Thyroid Hormone Regulates Hepatic Triglyceride Mobilization and Apolipoprotein B Messenger Ribonucleic Acid Editing in a Murine Model of Congenital Hy
Abstract|, http://www.100md.com
Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8-/-) that lacks thyroid follicular cells. Neonatal Pax8-/- mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8-/- mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8-/- mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.
Introduction5i[}*r, 百拇医药
IT HAS LONG been recognized that thyroid hormones (TH), specifically T3 and T4, regulate important elements of hepatic intermediary metabolism, particularly lipoprotein production and uptake (1, 2). For example, TH regulate the abundance of candidate genes involved in hepatic triglyceride production, including spot 14 and fatty acid transporter protein as well as a range of genes involved in hepatic lipogenesis, gluconeogenesis, and low density lipoprotein receptor expression (3, 4). The importance of these findings is underscored by the clinical findings that hypothyroidism in humans is associated with hyperlipidemia, which is reversible after TH supplementation (5).5i[}*r, 百拇医药
In attempting to elucidate the molecular mechanisms by which T3 and T4 modulate hepatic lipoprotein metabolism in vivo, numerous laboratories have employed pharmacological induction of hypothyroidism in rats followed by T3 administration to replicate the effects of TH deficiency and therapeutic supplementation, respectively, in vivo. This approach has yielded important information concerning the TH-dependent induction of low density lipoprotein receptor expression as well as hepatic apolipoprotein (apo) gene expression, notably that of apoA-I, apoA-IV, and apoB (6, 7, 8, 9). With regard to the regulation of hepatic apoB gene expression, this approach was particularly informative. Studies in hypothyroid rats demonstrated that hepatic apoB synthesis undergoes a T3-dependent switch in apoB isoform production after the administration of supraphysiological doses of TH (10). More specifically, these studies revealed the virtual elimination of apoB100 production in favor of the truncated species, apoB48 (10). The molecular mechanism underlying this switch in hepatic apoB isoform synthesis was later shown to reside in a T3-dependent induction of apoB mRNA editing, the posttranscriptional process by which a C to U change is introduced into the nuclear transcript encoding apoB and thereby directly alters the proportions of hepatic apoB isoforms synthesized (11). Exploration of the mechanisms involved is the subject of this report.
ApoB mRNA editing occurs in the mammalian intestine and liver of certain species (notably rats and mice) and requires a multicomponent enzyme complex that targets a single base presented in the context of a highly conserved RNA sequence (reviewed in Ref. 12). The core components of this complex include apobec-1, the RNA-specific catalytic deaminase, and apobec-1 complementation factor (ACF), the presumed RNA-binding subunit, each of which is indispensable to the reaction (13, 14, 15, 16, 17, 18). Although extensive information exists concerning the roles of these proteins in regulating apoB RNA editing in reconstituted systems in vitro, relatively little is known about their physiological regulation in vivo. As alluded to above, prior studies have demonstrated rat hepatic apoB RNA editing is regulated by TH and its active analogs, but the molecular mechanisms underlying this response have not been elucidated (9, 10, 19). Examination of the potential mechanisms underlying the TH-dependent increase in rat hepatic apoB mRNA editing revealed only that such regulation is independent of alterations in the abundance of apobec-1 mRNA (20).
The recent identification of ACF and its emergence as a critical core component of the apoB RNA editing holo-enzyme suggest that other possibilities need to be considered for the TH-dependent regulation of apoB RNA editing in rodent liver. We elected to approach this question using a murine model, because the availability of apobec-1-/- mice provides a convenient starting point for a dissection of the trans-acting factors involved (17, 18). However, our preliminary experiments with pharmacological models of hypothyroidism in wild-type (WT) mice indicated that changes in apoB RNA editing were more consistently noted during the neonatal period as opposed to adult animals (Plateroti, M., D. Mukhopadhyay, and N. O. Davidson, unpublished observations). Accordingly, in the present study we have examined the mechanisms of TH regulation of hepatic lipid metabolism and apoB RNA editing in a strain of congenitally hypothyroid mice, Pax8-/-, that lack thyroid follicular cells and are profoundly hypothyroid from birth (21). Because previous studies have demonstrated that hepatic apoB RNA editing is developmentally regulated (22), this was believed to represent a suitable model in which to analyze the molecular mechanisms involved in the developmental control of apoB RNA editing by TH. In addition, because Pax8-/- mice exhibit a severe intestinal phenotype, with extensive vacuolation of villus enterocytes and developmental immaturity (21), this model offers an opportunity to examine the tissue-specific effects of TH on potential target genes in both the small intestine and liver (23).
Materials and Methods2st%:), http://www.100md.com
Animals and tissue preparation2st%:), http://www.100md.com
Animals were housed and maintained according to published national guidelines and with approval from the animal experimental committee of the École Normale Supérieure de Lyon (Lyon, France). Two-week-old Pax8-/- animals were used (24) and were studied in comparison with their WT littermates. Groups of hypothyroid Pax8-/- mice were injected with a mixture of T4 and T3 (2.5 mg/kg T4 and 0.25 mg/kg T3 in 100 µl PBS daily for 2 or 4 d), as previously described (21). This regimen has been previously validated to produce hyperthyroidism in mice (25). At the conclusion of each experiment the animals were exsanguinated, serum was recovered, and the liver and small intestine were removed quickly, frozen in liquid nitrogen, and used for RNA and/or protein extraction and analysis.2st%:), http://www.100md.com
RNA extraction and analysis
RNA was extracted in TRI-reagent (Sigma-Aldrich, St. Louis, MO) and used for primer extension analysis of apolipoprotein B RNA editing as previously described (26). Briefly, samples (10 µg) of total RNA were treated with deoxyribonuclease I and subjected to RT, followed by PCR, using primers (5'-ATCTGACTGGGAGAGACAAGTAG-3' and 5'-CAAGCATTTTTAACTTTTCAATGATTCGATC-3') flanking a 253-bp region of murine apoB surrounding the edited base. Aliquots of apoB cDNA were annealed to a 32P end-labeled 35-mer antisense mouse apoB oligonucleotide (5'-AGTCATGTGGATCATAATTATCTTTAATATACTGA-3') and subjected to primer extension analysis using T7 DNA polymerase (26). The products were precipitated in ethanol, resolved by 8% polyacrylamide-urea gel electrophoresis, and subjected to autoradiography and phosphorimaging analysis (Molecular Dynamics, Inc., Sunnyvale, CA). The ratio of edited (apoB48) to unedited apoB (apoB100) cDNA was determined using ImageQuant software (BD Biosciences, Mountain View, CA). Real-time PCR analysis used random hexanucleotide priming and amplification in an iCycler (Bio-Rad Laboratories, Inc., Richmond, CA) using SYBR Green PCR master mix according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). The primers were: apoB, 5-ATGTACTAATTGCCATAGATAGTGCCA-3' and 5'-TAGTTCTTTTTAAGTCATGTGGATCATAATTAT-3' (product size, 129 bp); apobec-1, 5'-ACCACACGGATCAGCGAAA-3' and 5'-TCATGATCTGGATAGTCACACCG-3 (product size, 72 bp); ACF, 5'-AGCCAGAATCCTGCAATCC-3' and 5'-AGCATACCTCTTCGCTTCATCC-3' (product size, 75 bp); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GGCAAATTCAACGGCACAGT-3' and AGATGGTGATGGGCTTCCC-3' (product size, 70 bp). The data from the PCR were normalized to GAPDH levels in each sample. Analysis of the murine ACF gene will be reported in complete detail elsewhere, but primers were prepared to detect the major alternatively spliced mRNA species. The primer set was: P11, 5'-GGTGGGGAACCTCAGAAATTGC-3; and P12, 5'-TGGCTTCCTGCTTATTTGAGAATG-3'. The expected products from a PCR with this primer set are 399, 255, and 120 bp, respectively.
Nuclear and cytoplasmic protein extraction, Western blot, and editing activity analysis;, http://www.100md.com
Nuclear and cytoplasmic S100 extracts were prepared as originally detailed by Dignam et al. (27) with minor modifications as recently described (26). Briefly, 100 mg tissue were minced and suspended at 4 C in hypotonic buffer A [10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 mm EDTA, 1 mM dithiothreitol (DTT), containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonylfluoride (PMSF), and 0.5 mm benzamidine]; adjusted to 300 mM HEPES, 1.4 mM KCl, and 30 mM MgCl2; and homogenzied with a Dounce type B pestle (Kontes Co., Vineland, NJ). After a low speed spin at 3000 rpm for 5 min in an SS34 rotor (DuPont-Sorvall Instruments, Asheville, NC), the pellet was resuspended in 3 ml buffer C (20 mM HEPES, 500 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, and 1 mM DTT containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM PMSF, and 0.5 mm benzamidine), rehomogenized with a Dounce B pestle, and centrifuged at 15,000 rpm for 10 min in an SS 34 rotor to yield nuclear extracts. Cytoplasmic extracts were prepared using the supernatant fraction from a 100,000 x g ultracentrifugation spin of the initial homogenate. This supernatant was dialyzed overnight into 20 mM HEPES (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA. 0.5 mM DTT, 0.5 mM benzamidine, 0.5 mM PMSF, and stored at -70 C. Preparations of total S100 extract were generated as previously detailed (26). These extracts include both nuclear and cytoplasmic compartments. Western blot analysis was performed on aliquots of nuclear or cytoplasmic S100 extract and the indicated amount of recombinant ACF (14). Serum apoB analysis was conducted on 2 µl serum in a 4–12% sodium dodecyl sulfate gradient gel. Membranes were probed with antibodies against apoB (28), apobec-1 (26), ACF (14), 40-kDa heat shock protein (HSP40; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and HuR (29) and were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). In vitro editing assays were conducted using 75 µg S100 extract with 20 fmol of a 470-nucleotide rat apoB RNA template for 3 h at 30 C in the presence or absence of recombinant ACF (14). C to U editing activity was determined by phosphorimager analysis of primer extension products as described above.
Lipid accumulation and immunohistochemistry/7cg5z, 百拇医药
Dissected livers were immediately fixed by immersion in 4% paraformaldehyde in buffered PBS, pH 7.2. For lipid detection, 10-µm sections were stained with Oil Red-O (Sigma-Aldrich) after immersion of the sections in propylene glycol and were lightly counterstained in hematoxylin. Quantitative estimation of hepatic lipid accumulation was accomplished by extraction of hepatic lipids from cell homogenates using chloroform/methanol (2:1) and enzymatic assay of triglyceride mass using a commercial kit (Wako Pure Chemical Industries, Inc., Osaka, Japan). ACF immunohistochemistry was performed on 5-µm sections. The slides were first rehydrated and then microwaved for 15 min in citrate buffer (0.01 M, pH 6) before incubation with a 1:500 dilution of a polyclonal rabbit anti-ACF antibody (14), followed by incubation with a secondary goat antirabbit biotinylated antibody and streptavidin-peroxidase detection (Histomouse, Zymed Laboratories, Inc., San Francisco, CA).
Results46v, http://www.100md.com
Analysis of hepatic lipid accumulation in neonatal Pax8-/- animals46v, http://www.100md.com
Oil Red-O staining for neutral lipid revealed extensive accumulation of lipid droplets within hepatocytes of neonatal Pax8-/- animals compared with their WT littermates (compare Fig. 1, A and B), with progressive elimination after TH administration (Fig. 1C). These qualitative impressions were confirmed by assay of hepatic triglyceride mass, which demonstrated an approximately 10-fold increase in hepatic triglyceride content in hypothyroid animals and progressive reversal over 2 and 4 d of treatment with TH (Fig. 1D). There was a corresponding increase in serum triglyceride content after TH treatment for 4 d (Fig. 1E), although serum cholesterol content was not significantly altered (data not shown).46v, http://www.100md.com
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Figure 1. Hepatic lipid accumulation in Pax8-/-mice. A–C, Oil Red-O staining of liver sections. Sections of mouse liver from the indicated genotype were incubated with Oil Red-O, and neutral lipid was visualized as detailed in Materials and Methods. A, WT; B, Pax8-/-; C, Pax8-/- treated with TH for 96 h before death. Magnification, x200. These are representative sections from six animals per genotype and treatment. D, Hepatic triglyceride mass was determined after lipid extraction and enzymatic assay as described in Materials and Methods. The data are derived from six animals per group and are expressed as micrograms of triglyceride per milligram of cell protein (mean ± SEM). E, Serum triglyceride levels (n = 8–10 animals/group) determined by enzymatic assay.
ApoB gene expression in hypothyroid Pax8-/- mice@1h, http://www.100md.com
We next examined apoB mRNA abundance and the extent of C to U editing of apoB RNA in the liver and small intestine of neonatal Pax8-/- mice. In WT animals, apoB48 RNA accounted for approximately 60% of the hepatic apoB mRNA population (Fig. 2A, lanes 1–3), similar to previous findings (19, 22). By contrast, hypothyroid Pax8-/- mice showed a significant decrease in apoB editing (38% apoB48; Fig. 2A, lanes 4 and 5) with a return to WT levels after TH injection for 2 (Fig. 2A, lanes 6–8) or 4 d (Fig. 2A, lanes 9 and 10). The temporal pattern of response of hepatic apoB RNA editingto TH administration in hypothyroid neonatal mice is similar to that described in adult rats after TH administration (6, 9, 10, 11); these findings imply that this response is not confined to a single rodent species. This was an important demonstration, because previous studies of hepatic apoB mRNA editing in mice failed to demonstrate regulation after fasting (30), an established mechanism for modulating apoB mRNA editing in rat liver (12). The changes noted in hepatic apoB RNA editing and triglyceride mobilization were accompanied by a temporally coincident appearance of apoB48 in the plasma of TH-treated animals, particularly at 4 d of TH administration (Fig. 2B). Taken together, the data suggest that TH treatment of Pax8-/- mice regulates hepatic apoB mRNA editing in association with increased abundance of apoB48 in plasma.
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Figure 2. A, Endogenous hepatic apoB mRNA editing in WT and Pax8-/- mice. Upper panel, ApoB mRNA editing was determined by primer extension analysis. The primer extension products were separated on a 10% acrylamide gel containing 8 M urea and autoradiographed. A representative assay from three independent experiments is shown. Lanes 1–3, WT; lanes 4–10, Pax8-/- mice; lanes 4 and 5, untreated (Ctrl); lanes 6–8, TH-treated (48 h); lanes 9 and 10, TH-treated (96 h). The locations of the primer (P), unedited (C; apoB100), and edited (U; apoB48) products are indicated to the right of the gel. Lower panel, Bar graph summarizing the data (mean ± SEM; n = 6) for each group. B, Western blot analysis of apoB in serum. Serum (2 µl) was electrophoresed in 4% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. The membrane was blocked and incubated with rabbit antimouse apoB antiserum and visualized by enhanced chemiluminescence. Lanes 1–3, WT; lanes 4–11, Pax8-/- mice; lanes 4–6, untreated (Ctrl); lanes 7 and 8, TH-treated (48 h); lanes 9–11, TH-treated (96 h). The migrations of apoB 100 and apoB 48 are indicated by arrows. This is a representative of three independent experiments.
Because apoB48 arises from both the small intestine and the liver of mice, we examined the effects of TH administration on intestinal apoB RNA editing. Analysis of intestinal RNA from these same groups of animals showed that both apoB mRNA abundance and C to U RNA editing were unchanged despite alterations in TH status (data not shown). Consequently, our further studies focused on the liver to examine the mechanisms accounting for the tissue-specific regulation of apoB mRNA editing.1v1f7tp, 百拇医药
Alterations in the expression of the apoB RNA editing core components, apobec-1 and ACF1v1f7tp, 百拇医药
ApoB RNA editing requires a minimal core complex composed of apobec-1, the catalytic deaminase, and ACF, the RNA-binding complementation factor (12). We performed real-time PCR to quantify these mRNAs in the liver and small intestine of Pax8-/- mice compared with their WT littermates and with animals injected with TH. There was no change in apobec-1 mRNA abundance in hypothyroid Pax8-/- animals both in comparison with WT controls and after and injection of TH for 2 or 4 d (Fig. 3A). By contrast, ACF mRNA abundance was decreased in the liver of hypothyroid Pax8-/- mice, with a significant increase noted after TH administration (Fig. 3A). Total hepatic apoB mRNA abundance was also unchanged among the groups compared with WT levels (Fig. 3A).
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Figure 3. A, Effect of TH treatment on apoB, apobec-1, and ACF mRNA abundance in the liver. Transcript abundance was determined by real-time PCR analysis as described in Materials and Methods. The data (n = 6/group) were normalized to GAPDH levels in each sample. The data are expressed as normalized mRNA abundance relative to WT animals. *, Significant difference (P < 0.001) in ACF mRNA expression in livers of Pax8-/- mice compared with both WT and TH-treated Pax8-/- mice. B, Effect of TH treatment on apobec-1 and ACF protein expression. Liver S100 extract (75 µg) obtained from the indicated groups was resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with specific polyclonal antisera to apobec-1 and ACF or HSP40 (control) and visualized by enhanced chemiluminescence. Migration markers are indicated on the left. This is a representative of three independent experiments.!w)4m-, 百拇医药
Recent studies have demonstrated the presence of multiple, alternatively spliced forms of ACF mRNA in both human and rat liver, up to 25% of which encode functionally inactive variants (31, 32). PCR amplification of the major splice forms of ACF mRNA revealed no alteration in the proportion of the major forms in either liver or intestine (data not shown) under any condition examined. Thus, the decrease in total ACF mRNA abundance noted above does not reflect an altered transcript distribution between functional and inactive isoforms.
To examine apobec-1 and ACF protein expression, we also performed Western blot analysis on hepatic S100 extracts from animals in the different treatment groups. Apobec-1 protein content was comparable across all genotypes and treatment groups (Fig. 3B). On the other hand, ACF protein expression was decreased in the hypothyroid Pax8-/- mice and was increased after TH administration (Fig. 3B), findings consistent with the predictions from the mRNA quantitation above.4#, 百拇医药
Immunohistochemical localization of ACF in sections of liver tissue suggests that this protein is localized in both cytoplasmic and nuclear compartments in WT mice (Fig. 4A). In the Pax8-/- mutant animals a similar, but less intense, staining pattern was revealed (Fig. 4B) TH administration resulted in an increased staining intensity for ACF, particularly within the nucleus (Fig. 4C. Serial sections were examined to quantitate the distribution of nuclear staining in the respective genotypes and treatment groups. Nuclear staining was demonstrated in 10% of hepatocytes (40 of 390 hepatocytes counted) in WT mice compared with 4% in Pax8-/- (17 of 420 hepatocytes counted; P < 0.0005) vs. 40% of hepatocytes in TH-treated Pax8-/- animals (169 of 425 hepatocytes counted; P < 0.0001). These data raise the strong possibility that TH administration to Pax8-/- mice may modulate the intracellular location of ACF in addition to its abundance. This possibility was further addressed using Western blotting of isolated nuclear and cytoplasmic extracts from the various treatment groups. WT mice demonstrate immunoreactive ACF in both nuclear and cytoplasmic extracts. However, as shown in Fig. 4D, there was a marked induction of ACF protein expression in nuclear extracts at 48 and 96 h of TH treatment, which exceeds that observed in cytoplasmic extracts (compare also with Fig. 3B). These data collectively suggest that TH may regulate nuclear expression of ACF in murine liver.
fig.ommitteed+})mw, 百拇医药
Figure 4. A–C, Immunohistochemical localization of hepatic ACF in WT mice, Pax8-/- mice, and Pax8-/- mice treated with TH. Paraffin sections (5 µM) were prepared from the indicated animals and reacted with polyclonal ACF antisera as described in Materials and Methods. This is a representative illustration from three replicate experiments. Intense brown staining of nuclei is particularly evident in C. D, Western blot analysis of ACF abundance in nuclear (N) and cytoplasmic (C) extracts from WT mice, Pax8-/- mice, and Pax8-/- mice treated with TH for 48 or 96 h. One hundred micrograms of protein were used in each lane, and the blots were probed sequentially with anti-ACF antisera, followed by anti-HSP40 or anti-HuR to demonstrate equivalent loading and transfer of cytosolic and nuclear proteins, respectively. This is a representative experiment that was performed in duplicate.+})mw, 百拇医药
Analysis of apoB RNA editing activity in liver extracts: supplementation with recombinant ACF rescues the defect in C to U RNA editing
S100 extracts were prepared from WT as well as Pax8-/- animals, both untreated and treated with TH for 2 and 4 d. These extracts include both nuclear and cytoplasmic compartments, aliquots of which were used for in vitro editing assays where the extent of C to U RNA editing of a synthetic template is determined. The results of these studies, shown in Fig. 5, confirm the predictions emerging from the in vivo analysis of endogenous apoB RNA editing, namely an approximately 50% decrease in the enzymatic (in vitro) editing activity of liver S100 extracts prepared from Pax8-/- animals and recovery of editing activity after TH administration.3aj.9, http://www.100md.com
fig.ommitteed3aj.9, http://www.100md.com
Figure 5. In vitro ApoB RNA editing activity in liver S100 extracts from WT and Pax8-/- mice. In vitro conversions were performed using 75 µg S100 extract, 20 fmol of a 470-nucleotide rat apoB cRNA template, and incubation at 30 C for 3 h. The products were precipitated and analyzed by denaturing urea-acrylamide gel electrophoresis as described in Materials and Methods. The bar graph demonstrates RNA editing activity from each group of animals (n = 4/group). The data are presented as femtomoles of RNA deaminated per microgram of S100 extract per hour (mean ± SD).
To determine conclusively that this rescue was accounted for by the increase in ACF protein, the abundance of ACF protein was estimated from Western blots of S100 extracts prepared using increasing amounts of protein compared with standards of recombinant ACF (Fig. 6). S100 extracts from Pax8-/- mice were then supplemented with increasing amounts of recombinant ACF to supply the equivalent quantities of ACF estimated to be present in TH-treated S100 liver extracts, as judged in comparison with the recombinant protein standards analyzed on the same blots (Fig. 6, upper panel). Unsupplemented extracts from Pax8-/- mice yielded approximately 17% editing of a synthetic RNA template (Fig. 6, lower panel). Supplementation with increasing amounts of recombinant ACF (25–100 ng) completely restored editing activity to that found in WT and TH-treated animals (Fig. 6, lower panel, compare lanes 3–5 to lane 2), demonstrating that alterations in ACF alone account for the decrease in editing activity in Pax8 mutants. Accordingly, the data suggest that treatment with TH restores apoB RNA editing activity in hypothyroid mice most likely through a direct effect mediated by ACF.
