1. Volume 122, Issue 4, Pages 985-993 (April 2002)
Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line 
Jung–Hwan Yoon, Hajime Higuchi, Nathan W. Werneburg, Scott H. Kaufmann, Gregory J. Gores 
Gastroenterology 
Volume 122, Issue 4, Pages (April 2002)
DOI: /gast
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2. Fig. 1
EGFR is phosphorylated by bile acids. (A) KMBC cells, a human cholangiocyte-derived cell line, were cultured in the presence and absence of TCDC, and equivalent amounts of protein were immunoblotted (IB) with antiphosphotyrosine antibody (PTy). The membrane was stripped and reblotted with the anti-EGFR antibody. (B) Immunoprecipitation (IP) of the EGFR from cell lysates was performed followed by immunoblot analyses using either antiphosphotyrosine or anti-EGFR antibody. (C) EGFR tyrosine phosphorylation was compared in cells treated for 30 minutes with different bile acids of the same concentration (200 μmol/L) by immunoblot using antiphosphotyrosine antibody.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Both Src and EGFR kinase activity are necessary for bile acid–induced EGFR phosphorylation. Before TCDC (200 μmol/L) treatment, KMBC cells were preincubated for 1 hour in the presence or absence of (A) a specific Src kinase inhibitor (PP2) or (B) the EGFR kinase inhibitor (PD168393, 2 μmol/L). Cells were lysed after 30 minutes of exposure to TCDC, and immunoblot analysis was performed using antiphosphotyrosine antibody (PTy).
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Bile acid–induced EGFR activation results in p42/44, p38, and JNK activation. (A) KMBC cells were treated with TCDC (200 μmol/L) for each indicated time period, lysed, and immunoblot analysis was performed in the cell lysates using antisera specific for the phosphorylated forms of p42/p44 MAPK (Phospho p42/44), p38 MAPK (Phospho p38), or JNK (Phospho JNK). Immunoblots for total MAPK proteins (phosphorylated and unphosphorylated) were also performed. (B) KMBC cells were preincubated in the presence or absence of the EGFR kinase inhibitor (PD168393, 2 μmol/L) or Src kinase inhibitor (PP2, 10 μmol/L) for 1 hour before addition of TCDC (200 μmol/L). After 30 minutes of exposure to TCDC, cells were lysed, and immunoblot analysis was performed as described in A.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Bile acids induce COX-2. (A) KMBC cells were incubated with medium (control) or 200 μmol/L DC, TCDC, and GCDC. Cells were lysed after 24 hours, and immunoblot analysis was performed using anti–COX-2 antisera. Mouse denotes the whole cell lysate of a mouse macrophage cell line used as a positive control for COX-2 protein. Immunoblot analysis using anti–β-actin antisera was performed as a control for protein loading. (B) KMBC cells were treated with DC, GDC, and TDC for 24 hours, and then immunoblot analysis was performed for COX-2.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Bile acid–induced activation of the EGFR/MAPK cascade is responsible for COX-2 induction. (A) KMBC cells were preincubated in the presence or absence of the EGFR kinase inhibitor (PD168393, 2 μmol/L) or Src kinase inhibitor (PP2, 10 μmol/L) for 1 hour before bile acid treatment, and then further incubated with media (control) or selected bile acids (200 μmol/L) for 24 hours. Cells were lysed, and immunoblot analysis was performed using anti–COX-2 antisera. (B) KMBC cells were pretreated with the inhibitors of MAPK, PD (50 μmol/L) for MEK1 or SB (20 μmol/L) for p38. Raf-1 was inhibited with BAY (20 μmol/L) for 1 hour before bile acid treatment. Immunoblot analysis was performed in whole cell lysates using anti–COX-2 antisera.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
EGFR/JNK cascade is not responsible for COX-2 induction. (A) KMBC cells were infected with an adenovirus expressing a dominant negative TAK1 (Ad-TAK1-DN) at each indicated MOI. Twenty-four hours after transduction, cells were treated with DC (200 μmol/L) for 30 minutes. The cells were lysed, and JNK activity was assayed as described in Materials and Methods. (B) KMBC cells were transduced with an Ad-TAK1-DN at 100 MOI and cultured for 24 hours. Cells were then treated with DC (200 μmol/L) for an additional 24 hours. The cells were then lysed, and immunoblot analysis was performed for COX-2.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Proposed model of signaling pathways for bile acid–mediated COX-2 induction in human cholangiocytes. Bile acids (BA) induce EGFR phosphorylation, which can be blocked by both EGFR kinase (PD168393) and Src kinase (PP2) inhibitors. Activation of EGFR by bile acids results in subsequent activation of MAPK p42/44, p38, and JNK. The MAPK inhibitors, PD for MEK1, SB for p38, and BAY for Raf-1, can diminish COX-2 induction by bile acids, whereas inhibition of JNK activity by an adenovirus expressing the dominant negative TAK1 (Ad-TAK1-DN) does not block COX-2 induction.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions