1. Volume 131, Issue 2, Pages 554-567 (August 2006)
c-Fos Is a Critical Mediator of Inflammatory-Mediated Repression of the Apical Sodium- Dependent Bile Acid Transporter 
Ezequiel Neimark, Frank Chen, Xiaoping Li, Margret S. Magid, Teresa M. Alasio, Tamara Frankenberg, Jyoti Sinha, Paul A. Dawson, Benjamin L. Shneider 
Gastroenterology 
Volume 131, Issue 2, Pages (August 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Northern analysis of IL-1β–treated Caco-2 cells. Three separate sets of cells were treated with 20 pg/mL of IL-1β (lanes 2, 4, and 6) for 40 hours. mRNA levels were measured by phosphorimager analysis of a Northern blot of 10 μg of total RNA. Treatment with IL-1β yielded a 65% reduction in steady-state ASBT mRNA levels and 250% and 275% increases in c-jun and c-fos, respectively. Similar RNA loading was demonstrated by equivalent signals for 28S RNA.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Cytokine-induced repression of ASBT promoter activity. Caco-2 cells were cotransfected and treated for 40 hours with the human ASBT promoter plus 20 pg/mL of IL-1β, 100 ng/mL of TNF-α, or 2 ng/mL of IL-6. The activity of the promoter was reduced with IL-1β and TNF-α. There was no change in the promoter activity when the inflammatory cytokine IL-6 was used. All studies were performed in a medium containing 10% fetal calf serum.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Sequence analysis of the human ASBT promoter. The dAP-1 cis-element sequence is underlined. The exact sites of the point mutations in the dAP-1 element are marked by asterisks. Previous studies of the rat ASBT promoter have identified cis-acting elements that are important for basal transcription mediated by uAP1 and hepatocyte nuclear factor 1α (HNF-1α) and for cytokine-induced repression (dAP-1).25 These sites are conserved in the human promoter as indicated. Nucleotide numbering starts with 0 at the site of transcription initiation.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Effect of c-jun and c-fos overexpression on human ASBT promoter activity. The effect of 40 hours of overexpression of c-jun, c-fos, their dn constructs, and c-fos antisense on the ASBT promoter was assessed. Human ASBT promoter activity was induced by c-jun and repressed by c-fos, overexpression of dn c-jun repressed promoter activity, and dn c-fos also inhibited promoter activity. Antisense c-fos RNA oligonucleotide (c-fos[−]) treatment led to a 3-fold increase in promoter activity, similar to what was observed with c-jun overexpression.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Quantitative analysis of the effect of IL-1β on AP-1 DNA/protein complex formation. (A) The linear range of response for dAP-1 and uAP-1 was found to be between 0.1 and 5.0 μg of nuclear proteins. Quantitative electrophoretic mobility shift assay studies were performed using 1 μg of nuclear proteins from 3 different sets of Caco-2 cells treated with either vehicle (−) or with IL-1β (+). (B) Phosphorimager quantification of signal intensity revealed a 5-fold increase in the abundance of both the uAP-1 and dAP-1 DNA/protein complexes after treatment with IL-1β.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Effect of dAP-1 mutation on cytokine-mediated response of ASBT. The dAP-1 cis-element in the human ASBT that binds a c-fos/c-jun heterodimer was mutated by site-directed mutagenesis to yield a mutant luciferase reporter construct (ASBTμ) with approximately 400% of the activity of the control. Overexpression of c-fos for 40 hours did not decrease the activity of the mutated promoter, unlike the repression observed with the wild-type promoter. Treatment with IL-1β led to an increase and not a decrease in the activity of the mutated promoter, in contrast to the repression observed with the wild-type promoter. This is likely due to the activation by c-jun that is increased by IL-1β (Figure 1) and binds to the uAP-1 site.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Western blot analysis of the effect of c-fos antisense treatment. CT-26 (mouse), IEC-6 (rat), and Caco-2 (human) cells were either untreated (−), treated with c-fos antisense (si), or a scrambled control (sc). Nuclear proteins were then analyzed by Western blot analysis for the c-fos protein, which is shown to be knocked down in all 3 cell lines treated with the c-fos antisense. Scrambled antisense had no effect on c-fos protein levels.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Promoter activity in c-fos antisense-treated Caco-2 cells. Caco-2 cells were pretreated with c-fos antisense RNA (−) or c-fos scrambled RNA oligonucleotides (s) and then transfected with luciferase reporter constructs for human ASBT. Human ASBT promoter activity was enhanced in the absence of c-fos (c-fos [−]), while there was no effect when treating with the scrambled antisense oligonucleotide (c-fos [s]). IL-1β treatment was performed as indicated in the figure. In c-fos antisense-treated cells (c-fos [−]), ASBT promoter activity was enhanced by IL-1β, most probably by inducing c-jun in the absence of c-fos. IL-1β treatment of the scrambled antisense-treated cells yielded the expected reduction in ASBT promoter activity.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
