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121 ELF is a key adaptor for TGF-beta signaling, liver development and hepatocellular cancer
214A
AASLD ABSTRACTS
HEPATOLOGY, October 2003
120 NEUROLOGICAL CHARACTERIZATION OF A NOVEL MOUSE MODEL OF ACUTE LIVER FAILURE. Mireille
121
B~langer, Paul Desjardins, Roger F Butterworth, CHUM-H6pital Saint-Luc, Montreal, PQ, Canada
Yi Tang, Varalakshmi Katuri, Allan Dillner, Stuart Danovitch, Bibhuh" Mishra, Lopa Mishra, Laboratory of GI Developmental Biology, Fels Insh'tute for Cancer Research and Molecular Biology, Temple University, Philadelphia, DVAMC & Department of Medicine, Georgetown University, Washington, DC
Molecular biological approaches continue to lead to the identification of alterations in expression of genes coding for key central nervous system proteins involved in water homeostasis, energy metabolism and neurotransmitter regulation in acute liver failure (ALF). However, studies of the pathophysiological consequences of these changes in gene expression is i m p e d e d by the lack of a suitable mouse model of ALF. A recent report described a model of ALF resulting from the administration of the hepatotoxin azoxymethane (AOM), an active ingredient of the cycad palm nut, in mice (Matkowskyj et al., 1999). In a series of experiments to further assess this treatment as an effective model of ALF, the effects of treatment of male C57BI mice (25-30 g) with AOM (0.1 mg/g, i.p.) on hepatic and cerebral function were studied. With maintenance of body temperature at 37 °C and control of hypoglycemia, mice developed signs of encephalopathy (decreased locomotor activity followed by loss of righting and corneal reflexes) within 16 h of AOM treatment. Liver pathology consisted of steatosis, ballooning of centrolobular hepatocytes, the appearance of apoptotic bodies followed by severe centrolobular necrosis. AOM-treated mice were h y p e r a m m o n e m i c and developed spontaneous hypothermia and brain edema. Brain ammonia concentrations were increased to the 2.0 - 2.5 m M range compared to vehicle-treated littermate controls and brain water concentrations m e a s u r e d by the gravimetric procedure were significantly increased (AOM-treated : 82.54 ± 0.40 %; vehicle-treated : 81.28 ± 0.26 %, p < 0.01). Brain amino acid profiles determined by HPLC with fluorescence detection were typical of ALF in other species including h u m a n s (glutamine increased by 73% (p < 0.01), alanine increased by 48% (p < 0.01), histidine increased by 43% (p < 0.01) with concomitant decreases of brain glutamate and taurine). RTPCR and Western blot analyses showed significant increases in expression of the "peripheral-type" benzodiazepine receptor and of the astrocytic glucose transporter GLUT-1 in brains of AOMtreated mice; immunohistochemical evaluation using a monoclohal antibody to GLUT-1 revealed increased immunostaining of perivascular endothelial cells. Mild hypothermia (35°C) led to significant attenuation of brain ammonia, alanine and lactate concentrations and of GLUT-1 expression. These findings suggest that AOM treatment affords a simple, reproducible mouse model of ALF which is suitable for the study of the effects of gene manipulation (" knockdown" strategies) on the pathogenesis of brain e d e m a and hepatic encephalopathy in ALF. (Funded by CIHR and the Canadian Liver Foundation) Disclosures: Mireille B61anger - No relationships to disclose Roger F Butterworth - No relationships to disclose Paul Desjardins - No relationships to disclose
ELF IS A KEY ADAPTOR FOR TGF-BETA SIGNALING,
LIVER DEVELOPMENT AND HEPATOCELLULAR CANCER.
Background: TGF-/3 modulates angiogenesis, organogenesis, and promotes carcinogenesis. Signaling is mediated by Smad proteins and cytoplasmic adaptors. We have shown that disruption of the adaptor protein ELF, disrupts TGF-/3 signaling in mice (Science2002, in press). The e/f 4- mutant phenotype, is similar to that seen in smad2+~/ smad3 +/ mutants with defective liver formation. Also ELF interacts with Smad4 a gastrointestinal tumor suppressor. In addition, a major fi~Tcfion of spectrins is in organelle formation and to confer apicobasal polarity. Aims: To test: 1. disrupted angiogenesis and liver formation in e/f-/- mutants, 2. ELF association with Smadl or Smad5 3. If loss of ELF results in hepatocellular carcinogenesis. 4. To determine cell polarization abnormalities in e/f -/- mutants. Methods and Results: 1. Immunoblot and Immunohistochemical labeling of wild type and e/f mutant embryonic tissue showed reduced expression of ilk-1 and p e cam in e/f--/ embryonic liver. Reduced expression of a-fetoprotein and cytokeratin in e/f -/- embryonic liver along with abnormal hepatocytes and bile ducts indicated arrested differentiation and growth, resulting in liver hypoplasia. 2. Smadl and Smad5 expression were similar in the e/f -/- mutants to controls. There was no association between ELF and Smadl or Smad5.3. Follow up of 18 e/f +/ mutant mice for 12 months revealed multiple foci of hepatocyte dysplasia with 2 hepatocellular carcinomas and I renal cell carcinoma with none in the controls. Albumin and cY-fetoprotein expression increased in liver tissues of e/f +/ adult mice, especially with ~mors. 4.We next examined for abnormalities in gut epithelial cell morphology in the e/f -/-mutant embryos. Visualization of plasma membrane, Golgi and nudei by transfecfion of pEYFP-Mem, pECFP-Golgi and pEYFP-Nuc (Clontech) into e/f4-mutant fibroblasts, revealed all three to be normal. However, a marked distortion in Na,K ATPase, MAP-2 (Microtubule Associated Protein) and Actin with increased Vimentin, decreased E-cadherin, but normal Ankyrin B and G expression were observed in the e/f -/- gut epithelial cells: Na, K ATPase appeared irregular and punctate intracellularly, and absent at the plasma membrane in the e/f -/- cells. Condusions: 1. These studies support a role for ELF in liver organogenesis. 2. ELF involvement in angiogenesis may occur through the ALK5-Smad3/4 pathway 3. These results indicate that ELF Spectrins may behave as a ~ r n o r suppressor through TGF-/3 signaling in mice.
Conclusions: These results provide new and unexpected insights into both TGF-/3 signaling as well an essential role for the adaptor proteins of the /3-Spectrin gene family in this signaling process. Our intercross studies indicate that TGF-/3 signaling through ELF is important in suppression against hepatocellular cancer. Our studies also indicate a role for ELF in conferring specific aspects of cell polarization. Disclosures: Stuart Danovitch - No relationships to disclose Allan Dillner - No relationships to disclose Varalakshmi Katuri - No relationships to disclose Bibhuti Mishra - No relationships to disclose Lopa Mishra - No relationships to disclose Yi Tang - No relationships to disclose
AASLD ABSTRACTS
HEPATOLOGY, October 2003
120 NEUROLOGICAL CHARACTERIZATION OF A NOVEL MOUSE MODEL OF ACUTE LIVER FAILURE. Mireille
121
B~langer, Paul Desjardins, Roger F Butterworth, CHUM-H6pital Saint-Luc, Montreal, PQ, Canada
Yi Tang, Varalakshmi Katuri, Allan Dillner, Stuart Danovitch, Bibhuh" Mishra, Lopa Mishra, Laboratory of GI Developmental Biology, Fels Insh'tute for Cancer Research and Molecular Biology, Temple University, Philadelphia, DVAMC & Department of Medicine, Georgetown University, Washington, DC
Molecular biological approaches continue to lead to the identification of alterations in expression of genes coding for key central nervous system proteins involved in water homeostasis, energy metabolism and neurotransmitter regulation in acute liver failure (ALF). However, studies of the pathophysiological consequences of these changes in gene expression is i m p e d e d by the lack of a suitable mouse model of ALF. A recent report described a model of ALF resulting from the administration of the hepatotoxin azoxymethane (AOM), an active ingredient of the cycad palm nut, in mice (Matkowskyj et al., 1999). In a series of experiments to further assess this treatment as an effective model of ALF, the effects of treatment of male C57BI mice (25-30 g) with AOM (0.1 mg/g, i.p.) on hepatic and cerebral function were studied. With maintenance of body temperature at 37 °C and control of hypoglycemia, mice developed signs of encephalopathy (decreased locomotor activity followed by loss of righting and corneal reflexes) within 16 h of AOM treatment. Liver pathology consisted of steatosis, ballooning of centrolobular hepatocytes, the appearance of apoptotic bodies followed by severe centrolobular necrosis. AOM-treated mice were h y p e r a m m o n e m i c and developed spontaneous hypothermia and brain edema. Brain ammonia concentrations were increased to the 2.0 - 2.5 m M range compared to vehicle-treated littermate controls and brain water concentrations m e a s u r e d by the gravimetric procedure were significantly increased (AOM-treated : 82.54 ± 0.40 %; vehicle-treated : 81.28 ± 0.26 %, p < 0.01). Brain amino acid profiles determined by HPLC with fluorescence detection were typical of ALF in other species including h u m a n s (glutamine increased by 73% (p < 0.01), alanine increased by 48% (p < 0.01), histidine increased by 43% (p < 0.01) with concomitant decreases of brain glutamate and taurine). RTPCR and Western blot analyses showed significant increases in expression of the "peripheral-type" benzodiazepine receptor and of the astrocytic glucose transporter GLUT-1 in brains of AOMtreated mice; immunohistochemical evaluation using a monoclohal antibody to GLUT-1 revealed increased immunostaining of perivascular endothelial cells. Mild hypothermia (35°C) led to significant attenuation of brain ammonia, alanine and lactate concentrations and of GLUT-1 expression. These findings suggest that AOM treatment affords a simple, reproducible mouse model of ALF which is suitable for the study of the effects of gene manipulation (" knockdown" strategies) on the pathogenesis of brain e d e m a and hepatic encephalopathy in ALF. (Funded by CIHR and the Canadian Liver Foundation) Disclosures: Mireille B61anger - No relationships to disclose Roger F Butterworth - No relationships to disclose Paul Desjardins - No relationships to disclose
ELF IS A KEY ADAPTOR FOR TGF-BETA SIGNALING,
LIVER DEVELOPMENT AND HEPATOCELLULAR CANCER.
