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Ethanol Stimulates Apolipoprotein B mRNA Editing in the Absence of de Novo RNA or Protein Synthesis
Biochemical and Biophysical Research Communications 289, 1162–1167 (2001) doi:10.1006/bbrc.2001.6082, available online at http://www.idealibrary.com on
Ethanol Stimulates Apolipoprotein B mRNA Editing in the Absence of de Novo RNA or Protein Synthesis Adam Giangreco,* Mark P. Sowden,† ,‡ Igor Mikityansky,† and Harold C. Smith* ,† ,‡ ,§ ,1 †Department of Biochemistry and Biophysics, ‡Department of Pathology, *Department of Environmental Health Sciences, and §James P. Wilmot Cancer Centers, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14623
Received October 26, 2001
Apolipoprotein B (apoB) mRNA editing involves a site-specific modification of cytidine to form uridine. The reaction is catalyzed in the nucleus by a multiprotein editosome. Rat hepatic editing is regulated during development, metabolically and in response to ethanol. Ethanol stimulated editing in hepatocytes within minutes of exposure. In the present study, we show that ethanol stimulated apoB mRNA synthesis and apoB mRNA editing. Significantly, the proportion of edited apoB mRNA also increased following ethanol treatment of transcription or translation arrested cells. These data suggested that ethanol could regulate editing activity using pre-existing editosomal proteins. In addition, the presence of a suppressor of apoB mRNA editing activity was suggested by the finding that inhibition of mRNA or protein synthesis alone was sufficient to increase the proportion of edited RNA. It is proposed that the level of editing activity observed in hepatocytes may be the end result of positive and negative regulatory proteins. © 2001 Elsevier Science
Apolipoprotein B (apoB) mRNA editing involves a deamination of cytidine to form uridine at nucleotide 6666 (1, 2). The minimal requirement for apoB mRNA editing in vitro was the cytidine deaminase APOBEC-1 and an apoB editing site, RNA binding protein known as APOBEC-1 Complementation Factor (ACF) (3) or APOBEC-1 Stimulatory Protein (ASP) (4). Several lines of evidence suggested that APOBEC-1 functioned as a homodimer (5–7). Yeast two hybrid analysis and biochemical fractionation suggested that ACF/ASP and APOBEC-1 interact (3, 4). The minimal size of the editosome is likely to be therefore a heterotrimer. Analyses of editing complexes suggested that the editosome may be 27S, considerably more complex 1
To whom correspondence should be addressed. Fax: (716) 2756007. E-mail: [email protected]. 0006-291X/01 $35.00 © 2001 Elsevier Science All rights reserved.
than three proteins (8 –11). Several candidate auxiliary proteins have been identified through their ability to bind APOBEC-1 and/or apoB mRNA. Editing activity was enhanced (3, 4, 10, 12, 13) or inhibited (11, 14, 15) when these auxiliary proteins interacted with the editosome. Substantial evidence exists for the regulation of apoB mRNA editing during development, by hormones, metabolically and through changes in tissue microenvrionment (2, 16 –29). In some instances, the regulation of APOBEC-1 expression could account for the observed changes in editing activity (18, 19, 24, 28). Regulation of apoB mRNA editing could occur however in the absence of changes in APOBEC-1 expression (17, 27). The basis for these changes was presumed to be alterations in auxiliary protein expression and/or interactions. Recently, phosphorylation of APOBEC-1 at serine 47 was shown to be important for editing activity whereas phosphorylation of serine 72 inhibited editing activity (30). These studies suggested that apoB mRNA editing could be regulated through posttranslation modification of editing factors. The present study demonstrated that ethanol stimulated apoB mRNA editing in the absence of de novo mRNA or protein synthesis. The data also suggested a suppressor of apoB mRNA editing in that inhibition of mRNA or protein synthesis in and of itself stimulated editing. This was observed at low and high levels of APOBEC-1 expression, suggesting that pre-existing auxiliary proteins were not rate limiting. The ability of ethanol to further stimulate editing in cells that were inhibited in mRNA and protein synthesis suggested that positive and negative control factors were involved in establishing homeostasis in the proportion of edited apoB mRNAs. MATERIALS AND METHODS Hepatocytes and cell lines. Rat primary hepatocytes were prepared from male Sprague Dawley rats and grown on rat tail collagen Type I coated T25 flasks as described previously (26). For ethanol
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RESULTS AND DISCUSSION
FIG. 1. RNA synthesis in ethanol treated rat primary hepatocytes. Cultured cells were placed in media containing 3H uridine and treated with ethanol, actinomycin D (ActD) or both for the indicated times. Total cellular RNA was isolated and acid precipitable radioactivity determined by scintillation counting.
treatment, the flasks were filled with media containing the indicated concentration of ethanol and sealed for 12 h. McArdle 7777 rat hepatoma cells and McAPOBEC cells, a McArdle cell line expressing high levels of rat APOBEC-1 (31) were treated with 0.45% ethanol for 12 h in 60 mm Petri dishes (26). RNA synthesis was determined by labeling cells with 50 Ci of 3H uridine (Amersham) for the indicated times, isolating total cellular RNA (as described below) and determining by scintillation counting acid precipitable radioactivity from 5 g of RNA. The data represent the average of triplicate determinations from two separate experiments. Treatment with inhibitors. Cells were treated with actinomycin D (5 g/ml) or 5,6-dichloro-1--D ribo-furanosylbensimidazole, DRB (10 M) or emetine (300 nM) for 15 min prior to ethanol addition and the inhibitors were left in the media for the duration of the ethanol treatment. Control cells were either untreated or treated with inhibitors alone for 12 h. RNA analyses. Following each treatment, total cellular RNA was isolated using TriReagent (MRC). For RNase protection assays, a plasmid encoding a 286 nucleotide anti-sense apoB RNA probe complementary to sequence 5⬘ of nucleotide 6666 was constructed and transcribed in the presence of 32P-␣ATP using the mMessage mMachine kit (Ambion). A plasmid encoding a 124 nucleotide antisense actin RNA probe was purchased from Ambion and transcribed as described above. RNase protection was performed using the HybSpeed RPA system (Ambion) on 5 g of total cellular RNA and the products resolved on denaturing 10% polyacrylamide gels, autoradiographed and quantified by PhosphorImager analysis. RNase protection was dependent on input total cellular RNA and the amount of protected fragment was proportional to input total cellular RNA between 2–10 g. To analyze RNA editing, first strand cDNA was primed from 1 g of total cellular RNA and amplified by the PCR (31). Purified PCR products were quantified and 25 ng were analyzed by poisoned primer extension (31). Gel resolved primer extension products were quantified by PhosphorImager analysis and the percent editing was calculated from triplicate determinations as the density of the primer extension product corresponding to unedited apoB RNA (CAA) plus that of the edited apoB RNA (UAA) primer extension product, divided into UAA times 100. Determination of apoptosis. Apoptosis was determined by the Core Flow Cytometry facility in the James P. Wilmot Cancer Center using the TUNEL/Hoechst staining kit (Boehringer Mannheim) as per the manufacturer’s protocol using the negative and positive control cells provided.
Previous studies showed that hepatic apoB mRNA editing was stimulated in rats consuming ethanol as a fixed proportion of daily caloric intact (27). ApoB mRNA editing also increased in rat primary hepatocytes in response to 0.1% to 2.5% ethanol in the media (26). McArdle rat hepatoma cells and McArdle cells overexpressing rat APOBEC-1 (McAPOBEC cells) had to be treated with higher concentrations of ethanol (0.9% to 2.5%) before apoB mRNA editing activity increased. Ethanol specifically enhanced editing of apoB mRNA in the nucleus of cells and the change in editing was measurable within 15 min of treatment (9, 32). However, editing of apo B mRNA in HepG2 human hepatoma cells and HepG2 cells overexpressing human APOBEC-1 (HepG2APOBEC cells) was refractory to ethanol (26, 32). Ethanol induced RNA synthesis. Regulation of apoB mRNA editing involved de novo synthesis of APOBEC-1 in insulin stimulated hepatocytes (24, 28) but this did not appear to be the mechanism whereby ethanol stimulated editing (27). It was presumed that ethanol regulated editing involved de novo synthesis of auxiliary proteins and/or post-translation control of editosomal protein function. RNA synthesis, as measured by 3H uridine incorporation (Fig. 1) and apoB mRNA editing (Figs. 3 and 4) were markedly stimulated in ethanol treated McArdle cells. RNA synthesis in control and ethanol treated cells could be blocked by treating cells with 5 g/ml actinomycin D (a concentration that inhibits both RNA polymerase I and II). RNase protection analysis, designed to selectively quantify apoB mRNA, demonstrated a 2.5-fold increase in apoB mRNA abundance within 12 h of treatment with 0.1% ethanol. A 10-fold lower dose of ethanol (0.01% ethanol did not stimulate editing), did not alter apoB mRNA abundance over control cells (Fig. 2).
FIG. 2. Ethanol induced apolipoprotein B mRNA synthesis. RNase protection analysis was performed with either actin or apoB RNA probes annealed to 5 g of total cellular RNA isolated from rat primary hepatocytes grown on Type I collagen coated Petri dishes or in T-flasks and exposed to the indicated concentration of ethanol for 12 h. Ratios were calculated as the dpms in the protected fragment from the ethanol treated sample relative to that determined in untreated cells.
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FIG. 3. Ethanol stimulated editing in the absence of RNA synthesis. ApoB mRNA was amplified from wild type McArdle cells or McArdle cells expressing high levels of APOBEC-1 (McAPOBEC) that had been treated for 12 h with ethanol, ActD, DRB or ethanol plus an inhibitor. Editing was determined by poisoned primer extension and quantified from triplicate determinations as described in Materials and Methods. CAA, unedited RNA, UAA edited RNA and 1–3, promiscuous editing sites.
In contrast, the abundance of actin mRNA in cells treated with either 0.01% or 0.1% ethanol was equivalent to that in untreated cells. These data demonstrated that RNA synthesis is induced by ethanol and that transcription of the apoB gene may be more responsive to ethanol than that of house keeping genes. The data suggested a large activation of editing activity in the cell nucleus had occurred for not only had the proportion of edited apoB mRNAs increased but the size of the apoB mRNA population serving as editing substrates had also increased. RNA synthesis was not required for ethanol stimulated editing. If de novo synthesis of auxiliary proteins or apoB mRNA was required for ethanol stimulated editing activity then inhibition of RNA polymerase II should block the increase in edited apoB mRNA. To evaluate this, McArdle and McAPOBEC cells were treated with 0.4% ethanol or pretreated for 15 min with RNA polymerase II inhibitors (Act D or DRB) and then treated with ethanol. Surprisingly, cells pretreated with Act D or DRB and then treated with ethanol demonstrated marked stimulation of editing activity (Fig. 3). The proportion of edited apoB mRNA was higher in cells treated with inhibitor plus ethanol than that seen in cells treated with ethanol alone. An analysis of editing in cells treated with RNA polymerase II inhibitors alone showed that apoB mRNA editing was also markedly stimulated (Fig. 3). Treatment of cells with a low dose of Act D (0.04 g/ ml), which only inhibits ribosomal RNA synthesis, did not stimulate editing activity (data not shown). These data suggested that not only was mRNA synthesis not
required for ethanol to stimulate apoB mRNA editing but that the absence of mRNA synthesis was in itself permissive for enhanced editing activity. Promiscuous editing of apoB mRNA (the editing of additional cytidines 5⬘ of C6666 within apoB mRNA, evident as primer extension stops 1–3 in Fig. 3) was observed whenever APOBEC-1 was artificially overexpressed as in McAPOBEC cells (32–35). Interestingly, ethanol stimulated editing activity in wild type McArdle cells without inducing promiscuous editing. Promiscuous editing was not induced in McArdle cells by inhibiting RNA synthesis and remained unchanged in inhibitor treated McAPOBEC cells. The apparent reduction in promiscuous editing in ethanol treated McAPOBEC cells was not consistently observed. Protein synthesis is not required for ethanol stimulated editing. The stimulation of editing activity in RNA polymerase II inhibited and ethanol treated cells may have been due to increased translation of auxiliary proteins on pre-existing mRNAs. To evaluate the requirement of protein synthesis in ethanol regulated editing activity, McArdle and McAPOBEC cells were treated with either ethanol alone or pretreated with the protein synthesis inhibitor, emetine, and then treated with ethanol. Cells treated with emetine alone demonstrated a marked stimulation in apoB mRNA editing activity. Moreover, cells treated with emetine and ethanol demonstrated a more marked stimulation of apoB mRNA editing activity than that seen in cells treated with ethanol alone (Fig. 4). Moreover, These data demonstrate that de novo protein synthesis was not required for ethanol stimulated editing activity and suggested that pre-existing editing factors were suffi-
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FIG. 4. Ethanol stimulated editing in the absence of protein synthesis. ApoB mRNA was amplified from wild type McArdle cells or McAPOBEC cells that had been treated with ethanol, emetine or both for 12 h. Editing was quantified from triplicate determinations as described in Materials and Methods.