Northern blot analysis of c-fos antisense-treated Caco-2 cells. Two separate groups of cells were treated with c-fos antisense oligonucleotides (c-fos [−]) (lanes 3 and 5) and 2 separate groups with c-fos scrambled oligonucleotides (c-fos [s]) (lanes 4 and 6) as a control for the oligonucleotide treatment. Control cells were not treated with oligonucleotides (lanes 1 and 2). The effect of IL-1β treatment was assessed (lanes 2, 5, and 6). The number under the mRNA signal represents the intensity of the phosphorimager signal normalized to the signal for the untreated Caco-2 cells (lane 1). c-fos mRNA is minimal to undetectable in the c-fos antisense-treated cells (lanes 3 and 5), indicating the efficacy of the antisense treatment. Scrambled c-fos oligonucleotide had no effect on c-fos mRNA levels (lanes 4 and 6). IL-1β treatment (lanes 2, 5, and 6) yielded increased enhanced c-jun expression and enhanced c-fos expression in the cells that were not treated with c-fos antisense oligonucleotides (lanes 2 and 6). c-fos antisense-treated cells have enhanced ASBT mRNA expression (lanes 3 and 5). There was no change in ASBT mRNA level in the c-fos (s)-treated cells (lane 4) compared with that in the untreated cells (lane 1). Similar RNA loading was demonstrated by equivalent signals for 28S RNA.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
Positional effects of the dAP-1 cis-element on SV40 promoter activity. There was no change in the promoter activity of SV40 lacking dAP-1 when treated with IL-1β. Insertion of the dAP-1 element either downstream, SV40dAP-1, or upstream, dAP1SV40, endowed this construct with IL-1β responsiveness.
Gastroenterology  , DOI: ( /j.gastro )
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12. Figure 11
Positional effects of the dAP-1 cis-element on rat ASBT promoter activity. The wild-type rat ASBT promoter, P3, was significantly repressed by IL-1β. The rat ASBT promoter with a mutated dAP-1 site (M2) showed no change in expression when treated with IL-1β. The uAP-1 cis-element of the M2 promoter was mutated by site-directed mutagenesis to convert it to a dAP-1 site (M4) and once treated with IL-1β showed repression by IL-1β.
Gastroenterology  , DOI: ( /j.gastro )
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13. Figure 12
Northern analysis of indomethacin-treated rat ASBT promoter transgenic mice. Nucleotides −208 to +118 of the rat ASBT promoter were linked to an hGH reporter. Transgenic mice were treated with indomethacin (+) or vehicle control (−). Five different animals in each group were studied. Terminal ileum was isolated and Northern blotting of total RNA was performed. Consistent and significant reductions in both ASBT and hGH mRNA were observed. Equivalent loading was demonstrated by the 28S ribosomal RNA signal.
Gastroenterology  , DOI: ( /j.gastro )
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14. Figure 13
Northern analysis of indomethacin-treated wild-type and c-fos–null mice. Two separate sets of mice, wild-type (+/+) and c-fos null (−/−), were treated with indomethacin (lanes 3, 4, 7, and 8) or vehicle (lanes 1, 2, 5, and 6). mRNA levels were measured by phosphorimager analysis (B) of a Northern blot (A) of 10 μg of total RNA. Treatment with indomethacin yielded an increase in c-jun in both sets of mice (lanes 3, 4, 7, and 8). An increase in c-fos expression was seen in the indomethacin-treated c-fos+/+ mice (lanes 3 and 4) compared with the untreated c-fos+/+ mice (lanes 1 and 2). In contrast, there was no c-fos expression in the c-fos−/− mice untreated (lanes 5 and 6) or treated with indomethacin (lanes 7 and 8). A reduction in steady-state ASBT mRNA levels was observed in the indomethacin-treated c-fos+/+ mice (lanes 3 and 4). Paradoxically, the c-fos−/− mice treated with indomethacin showed a significant increase in the ASBT mRNA expression (lanes 7 and 8). Similar RNA loading was demonstrated by equivalent signals for 28S RNA.
Gastroenterology  , DOI: ( /j.gastro )
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15. Figure 14
Ileal histology of indomethacin-treated c-fos–null mice. Cross section of the distal 2 cm in the terminal ileum of a c-fos–null mouse treated with indomethacin. There is diffuse, transmural acute inflammation with villous blunting and focal ulceration (arrow). Histologic grade of acute inflammation = 12 (scoring: ulcer [1] × depth [muscularis propria] = 3 + acute inflammation extent = 2 [diffuse] + degree = 3 [severe] + depth = 4 [transmural]). (H & E; original magnification 6×.)
Gastroenterology  , DOI: ( /j.gastro )
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16. Figure 15
Northern analysis of indomethacin-treated wild-type and c-fos heterozygote null mice. Two separate sets of mice, c-fos+/+ (+/+: lanes 1, 2, 5, 6, 9, and 10) and c-fos+/− (+/−: lanes 3, 4, 7, 8, 11, and 12), were treated with indomethacin (lanes 2, 4, 6, 8, 10, and 12) or vehicle (lanes 1, 3, 5, 7, 9, and 11). mRNA levels were measured by phosphorimager analysis (B) of a Northern blot (A) of 10 μg of total RNA. Treatment with indomethacin yielded an increase in IL-1β and c-jun in both sets of mice. An increase in c-fos expression was seen in both sets of mice, although the basal level in the c-fos+/− mice was approximately 50% of the c-fos+/+ mice. A reduction in steady-state ASBT mRNA levels was observed in the indomethacin-treated c-fos+/+ mice (lanes 2, 6, and 10), while there was no reduction in ASBT expression in the c-fos+/− mice (lanes 4, 8, and 12). Similarly, Ostα and Ostβ mRNA levels were reduced after indomethacin treatment in c-fos+/+ mice, while the effect was abrogated in the c-fos+/− mice. Similar RNA loading was demonstrated by equivalent signals for 28S RNA.
Gastroenterology  , DOI: ( /j.gastro )
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17. Figure 16
Model for regulation of ASBT by AP-1. (A) Under normal conditions, the upstream (uAP-1) and downstream (dAP-1) AP-1 elements are bound by c-jun/c-jun (arrows) and c-jun/c-fos (octagon), respectively. (B) In the setting of IL-1β treatment or in response to inflammation, there are increases in the concentration of c-jun and c-fos coupled with translocation of c-fos into the nucleus. This leads to increased occupancy of both uAP-1 and dAP-1, with a predominant effect on dAP-1 yielding relative inhibition of the promoter activity. (C) Overexpression of c-fos inhibits the promoter via occupancy of dAP-1. (D) Transfection with a dn c-fos that binds to the dAP-1 element also inhibits the promoter activity. (E) In c-fos–negative cells, the activity of the ASBT promoter increases presumably to the endogenous c-jun binding to the uAP-1 element unopposed by c-fos binding to dAP-1. (F) The promoter activity is further up-regulated when IL-1β is added to c-fos–negative cells, due to the higher concentration of c-jun seen after cytokine treatment. (G) A dAP-1 site-mutated promoter showed a higher basal activity, presumably due to the lack of binding by c-fos, yielding an unopposed activation of c-jun. (H) IL-1β treatment of the mutated promoter increased the activity, consistent with the effect seen in c-fos–negative cells. Block arrow, c-jun homodimer; octagon, c-jun/c-fos heterodimer; shaded octagon with slash, dn c-fos; curved cis-element, mutated dAP-1 cis-element; +, activity of the human ASBT promoter. Experimental figures that correlate with these models are indicated.
Gastroenterology  , DOI: ( /j.gastro )
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