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• W2102457486 abstract "Background & Aims: This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine. Methods: Apobec-1-deleted mice (APOBEC-1−/−) and wild-type controls were treated with 12 Gy of whole-body γ-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3′ untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3′ untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays. Results: After γ-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC-1−/− mice. In wild-type mice treated with lipopolysaccharide before γ-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E2 mediated by cyclooxygenase-2. No such effect was observed in the APOBEC-1−/− mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3′ untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways. Conclusions: Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability. Background & Aims: This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine. Methods: Apobec-1-deleted mice (APOBEC-1−/−) and wild-type controls were treated with 12 Gy of whole-body γ-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3′ untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3′ untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays. Results: After γ-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC-1−/− mice. In wild-type mice treated with lipopolysaccharide before γ-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E2 mediated by cyclooxygenase-2. No such effect was observed in the APOBEC-1−/− mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3′ untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways. Conclusions: Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability. Apobec-1 is a sequence-specific cytidine deaminase that catalyzes C to U editing of nuclear apolipoprotein B (apoB) messenger RNA (mRNA) in mammalian small-intestinal enterocytes.1Young S.G. Recent progress in understanding apolipoprotein B.Circulation. 1990; 82: 1574-1594Crossref PubMed Scopus (322) Google Scholar, 2Davidson N.O. Anant S. MacGinnitie A.J. Apolipoprotein B messenger RNA editing insights into the molecular regulation of post-transcriptional cytidine deamination.Curr Opin Lipidol. 1995; 6: 70-74Crossref PubMed Scopus (34) Google Scholar ApoB mRNA editing introduces an in-frame UAA stop codon that generates a truncated protein, apoB48, required for intestinal lipid export. Apobec-1 shows broad enzymatic activity directed at free monomeric nucleoside substrates, cytidine bases within specific RNA targets (including apoB), and deoxycytidine bases within DNA.3Harris S.G. Sabio I. Mayer E. Steinberg M.F. Backus J.W. Sparks J.D. Sparks C.E. Smith H.C. Extract-specific heterogeneity in high-order complexes containing apolipoprotein B mRNA editing activity and RNA-binding proteins.J Biol Chem. 1993; 268: 7382-7392Abstract Full Text PDF PubMed Google Scholar, 4MacGinnitie A.J. Anant S. Davidson N.O. Mutagenesis of apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, reveals distinct domains that mediate cytosine nucleoside deaminase, RNA binding, and RNA editing activity.J Biol Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 5Navaratnam N. Morrison J.R. Bhattacharya S. Patel D. Funahashi T. Giannoni F. Teng B.B. Davidson N.O. Scott J. The p27 catalytic subunit of the apolipoprotein B mRNA editing enzyme is a cytidine deaminase.J Biol Chem. 1993; 268: 20709-20712Abstract Full Text PDF PubMed Google Scholar, 6Yamanaka S. Poksay K.S. Balestra M.E. Zeng G.Q. Innerarity T.L. Cloning and mutagenesis of the rabbit ApoB mRNA editing protein A zinc motif is essential for catalytic activity, and noncatalytic auxiliary factor(s) of the editing complex are widely distributed.J Biol Chem. 1994; 269: 21725-21734Abstract Full Text PDF PubMed Google Scholar, 7Mukhopadhyay D. Anant S. Lee R.M. Kennedy S. Viskochil D. Davidson N.O. C→U editing of neurofibromatosis 1 mRNA occurs in tumors that express both the type II transcript and apobec-1, the catalytic subunit of the apolipoprotein B mRNA-editing enzyme.Am J Hum Genet. 