cient for the cellular response to ethanol. An intact cell system was required for regulating editing as in vitro editing activity was not affected by treating McArdle cell extracts with ethanol and/or RNA and protein synthesis inhibitors (data not shown). In contrast to wild type McArdle cells that were inhibited in RNA synthesis, McArdle cells inhibited in protein synthesis demonstrated a very weak band at position 1, suggesting a low level of promiscuous editing. Furthermore, ethanol treatment of cells which were inhibited in protein synthesis, not only stimulated editing at the wild type site but also stimulated promiscuous editing. Considering that promiscuous editing in wild type McArdle cells only occurred when protein synthesis was inhibited, and not when RNA synthesis was inhibited, the data suggested that a protein factor(s) translated from pre-existing mRNA(s) controlled promiscuous editing. The protein(s) became rate limiting however when protein synthesis was inhibited. This would be consistent with earlier studies suggesting that auxiliary proteins and cis-acting RNA sequences were responsible for the control of promiscuous editing (35).
McArdle cells induced apoptosis (Table 1). The data suggested that significant changes in cell viability were not induced in inhibitor treated cells. Taken together with data that demonstrated ethanol stimulated editing activity only in the nucleus (32), our findings indicated that the proportion of edited apoB mRNAs could be increased by modulating the activity of pre-existing editing factors. Activation of apoB mRNA editing by inhibitors of RNA polymerase II or protein synthesis may have resulted from a permissive condition established by the reduction of a protein(s) that suppressed efficiency. Several proteins with the capacity to inhibit apoB mRNA editing have been identified (11, 14, 15), yet their mechanism of action is unknown. These proteins may sequester auxiliary proteins and/or APOBEC-1 as inactive 60S complexes on the endoplasmic reticulum (9). In the absence of these editing suppressor proteins, more APOBEC-1 and/or auxiliary proteins may have translocated to the cell nucleus (9). Alternatively, the reduction in the suppressor protein(s) may have activated cytoplasmic editing factors, resulting in editing of cytoplasmic mRNAs. We cannot rule out the possibility that the increased proportion of edited apoB mRNAs in drug treated cells was due to a selective degradation of unedited apoB mRNAs. Other studies have suggested that nuclear apoB mRNA editing activity was activated by its uncoupling from the apoB pre-mRNA splicing pathway (36). In addition, recent data have suggested that protein kinases and protein phosphatases might regulate apoB mRNA editing through inhibitory and stimulatory phosphorylation sites on APOBEC-1 (30). Stimulation of editing activity by RNA and protein synthesis inhibitors or ethanol may have resulted from the decreased expression or activity of kinases or phosphatases that modify APOBEC-1 and/or the auxiliary proteins. Changes in post-translation modification could also affect editosome assembly and/or translocation of editing factors from the cytoplasm to the nucleus. Given the ability of ethanol to additionally stimulate editing activity in RNA or protein synthesis inhibited cells, it is possible that ethanol’s effect on editing involved a different set of pathways than those affected by RNA and protein synthesis inhibitors. Whether changes in the cellular microenvironment such as increased calcium
RNA and protein synthesis inhibited cells were viable. One explanation for the activation of apoB mRNA editing in RNA and protein synthesis inhibited cells is that cell death had enabled nuclear editing factors to leak into the cytoplasm where they acted upon cytoplasmic apoB mRNA. To evaluate this, cells were treated for 12 h with actinomycin D, DRB or emetine and then assayed for apoptosis using a flow cytometricbased TUNEL assay. Relative to negative and positive controls for apoptosis, none of the treatments of 1165
TABLE 1 Cell treatment
Percent apoptosis
Negative control Positive control McArdle control Act D DRB Emetine
0.4 30 0.9 0.9 1.5 1.3
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(37) or hypertonicity (23), which also increased the proportion of edited apoB mRNA, exerted their effect through pre-existing editing factors remains to be determined. In conclusion, although ethanol can stimulate macromolecular synthesis, it increased the proportion of edited apoB mRNA independent of de novo RNA or protein synthesis. The data also demonstrated that editing activity in cells expressing low or high APOBEC-1 can be stimulated by inhibiting the translation of proteins. An intact cellular system was required for this effect. These data suggested that in addition activators of apoB mRNA editing, homeostasis in the proportion of edited apoB mRNAs also required the expression of protein suppressors of apoB mRNA editing. We propose that the assembly of editosomes and/or their activity may be regulated through post-translational modification of editing factors and the expression of activator or suppressor auxiliary proteins. ACKNOWLEDGMENTS
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The authors thank Dr. Nazzareno Ballatori from the Environmental Health Sciences Center for the preparation of rat primary hepatocytes, Dr. Peter Keng from the James P. Wilmot Cancer Center for performing the TUNEL analyses, and Jenny M.L. Smith for the preparation of figures. This work was supported in part through a Public Health Service Grant DK43739 and a grant from the Alcohol Beverage Medical Research Foundation awarded to HCS.
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Ethanol Stimulates Apolipoprotein B mRNA Editing in the Absence of de Novo RNA or Protein Synthesis Adam Giangreco,* Mark P. Sowden,† ,‡ Igor Mikityansky,† and Harold C. Smith* ,† ,‡ ,§ ,1 †Department of Biochemistry and Biophysics, ‡Department of Pathology, *Department of Environmental Health Sciences, and §James P. Wilmot Cancer Centers, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14623
Received October 26, 2001
Apolipoprotein B (apoB) mRNA editing involves a site-specific modification of cytidine to form uridine. The reaction is catalyzed in the nucleus by a multiprotein editosome. Rat hepatic editing is regulated during development, metabolically and in response to ethanol. Ethanol stimulated editing in hepatocytes within minutes of exposure. In the present study, we show that ethanol stimulated apoB mRNA synthesis and apoB mRNA editing. Significantly, the proportion of edited apoB mRNA also increased following ethanol treatment of transcription or translation arrested cells. These data suggested that ethanol could regulate editing activity using pre-existing editosomal proteins. In addition, the presence of a suppressor of apoB mRNA editing activity was suggested by the finding that inhibition of mRNA or protein synthesis alone was sufficient to increase the proportion of edited RNA. It is proposed that the level of editing activity observed in hepatocytes may be the end result of positive and negative regulatory proteins. © 2001 Elsevier Science
Apolipoprotein B (apoB) mRNA editing involves a deamination of cytidine to form uridine at nucleotide 6666 (1, 2). The minimal requirement for apoB mRNA editing in vitro was the cytidine deaminase APOBEC-1 and an apoB editing site, RNA binding protein known as APOBEC-1 Complementation Factor (ACF) (3) or APOBEC-1 Stimulatory Protein (ASP) (4). Several lines of evidence suggested that APOBEC-1 functioned as a homodimer (5–7). Yeast two hybrid analysis and biochemical fractionation suggested that ACF/ASP and APOBEC-1 interact (3, 4). The minimal size of the editosome is likely to be therefore a heterotrimer. Analyses of editing complexes suggested that the editosome may be 27S, considerably more complex 1
To whom correspondence should be addressed. Fax: (716) 2756007. E-mail: [email protected]. 0006-291X/01 $35.00 © 2001 Elsevier Science All rights reserved.
than three proteins (8 –11). Several candidate auxiliary proteins have been identified through their ability to bind APOBEC-1 and/or apoB mRNA. Editing activity was enhanced (3, 4, 10, 12, 13) or inhibited (11, 14, 15) when these auxiliary proteins interacted with the editosome. Substantial evidence exists for the regulation of apoB mRNA editing during development, by hormones, metabolically and through changes in tissue microenvrionment (2, 16 –29). In some instances, the regulation of APOBEC-1 expression could account for the observed changes in editing activity (18, 19, 24, 28). Regulation of apoB mRNA editing could occur however in the absence of changes in APOBEC-1 expression (17, 27). The basis for these changes was presumed to be alterations in auxiliary protein expression and/or interactions. Recently, phosphorylation of APOBEC-1 at serine 47 was shown to be important for editing activity whereas phosphorylation of serine 72 inhibited editing activity (30). These studies suggested that apoB mRNA editing could be regulated through posttranslation modification of editing factors. The present study demonstrated that ethanol stimulated apoB mRNA editing in the absence of de novo mRNA or protein synthesis. The data also suggested a suppressor of apoB mRNA editing in that inhibition of mRNA or protein synthesis in and of itself stimulated editing. This was observed at low and high levels of APOBEC-1 expression, suggesting that pre-existing auxiliary proteins were not rate limiting. The ability of ethanol to further stimulate editing in cells that were inhibited in mRNA and protein synthesis suggested that positive and negative control factors were involved in establishing homeostasis in the proportion of edited apoB mRNAs. MATERIALS AND METHODS Hepatocytes and cell lines. Rat primary hepatocytes were prepared from male Sprague Dawley rats and grown on rat tail collagen Type I coated T25 flasks as described previously (26). For ethanol
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RESULTS AND DISCUSSION
FIG. 1. RNA synthesis in ethanol treated rat primary hepatocytes. Cultured cells were placed in media containing 3H uridine and treated with ethanol, actinomycin D (ActD) or both for the indicated times. Total cellular RNA was isolated and acid precipitable radioactivity determined by scintillation counting.
treatment, the flasks were filled with media containing the indicated concentration of ethanol and sealed for 12 h. McArdle 7777 rat hepatoma cells and McAPOBEC cells, a McArdle cell line expressing high levels of rat APOBEC-1 (31) were treated with 0.45% ethanol for 12 h in 60 mm Petri dishes (26). RNA synthesis was determined by labeling cells with 50 Ci of 3H uridine (Amersham) for the indicated times, isolating total cellular RNA (as described below) and determining by scintillation counting acid precipitable radioactivity from 5 g of RNA. The data represent the average of triplicate determinations from two separate experiments. Treatment with inhibitors. Cells were treated with actinomycin D (5 g/ml) or 5,6-dichloro-1--D ribo-furanosylbensimidazole, DRB (10 M) or emetine (300 nM) for 15 min prior to ethanol addition and the inhibitors were left in the media for the duration of the ethanol treatment. Control cells were either untreated or treated with inhibitors alone for 12 h. RNA analyses. Following each treatment, total cellular RNA was isolated using TriReagent (MRC). For RNase protection assays, a plasmid encoding a 286 nucleotide anti-sense apoB RNA probe complementary to sequence 5⬘ of nucleotide 6666 was constructed and transcribed in the presence of 32P-␣ATP using the mMessage mMachine kit (Ambion). A plasmid encoding a 124 nucleotide antisense actin RNA probe was purchased from Ambion and transcribed as described above. RNase protection was performed using the HybSpeed RPA system (Ambion) on 5 g of total cellular RNA and the products resolved on denaturing 10% polyacrylamide gels, autoradiographed and quantified by PhosphorImager analysis. RNase protection was dependent on input total cellular RNA and the amount of protected fragment was proportional to input total cellular RNA between 2–10 g. To analyze RNA editing, first strand cDNA was primed from 1 g of total cellular RNA and amplified by the PCR (31). Purified PCR products were quantified and 25 ng were analyzed by poisoned primer extension (31). Gel resolved primer extension products were quantified by PhosphorImager analysis and the percent editing was calculated from triplicate determinations as the density of the primer extension product corresponding to unedited apoB RNA (CAA) plus that of the edited apoB RNA (UAA) primer extension product, divided into UAA times 100. Determination of apoptosis. Apoptosis was determined by the Core Flow Cytometry facility in the James P. Wilmot Cancer Center using the TUNEL/Hoechst staining kit (Boehringer Mannheim) as per the manufacturer’s protocol using the negative and positive control cells provided.
Previous studies showed that hepatic apoB mRNA editing was stimulated in rats consuming ethanol as a fixed proportion of daily caloric intact (27). ApoB mRNA editing also increased in rat primary hepatocytes in response to 0.1% to 2.5% ethanol in the media (26). McArdle rat hepatoma cells and McArdle cells overexpressing rat APOBEC-1 (McAPOBEC cells) had to be treated with higher concentrations of ethanol (0.9% to 2.5%) before apoB mRNA editing activity increased. Ethanol specifically enhanced editing of apoB mRNA in the nucleus of cells and the change in editing was measurable within 15 min of treatment (9, 32). However, editing of apo B mRNA in HepG2 human hepatoma cells and HepG2 cells overexpressing human APOBEC-1 (HepG2APOBEC cells) was refractory to ethanol (26, 32). Ethanol induced RNA synthesis. Regulation of apoB mRNA editing involved de novo synthesis of APOBEC-1 in insulin stimulated hepatocytes (24, 28) but this did not appear to be the mechanism whereby ethanol stimulated editing (27). It was presumed that ethanol regulated editing involved de novo synthesis of auxiliary proteins and/or post-translation control of editosomal protein function. RNA synthesis, as measured by 3H uridine incorporation (Fig. 1) and apoB mRNA editing (Figs. 3 and 4) were markedly stimulated in ethanol treated McArdle cells. RNA synthesis in control and ethanol treated cells could be blocked by treating cells with 5 g/ml actinomycin D (a concentration that inhibits both RNA polymerase I and II). RNase protection analysis, designed to selectively quantify apoB mRNA, demonstrated a 2.5-fold increase in apoB mRNA abundance within 12 h of treatment with 0.1% ethanol. A 10-fold lower dose of ethanol (0.01% ethanol did not stimulate editing), did not alter apoB mRNA abundance over control cells (Fig. 2).
FIG. 2. Ethanol induced apolipoprotein B mRNA synthesis. RNase protection analysis was performed with either actin or apoB RNA probes annealed to 5 g of total cellular RNA isolated from rat primary hepatocytes grown on Type I collagen coated Petri dishes or in T-flasks and exposed to the indicated concentration of ethanol for 12 h. Ratios were calculated as the dpms in the protected fragment from the ethanol treated sample relative to that determined in untreated cells.
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FIG. 3. Ethanol stimulated editing in the absence of RNA synthesis. ApoB mRNA was amplified from wild type McArdle cells or McArdle cells expressing high levels of APOBEC-1 (McAPOBEC) that had been treated for 12 h with ethanol, ActD, DRB or ethanol plus an inhibitor. Editing was determined by poisoned primer extension and quantified from triplicate determinations as described in Materials and Methods. CAA, unedited RNA, UAA edited RNA and 1–3, promiscuous editing sites.
In contrast, the abundance of actin mRNA in cells treated with either 0.01% or 0.1% ethanol was equivalent to that in untreated cells. These data demonstrated that RNA synthesis is induced by ethanol and that transcription of the apoB gene may be more responsive to ethanol than that of house keeping genes. The data suggested a large activation of editing activity in the cell nucleus had occurred for not only had the proportion of edited apoB mRNAs increased but the size of the apoB mRNA population serving as editing substrates had also increased. RNA synthesis was not required for ethanol stimulated editing. If de novo synthesis of auxiliary proteins or apoB mRNA was required for ethanol stimulated editing activity then inhibition of RNA polymerase II should block the increase in edited apoB mRNA. To evaluate this, McArdle and McAPOBEC cells were treated with 0.4% ethanol or pretreated for 15 min with RNA polymerase II inhibitors (Act D or DRB) and then treated with ethanol. Surprisingly, cells pretreated with Act D or DRB and then treated with ethanol demonstrated marked stimulation of editing activity (Fig. 3). The proportion of edited apoB mRNA was higher in cells treated with inhibitor plus ethanol than that seen in cells treated with ethanol alone. An analysis of editing in cells treated with RNA polymerase II inhibitors alone showed that apoB mRNA editing was also markedly stimulated (Fig. 3). Treatment of cells with a low dose of Act D (0.04 g/ ml), which only inhibits ribosomal RNA synthesis, did not stimulate editing activity (data not shown). These data suggested that not only was mRNA synthesis not
required for ethanol to stimulate apoB mRNA editing but that the absence of mRNA synthesis was in itself permissive for enhanced editing activity. Promiscuous editing of apoB mRNA (the editing of additional cytidines 5⬘ of C6666 within apoB mRNA, evident as primer extension stops 1–3 in Fig. 3) was observed whenever APOBEC-1 was artificially overexpressed as in McAPOBEC cells (32–35). Interestingly, ethanol stimulated editing activity in wild type McArdle cells without inducing promiscuous editing. Promiscuous editing was not induced in McArdle cells by inhibiting RNA synthesis and remained unchanged in inhibitor treated McAPOBEC cells. The apparent reduction in promiscuous editing in ethanol treated McAPOBEC cells was not consistently observed. Protein synthesis is not required for ethanol stimulated editing. The stimulation of editing activity in RNA polymerase II inhibited and ethanol treated cells may have been due to increased translation of auxiliary proteins on pre-existing mRNAs. To evaluate the requirement of protein synthesis in ethanol regulated editing activity, McArdle and McAPOBEC cells were treated with either ethanol alone or pretreated with the protein synthesis inhibitor, emetine, and then treated with ethanol. Cells treated with emetine alone demonstrated a marked stimulation in apoB mRNA editing activity. Moreover, cells treated with emetine and ethanol demonstrated a more marked stimulation of apoB mRNA editing activity than that seen in cells treated with ethanol alone (Fig. 4). Moreover, These data demonstrate that de novo protein synthesis was not required for ethanol stimulated editing activity and suggested that pre-existing editing factors were suffi-
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FIG. 4. Ethanol stimulated editing in the absence of protein synthesis. ApoB mRNA was amplified from wild type McArdle cells or McAPOBEC cells that had been treated with ethanol, emetine or both for 12 h. Editing was quantified from triplicate determinations as described in Materials and Methods.
cient for the cellular response to ethanol. An intact cell system was required for regulating editing as in vitro editing activity was not affected by treating McArdle cell extracts with ethanol and/or RNA and protein synthesis inhibitors (data not shown). In contrast to wild type McArdle cells that were inhibited in RNA synthesis, McArdle cells inhibited in protein synthesis demonstrated a very weak band at position 1, suggesting a low level of promiscuous editing. Furthermore, ethanol treatment of cells which were inhibited in protein synthesis, not only stimulated editing at the wild type site but also stimulated promiscuous editing. Considering that promiscuous editing in wild type McArdle cells only occurred when protein synthesis was inhibited, and not when RNA synthesis was inhibited, the data suggested that a protein factor(s) translated from pre-existing mRNA(s) controlled promiscuous editing. The protein(s) became rate limiting however when protein synthesis was inhibited. This would be consistent with earlier studies suggesting that auxiliary proteins and cis-acting RNA sequences were responsible for the control of promiscuous editing (35).
McArdle cells induced apoptosis (Table 1). The data suggested that significant changes in cell viability were not induced in inhibitor treated cells. Taken together with data that demonstrated ethanol stimulated editing activity only in the nucleus (32), our findings indicated that the proportion of edited apoB mRNAs could be increased by modulating the activity of pre-existing editing factors. Activation of apoB mRNA editing by inhibitors of RNA polymerase II or protein synthesis may have resulted from a permissive condition established by the reduction of a protein(s) that suppressed efficiency. Several proteins with the capacity to inhibit apoB mRNA editing have been identified (11, 14, 15), yet their mechanism of action is unknown. These proteins may sequester auxiliary proteins and/or APOBEC-1 as inactive 60S complexes on the endoplasmic reticulum (9). In the absence of these editing suppressor proteins, more APOBEC-1 and/or auxiliary proteins may have translocated to the cell nucleus (9). Alternatively, the reduction in the suppressor protein(s) may have activated cytoplasmic editing factors, resulting in editing of cytoplasmic mRNAs. We cannot rule out the possibility that the increased proportion of edited apoB mRNAs in drug treated cells was due to a selective degradation of unedited apoB mRNAs. Other studies have suggested that nuclear apoB mRNA editing activity was activated by its uncoupling from the apoB pre-mRNA splicing pathway (36). In addition, recent data have suggested that protein kinases and protein phosphatases might regulate apoB mRNA editing through inhibitory and stimulatory phosphorylation sites on APOBEC-1 (30). Stimulation of editing activity by RNA and protein synthesis inhibitors or ethanol may have resulted from the decreased expression or activity of kinases or phosphatases that modify APOBEC-1 and/or the auxiliary proteins. Changes in post-translation modification could also affect editosome assembly and/or translocation of editing factors from the cytoplasm to the nucleus. Given the ability of ethanol to additionally stimulate editing activity in RNA or protein synthesis inhibited cells, it is possible that ethanol’s effect on editing involved a different set of pathways than those affected by RNA and protein synthesis inhibitors. Whether changes in the cellular microenvironment such as increased calcium
RNA and protein synthesis inhibited cells were viable. One explanation for the activation of apoB mRNA editing in RNA and protein synthesis inhibited cells is that cell death had enabled nuclear editing factors to leak into the cytoplasm where they acted upon cytoplasmic apoB mRNA. To evaluate this, cells were treated for 12 h with actinomycin D, DRB or emetine and then assayed for apoptosis using a flow cytometricbased TUNEL assay. Relative to negative and positive controls for apoptosis, none of the treatments of 1165
TABLE 1 Cell treatment
Percent apoptosis
Negative control Positive control McArdle control Act D DRB Emetine
0.4 30 0.9 0.9 1.5 1.3
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(37) or hypertonicity (23), which also increased the proportion of edited apoB mRNA, exerted their effect through pre-existing editing factors remains to be determined. In conclusion, although ethanol can stimulate macromolecular synthesis, it increased the proportion of edited apoB mRNA independent of de novo RNA or protein synthesis. The data also demonstrated that editing activity in cells expressing low or high APOBEC-1 can be stimulated by inhibiting the translation of proteins. An intact cellular system was required for this effect. These data suggested that in addition activators of apoB mRNA editing, homeostasis in the proportion of edited apoB mRNAs also required the expression of protein suppressors of apoB mRNA editing. We propose that the assembly of editosomes and/or their activity may be regulated through post-translational modification of editing factors and the expression of activator or suppressor auxiliary proteins. ACKNOWLEDGMENTS
8.
9.
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11.
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The authors thank Dr. Nazzareno Ballatori from the Environmental Health Sciences Center for the preparation of rat primary hepatocytes, Dr. Peter Keng from the James P. Wilmot Cancer Center for performing the TUNEL analyses, and Jenny M.L. Smith for the preparation of figures. This work was supported in part through a Public Health Service Grant DK43739 and a grant from the Alcohol Beverage Medical Research Foundation awarded to HCS.
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15.
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