1. A Novel Role of Transforming Growth Factor β1 in Transcriptional Repression of Human Cholesterol 7α-Hydroxylase Gene 
Tiangang Li, John Y.L. Chiang 
Gastroenterology 
Volume 133, Issue 5, Pages (November 2007)
DOI: /j.gastro
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2. Figure 1
TGFβ1 specifically inhibits CYP7A1 mRNA expressions in hepatocytes. (A and B) HepG2 cells were treated with 0.1 nmol/L TGFβ1 as indicated, and mRNA expression levels were determined by real-time PCR. Assays were done in triplicate. Relative expression was expressed as mean ± SD. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), treated versus control. (C) Primary human hepatocytes isolated from 10 human donors (indicated by PHH#) were treated with TGFβ1 (0.1 nmol/L), and mRNA expression levels were determined by real-time PCR as in A. Bars connected by a dotted line indicate the means of relative CYP7A1 mRNA expression levels from 10 PHHs. Statistical analysis was performed with paired t test. An asterisk (*) indicates statistically significant difference (P < .005, n = 10), treated versus control. (D) Cells were treated with TGFβ1 (0.1 nmol/L) for 24 hours, and total bile acids were measured and expressed as percentage of untreated control.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Smad3 inhibits CYP7A1 reporter activity. (A) CYP7A1 promoter/reporter construct ph-1887-luc (0.2 μg) was cotransfected with Smad2, Smad 3, and/or Smad 4 plasmids (0.1 μg) into HepG2 cells. After transfection for 24 h, TGFβ1 (0.1 nmol/L) was added as indicated for 6 hours before cells were harvested for luciferase activity. (B) Human CYP7A1 promoter construct (0.2 μg) was cotransfected with increasing amount of Smad3. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), pcDNA3 control plasmid versus Smad3.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Smad3 interacts with HNF4α to inhibit CYP7A1. (A) Human CYP7A1 promoter deletion constructs (0.2 μg) was cotransfected with pcDNA3 or Smad3 (0.1 μg). An asterisk (*) indicates statistically significant difference (P < .05, n = 3), pcDNA3 control plasmid versus Smad3. (B) Human CYP7A1 HNF4α site mutant reporter construct (mHNF4-ph-1887-luc, 0.2 μg) was cotransfected with Smad3 (0.1 μg) into hepG2 cells for reporter assay. (C) glutathione S transferase (GST) pull-down assay. Bacterially expressed GST or GST-HNF4α full-length fusion protein and [S35] labeled Smad2, Smad 3, and Smad 4 were used. (D) A heterologous promoter reporter (0.2 μg) containing 4 HNF4α binding sites was cotransfected with 0.1 μg HNF4α and/or Smad2, Smad3, and/or Smad4 expression plasmids. (E) As indicated, 5 X UAS luciferase reporter (0.2 μg) containing 5 Gal4 binding sites was cotransfected with 0.1 μg Gal4-HNF4-LBD fusion plasmid, PGC-1α and/or Smad2, Smad 3, and Smad 4. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), TGFβ1-treated or plasmid-transfected versus nontreated or empty plasmid-transfected control.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
TGFβ1 and adenovirus-mediated transduction of Smad3 inhibits CYP7A1 expression in HepG2 cells. (A) HepG2 cells were treated with TGFβ1 (0.1 nmol/L) for time indicated. Antibodies against Smad2/3, phospho-Smad2/3, and Actin were used in Western blot. (B) HepG2 cells were treated with TGFβ1 (0.1 nmol/L) for 30 minutes, and nuclei were isolated for detection of Smad2/3 by Western blot analysis. (C) HepG2 cells were infected with adenovirus expressing enhanced green fluorescent protein (EGFP) or wild-type Smad3 at MOI of 30 for 36 hours. Antibody against Smad2/3 was used to detect Smad2 and Smad3 protein levels in cell nuclear fraction. (D) HepG2 cells were infected with adenovirus expressing EGFP or Smad3 at multiplicity of infection (MOI) of 30 for 36 hours. mRNA expressions of CYP7A1, CYP8B1, and CYP27A1 were determined by real-time PCR. Assays were done in triplicate and expressed as mean ± SD. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), AdSmad3 versus AdEGFP control.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
TGFβ1 inhibition of CYP7A1 involves HDAC activity. (A) HepG2 cells were pretreated with either tricostatin A (TSA) (100 nmol/L) or vehicle (DMSO) for 1 hour followed by either TGFβ1 (0.1 nmol/L) or vehicle treatment for 2 h. Relative CYP7A1 mRNA expression was determined by real-time PCR. TGFβ1 inhibition of CYP7A1 mRNA in either dimethylsulphoxide (DMSO) pretreated or TSA pretreated cells were expressed as percentage of corresponding controls (DMSA alone or TSA alone) set as 100%. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), TGFβ1-treated versus TSA + TGFβ1-treated. (B) Bacterially expressed GST or GST-HNF4α full-length fusion protein and HepG2 cell lysates were used in GST pull-down assay, and interacting proteins were detected by antibodies in Western blot analysis. (C) HepG2 cells were treated with TGFβ1 (0.1 nmol/L) for the time indicated. Anti-acetyl-histone3 antibody was used in ChIP assay to detect the amount of acetylated histone 3 in CYP7A1 and CYP27A1 chromatin.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
ChIP of effect of TGFβ1 on CYP7A1 chromatin structure. HepG2 cells were treated with TGFβ1 (0.1 nmol/L) for 2 hours. ChIP assays were performed with indicated antibodies. Taqman primers/probe sets were used in real-time PCR to detect CYP7A1 promoter region as described in Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
ChIP of effect of Smad3 overexpression on CYP7A1 chromatin structure. HepG2 cells were infected with adenovirus expressing either EGFP or Smad3 at MOI 30 for 36 hours. ChIP assay was performed as described in Materials and Methods.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Mechanisms of TGFβ1 inhibition of human CYP7A1 gene transcription. Bile acids, reactive oxidizing species, and cytokines released from hepatocytes and Kupffer cells activate HSC cells to release TGFβ1, which activates its receptor TRβI/II and Smad signaling. Smad3 enters the nucleus and inhibits HNF4α transactivation of the CYP7A1 gene by 2 possible mechanisms: (1) inhibiting HNF4α DNA binding activity, and (2) blocking HNF4α interaction with its coactivators such as PGC-1α and SRC1 and recruiting corepressors such as mSin3A and chromatin-modifying enzyme HDAC, which deacetylates CYP7A1 chromatin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions