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Induction of hepatic apolipoprotein B mRNA editing for reducing serum cholesterol levels: A breakthrough or a disaster
HEPATOLOGY Elsewhere T. JAKE LIANG, EDITOR
ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Liver Diseases Section NIH/NIDDK, Building 10-9 B16 10 Center Dr Bethesda, MD 20892-1800
INDUCTION OF HEPATIC APOLIPOPROTEIN B mRNA EDITING FOR REDUCING SERUM CHOLESTEROL LEVELS: A BREAKTHROUGH OR A DISASTER?
Yamanaka S, Balestra ME, Ferrell LD, Fan J, Arnold KS, Taylor S, Taylor JM, et al. Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals. Proc Natl Acad Sci U S A 1995;92:84838487. ABSTRACT
Apolipoprotein (apo-) B messenger RNA (mRNA) editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo B (apo-B48). The cytidine deaminase catalytic subunit (apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)) of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower lowdensity lipoprotein cholesterol concentrations. The apoB mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and lowdensity lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and one transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to those of apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in the livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed, editing enzyme, and suggests that aberrant editing of hepatic mRNAs is involved in cell growth and that regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower the plasma levels of low-density lipoproteins. COMMENTS
Apolipoprotein (apo) B, the essential core protein of all lipoproteins except the high-density lipoproteins, exists in two forms: the full-length protein, apo B-100 (512 kd), and the carboxy terminal truncated apo B-48 (248 kd).1 The human liver secretes triglyceride-rich), very-low-density lipoproteins (VLDL). Each VLDL particle contains one molecule of apo B-100.1,2 After triglyceride hydrolysis by lipoprotein lipases, most of the VLDL-remnants are cleared within minutes by the liver, yet some VLDL particles are transformed by incompletely understood processes into low-density lipoproteins (LDL).2 LDL are enriched in cholesterol-esters, have a plasma half-life of about 1 day, and, if elevated, represent an important cause of development of atherosclerosis and
coronary artery disease.2 The intestine, the second major source of triglyceride-rich lipoproteins, secretes chylomicrons, which contain apo B-48 as the core protein.2 Unlike VLDL, chylomicrons are rapidly and completely cleared by the liver after triglyceride hydrolysis, without giving rise to long-lived apo B–containing lipoproteins.2 The synthesis of apo B-48 is the result of mRNA editing, which consists of a specific base change from C to U in the apo B mRNA, creating a premature stop codon.3 The editing is mediated by the apo B–mRNA editing enzyme, which specifically deaminates the cytidine residue at nucleotide 6666 during the processing of apo B pre-mRNA.4 The editing enzyme is a complex of several subunits. The catalytic subunit, APOBEC-1 (apo B–mRNA editing enzyme catalytic polypeptide 1,)5 is a member of the cytidine deaminases gene family and contains a novel RNA binding motif, but requires additional ‘‘auxiliary’’ factors for expressing the apo B–mRNA editing reaction.6 Extracts from human, baboon, or rabbit liver that do not exhibit the editing activity can nonetheless supplement APOBEC-1 to reconstitute apo B–mRNA editing in vitro, suggesting that the livers of these species contain the additional ‘‘auxiliary’’ components of the apo B–mRNA editing enzyme complex.7 Almost 100% of the apo B mRNA is edited in the intestine of all mammalian species that are studied.8 APOBEC-1 is absent in rabbit, baboon, or human liver,5,7 and consequently, the liver of these species secrete VLDL containing the full-length apo B-100.8 In horses, rats, mice, and dogs, however, hepatic apo B mRNA is also substantially edited.8 These species have low plasma LDL levels,4 probably because their livers secrete apo B-48–containing VLDL, which are turned over much faster than apo B-100–containing lipoproteins.9 In mice10 and rats (Greeve et al., manuscript in preparation), two different forms of APOBEC-1 are expressed in the liver and in the intestine as a result of differential promoter usage and of alternative splicing. In these species, hepatic apo B–mRNA editing appears to be a genetic mechanism to regulate the metabolism and to modulate the plasma concentrations of the atherogenic apo B-100–containing LDL. The mechanism of the lack of APOBEC-1 expression in the liver of species, such as in the livers of humans and rabbits, remains to be uncovered. By establishing APOBEC-1 transgenic rabbits, Yamanaka et al. have directly addressed the question of how the induction of apo B–mRNA editing in the liver of a species without natural hepatic editing influences plasma LDL levels. The generation of transgenic rabbits required major refinements of the transgene technology. Simultaneously, Yamanaka et al. also generated transgenic mice that expressed active rabbit APOBEC-1 or a mutant, enzymatically inactive form of the protein. To achieve liver-specific expression of the APOBEC-1 transgene, they used a vector containing a promoter, an intron, and a hepatic control-region of the human apolipoprotein E gene. Four independent transgenic mouse lines were established from three transgenic mouse founders that expressed wild-type rabbit APOBEC-1 with estimated transgene copy numbers of 3, 7, 10, and 17, respectively. In
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addition, 2 transgenic rabbit founders were established with 17 copies and 1 copy of the APOBEC-1 transgene, respectively. In the transgenic mouse lines, APOBEC-1 was highly expressed in the liver and was accompanied by an increase in hepatic apo B–mRNA editing to an average of 92%, as compared with an average of 83% in the nontransgenic mice and 82% in the mice transgenic for the mutant inactive APOBEC-1. Cytosolic extracts from the liver of the transgenic mice were 15-fold more active in editing a synthetic apo B mRNA in vitro than extracts from matched control rats, further demonstrating that APOBEC-1 was highly overexpressed in the liver. In the transgenic rabbit founder with 17 copies of the transgene, APOBEC-1 was highly expressed in the liver, and 78% of the hepatic apo B mRNA was found to be edited, whereas, in normal rabbits, editing of the hepatic apo B mRNA is not detectable. APOBEC-1 expression and hepatic apo B–RNA editing were not measured in the liver of the rabbit founder that had only 1 copy of the transgene. LDL was undetectable in the plasma of the APOBEC-1 transgenic mice. Apo B-100 was absent in these mice and apo B-48 was the only apolipoprotein found in the plasma. Initial observations in the APOBEC-1 transgenic rabbit that had 1 copy of the transgene showed a 50% reduction of plasma LDL and a small increase in HDL compared with the levels found in the nontransgenic littermates. The most striking observation of this study was the marked enlargement of the livers with modest to severe hepatic dysplasia in all of the transgenic mouse lines, and the occurrence of hepatocellular carcinoma (HCC) in some. One of four mice with 3 copies of the transgene, one of 20 mice with a copy number of 7, 4 of 5 mice with a copy number of 10, and 2 of 5 mice with a copy number of 17 all developed full-blown HCC by histological criteria. In contrast, none of the 8 transgenic mice that expressed the mutant inactive APOBEC-1 developed severe hepatic dysplasia or HCC. The rabbit founder with a copy number of 17 grew slowly and was euthanized at the age of 8 weeks because of progressive weakness. Necropsy revealed an enlarged liver with fibrosis and histologically distorted architecture with intracytoplasmic lipid droplets; however, severe hepatic dysplasia or HCC was not found. The occurrence of hepatic dysplasia and HCC in all APOBEC-1 transgenic mouse lines and in the absence of dysplasia in mice transgenic for the mutant inactive APOBEC-1 make it very unlikely that the site of transgene insertion is related to the observed effects on hepatocyte proliferation and differentiation. Moreover, the authors do not believe that a lack of the full-length protein apo B-100 caused the observed hepatic abnormalities, because, in ‘‘apo B-48 only mice,’’ generated by gene-targeting in embryonic stem cells, no such effect has been observed. Instead, the authors speculate that APOBEC-1 can act as an oncogene leading to HCC by editing of mRNAs other than apo B. Indeed, in the transgenic mice, they identified such aberrant C-to-U editing at codon 411 of the mouse tyrosine kinase gene. This nucleotide change, however, is not predicted to change the amino acid sequence as both CUG and UUG code for leucine. Is the observation of HCC in APOBEC-1 transgenic animals the death-blow to the idea that the induction of hepatic apo B–mRNA editing, possibly by gene transfer of APOBEC1, could be a genetic approach to the treatment of hypercholesterolemia? By generating the APOBEC-1 transgenic mice and rabbits, the authors have provided compelling evidence that the expression of apo B–mRNA editing in the liver results in a reduction, or even with the absence of plasma LDL. This conclusion is further supported by recent studies using APOBEC-1 recombinant adenoviruses to induce hepatic apo B–mRNA editing. Both in mice and rabbits, the injection of APOBEC-1 recombinant adenoviruses resulted in the substantial reduction of plasma LDL levels. In the LDL-receptor–deficient Watanabe heritable hyperlipidemic rabbit, we
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achieved a 40% reduction of the high plasma concentrations of LDL by this approach.11 The development of HCC in APOBEC-1 transgenic animals severely compromises these promising findings. However, it should be noted that rats, mice, dogs, and horses express APOBEC-1 in the liver but are not known to be excessively susceptible to the development of HCC. Thus, in general, the hepatic expression of APOBEC1 is not oncogenic, although the type of expression achieved in this study resulted in HCC. What could explain this discrepancy? First, the complementary DNA (cDNA) for rabbit APOBEC-1 was introduced into the germ-line of mice. The comparison of the cDNA sequence from rat, mice, rabbit, baboon, and human has revealed a great deal of interspecies sequence divergence.5,7 The sequence-specificity of the editing function of APOBEC-1 most likely requires the ‘‘auxiliary’’ regulatory factors present in the editing enzyme complex, which has been termed the editosome. By introducing rabbit APOBEC-1 in mice, this interaction could be impaired, resulting in the observed loss of editing specificity. It should be pointed out that the (rabbit) APOBEC-1 transgenic rabbits had derangement of the liver structure, but did not develop severe hepatocyte dysplasia or HCC. Importantly, the rabbit founder with one transgene copy had no obvious hepatic abnormality. Establishing a rabbit line from this particular rabbit founder will be helpful in further evaluating the oncogenic potential of APOBEC-1. Second, in the human intestine and in the rat liver, the expression of APOBEC-1 is upregulated immediately before birth and correlates with the editing of apo B mRNA.12 The heterologous promoter of apo E, which has important functions in the embryonic lipid metabolism, may lead to APOBEC-1 expression in the early embryonic liver possibly without the accompanying expression of the ‘‘auxiliary’’ components of the editosome, which are responsible for the normal ‘‘quenching’’ of the nonspecific editing capacity of APOBEC-1. Aberrant mRNA editing of genes that are involved in the regulation of proliferation and differentiation in the prenatal liver may initiate the tumorigenesis at early stages of hepatic development. One potential auxiliary factor has been identified recently13 and others are expected to follow. Further biochemical characterization of these candidates may unravel the structure of the apo B–mRNA editosome. Using the species-specific APOBEC-1 cDNA under the control of its own promoter in transgenic animals, however, may well avoid the observed oncogenic effects of APOBEC-1. In conclusion, the study by Innerarity et al. is a cornerstone in our understanding of the benefits of APOBEC-1 expression in liver, namely the reduction of plasma LDL levels, as well as its major potential pitfall, carcinogenesis. Further investigation into the regulation of APOBEC-1 expression and apo B–mRNA editing may teach us how nature keeps this enzyme in control, allowing us to harness the power of the editing enzyme complex to reduce the level of plasma LDL, the best known factor in atherogenesis. JOBST GREEVE, M.D. Medizinische Klinik, Universitats-Krankenhaus Eppendorf, Hamburg, Germany JAYANTA ROY CHOWDHURY, M.D. NAMITA ROY CHOWDHURY, Ph.D. Department of Medicine and Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, NY REFERENCES 1. Chan L. Apolipoprotein B, the major protein component of triglyceriderich and low density lipoproteins. J Biol Chem 1992;267:25621-25624. 2. Havel RH, Kane JP, Goldstein JL, Hobbs HH, Brown MS. Lipoprotein and lipid metabolism disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D,
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3. 4. 5. 6.
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9.
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eds. The metabolic and molecular bases of inherited diseases. Vol 2, Ed 7. New York: McGraw-Hill, 1995:1841-1885, 1981-2030. Powell LM, Wallis SC, Pease RJ, Edwards YH, Knott TJ, Scott J. A novel form of tissue-specific RNA processing produces apo B-48 in the intestine. Cell 1987;50:831-840. Scott J, Navaratnam N, Bhattacharya S, Morrison JR. The apolipoprotein messenger RNA editing enzyme. Curr Opin Lipidol 1994;5:87-93. Teng BB, Burand CF, Davidson NO. Molecular cloning of an apolipoprotein B messenger RNA editing protein. Science 1993;260:1816-1819. Navaratnam N, Bhattacharya S, Fufino T, Patel D, Jaumuz AL, Scott J. Evolutionary origins of apo B mRNA editing: catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif as its active site. Cell 1995;81:187-195. Giannoni F, Bonen DK, Funahashi T, Hadjiagapiou C, Burant CF, Davidson NO. Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B-48. J Biol Chem 1994; 269:5932-5936. Greeve J, Altkemper I, Dieterich JH, Greten H, Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apo B-containing plasma lipoproteins. J Lipid Res 1993;34:1367-1382. Li X, Catalina F, Grundy SM, Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48 relative to B100-containing lipoproteins. J Lipid Res 1996;37:210-220. Nakamuta M, Oka K, Krushkal J, Kobayashi K, Yamamoto M, Li WH, Chan L. Alternative mRNA splicing and differential promoter utilization determine tissue-specific of the apolipoprotein B mRNA-editing protein (APOBEC-1) gene in mice. Structure and evolution of APOBEC-1 and related nucleoside/nucleotide deaminases. J Biol Chem 1995;270:1304213056. Greeve J, Jona VK, Roy Chowdhury N, Horwitz MS, Roy Chowdhury J. Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme, APOBEC-1, leads to reduction of LDL in normal and Watanabe heritable hyperlipidemic rabbits. J Lipid Res 1996;37:20012017. Giannoni F, Chow S-C, Skarosi SF, Verp MS, Field FH, Coleman RA, Davidson NO. Developmental regulation of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (APOBEC-1) in human small intestine. J Lipid Res 1995;36:1664-1675. Schock D, Kuo S-R, Steinburg MF, Bolognino M, Sparks JD, Sparks CE, Smith HC. An auxiliary factor containing a 240 kDa protein complex is involved in apolipoprotein B RNA editing. Proc Natl Acad Sci U S A 1996; 93:1097-1102.
REGULATION OF HEPATOCELLULAR BILE SALT SECRETION: WHO SHOOTS THE MESSENGERS?
Boyer JL, Soroka CJ. Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets. Gastroenterology 1995;109:1600-1611. ABSTRACT
Background and Aims. Plasma membrane solute transport may be regulated in many epithelial cells by vesicle traffic to and from the site of residence of the transporter. The aim of this study is to determine if this phenomenon may also play a role in the regulation of the canalicular transport of bile acids. Methods. Confocal microscopy and image analysis were performed to quantitatively assess changes in secretory capacity and vesicle targeting in isolated rat hepatocyte couplets that had been exposed to fluorescent bile acid after pretreatment with dibutyryl adenosine 3*,5*-cyclic monophosphate (DBcAMP) and/or nocodazole. Results. DBcAMP stimulated bile acid secretion by 240% while significantly increasing canalicular circumference. Nocodazole decreased secretion by 410% and significantly decreased canalicular circumference. When DBcAMP was added to nocodazole-treated couplets, a slight but significant increase was found in both fluorescent bile acid secretion and canalicular circumference as compared with nocodazole alone. Finally, DBcAMP stimulated the translocation of vesicles to the canalicular membrane as determined by immunocytochemical localization of a putative bile acid transporter, Ca2/, Mg2/-ecto-adenosine triphosphatases. Conclusions. The findings support the view that apical membrane transport activity in the rat
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hepatocyte is highly regulated by the insertion of vesicles into this domain and that this process involves both microtubule-dependent and microtubule-independent mechanisms. Noe´ B, Schliess F, Wettstein M, Heinrich S, Ha¨ussinger D. Regulation of taurocholate excretion by a hypo-osmolarityactivated signal transduction pathway in rat liver. Gastroenterology 1996;110:858-865. ABSTRACT
Background and Aims. Hypo-osmotic cell swelling increases the capacity of taurocholate excretion into bile in the perfused rat liver. The aim of this study was to clarify the mechanisms linking cell swelling to bile acid secretion. Methods. The influence of hypo-osmotic cell swelling on intracellular signal transduction and bile acid secretion was studied in isolated rat hepatocytes and the perfused rat liver. Results. In rat livers perfused with hypo-osmotic buffer (225 mOsm/L), the maximum velocity of taurocholate excretion into bile is increased by 135% within 10 to 20 minutes. To unravel the signaling events mediating this effect, the activities of the mitogen-activated protein kinases, extracellular signal-regulated kinases (Erk)-1 and Erk-2, were measured after hypo-osmotic treatment in cultured rat hepatocytes. A rapid parallel activation of Erk-1 and Erk-2 was observed within 1 minute, which became maximal after 10 minutes and returned to the basal level within 60 minutes. The hypo-osmolarity-induced Erk activation and the increase in bile flow after hypo-osmotic liver perfusion were completely abolished by inhibitors of signal transduction at the level of G proteins and tyrosine kinases but remained unaffected by the inhibition of the protein kinase C. Conclusions. A G protein and tyrosine kinase-dependent but protein kinase C-independent activation of mitogen-activated protein kinases is involved in the regulation of taurocholate excretion by liver cell hydration changes. Beuers U, Throckmorton DC, Anderson MS, Isales CM, Thasler W, Kullak-Ublick G-A, Sauter G, et al. Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996;110:1553-1563. ABSTRACT
Background and Aims. Ursodeoxycholic acid improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. Ursodeoxycholic acid is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2/ agonist and stimulates biliary exocytosis and hepatocellular Ca2/ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates the stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. Methods. The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accumulation of sn-1,2-diacylglycerol, the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. Results. Immunoblotting studies showed the presence of four isoenzymes (a, d, e, and z). The phorbol ester phorbol 12-myristate 13-acetate (1 mmol/L) induced translocation of a-PKC, d-PKC, and e-PKC from cytosol to a particulate
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ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Liver Diseases Section NIH/NIDDK, Building 10-9 B16 10 Center Dr Bethesda, MD 20892-1800
INDUCTION OF HEPATIC APOLIPOPROTEIN B mRNA EDITING FOR REDUCING SERUM CHOLESTEROL LEVELS: A BREAKTHROUGH OR A DISASTER?
