma cell line which is stably transfected with a bile salt transporter and undergoes bile saltinduced apoptosis. Apoptosis was quantitated using the nueleic acid binding dye DAPI and fluorescent microscopy. Akt activation was assessed using a specific antibody against the phosphorylated form of Akt and immunoblot analysis. PKC-( was immunoprecipitated from cell Iysates and its activity measured using the substrate myelin basic protein and [y_32p]_ATP. Transfections with dominant negative and wildtype PKC-( were used to alter PKC-( activity. NF-KB transcriptional activity was assessed by a luciferase reporter gene assay. RESULTS: Akt was not activated by TCDC. In contrast, PKC-( activity was increased by more than 100% after stimulation with 200 pM TCDC. Furthermore, PKC-( activation was blocked by the PI 3-kinase inhibitor wortmannin (250 nM), suggesting that PKC-( is downstream of PI 3-kinase. In cells transfected with wildtype PKC-(, TCDC-induced NF-KB transcriptional activity was more than doubled as compared to unstimulated controls. This TCDCinduced activation was blocked in cells transfected with the dominant negative PKC-(. These data suggest that PKC-( can activate NF-KB in hepatocytes. Although transfection of McNtcp.24 cells with dominant negative PKC-( did not induce significant apoptosis by itself, TCDC (50 f.LM) increased apoptosis 4-fold above control in cells expressing this plasmid. These data demonstrate the importance of PKC-( in TCDCmediated survival signals. CONCLUSIONS: Collectively, these results indicate that PKC-(, but not Akt, is mediating TCDC-induced survival signaling downstream of PI 3-kinase and upstream of NF-KB. PKC-( appears to be a key kinase mediating survival signals in hepatocytes. Activation of this signaling cascade could prove useful in decreasing the proapoptotic effects of toxic bile salts in cholestasis.
4 PROTECTIVE PATHWAYS IN TNFA- AND FAS-MEDIATED APOPTOSIS IN HEPATOCYTES. Etsuro Hatano, Cynthia A. Bradham, Alexandre Mignon, David A. Brenner, Univ of North Carolina, Chapel Hill, NC; Univ, Paris, France. NFKB-mediated upregulation of iNOS and nitric oxide protect mouse hepatocytes from TNFa- and Fas-mediated apoptosis. Because iNOS is not sufficient for complete protection from TNFa- and Fas-mediated apoptosis, additional NFKB-responsive genes are required. Recent reports have shown that phosphorylation of Bad by PI-3KJAkt abolishes its binding to Bcl-xl and results in Bcl-xl, homodimer formation and anti-apoptotic activity. Our AIMS are to elarify the effect of Bcl-xl, and to investigate NFKB-independent pathways in TNFa- and Fas-mediated apoptosis. METHODS: Primary hepatocytes were isolated from wild type (wt)-mice, Bcl-xl., or Bel-2 transgenic mice. Hepatocytes were treated with TNFaor agonistic anti-Fas antibody (102) with actinomycinD (ActD) or after infection with an adenoviral IKB superrepressor. To block PI3K, p38, or JNK, hepatocytes were treated with LY294002, SB203580, or dominant-negative TAK adenovirus, respectively. Apoptosis was assessed by propidium iodide and trypan blue staining. JNK activity was determined by kinase assay. RESULTS: J02 or TNFainduced massive apoptosis in ActD-sensitized or IKB super-repressor expressing hepatocytes (12± I % of control viability after 17 h). Overexpression of Bcl-xl, suppressed Pas-mediated apoptosis in ActD-sensitized hepatocytes (34±4%) and hepatocytes expressing IKB super-repressor (53 ±6%). In contrast, Bel-xL failed to protect from TNFamediated apoptosis in ActD-sensitized (l.l±l.l%) or Ad5IKB-infected (l.7±0.7%) hepatocytes. Bel-2 overexpression also protected AcID-sensitized hepatocytes from Fas, but not TNFa-, mediated cytotoxicity. Inhibition of PI3K by LY294002 sensitized wt hepatocytes to J02 (27.1 ±2.5%) and, to lesser extent, to TNFa(62.2±4.4%). TNFatreatment induced JNK activation, whereas Fas-induced JNK activation was minimal. However, blockage of JNK by dnTAK increased both TNFa- (27.4%) and Fas(36.8%) mediated cytotoxicity. SB203580 treatment promoted TNFa- and Fas-mediated cytotoxicity (65.3±3.3%, 41.2±4.5%, respectively). CONCLUSION: Bcl-xl, or Bel-2 protects hepatocytes from Fas-, but not TNFa-, mediated apoptosis, suggesting that TNFa- and Fas-signaling pathways are differentially regulated by anti-apoptotic Bel-2 family members in hepatocytes. Thus the Bel-2 family must have other functions than inhibiting the MPT. The PI3K1Akt pathway, an activator of Bel-xL, has a protective role in both TNFa- and Fas-mediated apoptosis. Furthermore, JNK and p38 activation are anti-apoptotic in hepatocytes.