2002; 70: 38-50Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Apobec-1 has an affinity for AU-rich elements (AREs) in the 3′ untranslated region (UTR) of rapidly degraded mRNAs, including c-myc, tumor necrosis factor-α, interleukin-2, and granulocyte-macrophage colony-stimulating factor, and it alters c-myc mRNA stability in vivo.8Anant S. Davidson N.O. An AU-rich sequence element (UUUN[A/U]U) downstream of the edited C in apolipoprotein B mRNA is a high-affinity binding site for Apobec-1 binding of Apobec-1 to this motif in the 3′ untranslated region of c-myc increases mRNA stability.Mol Cell Biol. 2000; 20: 1982-1992Crossref PubMed Scopus (80) Google Scholar These data suggest that apobec-1 regulates aspects of mRNA metabolism through sequence-specific binding to AU-rich targets. Cytokine induction plays a central role in mediating the response to injury and repair in gastrointestinal epithelial cells.9Neta R. Douches S. Oppenheim J.J. Interleukin 1 is a radioprotector.J Immunol. 1986; 136: 2483-2485PubMed Google Scholar, 10Neta R. Vogel S.N. Oppenheim J.J. Douches S.D. Cytokines in radioprotection Comparison of the radioprotective effects of IL-1 to IL-2, GM-CSF and IFN gamma.Lymphokine Res. 1986; 5: S105-S110PubMed Google Scholar After sublethal γ-irradiation, rapidly proliferating enterocytes either undergo apoptosis or stop replicating and are later shed from the villus. Surviving stem cells subsequently proliferate to form regenerative crypts, which eventually repopulate the epithelium.11Potten C.S. The role of stem cells in the regeneration of intestinal crypts after cytotoxic exposure.Prog Clin Biol Res. 1991; 369: 155-171PubMed Google Scholar, 12Bach S.P. Renehan A.G. Potten C.S. Stem cells the intestinal stem cell as a paradigm.Carcinogenesis. 2000; 21: 469-476Crossref PubMed Scopus (268) Google Scholar Intestinal prostaglandin synthesis plays an important role in this response, and treatment of mice with indomethacin, a nonselective cyclooxygenase (COX) inhibitor, reduces stem cell survival after radiation injury.13Milas L. Nishiguchi I. Hunter N. Murray D. Fleck R. Ito H. Travis E. Radiation protection against early and late effects of ionizing irradiation by the prostaglandin inhibitor indomethacin.Adv Space Res. 1992; 12: 265-271Crossref PubMed Scopus (19) Google Scholar, 14Cohn S.M. Schloemann S. Tessner T. Seibert K. Stenson W.F. Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1.J Clin Invest. 1997; 99: 1367-1379Crossref PubMed Scopus (201) Google Scholar Evidence supports a major role of COX-1 in radioprotection, but a role for COX-2 has emerged from studies in which lipopolysaccharide (LPS) administered before irradiation induced COX-2 gene expression and increased prostaglandin E2 (PGE2) synthesis, which in turn afforded protection of intestinal stem cells against radiation injury.15Riehl T. Cohn S. Tessner T. Schloemann S. Stenson W.F. Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism.Gastroenterology. 2000; 118: 1106-1116Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar COX-2 is regulated through multiple mechanisms, including posttranscriptional mRNA stability.16Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade.Mol Cell Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (366) Google Scholar, 17Gou Q. Liu C.H. Ben-Av P. Hla T. Dissociation of basal turnover and cytokine-induced transcript stabilization of the human cyclooxygenase-2 mRNA by mutagenesis of the 3′-untranslated region.Biochem Biophys Res Commun. 1998; 242: 508-512Crossref PubMed Scopus (70) Google Scholar, 18Cok S.J. Morrison A.R. The 3′-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency.J Biol Chem. 2001; 276: 23179-23185Crossref PubMed Scopus (188) Google Scholar, 19Dixon D.A. Tolley N.D. King P.H. Nabors L.B. McIntyre T.M. Zimmerman G.A. Prescott S.M. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.J Clin Invest. 2001; 108: 1657-1665Crossref PubMed Scopus (371) Google Scholar In COX-2 mRNA, AREs in the 3′ UTR function as cis-acting elements that regulate both mRNA stability and translation.18Cok S.J. Morrison A.R. The 3′-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency.J Biol Chem. 2001; 276: 23179-23185Crossref PubMed Scopus (188) Google Scholar We now show that apobec-1 binds to AREs in the 3′ UTR of COX-2 mRNA and increases the stability of COX-2 mRNA, thereby facilitating COX-2-mediated PGE2 synthesis. Furthermore, apobec-1-mediated stabilization is not regulated by p38 mitogen-activated protein kinase (MAPK). We also show that APOBEC-1−/− mice are susceptible to radiation injury despite LPS treatment as a result of decreased COX-2 mRNA stability and a consequent reduction in PGE2 production. These findings illustrate an unanticipated role of apobec-1 in the regulation of mRNA stability in response to acute intestinal radiation injury. APOBEC-1−/− mice and wild-type (WT) littermates of the C57BL/6 background20Hirano K. Young S.G. Farese Jr, R.V. Ng J. Sande E. Warburton C. Powell-Braxton L.M. Davidson N.O. Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48.J Biol Chem. 1996; 271: 9887-9890Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar and apoB100-only mice on a mixed C57BL/6/129SvJae background21Veniant M.M. Zlot C.H. Walzem R.L. Pierotti V. Driscoll R. Dichek D. Herz J. Young S.G. Lipoprotein clearance mechanisms in LDL receptor-deficient “Apo-B48-only” “and Apo-B100-only” mice.J Clin Invest. 1998; 102: 1559-1568Crossref PubMed Scopus (118) Google Scholar were maintained on a 12-hour light/12-hour dark schedule and fed standard laboratory chow. At 8 weeks of age, mice were subjected to 12 Gy of whole-body γ-irradiation in a Gammacel 40 irradiator (Atomic Energy Canada, Ottawa, Canada) at 0.96 cGy/min. The mice were killed 6 hours after radiation and rapidly dissected as described previously.15Riehl T. Cohn S. Tessner T. Schloemann S. Stenson W.F. Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism.Gastroenterology. 2000; 118: 1106-1116Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Where indicated, LPS and dimethyl PGE2 were administered intraperitoneally 14 hours before radiation treatment. The distal jejunum was isolated and divided in 2 portions. One portion was fixed in Bouin’s solution for histochemistry analyses by H&E and terminal deoxynucleotidyltransferase-mediated uridine triphosphate nick-end labeling staining. Apoptosis scoring was determined as previously described.22Potten C.S. Li Y.Q. O’Connor P.J. Winton D.J. A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in the murine large bowel.Carcinogenesis. 1992; 13: 2305-2312Crossref PubMed Scopus (145) Google Scholar, 23Li Y.Q. Fan C.Y. O’Connor P.J. Winton D.J. Potten C.S. Target cells for the cytotoxic effects of carcinogens in the murine small bowel.Carcinogenesis. 1992; 13: 361-368Crossref PubMed Scopus (56) Google Scholar, 24Ijiri K. Potten C.S. Response of intestinal cells of differing topographical and hierarchical status to ten cytotoxic drugs and five sources of radiation.Br J Cancer. 1983; 47: 175-185Crossref PubMed Scopus (203) Google Scholar Scoring was restricted to good longitudinal sections of the crypt in which the base of the crypt was aligned with all the other crypt bases. At least 50 half-crypts were counted from each individual mouse. Apoptosis was assessed by terminal deoxynucleotidyltransferase-mediated uridine triphosphate nick-end labeling staining and morphological characteristics, such as cell shrinkage, condensed chromatin, and nuclear fragmentation. We have expressed the data as percentage apoptosis at each location from the base of the crypt. The second portion of the distal jejunum was snap-frozen in liquid nitrogen for RNA isolation and analysis of PGE2 levels, which were determined by enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s recommendations. LPS and dimethyl PGE2 were dissolved in pyrogen-free phosphate-buffered saline and injected at a dose of 0.5 mg/kg each. The c-myc and COX-2 mRNA and protein levels were determined by real-time polymerase chain reaction and Western blot analysis, respectively, as previously described.25Mukhopadhyay D. Houchen C.W. Kennedy S. Dieckgraefe B.K. Anant S. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2.Mol Cell. 2003; 11: 113-126Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Mice were killed at the indicated times after irradiation, and crypt stem cell survival was measured with the microcolony assay.26Cai W.B. Roberts S.A. Bowley E. Hendry J.H. Potten C.S. Differential survival of murine small and large intestinal crypts following ionizing radiation.Int J Radiat Biol. 1997; 71: 145-155Crossref PubMed Scopus (42) Google Scholar, 27Potten C.S. Protection of the small intestinal clonogenic stem cells from radiation-induced damage by pretreatment with interleukin 11 also increases murine survival time.Stem Cells. 1996; 14: 452-459Crossref PubMed Scopus (81) Google Scholar The mice received bromodeoxyuridine (BrdU; 120 mg/kg) and fluorodeoxyuridine (12 mg/kg) 2 hours before death to label replicating DNA. BrdU incorporation in distal jejunum sections was determined immunohistochemically as previously described.14Cohn S.M. Schloemann S. Tessner T. Seibert K. Stenson W.F. Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1.J Clin Invest. 1997; 99: 1367-1379Crossref PubMed Scopus (201) Google Scholar Regenerating crypts were defined as those containing at least 5 BrdU-positive cells. Recombinant glutathione S-transferase/APOBEC-1 (250 ng) was incubated with 32P-labeled RNA (50,000 cpm) in a buffer containing 10 mmol/L HEPES HCl (pH 7.9), 100 mmol/L KCl, 1 mmol/L dithiothreitol, and 10% glycerol for 20 minutes at room temperature. The reactions were then sequentially treated with heparin (final concentration, 5 mg/mL) and ribonuclease T1 (final concentration, 1 U/mL). For electrophoretic mobility shift assays, the samples were resolved in a native 5% polyacrylamide gel. For UV crosslinking assays, the reaction mixture was further treated with UV in a Stratalinker (Stratagene, La Jolla, CA) at 250 J/m2 and resolved by sodium dodecyl sulfate containing 10% polyacrylamide gel under reducing conditions. Substitution mutants were generated as described previously.25Mukhopadhyay D. Houchen C.W. Kennedy S. Dieckgraefe B.K. Anant S. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2.Mol Cell. 2003; 11: 113-126Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar For in vivo RNA binding, isolated epithelial cells from the distal jejunum28Weiser M.M. Intestinal epithelial cell surface membrane glycoprotein synthesis II. Glycosyltransferases and endogenous acceptors of the undifferentiated cell surface membrane.J Biol Chem. 1973; 248: 2542-2548Abstract Full Text PDF PubMed Google Scholar were subjected to in vivo crosslinking as described previously.29Niranjanakumari S. Lasda E. Brazas R. Garcia-Blanco M.A. Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo.Methods. 2002; 26: 182-190Crossref PubMed Scopus (325) Google Scholar Lysates were immunoprecipitated with affinity-purified rabbit anti-apobec-1 immunoglobulin G and subjected to reverse-transcription polymerase chain reaction for COX-2 and glyceraldehyde phosphate dehydrogenase, as described previously.25Mukhopadhyay D. Houchen C.W. Kennedy S. Dieckgraefe B.K. Anant S. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2.Mol Cell. 2003; 11: 113-126Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Full-length and various deletions of the COX-2 3′ UTR were cloned downstream of the luciferase gene in pGL3-control plasmids.18Cok S.J. Morrison A.R. The 3′-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency.J Biol Chem. 2001; 276: 23179-23185Crossref PubMed Scopus (188) Google Scholar The plasmids were transfected along with either plasmid vector pCMV-Tag 3 B or the vector containing apobec-1 with an N-terminal FLAG tag into HEK293 cells, a human embryonic kidney adenocarcinoma cell line that does not express apobec-1. Actinomycin D (10 μg/mL) was added 48 hours after transfection, and the cells were incubated for the indicated times. RNA from the cells was isolated and subjected to Northern blot analysis for luciferase and β-actin mRNA. The data from luciferase mRNA were normalized to β-actin mRNA, expressed as percentages of initial amounts, and the half-life was determined. Recombinant adenoviruses expressing either rat apobec-1 or a control, lacZ complementary DNA, have been described previously30Kozarsky K.F. Bonen D.K. Giannoni F. Funahashi T. Wilson J.M. Davidson N.O. Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (apobec-1) ameliorates hypercholesterolemia in LDL receptor-deficient rabbits.Hum Gene Ther. 1996; 7: 943-957Crossref PubMed Scopus (51) Google Scholar and were titered to a multiplicity of infection of 109 to 1010 plaque-forming units per milliliter. HeLa cells, a human cervical carcinoma cell line, were infected with 3 × 103 multiplicity of infection of the indicated adenovirus for 16 hours, and cell lysates were analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting by rabbit anti-apobec-1 immunoglobulin G to show the presence of apobec-1.8Anant S. Davidson N.O. An AU-rich sequence element (UUUN[A/U]U) downstream of the edited C in apolipoprotein B mRNA is a high-affinity binding site for Apobec-1 binding of Apobec-1 to this motif in the 3′ untranslated region of c-myc increases mRNA stability.Mol Cell Biol. 2000; 20: 1982-1992Crossref PubMed Scopus (80) Google Scholar To determine the effect of p38 MAPK on apobec-1-mediated stabilization of COX-2 mRNA, a p38-specific inhibitor (SB202190) was added to the cells at a final concentration of 20 μmol/L, and cells were incubated for 1 hour. After the addition of actinomycin D, RNA stability was determined as described previously. WT and APOBEC-1−/− mice were killed 84 hours after a single dose of whole-body γ-radiation (12 Gy) to determine the number of regenerating crypts (Figure 1A). WT mice had more than twice as many regenerating crypts per cross section at 3.5 days after radiation as APOBEC-1−/− mice (≈22.5 vs. 10; Figure 1B and C). As a control, we also treated mice expressing apobec-1 but producing only apoB100 in the intestine (apoB100-only mice). The apoB100 mouse was generated by converting the apoB48-editing codon (codon 2153) from CAA to CTA. In WT cells, the cytidine in the CAA codon is edited to uridine, resulting in a UAA stop codon. However, editing of the CUA codon (coding for leucine) results in a UUA codon, which also encodes leucine.21Veniant M.M. Zlot C.H. Walzem R.L. Pierotti V. Driscoll R. Dichek D. Herz J. Young S.G. Lipoprotein clearance mechanisms in LDL receptor-deficient “Apo-B48-only” “and Apo-B100-only” mice.J Clin Invest. 1998; 102: 1559-1568Crossref PubMed Scopus (118) Google Scholar The consequence of this change is that only the full-length apoB100, but no apoB48, is produced in these mice. In contrast to APOBEC-1−/− mice, the apoB100 mice had the same number of regenerating crypts as WT mice (Figure 1C). These data suggest that apobec-1, rather than intestinal apoB48, plays an important role in protecting intestinal stem cells from the effects of radiation.Figure 1Stem cell survival in APOBEC-1−/− mice is reduced after radiation injury. (A) Experimental protocol. WT, apoB100-only, and APOBEC-1−/− mice were treated with 12 Gy of γ-irradiation; 82 hours after radiation, BrdU was administered, and the mice were killed 2 hours later. (B) The distal jejunum was stained with H&E for morphological analysis. To identify dividing cells, the tissue was immunostained for BrdU and counterstained with eosin. (C) Total number of regenerating crypts per cross section; values are mean ± SEM (n = 5 mice per genotype). (D) Number of BrdU-positive cells. Values are mean ± SEM (n = 5 mice per genotype). (E) Total number of regenerating crypts per cross section/time course. Values are mean ± SEM (n = 4 mice per genotype). (F) Total number of regenerating crypts/dose curve. Values are mean ± SEM (n = 4 mice per genotype). (G) Epithelial cells at positions 3 to 8 from the base of the crypts are preferentially protected from apoptosis in WT mice.View Large Image Figure ViewerDownload (PPT) Next, to determine whether apobec-1 is necessary for efficient crypt regeneration, we used BrdU labeling to identify proliferating crypt cells. As shown by immunostaining, WT and apoB100-only mice had more than twice as many BrdU-positive cells per crypt as APOBEC-1−/− mice (38% vs. 15%; Figure 1D). To determine whether there are differences in the time course of crypt survival between WT and APOBEC-1−/− mice, the number of surviving crypts after 12 Gy of γ-irradiation was determined for up to 120 hours. The number of regenerating crypts did not change during this time (Figure 1E). As expected, fewer surviving crypts were observed in both genotypes with increasing doses of radiation (Figure 1F). At all times examined and at each dose of radiation, APOBEC-1−/− mice contained fewer regenerating crypts. Each small-intestinal crypt contains approximately 20 epithelial cells along the long axis and approximately 16 cells in the circumference.31Potten C.S. Owen G. Hewitt D. Chadwick C.A. Hendry H. Lord B.I. Woolford L.B. Stimulation and inhibition of proliferation in the small intestinal crypts of the mouse after in vivo administration of growth factors.Gut. 1995; 36: 864-873Crossref PubMed Scopus (127) Google Scholar Although multiple stem cells may be present in a small-intestinal crypt, it is suggested that potential stem cells may reside at positions 4 to 6 from the base of the crypt.32Potten C.S. Stem cells in gastrointestinal epithelium numbers, characteristics and death.Philos Trans R Soc Lond B Biol Sci. 1998; 353: 821-830Crossref PubMed Scopus (432) Google Scholar The remainder of proliferating crypt epithelial cells transit up and differentiate to become villus epithelial cells.32Potten C.S. Stem cells in gastrointestinal epithelium numbers, characteristics and death.Philos Trans R Soc Lond B Biol Sci. 1998; 353: 821-830Crossref PubMed Scopus (432) Google Scholar To determine whether radiation affects any specific position in the crypts in APOBEC-1−/− mice, apoptosis at every position was scored. When compared with WT mice, there was a significantly higher level of apoptosis in cells located at positions 3 to 8 from the base of the crypt in APOBEC-1−/− mice, with the highest levels observed between positions 4 and 6 (Figure 1G). Together, these data suggest that apobec-1 is required for both cell survival and efficient proliferation of daughter cells after radiation injury. In the setting of radiation injury, the COX-2 gene undergoes posttranscriptional regulation.25Mukhopadhyay D. Houchen C.W. Kennedy S. Dieckgraefe B.K. Anant S. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2.Mol Cell. 2003; 11: 113-126Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar To explore the mechanisms underlying apobec-1-dependent radioprotection, we therefore measured COX-2 protein levels. As shown by Western blots, COX-2 protein levels were 50% lower in APOBEC-1−/− mice than in WT and apoB100 mice after 12 Gy of γ-irradiation (Figure 2A). To determine whether the increased susceptibility of APOBEC-1−/− mice to radiation injury reflects decreased PGE2 levels resulting from the reduced COX-2 expression, we pretreated the mice with LPS and measured COX-2-dependent prostaglandin production. LPS or dimethyl PGE2 was administered intraperitoneally 14 hours before irradiation, and the animals were allowed to recover for 6 hours (Figure 2B). Radiation alone induced a 2.8- and 5-fold increase in crypt epithelial cell apoptosis in WT and APOBEC-1−/− mice, respectively (Figure 2B). LPS and dimethyl PGE2 provided complete radioprotection in WT mice; in APOBEC-1−/− mice, however, only dimethyl PGE2 was radioprotective (Figure 2C). These findings suggest that LPS-mediated PGE2 production is impaired in APOBEC-1−/− mice. We next assessed PGE2 expression in the intestine after LPS and irradiation. PGE2 levels increased 8-fold in WT mice but did not change significantly in APOBEC-1−/− mice (Figure 2D). Thus, the increased crypt cell apoptosis in APOBEC-1−/− mice seems to reflect diminished PGE2 production. Because LPS increases PGE2 synthesis by inducing COX-2 expression, we next examined whether LPS-induced COX-2 expression is altered in APOBEC-1−/− mice. COX-2 mRNA levels increased 3.2 ± 0.4-fold in response to LPS treatment alone and 3.9 ± 0.6-fold in response to both LPS and γ-radiation in WT mice but were unaffected in APOBEC-1−/− mice (Figure 3A and B). Mimicking the RNA expression profiles in both WT and APOBEC-1−/− mice, COX-2 protein expression increased 4-fold in LPS-treated WT mice compared with untreated controls but was unchanged in APOBEC-1−/− mice (Figure 3B and C). Apobec-1 protein levels in WT mice were similar under all the conditions tested, whereas no apobec-1 protein was observed in APOBEC-1−/− mice (Figure 3C). We have previously shown that apobec-1 stabilizes c-myc mRNA in F442A cells by binding to AU-rich sequences in the 3′ UTR. Accordingly, we examined whether c-myc expression is affected in APOBEC-1−/− mice at baseline and after radiation treatment. The steady-state level of c-myc mRNA in the APOBEC-1−/− mice is 50% less than that observed in WT mice (Figure 3D). Furthermore, in WT mice, c-myc mRNA levels increased 1.6- and 2.3-fold at 6 and 84 hours, respectively, after 12 Gy of γ-irradiation (Figure 3E). However, in APOBEC-1−/− mice, the induction of c-myc mRNA expression was abrogated, and the small increases observed did not achieve statistical significance (Figure 3E). Thus, apobec-1 seems to affect expression of multiple transcripts, including COX-2 and c-myc. We next determined whether apobec-1 interacts with the 3′ UTR of COX-2 mRNA. In electrophoretic mobility shift assays, recombinant apobec-1 bound to the 3′ UTRs of COX-2, vascular endothelial growth factor, and interleukin-8 (Figure 4A). To show the presence of specific interactions between apobec-1 and COX-2 mRNA in vivo, cytoplasmic S-100 extracts of intestinal epithelial cells from both irradiated and unirradiated mice were immunoprecipitated with anti-apobec-1 antibody. RNA extracted from the immune complex showed COX-2 mRNA (Figure 4B). No C to U change was detected in the bound transcript (data not shown). The first 60 nucleotides (nt) of the COX-2 3′ UTR contain the canonical apobec-1 binding site, UUUN[A/U]U (Figure 4C). This region is important in the regulation of COX-2 mRNA stability.16Lasa M. Mahtani K.R. Finch A. Brewer G. Sakl" @default.
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• W2102457486 date "2004-10-01" @default.
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• W2102457486 title "Apobec-1 protects intestine from radiation injury through posttranscriptional regulation of cyclooxygenase-2 expression" @default.
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