Yamanaka S, Balestra ME, Ferrell LD, Fan J, Arnold KS, Taylor S, Taylor JM, et al. Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals. Proc Natl Acad Sci U S A 1995;92:84838487. ABSTRACT
Apolipoprotein (apo-) B messenger RNA (mRNA) editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo B (apo-B48). The cytidine deaminase catalytic subunit (apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)) of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower lowdensity lipoprotein cholesterol concentrations. The apoB mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and lowdensity lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and one transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to those of apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in the livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed, editing enzyme, and suggests that aberrant editing of hepatic mRNAs is involved in cell growth and that regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower the plasma levels of low-density lipoproteins. COMMENTS
Apolipoprotein (apo) B, the essential core protein of all lipoproteins except the high-density lipoproteins, exists in two forms: the full-length protein, apo B-100 (512 kd), and the carboxy terminal truncated apo B-48 (248 kd).1 The human liver secretes triglyceride-rich), very-low-density lipoproteins (VLDL). Each VLDL particle contains one molecule of apo B-100.1,2 After triglyceride hydrolysis by lipoprotein lipases, most of the VLDL-remnants are cleared within minutes by the liver, yet some VLDL particles are transformed by incompletely understood processes into low-density lipoproteins (LDL).2 LDL are enriched in cholesterol-esters, have a plasma half-life of about 1 day, and, if elevated, represent an important cause of development of atherosclerosis and
coronary artery disease.2 The intestine, the second major source of triglyceride-rich lipoproteins, secretes chylomicrons, which contain apo B-48 as the core protein.2 Unlike VLDL, chylomicrons are rapidly and completely cleared by the liver after triglyceride hydrolysis, without giving rise to long-lived apo B–containing lipoproteins.2 The synthesis of apo B-48 is the result of mRNA editing, which consists of a specific base change from C to U in the apo B mRNA, creating a premature stop codon.3 The editing is mediated by the apo B–mRNA editing enzyme, which specifically deaminates the cytidine residue at nucleotide 6666 during the processing of apo B pre-mRNA.4 The editing enzyme is a complex of several subunits. The catalytic subunit, APOBEC-1 (apo B–mRNA editing enzyme catalytic polypeptide 1,)5 is a member of the cytidine deaminases gene family and contains a novel RNA binding motif, but requires additional ‘‘auxiliary’’ factors for expressing the apo B–mRNA editing reaction.6 Extracts from human, baboon, or rabbit liver that do not exhibit the editing activity can nonetheless supplement APOBEC-1 to reconstitute apo B–mRNA editing in vitro, suggesting that the livers of these species contain the additional ‘‘auxiliary’’ components of the apo B–mRNA editing enzyme complex.7 Almost 100% of the apo B mRNA is edited in the intestine of all mammalian species that are studied.8 APOBEC-1 is absent in rabbit, baboon, or human liver,5,7 and consequently, the liver of these species secrete VLDL containing the full-length apo B-100.8 In horses, rats, mice, and dogs, however, hepatic apo B mRNA is also substantially edited.8 These species have low plasma LDL levels,4 probably because their livers secrete apo B-48–containing VLDL, which are turned over much faster than apo B-100–containing lipoproteins.9 In mice10 and rats (Greeve et al., manuscript in preparation), two different forms of APOBEC-1 are expressed in the liver and in the intestine as a result of differential promoter usage and of alternative splicing. In these species, hepatic apo B–mRNA editing appears to be a genetic mechanism to regulate the metabolism and to modulate the plasma concentrations of the atherogenic apo B-100–containing LDL. The mechanism of the lack of APOBEC-1 expression in the liver of species, such as in the livers of humans and rabbits, remains to be uncovered. By establishing APOBEC-1 transgenic rabbits, Yamanaka et al. have directly addressed the question of how the induction of apo B–mRNA editing in the liver of a species without natural hepatic editing influences plasma LDL levels. The generation of transgenic rabbits required major refinements of the transgene technology. Simultaneously, Yamanaka et al. also generated transgenic mice that expressed active rabbit APOBEC-1 or a mutant, enzymatically inactive form of the protein. To achieve liver-specific expression of the APOBEC-1 transgene, they used a vector containing a promoter, an intron, and a hepatic control-region of the human apolipoprotein E gene. Four independent transgenic mouse lines were established from three transgenic mouse founders that expressed wild-type rabbit APOBEC-1 with estimated transgene copy numbers of 3, 7, 10, and 17, respectively. In
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addition, 2 transgenic rabbit founders were established with 17 copies and 1 copy of the APOBEC-1 transgene, respectively. In the transgenic mouse lines, APOBEC-1 was highly expressed in the liver and was accompanied by an increase in hepatic apo B–mRNA editing to an average of 92%, as compared with an average of 83% in the nontransgenic mice and 82% in the mice transgenic for the mutant inactive APOBEC-1. Cytosolic extracts from the liver of the transgenic mice were 15-fold more active in editing a synthetic apo B mRNA in vitro than extracts from matched control rats, further demonstrating that APOBEC-1 was highly overexpressed in the liver. In the transgenic rabbit founder with 17 copies of the transgene, APOBEC-1 was highly expressed in the liver, and 78% of the hepatic apo B mRNA was found to be edited, whereas, in normal rabbits, editing of the hepatic apo B mRNA is not detectable. APOBEC-1 expression and hepatic apo B–RNA editing were not measured in the liver of the rabbit founder that had only 1 copy of the transgene. LDL was undetectable in the plasma of the APOBEC-1 transgenic mice. Apo B-100 was absent in these mice and apo B-48 was the only apolipoprotein found in the plasma. Initial observations in the APOBEC-1 transgenic rabbit that had 1 copy of the transgene showed a 50% reduction of plasma LDL and a small increase in HDL compared with the levels found in the nontransgenic littermates. The most striking observation of this study was the marked enlargement of the livers with modest to severe hepatic dysplasia in all of the transgenic mouse lines, and the occurrence of hepatocellular carcinoma (HCC) in some. One of four mice with 3 copies of the transgene, one of 20 mice with a copy number of 7, 4 of 5 mice with a copy number of 10, and 2 of 5 mice with a copy number of 17 all developed full-blown HCC by histological criteria. In contrast, none of the 8 transgenic mice that expressed the mutant inactive APOBEC-1 developed severe hepatic dysplasia or HCC. The rabbit founder with a copy number of 17 grew slowly and was euthanized at the age of 8 weeks because of progressive weakness. Necropsy revealed an enlarged liver with fibrosis and histologically distorted architecture with intracytoplasmic lipid droplets; however, severe hepatic dysplasia or HCC was not found. The occurrence of hepatic dysplasia and HCC in all APOBEC-1 transgenic mouse lines and in the absence of dysplasia in mice transgenic for the mutant inactive APOBEC-1 make it very unlikely that the site of transgene insertion is related to the observed effects on hepatocyte proliferation and differentiation. Moreover, the authors do not believe that a lack of the full-length protein apo B-100 caused the observed hepatic abnormalities, because, in ‘‘apo B-48 only mice,’’ generated by gene-targeting in embryonic stem cells, no such effect has been observed. Instead, the authors speculate that APOBEC-1 can act as an oncogene leading to HCC by editing of mRNAs other than apo B. Indeed, in the transgenic mice, they identified such aberrant C-to-U editing at codon 411 of the mouse tyrosine kinase gene. This nucleotide change, however, is not predicted to change the amino acid sequence as both CUG and UUG code for leucine. Is the observation of HCC in APOBEC-1 transgenic animals the death-blow to the idea that the induction of hepatic apo B–mRNA editing, possibly by gene transfer of APOBEC1, could be a genetic approach to the treatment of hypercholesterolemia? By generating the APOBEC-1 transgenic mice and rabbits, the authors have provided compelling evidence that the expression of apo B–mRNA editing in the liver results in a reduction, or even with the absence of plasma LDL. This conclusion is further supported by recent studies using APOBEC-1 recombinant adenoviruses to induce hepatic apo B–mRNA editing. Both in mice and rabbits, the injection of APOBEC-1 recombinant adenoviruses resulted in the substantial reduction of plasma LDL levels. In the LDL-receptor–deficient Watanabe heritable hyperlipidemic rabbit, we
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achieved a 40% reduction of the high plasma concentrations of LDL by this approach.11 The development of HCC in APOBEC-1 transgenic animals severely compromises these promising findings. However, it should be noted that rats, mice, dogs, and horses express APOBEC-1 in the liver but are not known to be excessively susceptible to the development of HCC. Thus, in general, the hepatic expression of APOBEC1 is not oncogenic, although the type of expression achieved in this study resulted in HCC. What could explain this discrepancy? First, the complementary DNA (cDNA) for rabbit APOBEC-1 was introduced into the germ-line of mice. The comparison of the cDNA sequence from rat, mice, rabbit, baboon, and human has revealed a great deal of interspecies sequence divergence.5,7 The sequence-specificity of the editing function of APOBEC-1 most likely requires the ‘‘auxiliary’’ regulatory factors present in the editing enzyme complex, which has been termed the