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Figure 6. Addition of recombinant ACF to liver S100 extracts from Pax8-/- mice rescues in vitro apoB RNA editing. Upper panel, Quantitation of ACF protein levels in hepatic S100 extracts. Seventy-five micrograms of S100 extract from WT (lane 1), Pax8-/- untreated (lane 2), and Pax8-/- TH-treated (lane 3) mice were analyzed by SDS-PAGE and Western blot analysis with anti-ACF polyclonal antisera. Increasing amounts (5–50 µg) of recombinant ACF protein (lanes 4–6) were included on the gel to estimate the amount of ACF protein present in the S100 extracts. Lower panel, Rescue of editing activity in Pax8-/- liver S100 extract by supplementation with recombinant ACF. C to U editing assays were performed using 75 µg Pax8-/- S100 extract, either unsupplemented (lane 2) or supplemented with recombinant ACF (25–100 ng; lanes 3–5). Control incubations, shown in lane 1, contained only apoB RNA. This is a representative of three independent experiments.&5#(], 百拇医药
Discussion&5#(], 百拇医药
Regulation of murine hepatic apoB mRNA editing by TH provides an important opportunity to understand the molecular basis by which this posttranscriptional process is modulated in vivo. Such insight may provide clues to the presumed metabolic advantage of apoB48, the product of the edited mRNA, in comparison with apoB100. The current studies provide several lines of evidence to suggest that TH regulates hepatic apoB mRNA editing in neonatal mice through alterations in the abundance of ACF. Furthermore, this mechanism is sufficient to account for the alterations in editing efficiency noted both in vivo and in vitro. Several aspects of these findings merit additional consideration.
The data indicate that Pax8-/- mice exhibit hepatic triglyceride accumulation, which is reversed upon administration of TH. These findings taken together with the temporally coincident increase in apoB mRNA editing lend support to the possibility that hepatic triglyceride secretion may be facilitated in association with the switch in production of the apoB isoforms to favor apoB48. Although the current findings demonstrate that hepatic triglyceride content is decreased along with an increase in plasma triglyceride concentration, further analysis will be necessary to determine whether hepatic very low density lipoprotein assembly and secretion are augmented in association with this change in apoB100 and apoB48 mRNA populations. Such a prediction, however, would be consistent with studies from rat models (6, 7, 8, 9). Data from apobec-1-targeted mice, which express only apoB100, suggest that there is no obvious phenotype with regard to intestinal lipid uptake (17, 18), although more recent findings suggest that there may be some preference for preformed vs. newly synthesized triglyceride that distinguishes the different isoforms in mouse enterocytes (33). The implications of these findings in relation to hepatic triglyceride mobilization in TH-treated neonatal mice will clearly require thorough analysis in the future.
The current studies demonstrate that ACF, the RNA-binding subunit of the core apoB RNA editing holo-enzyme, undergoes metabolic regulation in response to changes in TH status in vivo. This is the first demonstration of physiological metabolic regulation of this gene in any mammalian species. Recent findings revealed that ACF expression in human intestine was unchanged during fetal development, over a period during which endogenous apoB RNA editing undergoes a significant increase (31). These findings taken together with the current demonstration that hepatic (but not intestinal) apoB RNA editing is regulated, with accompanying changes in ACF gene expression, by TH suggest that this metabolic regulation is tissue specific. The data further suggest a concordant temporal response in both mRNA and protein abundance for ACF along with the suggestion, from immunochemical data, that TH treatment results in increased intensity of nuclear staining for ACF. These qualitative findings were complemented by Western blot analysis of nuclear extracts from TH-treated animals, which confirmed the increased abundance of ACF protein more directly. It remains to be determined whether there is regional or zonal regulation of ACF expression within the hepatic lobule. Recent studies (34) in rat liver suggested that ACF expression was localized to a centrizonal rim of cells surrounding the hepatic vein. This particular distribution pattern was not specifically noted in our studies, but we suspect that more extensive evaluation will be necessary to comment with certainty upon the regional expression patterns of ACF within hepatocytes.
Regardless of the possible regional or zonal patterns of ACF expression within the hepatocyte lobule, our studies raise the intriguing possibility of hormonal regulation of ACF distribution between nuclear and cytoplasmic compartments of the cell. The current findings demonstrating a shift in hepatic ACF distribution to the nucleus after TH administration are consistent with a recent report demonstrating ACF redistribution after exposure of primary rat hepatocytes to either insulin or ethanol (34). The current evidence is somewhat conflicting concerning the normal distribution of ACF within cultured cells other than of hepatic origin. Our own data in COS-7 cells suggest that ACF is predominantly nuclear (14, 35), whereas data from other studies have suggested both a cytoplasmic and a nuclear localization pattern (34). Differences in antisera and in the preparation of cells used for these studies may underlie the apparent discrepancies, but this is an issue that will require further study.4a6k${5, http://www.100md.com
Finally, as alluded to above, the increased abundance of ACF mRNA in response to TH treatment suggests, among other possibilities, that a TH response element (TRE) may be present in the murine acf gene. Preliminary analysis of the murine chromosomal acf locus using available databases indicates the presence of two imperfect TRE half-sites (TGACCcaA and TGgCCTGA at nucleotide positions -26 and -397, respectively) and a perfect TRE half-site (TGACCTGA at nucleotide position -727) upstream of the initiator methionine (Newberry, E., and N. Davidson, unpublished observations). These findings will require formal experimental analysis to assess their functional relevance. In addition, the active form of TH, T3, mediates its actions by high affinity interaction with distinctive subtypes of TH receptor, all of which are members of a superfamily of nuclear hormone receptors. TH receptors are encoded by two distinct genes, with further heterogeneity imposed by the use of alternative promoters and differential splicing (25, 36). The demonstration of TH modulation of an important metabolic process in relation to hepatic lipid processing naturally leads to the question of which receptor pathway may be involved in this process. This and other related issues will be the focus of future reports.
Acknowledgments\:/'#-., http://www.100md.com
In particular, we acknowledge Karen Hutton and Randal May in the Morphology Core of the Digestive Disease Research Core Center. We thank Denise Aubert, who manages the transgenic facility at École Normale Supérieure de Lyon, and N. Aguilera and C. Morin for animal care and breeding and Imed Gallouzi (University of Montréal) for the gift of HuR antisera.\:/'#-., http://www.100md.com
Received July 22, 2002.\:/'#-., http://www.100md.com
Accepted for publication October 29, 2002.\:/'#-., http://www.100md.com
References\:/'#-., http://www.100md.com
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Anant S, Henderson JO, Mukhopadhyay D, Navaratnam N, Kennedy S, Min J, Davidson NO 2001 Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing: CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor. J Biol Chem 276:47338–47351)8}+2f', 百拇医药
Blanc V, Navaratnam N, Henderson JO, Anant S, Kennedy S, Jarmuz A, Scott J, Davidson NO 2001 Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing. J Biol Chem 276:10272–10283
Mehta A, Kinter MT, Sherman NE, Driscoll DM 2000 Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol Cell Biol 20:1846–1854cl!8v, http://www.100md.com
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Hirano K, Young SG, Farese Jr RV, Ng J, Sande E, Warburton C, Powell-Braxton LM, Davidson NO 1996 Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48. J Biol Chem 271:9887–9890cl!8v, http://www.100md.com
Morrison JR, Paszty C, Stevens ME, Hughes SD, Forte T, Scott J, Rubin EM 1996 Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism. Proc Natl Acad Sci USA 93:7154–7159cl!8v, http://www.100md.com
Funahashi T, Giannoni F, DePaoli AM, Skarosi SF, Davidson NO 1995 Tissue-specific, developmental and nutritional regulation of the gene encoding the catalytic subunit of the rat apolipoprotein B mRNA editing enzyme: functional role in the modulation of apoB mRNA editing. J Lipid Res 36:414–428
Inui Y, Hausman AM, Nanthakumar N, Henning SJ, Davidson NO 1992 Apolipoprotein B messenger RNA editing in rat liver: developmental and hormonal modulation is divergent from apolipoprotein A-IV gene expression despite increased hepatic lipogenesis. J Lipid Res 33:1843–18569';)63u, http://www.100md.com
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Henderson JO, Blanc V, Davidson NO 2001 Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene. Biochim Biophys Acta 1522:22–304&hyc, http://www.100md.com
Dance GS, Sowden MP, Cartegni L, Cooper E, Krainer AR, Smith HC 2002 Two proteins essential for apolipoprotein B mRNA editing are expressed from a single gene through alternative splicing. J Biol Chem 277:12703–127094&hyc, http://www.100md.com
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Blanc V, Henderson JO, Kennedy S, Davidson NO 2001 Mutagenesis of apobec-1 complementation factor reveals distinct domains that modulate RNA binding, protein-protein interaction with apobec-1, and complementation of C to U RNA-editing activity. J Biol Chem 276:46386–463934&hyc, http://www.100md.com
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Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8-/-) that lacks thyroid follicular cells. Neonatal Pax8-/- mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8-/- mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8-/- mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.
Introduction5i[}*r, 百拇医药
IT HAS LONG been recognized that thyroid hormones (TH), specifically T3 and T4, regulate important elements of hepatic intermediary metabolism, particularly lipoprotein production and uptake (1, 2). For example, TH regulate the abundance of candidate genes involved in hepatic triglyceride production, including spot 14 and fatty acid transporter protein as well as a range of genes involved in hepatic lipogenesis, gluconeogenesis, and low density lipoprotein receptor expression (3, 4). The importance of these findings is underscored by the clinical findings that hypothyroidism in humans is associated with hyperlipidemia, which is reversible after TH supplementation (5).5i[}*r, 百拇医药
In attempting to elucidate the molecular mechanisms by which T3 and T4 modulate hepatic lipoprotein metabolism in vivo, numerous laboratories have employed pharmacological induction of hypothyroidism in rats followed by T3 administration to replicate the effects of TH deficiency and therapeutic supplementation, respectively, in vivo. This approach has yielded important information concerning the TH-dependent induction of low density lipoprotein receptor expression as well as hepatic apolipoprotein (apo) gene expression, notably that of apoA-I, apoA-IV, and apoB (6, 7, 8, 9). With regard to the regulation of hepatic apoB gene expression, this approach was particularly informative. Studies in hypothyroid rats demonstrated that hepatic apoB synthesis undergoes a T3-dependent switch in apoB isoform production after the administration of supraphysiological doses of TH (10). More specifically, these studies revealed the virtual elimination of apoB100 production in favor of the truncated species, apoB48 (10). The molecular mechanism underlying this switch in hepatic apoB isoform synthesis was later shown to reside in a T3-dependent induction of apoB mRNA editing, the posttranscriptional process by which a C to U change is introduced into the nuclear transcript encoding apoB and thereby directly alters the proportions of hepatic apoB isoforms synthesized (11). Exploration of the mechanisms involved is the subject of this report.
ApoB mRNA editing occurs in the mammalian intestine and liver of certain species (notably rats and mice) and requires a multicomponent enzyme complex that targets a single base presented in the context of a highly conserved RNA sequence (reviewed in Ref. 12). The core components of this complex include apobec-1, the RNA-specific catalytic deaminase, and apobec-1 complementation factor (ACF), the presumed RNA-binding subunit, each of which is indispensable to the reaction (13, 14, 15, 16, 17, 18). Although extensive information exists concerning the roles of these proteins in regulating apoB RNA editing in reconstituted systems in vitro, relatively little is known about their physiological regulation in vivo. As alluded to above, prior studies have demonstrated rat hepatic apoB RNA editing is regulated by TH and its active analogs, but the molecular mechanisms underlying this response have not been elucidated (9, 10, 19). Examination of the potential mechanisms underlying the TH-dependent increase in rat hepatic apoB mRNA editing revealed only that such regulation is independent of alterations in the abundance of apobec-1 mRNA (20).
The recent identification of ACF and its emergence as a critical core component of the apoB RNA editing holo-enzyme suggest that other possibilities need to be considered for the TH-dependent regulation of apoB RNA editing in rodent liver. We elected to approach this question using a murine model, because the availability of apobec-1-/- mice provides a convenient starting point for a dissection of the trans-acting factors involved (17, 18). However, our preliminary experiments with pharmacological models of hypothyroidism in wild-type (WT) mice indicated that changes in apoB RNA editing were more consistently noted during the neonatal period as opposed to adult animals (Plateroti, M., D. Mukhopadhyay, and N. O. Davidson, unpublished observations). Accordingly, in the present study we have examined the mechanisms of TH regulation of hepatic lipid metabolism and apoB RNA editing in a strain of congenitally hypothyroid mice, Pax8-/-, that lack thyroid follicular cells and are profoundly hypothyroid from birth (21). Because previous studies have demonstrated that hepatic apoB RNA editing is developmentally regulated (22), this was believed to represent a suitable model in which to analyze the molecular mechanisms involved in the developmental control of apoB RNA editing by TH. In addition, because Pax8-/- mice exhibit a severe intestinal phenotype, with extensive vacuolation of villus enterocytes and developmental immaturity (21), this model offers an opportunity to examine the tissue-specific effects of TH on potential target genes in both the small intestine and liver (23).
Materials and Methods2st%:), http://www.100md.com
Animals and tissue preparation2st%:), http://www.100md.com
Animals were housed and maintained according to published national guidelines and with approval from the animal experimental committee of the École Normale Supérieure de Lyon (Lyon, France). Two-week-old Pax8-/- animals were used (24) and were studied in comparison with their WT littermates. Groups of hypothyroid Pax8-/- mice were injected with a mixture of T4 and T3 (2.5 mg/kg T4 and 0.25 mg/kg T3 in 100 µl PBS daily for 2 or 4 d), as previously described (21). This regimen has been previously validated to produce hyperthyroidism in mice (25). At the conclusion of each experiment the animals were exsanguinated, serum was recovered, and the liver and small intestine were removed quickly, frozen in liquid nitrogen, and used for RNA and/or protein extraction and analysis.2st%:), http://www.100md.com
RNA extraction and analysis
RNA was extracted in TRI-reagent (Sigma-Aldrich, St. Louis, MO) and used for primer extension analysis of apolipoprotein B RNA editing as previously described (26). Briefly, samples (10 µg) of total RNA were treated with deoxyribonuclease I and subjected to RT, followed by PCR, using primers (5'-ATCTGACTGGGAGAGACAAGTAG-3' and 5'-CAAGCATTTTTAACTTTTCAATGATTCGATC-3') flanking a 253-bp region of murine apoB surrounding the edited base. Aliquots of apoB cDNA were annealed to a 32P end-labeled 35-mer antisense mouse apoB oligonucleotide (5'-AGTCATGTGGATCATAATTATCTTTAATATACTGA-3') and subjected to primer extension analysis using T7 DNA polymerase (26). The products were precipitated in ethanol, resolved by 8% polyacrylamide-urea gel electrophoresis, and subjected to autoradiography and phosphorimaging analysis (Molecular Dynamics, Inc., Sunnyvale, CA). The ratio of edited (apoB48) to unedited apoB (apoB100) cDNA was determined using ImageQuant software (BD Biosciences, Mountain View, CA). Real-time PCR analysis used random hexanucleotide priming and amplification in an iCycler (Bio-Rad Laboratories, Inc., Richmond, CA) using SYBR Green PCR master mix according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). The primers were: apoB, 5-ATGTACTAATTGCCATAGATAGTGCCA-3' and 5'-TAGTTCTTTTTAAGTCATGTGGATCATAATTAT-3' (product size, 129 bp); apobec-1, 5'-ACCACACGGATCAGCGAAA-3' and 5'-TCATGATCTGGATAGTCACACCG-3 (product size, 72 bp); ACF, 5'-AGCCAGAATCCTGCAATCC-3' and 5'-AGCATACCTCTTCGCTTCATCC-3' (product size, 75 bp); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GGCAAATTCAACGGCACAGT-3' and AGATGGTGATGGGCTTCCC-3' (product size, 70 bp). The data from the PCR were normalized to GAPDH levels in each sample. Analysis of the murine ACF gene will be reported in complete detail elsewhere, but primers were prepared to detect the major alternatively spliced mRNA species. The primer set was: P11, 5'-GGTGGGGAACCTCAGAAATTGC-3; and P12, 5'-TGGCTTCCTGCTTATTTGAGAATG-3'. The expected products from a PCR with this primer set are 399, 255, and 120 bp, respectively.
Nuclear and cytoplasmic protein extraction, Western blot, and editing activity analysis;, http://www.100md.com
Nuclear and cytoplasmic S100 extracts were prepared as originally detailed by Dignam et al. (27) with minor modifications as recently described (26). Briefly, 100 mg tissue were minced and suspended at 4 C in hypotonic buffer A [10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 mm EDTA, 1 mM dithiothreitol (DTT), containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonylfluoride (PMSF), and 0.5 mm benzamidine]; adjusted to 300 mM HEPES, 1.4 mM KCl, and 30 mM MgCl2; and homogenzied with a Dounce type B pestle (Kontes Co., Vineland, NJ). After a low speed spin at 3000 rpm for 5 min in an SS34 rotor (DuPont-Sorvall Instruments, Asheville, NC), the pellet was resuspended in 3 ml buffer C (20 mM HEPES, 500 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, and 1 mM DTT containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM PMSF, and 0.5 mm benzamidine), rehomogenized with a Dounce B pestle, and centrifuged at 15,000 rpm for 10 min in an SS 34 rotor to yield nuclear extracts. Cytoplasmic extracts were prepared using the supernatant fraction from a 100,000 x g ultracentrifugation spin of the initial homogenate. This supernatant was dialyzed overnight into 20 mM HEPES (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA. 0.5 mM DTT, 0.5 mM benzamidine, 0.5 mM PMSF, and stored at -70 C. Preparations of total S100 extract were generated as previously detailed (26). These extracts include both nuclear and cytoplasmic compartments. Western blot analysis was performed on aliquots of nuclear or cytoplasmic S100 extract and the indicated amount of recombinant ACF (14). Serum apoB analysis was conducted on 2 µl serum in a 4–12% sodium dodecyl sulfate gradient gel. Membranes were probed with antibodies against apoB (28), apobec-1 (26), ACF (14), 40-kDa heat shock protein (HSP40; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and HuR (29) and were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). In vitro editing assays were conducted using 75 µg S100 extract with 20 fmol of a 470-nucleotide rat apoB RNA template for 3 h at 30 C in the presence or absence of recombinant ACF (14). C to U editing activity was determined by phosphorimager analysis of primer extension products as described above.
Lipid accumulation and immunohistochemistry/7cg5z, 百拇医药
Dissected livers were immediately fixed by immersion in 4% paraformaldehyde in buffered PBS, pH 7.2. For lipid detection, 10-µm sections were stained with Oil Red-O (Sigma-Aldrich) after immersion of the sections in propylene glycol and were lightly counterstained in hematoxylin. Quantitative estimation of hepatic lipid accumulation was accomplished by extraction of hepatic lipids from cell homogenates using chloroform/methanol (2:1) and enzymatic assay of triglyceride mass using a commercial kit (Wako Pure Chemical Industries, Inc., Osaka, Japan). ACF immunohistochemistry was performed on 5-µm sections. The slides were first rehydrated and then microwaved for 15 min in citrate buffer (0.01 M, pH 6) before incubation with a 1:500 dilution of a polyclonal rabbit anti-ACF antibody (14), followed by incubation with a secondary goat antirabbit biotinylated antibody and streptavidin-peroxidase detection (Histomouse, Zymed Laboratories, Inc., San Francisco, CA).
Results46v, http://www.100md.com
Analysis of hepatic lipid accumulation in neonatal Pax8-/- animals46v, http://www.100md.com
Oil Red-O staining for neutral lipid revealed extensive accumulation of lipid droplets within hepatocytes of neonatal Pax8-/- animals compared with their WT littermates (compare Fig. 1, A and B), with progressive elimination after TH administration (Fig. 1C). These qualitative impressions were confirmed by assay of hepatic triglyceride mass, which demonstrated an approximately 10-fold increase in hepatic triglyceride content in hypothyroid animals and progressive reversal over 2 and 4 d of treatment with TH (Fig. 1D). There was a corresponding increase in serum triglyceride content after TH treatment for 4 d (Fig. 1E), although serum cholesterol content was not significantly altered (data not shown).46v, http://www.100md.com
fig.ommitteed46v, http://www.100md.com
Figure 1. Hepatic lipid accumulation in Pax8-/-mice. A–C, Oil Red-O staining of liver sections. Sections of mouse liver from the indicated genotype were incubated with Oil Red-O, and neutral lipid was visualized as detailed in Materials and Methods. A, WT; B, Pax8-/-; C, Pax8-/- treated with TH for 96 h before death. Magnification, x200. These are representative sections from six animals per genotype and treatment. D, Hepatic triglyceride mass was determined after lipid extraction and enzymatic assay as described in Materials and Methods. The data are derived from six animals per group and are expressed as micrograms of triglyceride per milligram of cell protein (mean ± SEM). E, Serum triglyceride levels (n = 8–10 animals/group) determined by enzymatic assay.
ApoB gene expression in hypothyroid Pax8-/- mice@1h, http://www.100md.com
We next examined apoB mRNA abundance and the extent of C to U editing of apoB RNA in the liver and small intestine of neonatal Pax8-/- mice. In WT animals, apoB48 RNA accounted for approximately 60% of the hepatic apoB mRNA population (Fig. 2A, lanes 1–3), similar to previous findings (19, 22). By contrast, hypothyroid Pax8-/- mice showed a significant decrease in apoB editing (38% apoB48; Fig. 2A, lanes 4 and 5) with a return to WT levels after TH injection for 2 (Fig. 2A, lanes 6–8) or 4 d (Fig. 2A, lanes 9 and 10). The temporal pattern of response of hepatic apoB RNA editingto TH administration in hypothyroid neonatal mice is similar to that described in adult rats after TH administration (6, 9, 10, 11); these findings imply that this response is not confined to a single rodent species. This was an important demonstration, because previous studies of hepatic apoB mRNA editing in mice failed to demonstrate regulation after fasting (30), an established mechanism for modulating apoB mRNA editing in rat liver (12). The changes noted in hepatic apoB RNA editing and triglyceride mobilization were accompanied by a temporally coincident appearance of apoB48 in the plasma of TH-treated animals, particularly at 4 d of TH administration (Fig. 2B). Taken together, the data suggest that TH treatment of Pax8-/- mice regulates hepatic apoB mRNA editing in association with increased abundance of apoB48 in plasma.
fig.ommitteed*&f, 百拇医药
Figure 2. A, Endogenous hepatic apoB mRNA editing in WT and Pax8-/- mice. Upper panel, ApoB mRNA editing was determined by primer extension analysis. The primer extension products were separated on a 10% acrylamide gel containing 8 M urea and autoradiographed. A representative assay from three independent experiments is shown. Lanes 1–3, WT; lanes 4–10, Pax8-/- mice; lanes 4 and 5, untreated (Ctrl); lanes 6–8, TH-treated (48 h); lanes 9 and 10, TH-treated (96 h). The locations of the primer (P), unedited (C; apoB100), and edited (U; apoB48) products are indicated to the right of the gel. Lower panel, Bar graph summarizing the data (mean ± SEM; n = 6) for each group. B, Western blot analysis of apoB in serum. Serum (2 µl) was electrophoresed in 4% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. The membrane was blocked and incubated with rabbit antimouse apoB antiserum and visualized by enhanced chemiluminescence. Lanes 1–3, WT; lanes 4–11, Pax8-/- mice; lanes 4–6, untreated (Ctrl); lanes 7 and 8, TH-treated (48 h); lanes 9–11, TH-treated (96 h). The migrations of apoB 100 and apoB 48 are indicated by arrows. This is a representative of three independent experiments.
Because apoB48 arises from both the small intestine and the liver of mice, we examined the effects of TH administration on intestinal apoB RNA editing. Analysis of intestinal RNA from these same groups of animals showed that both apoB mRNA abundance and C to U RNA editing were unchanged despite alterations in TH status (data not shown). Consequently, our further studies focused on the liver to examine the mechanisms accounting for the tissue-specific regulation of apoB mRNA editing.1v1f7tp, 百拇医药
Alterations in the expression of the apoB RNA editing core components, apobec-1 and ACF1v1f7tp, 百拇医药
ApoB RNA editing requires a minimal core complex composed of apobec-1, the catalytic deaminase, and ACF, the RNA-binding complementation factor (12). We performed real-time PCR to quantify these mRNAs in the liver and small intestine of Pax8-/- mice compared with their WT littermates and with animals injected with TH. There was no change in apobec-1 mRNA abundance in hypothyroid Pax8-/- animals both in comparison with WT controls and after and injection of TH for 2 or 4 d (Fig. 3A). By contrast, ACF mRNA abundance was decreased in the liver of hypothyroid Pax8-/- mice, with a significant increase noted after TH administration (Fig. 3A). Total hepatic apoB mRNA abundance was also unchanged among the groups compared with WT levels (Fig. 3A).
fig.ommitteed!w)4m-, 百拇医药
Figure 3. A, Effect of TH treatment on apoB, apobec-1, and ACF mRNA abundance in the liver. Transcript abundance was determined by real-time PCR analysis as described in Materials and Methods. The data (n = 6/group) were normalized to GAPDH levels in each sample. The data are expressed as normalized mRNA abundance relative to WT animals. *, Significant difference (P < 0.001) in ACF mRNA expression in livers of Pax8-/- mice compared with both WT and TH-treated Pax8-/- mice. B, Effect of TH treatment on apobec-1 and ACF protein expression. Liver S100 extract (75 µg) obtained from the indicated groups was resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with specific polyclonal antisera to apobec-1 and ACF or HSP40 (control) and visualized by enhanced chemiluminescence. Migration markers are indicated on the left. This is a representative of three independent experiments.!w)4m-, 百拇医药
Recent studies have demonstrated the presence of multiple, alternatively spliced forms of ACF mRNA in both human and rat liver, up to 25% of which encode functionally inactive variants (31, 32). PCR amplification of the major splice forms of ACF mRNA revealed no alteration in the proportion of the major forms in either liver or intestine (data not shown) under any condition examined. Thus, the decrease in total ACF mRNA abundance noted above does not reflect an altered transcript distribution between functional and inactive isoforms.
To examine apobec-1 and ACF protein expression, we also performed Western blot analysis on hepatic S100 extracts from animals in the different treatment groups. Apobec-1 protein content was comparable across all genotypes and treatment groups (Fig. 3B). On the other hand, ACF protein expression was decreased in the hypothyroid Pax8-/- mice and was increased after TH administration (Fig. 3B), findings consistent with the predictions from the mRNA quantitation above.4#, 百拇医药
Immunohistochemical localization of ACF in sections of liver tissue suggests that this protein is localized in both cytoplasmic and nuclear compartments in WT mice (Fig. 4A). In the Pax8-/- mutant animals a similar, but less intense, staining pattern was revealed (Fig. 4B) TH administration resulted in an increased staining intensity for ACF, particularly within the nucleus (Fig. 4C. Serial sections were examined to quantitate the distribution of nuclear staining in the respective genotypes and treatment groups. Nuclear staining was demonstrated in 10% of hepatocytes (40 of 390 hepatocytes counted) in WT mice compared with 4% in Pax8-/- (17 of 420 hepatocytes counted; P < 0.0005) vs. 40% of hepatocytes in TH-treated Pax8-/- animals (169 of 425 hepatocytes counted; P < 0.0001). These data raise the strong possibility that TH administration to Pax8-/- mice may modulate the intracellular location of ACF in addition to its abundance. This possibility was further addressed using Western blotting of isolated nuclear and cytoplasmic extracts from the various treatment groups. WT mice demonstrate immunoreactive ACF in both nuclear and cytoplasmic extracts. However, as shown in Fig. 4D, there was a marked induction of ACF protein expression in nuclear extracts at 48 and 96 h of TH treatment, which exceeds that observed in cytoplasmic extracts (compare also with Fig. 3B). These data collectively suggest that TH may regulate nuclear expression of ACF in murine liver.
fig.ommitteed+})mw, 百拇医药
Figure 4. A–C, Immunohistochemical localization of hepatic ACF in WT mice, Pax8-/- mice, and Pax8-/- mice treated with TH. Paraffin sections (5 µM) were prepared from the indicated animals and reacted with polyclonal ACF antisera as described in Materials and Methods. This is a representative illustration from three replicate experiments. Intense brown staining of nuclei is particularly evident in C. D, Western blot analysis of ACF abundance in nuclear (N) and cytoplasmic (C) extracts from WT mice, Pax8-/- mice, and Pax8-/- mice treated with TH for 48 or 96 h. One hundred micrograms of protein were used in each lane, and the blots were probed sequentially with anti-ACF antisera, followed by anti-HSP40 or anti-HuR to demonstrate equivalent loading and transfer of cytosolic and nuclear proteins, respectively. This is a representative experiment that was performed in duplicate.+})mw, 百拇医药
Analysis of apoB RNA editing activity in liver extracts: supplementation with recombinant ACF rescues the defect in C to U RNA editing
S100 extracts were prepared from WT as well as Pax8-/- animals, both untreated and treated with TH for 2 and 4 d. These extracts include both nuclear and cytoplasmic compartments, aliquots of which were used for in vitro editing assays where the extent of C to U RNA editing of a synthetic template is determined. The results of these studies, shown in Fig. 5, confirm the predictions emerging from the in vivo analysis of endogenous apoB RNA editing, namely an approximately 50% decrease in the enzymatic (in vitro) editing activity of liver S100 extracts prepared from Pax8-/- animals and recovery of editing activity after TH administration.3aj.9, http://www.100md.com
fig.ommitteed3aj.9, http://www.100md.com
Figure 5. In vitro ApoB RNA editing activity in liver S100 extracts from WT and Pax8-/- mice. In vitro conversions were performed using 75 µg S100 extract, 20 fmol of a 470-nucleotide rat apoB cRNA template, and incubation at 30 C for 3 h. The products were precipitated and analyzed by denaturing urea-acrylamide gel electrophoresis as described in Materials and Methods. The bar graph demonstrates RNA editing activity from each group of animals (n = 4/group). The data are presented as femtomoles of RNA deaminated per microgram of S100 extract per hour (mean ± SD).
To determine conclusively that this rescue was accounted for by the increase in ACF protein, the abundance of ACF protein was estimated from Western blots of S100 extracts prepared using increasing amounts of protein compared with standards of recombinant ACF (Fig. 6). S100 extracts from Pax8-/- mice were then supplemented with increasing amounts of recombinant ACF to supply the equivalent quantities of ACF estimated to be present in TH-treated S100 liver extracts, as judged in comparison with the recombinant protein standards analyzed on the same blots (Fig. 6, upper panel). Unsupplemented extracts from Pax8-/- mice yielded approximately 17% editing of a synthetic RNA template (Fig. 6, lower panel). Supplementation with increasing amounts of recombinant ACF (25–100 ng) completely restored editing activity to that found in WT and TH-treated animals (Fig. 6, lower panel, compare lanes 3–5 to lane 2), demonstrating that alterations in ACF alone account for the decrease in editing activity in Pax8 mutants. Accordingly, the data suggest that treatment with TH restores apoB RNA editing activity in hypothyroid mice most likely through a direct effect mediated by ACF.
fig.ommitteed&5#(], 百拇医药
Figure 6. Addition of recombinant ACF to liver S100 extracts from Pax8-/- mice rescues in vitro apoB RNA editing. Upper panel, Quantitation of ACF protein levels in hepatic S100 extracts. Seventy-five micrograms of S100 extract from WT (lane 1), Pax8-/- untreated (lane 2), and Pax8-/- TH-treated (lane 3) mice were analyzed by SDS-PAGE and Western blot analysis with anti-ACF polyclonal antisera. Increasing amounts (5–50 µg) of recombinant ACF protein (lanes 4–6) were included on the gel to estimate the amount of ACF protein present in the S100 extracts. Lower panel, Rescue of editing activity in Pax8-/- liver S100 extract by supplementation with recombinant ACF. C to U editing assays were performed using 75 µg Pax8-/- S100 extract, either unsupplemented (lane 2) or supplemented with recombinant ACF (25–100 ng; lanes 3–5). Control incubations, shown in lane 1, contained only apoB RNA. This is a representative of three independent experiments.&5#(], 百拇医药
Discussion&5#(], 百拇医药
Regulation of murine hepatic apoB mRNA editing by TH provides an important opportunity to understand the molecular basis by which this posttranscriptional process is modulated in vivo. Such insight may provide clues to the presumed metabolic advantage of apoB48, the product of the edited mRNA, in comparison with apoB100. The current studies provide several lines of evidence to suggest that TH regulates hepatic apoB mRNA editing in neonatal mice through alterations in the abundance of ACF. Furthermore, this mechanism is sufficient to account for the alterations in editing efficiency noted both in vivo and in vitro. Several aspects of these findings merit additional consideration.
The data indicate that Pax8-/- mice exhibit hepatic triglyceride accumulation, which is reversed upon administration of TH. These findings taken together with the temporally coincident increase in apoB mRNA editing lend support to the possibility that hepatic triglyceride secretion may be facilitated in association with the switch in production of the apoB isoforms to favor apoB48. Although the current findings demonstrate that hepatic triglyceride content is decreased along with an increase in plasma triglyceride concentration, further analysis will be necessary to determine whether hepatic very low density lipoprotein assembly and secretion are augmented in association with this change in apoB100 and apoB48 mRNA populations. Such a prediction, however, would be consistent with studies from rat models (6, 7, 8, 9). Data from apobec-1-targeted mice, which express only apoB100, suggest that there is no obvious phenotype with regard to intestinal lipid uptake (17, 18), although more recent findings suggest that there may be some preference for preformed vs. newly synthesized triglyceride that distinguishes the different isoforms in mouse enterocytes (33). The implications of these findings in relation to hepatic triglyceride mobilization in TH-treated neonatal mice will clearly require thorough analysis in the future.
The current studies demonstrate that ACF, the RNA-binding subunit of the core apoB RNA editing holo-enzyme, undergoes metabolic regulation in response to changes in TH status in vivo. This is the first demonstration of physiological metabolic regulation of this gene in any mammalian species. Recent findings revealed that ACF expression in human intestine was unchanged during fetal development, over a period during which endogenous apoB RNA editing undergoes a significant increase (31). These findings taken together with the current demonstration that hepatic (but not intestinal) apoB RNA editing is regulated, with accompanying changes in ACF gene expression, by TH suggest that this metabolic regulation is tissue specific. The data further suggest a concordant temporal response in both mRNA and protein abundance for ACF along with the suggestion, from immunochemical data, that TH treatment results in increased intensity of nuclear staining for ACF. These qualitative findings were complemented by Western blot analysis of nuclear extracts from TH-treated animals, which confirmed the increased abundance of ACF protein more directly. It remains to be determined whether there is regional or zonal regulation of ACF expression within the hepatic lobule. Recent studies (34) in rat liver suggested that ACF expression was localized to a centrizonal rim of cells surrounding the hepatic vein. This particular distribution pattern was not specifically noted in our studies, but we suspect that more extensive evaluation will be necessary to comment with certainty upon the regional expression patterns of ACF within hepatocytes.
Regardless of the possible regional or zonal patterns of ACF expression within the hepatocyte lobule, our studies raise the intriguing possibility of hormonal regulation of ACF distribution between nuclear and cytoplasmic compartments of the cell. The current findings demonstrating a shift in hepatic ACF distribution to the nucleus after TH administration are consistent with a recent report demonstrating ACF redistribution after exposure of primary rat hepatocytes to either insulin or ethanol (34). The current evidence is somewhat conflicting concerning the normal distribution of ACF within cultured cells other than of hepatic origin. Our own data in COS-7 cells suggest that ACF is predominantly nuclear (14, 35), whereas data from other studies have suggested both a cytoplasmic and a nuclear localization pattern (34). Differences in antisera and in the preparation of cells used for these studies may underlie the apparent discrepancies, but this is an issue that will require further study.4a6k${5, http://www.100md.com
Finally, as alluded to above, the increased abundance of ACF mRNA in response to TH treatment suggests, among other possibilities, that a TH response element (TRE) may be present in the murine acf gene. Preliminary analysis of the murine chromosomal acf locus using available databases indicates the presence of two imperfect TRE half-sites (TGACCcaA and TGgCCTGA at nucleotide positions -26 and -397, respectively) and a perfect TRE half-site (TGACCTGA at nucleotide position -727) upstream of the initiator methionine (Newberry, E., and N. Davidson, unpublished observations). These findings will require formal experimental analysis to assess their functional relevance. In addition, the active form of TH, T3, mediates its actions by high affinity interaction with distinctive subtypes of TH receptor, all of which are members of a superfamily of nuclear hormone receptors. TH receptors are encoded by two distinct genes, with further heterogeneity imposed by the use of alternative promoters and differential splicing (25, 36). The demonstration of TH modulation of an important metabolic process in relation to hepatic lipid processing naturally leads to the question of which receptor pathway may be involved in this process. This and other related issues will be the focus of future reports.
Acknowledgments\:/'#-., http://www.100md.com
In particular, we acknowledge Karen Hutton and Randal May in the Morphology Core of the Digestive Disease Research Core Center. We thank Denise Aubert, who manages the transgenic facility at École Normale Supérieure de Lyon, and N. Aguilera and C. Morin for animal care and breeding and Imed Gallouzi (University of Montréal) for the gift of HuR antisera.\:/'#-., http://www.100md.com
Received July 22, 2002.\:/'#-., http://www.100md.com
Accepted for publication October 29, 2002.\:/'#-., http://www.100md.com
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