AASLDA895
5 NCX-IOOO, AN NO·DERIVATIVE OF URSODEOXYCHOLIC ACID PROTECTS AGAINST THI-MEDIATED LIVER DAMAGE IN MICE. Fiorucci Stefano, Santucci Luca, Mencarelli Andrea, Oliviai Morell. Piero Del Soldato, Antonio Morelli, Univ of Perugia, Perugia, Italy. Background: Concanavalin A (Con A)-induced hepatitis is an immunomediated disease in which assembly of CD4+ T cells and Thl-like cytokines causes a Fas-mediated liver cell death. The Con A-induced hepatitis is an useful model to test irnmuno-modulatory drugs. Nitric oxide (NO) modulates Thl response in vitro. NCX-looo is an NO derivative of ursodeoxycholic acid (URSO)generated by adding an NO-releasing mojety to the steroid molecule. Aims: to investigate whether NCX-l 000 modulates Thl-like response in Con A-treated mice and protects from acute liver injury induced by this lectin. Methods: Balb/c mice were injected with 0.3 mg/mouse Con A alone or in combination with NCX-looo (5, 15 or 30 mg/kg) or equimolar doses of URSO. To assess whether protection was maintained in established disease both drugs (15 mg/kg) were administered 2,4 and 8 hours after Con A. Results. NCX-looo, but not URSO, caused a dose-dependent protection against liver damage induced by Con A. At the dose of 30 and 15/mg. NCX-looo caused a =80% reduction of aminotransferase plasma levels (AST levels were: 121.4 ± 11 in control mice; 2456.3 ± 234 in mice treated with Con A and 78.4 ± 21 UIIL in mice treated with Con a in combination with NCX-IOOO 15/mg/kg. P>O.OOI versus Con A). and 40-805 reduction of IL-lf3. IL-12. IL-18, IFNy and TNFa plasma concentrations without affecting liver cytokine mRNA expression. NCX-lOOO administration completely protected from liver damage induced by Con A as assessed by liver histology and prevented Fas, Fas ligand (L) and IL-2 receptor upregulation on spleen lymphocytes and Fas L expression on hepatocyte. NCX-lOOO but not URSO inhibited IL-lf3, IL-12. IL-18 and IFNyrelease from Con A-stimulated spleen lymphocytes. Con A administration resulted in a time-dependent increase in caspase 3, 8 and 9 activity in liver tissues. NCX-looo, but not URSO, prevented caspase 3, 8 and 9 activation induced by Con A. The inhibition of caspase-3, 8 and 9 by NCX-looo was reversible by the addition of DTT. which is consistent with S-nitrosylation as the mechanism of caspase inhibition. In contrast to URSO. NCX-lOOO administered 2 hours after Con A was still able to protect against liver damage induced by Con A. Conclusions. Thl-like response induced by Con A results in activates a caspase cascade in the liver. NCX-looo an NO derivative of ursodeoxycholic acid causes the S-nitrosylationlinhibition of caspases involved in apoptosis and inhibit Thl-like response induced by Con A injection.
6 HETEROZYGOSITY FOR MITOCHONDRIAL TRIFUNCTIONAL PROTEIN DEFECT CAUSES HEPATIC STEATOSIS AND ELEVATED LIVER ENZYMES IN MICE. Jamal A. Ibdah, Hyacinth Paul. Yiwen Zhao, Scott Binford, Ken Salleng, Mark Cline. Wake Forest Univ Sch of Medicine. Winston-Salem. NC. Background: Little is known about the potential role of recessively inherited fatty acid oxidation disorders in development of fatty liver disease in the adult carriers. Affected children develop micro- and macro-vesicular steatosis associated with hepatic. cardiac, and muscular abnormalities often leading to death if untreated. Mitochondrial trifunctional protein (TFP) is a hetero-octamer of 4 a and 4 f3 subunits that catalyze the final 3 steps of long chain fatty acid oxidation. Heterozygosity for a TFP mutation is :f 1% in the general population. Hypothesis: Heterozygosity for TFP predisposes to development of fatty liver disease. Methods: We used gene targeting replacement strategy to generate a-subunit null allele. Routine histology and Oil 0 Red stain were used to assess hepatic changes in 6-month old +/- and +1+ littermates fed regular chow or 3-week 35% calorie fat diet with high content of long chain saturated fatty acids. Histological changes were correlated with serum ALT levels. Results: -/- mice were TFP deficient by enzymatic assays and lack both a and f3 subunits by Western blot analysis. All -/- mice suffered an early sudden death within 6-36 hours after birth with significant micro- and macro-vesicular fatty infiltration of the liver and acute degenerative changes in the muscle and heart. Table I lists the extent of hepatic steatosis and ALT levels in +/- and +1+ mice fed regular chow or high fat diet. +/- mice developed mild hepatic steatosis and elevated ALT on regular chow diet. High fat diet caused extensive hepatic steatosis without necrosis and significantly higher ALT levels in +/- mice compared to +/+ mice. Conelusions: Heterozygosity for TFP mutations can be important in development of fatty liver disease particularly under oxidative stress. Table 1 Diet
Genotype
Regular Chow
+/+(n:6) +/-(n=61 +/+ (n=6) +/-(n=6)
High Fat
a:0= nolipidosis, 4= extensive lipidosis b:P= 0.052 c: P= 0.016
AlTlUlL) b
51±22 79±27b 107±37' 225+93'
Fat Stain Score'
0 1 2 3.5