Background: TGF-/3 modulates angiogenesis, organogenesis, and promotes carcinogenesis. Signaling is mediated by Smad proteins and cytoplasmic adaptors. We have shown that disruption of the adaptor protein ELF, disrupts TGF-/3 signaling in mice (Science2002, in press). The e/f 4- mutant phenotype, is similar to that seen in smad2+~/ smad3 +/ mutants with defective liver formation. Also ELF interacts with Smad4 a gastrointestinal tumor suppressor. In addition, a major fi~Tcfion of spectrins is in organelle formation and to confer apicobasal polarity. Aims: To test: 1. disrupted angiogenesis and liver formation in e/f-/- mutants, 2. ELF association with Smadl or Smad5 3. If loss of ELF results in hepatocellular carcinogenesis. 4. To determine cell polarization abnormalities in e/f -/- mutants. Methods and Results: 1. Immunoblot and Immunohistochemical labeling of wild type and e/f mutant embryonic tissue showed reduced expression of ilk-1 and p e cam in e/f--/ embryonic liver. Reduced expression of a-fetoprotein and cytokeratin in e/f -/- embryonic liver along with abnormal hepatocytes and bile ducts indicated arrested differentiation and growth, resulting in liver hypoplasia. 2. Smadl and Smad5 expression were similar in the e/f -/- mutants to controls. There was no association between ELF and Smadl or Smad5.3. Follow up of 18 e/f +/ mutant mice for 12 months revealed multiple foci of hepatocyte dysplasia with 2 hepatocellular carcinomas and I renal cell carcinoma with none in the controls. Albumin and cY-fetoprotein expression increased in liver tissues of e/f +/ adult mice, especially with ~mors. 4.We next examined for abnormalities in gut epithelial cell morphology in the e/f -/-mutant embryos. Visualization of plasma membrane, Golgi and nudei by transfecfion of pEYFP-Mem, pECFP-Golgi and pEYFP-Nuc (Clontech) into e/f4-mutant fibroblasts, revealed all three to be normal. However, a marked distortion in Na,K ATPase, MAP-2 (Microtubule Associated Protein) and Actin with increased Vimentin, decreased E-cadherin, but normal Ankyrin B and G expression were observed in the e/f -/- gut epithelial cells: Na, K ATPase appeared irregular and punctate intracellularly, and absent at the plasma membrane in the e/f -/- cells. Condusions: 1. These studies support a role for ELF in liver organogenesis. 2. ELF involvement in angiogenesis may occur through the ALK5-Smad3/4 pathway 3. These results indicate that ELF Spectrins may behave as a ~ r n o r suppressor through TGF-/3 signaling in mice.
Conclusions: These results provide new and unexpected insights into both TGF-/3 signaling as well an essential role for the adaptor proteins of the /3-Spectrin gene family in this signaling process. Our intercross studies indicate that TGF-/3 signaling through ELF is important in suppression against hepatocellular cancer. Our studies also indicate a role for ELF in conferring specific aspects of cell polarization. Disclosures: Stuart Danovitch - No relationships to disclose Allan Dillner - No relationships to disclose Varalakshmi Katuri - No relationships to disclose Bibhuti Mishra - No relationships to disclose Lopa Mishra - No relationships to disclose Yi Tang - No relationships to disclose