editosome. By introducing rabbit APOBEC-1 in mice, this interaction could be impaired, resulting in the observed loss of editing specificity. It should be pointed out that the (rabbit) APOBEC-1 transgenic rabbits had derangement of the liver structure, but did not develop severe hepatocyte dysplasia or HCC. Importantly, the rabbit founder with one transgene copy had no obvious hepatic abnormality. Establishing a rabbit line from this particular rabbit founder will be helpful in further evaluating the oncogenic potential of APOBEC-1. Second, in the human intestine and in the rat liver, the expression of APOBEC-1 is upregulated immediately before birth and correlates with the editing of apo B mRNA.12 The heterologous promoter of apo E, which has important functions in the embryonic lipid metabolism, may lead to APOBEC-1 expression in the early embryonic liver possibly without the accompanying expression of the ‘‘auxiliary’’ components of the editosome, which are responsible for the normal ‘‘quenching’’ of the nonspecific editing capacity of APOBEC-1. Aberrant mRNA editing of genes that are involved in the regulation of proliferation and differentiation in the prenatal liver may initiate the tumorigenesis at early stages of hepatic development. One potential auxiliary factor has been identified recently13 and others are expected to follow. Further biochemical characterization of these candidates may unravel the structure of the apo B–mRNA editosome. Using the species-specific APOBEC-1 cDNA under the control of its own promoter in transgenic animals, however, may well avoid the observed oncogenic effects of APOBEC-1. In conclusion, the study by Innerarity et al. is a cornerstone in our understanding of the benefits of APOBEC-1 expression in liver, namely the reduction of plasma LDL levels, as well as its major potential pitfall, carcinogenesis. Further investigation into the regulation of APOBEC-1 expression and apo B–mRNA editing may teach us how nature keeps this enzyme in control, allowing us to harness the power of the editing enzyme complex to reduce the level of plasma LDL, the best known factor in atherogenesis. JOBST GREEVE, M.D. Medizinische Klinik, Universitats-Krankenhaus Eppendorf, Hamburg, Germany JAYANTA ROY CHOWDHURY, M.D. NAMITA ROY CHOWDHURY, Ph.D. Department of Medicine and Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, NY REFERENCES 1. Chan L. Apolipoprotein B, the major protein component of triglyceriderich and low density lipoproteins. J Biol Chem 1992;267:25621-25624. 2. Havel RH, Kane JP, Goldstein JL, Hobbs HH, Brown MS. Lipoprotein and lipid metabolism disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D,
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HEPATOLOGY Elsewhere
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eds. The metabolic and molecular bases of inherited diseases. Vol 2, Ed 7. New York: McGraw-Hill, 1995:1841-1885, 1981-2030. Powell LM, Wallis SC, Pease RJ, Edwards YH, Knott TJ, Scott J. A novel form of tissue-specific RNA processing produces apo B-48 in the intestine. Cell 1987;50:831-840. Scott J, Navaratnam N, Bhattacharya S, Morrison JR. The apolipoprotein messenger RNA editing enzyme. Curr Opin Lipidol 1994;5:87-93. Teng BB, Burand CF, Davidson NO. Molecular cloning of an apolipoprotein B messenger RNA editing protein. Science 1993;260:1816-1819. Navaratnam N, Bhattacharya S, Fufino T, Patel D, Jaumuz AL, Scott J. Evolutionary origins of apo B mRNA editing: catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif as its active site. Cell 1995;81:187-195. Giannoni F, Bonen DK, Funahashi T, Hadjiagapiou C, Burant CF, Davidson NO. Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B-48. J Biol Chem 1994; 269:5932-5936. Greeve J, Altkemper I, Dieterich JH, Greten H, Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apo B-containing plasma lipoproteins. J Lipid Res 1993;34:1367-1382. Li X, Catalina F, Grundy SM, Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48 relative to B100-containing lipoproteins. J Lipid Res 1996;37:210-220. Nakamuta M, Oka K, Krushkal J, Kobayashi K, Yamamoto M, Li WH, Chan L. Alternative mRNA splicing and differential promoter utilization determine tissue-specific of the apolipoprotein B mRNA-editing protein (APOBEC-1) gene in mice. Structure and evolution of APOBEC-1 and related nucleoside/nucleotide deaminases. J Biol Chem 1995;270:1304213056. Greeve J, Jona VK, Roy Chowdhury N, Horwitz MS, Roy Chowdhury J. Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme, APOBEC-1, leads to reduction of LDL in normal and Watanabe heritable hyperlipidemic rabbits. J Lipid Res 1996;37:20012017. Giannoni F, Chow S-C, Skarosi SF, Verp MS, Field FH, Coleman RA, Davidson NO. Developmental regulation of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (APOBEC-1) in human small intestine. J Lipid Res 1995;36:1664-1675. Schock D, Kuo S-R, Steinburg MF, Bolognino M, Sparks JD, Sparks CE, Smith HC. An auxiliary factor containing a 240 kDa protein complex is involved in apolipoprotein B RNA editing. Proc Natl Acad Sci U S A 1996; 93:1097-1102.
REGULATION OF HEPATOCELLULAR BILE SALT SECRETION: WHO SHOOTS THE MESSENGERS?
Boyer JL, Soroka CJ. Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets. Gastroenterology 1995;109:1600-1611. ABSTRACT
Background and Aims. Plasma membrane solute transport may be regulated in many epithelial cells by vesicle traffic to and from the site of residence of the transporter. The aim of this study is to determine if this phenomenon may also play a role in the regulation of the canalicular transport of bile acids. Methods. Confocal microscopy and image analysis were performed to quantitatively assess changes in secretory capacity and vesicle targeting in isolated rat hepatocyte couplets that had been exposed to fluorescent bile acid after pretreatment with dibutyryl adenosine 3*,5*-cyclic monophosphate (DBcAMP) and/or nocodazole. Results. DBcAMP stimulated bile acid secretion by 240% while significantly increasing canalicular circumference. Nocodazole decreased secretion by 410% and significantly decreased canalicular circumference. When DBcAMP was added to nocodazole-treated couplets, a slight but significant increase was found in both fluorescent bile acid secretion and canalicular circumference as compared with nocodazole alone. Finally, DBcAMP stimulated the translocation of vesicles to the canalicular membrane as determined by immunocytochemical localization of a putative bile acid transporter, Ca2/, Mg2/-ecto-adenosine triphosphatases. Conclusions. The findings support the view that apical membrane transport activity in the rat
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hepatocyte is highly regulated by the insertion of vesicles into this domain and that this process involves both microtubule-dependent and microtubule-independent mechanisms. Noe´ B, Schliess F, Wettstein M, Heinrich S, Ha¨ussinger D. Regulation of taurocholate excretion by a hypo-osmolarityactivated signal transduction pathway in rat liver. Gastroenterology 1996;110:858-865. ABSTRACT
Background and Aims. Hypo-osmotic cell swelling increases the capacity of taurocholate excretion into bile in the perfused rat liver. The aim of this study was to clarify the mechanisms linking cell swelling to bile acid secretion. Methods. The influence of hypo-osmotic cell swelling on intracellular signal transduction and bile acid secretion was studied in isolated rat hepatocytes and the perfused rat liver. Results. In rat livers perfused with hypo-osmotic buffer (225 mOsm/L), the maximum velocity of taurocholate excretion into bile is increased by 135% within 10 to 20 minutes. To unravel the signaling events mediating this effect, the activities of the mitogen-activated protein kinases, extracellular signal-regulated kinases (Erk)-1 and Erk-2, were measured after hypo-osmotic treatment in cultured rat hepatocytes. A rapid parallel activation of Erk-1 and Erk-2 was observed within 1 minute, which became maximal after 10 minutes and returned to the basal level within 60 minutes. The hypo-osmolarity-induced Erk activation and the increase in bile flow after hypo-osmotic liver perfusion were completely abolished by inhibitors of signal transduction at the level of G proteins and tyrosine kinases but remained unaffected by the inhibition of the protein kinase C. Conclusions. A G protein and tyrosine kinase-dependent but protein kinase C-independent activation of mitogen-activated protein kinases is involved in the regulation of taurocholate excretion by liver cell hydration changes. Beuers U, Throckmorton DC, Anderson MS, Isales CM, Thasler W, Kullak-Ublick G-A, Sauter G, et al. Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996;110:1553-1563. ABSTRACT
Background and Aims. Ursodeoxycholic acid improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. Ursodeoxycholic acid is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2/ agonist and stimulates biliary exocytosis and hepatocellular Ca2/ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates the stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. Methods. The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accumulation of sn-1,2-diacylglycerol, the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. Results. Immunoblotting studies showed the presence of four isoenzymes (a, d, e, and z). The phorbol ester phorbol 12-myristate 13-acetate (1 mmol/L) induced translocation of a-PKC, d-PKC, and e-PKC from cytosol to a particulate
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