1. Involvement of the Src family kinase yes in bile salt-induced apoptosis 
Roland Reinehr, Stephan Becker, Matthias Wettstein, Dieter Häussinger 
Gastroenterology 
Volume 127, Issue 5, Pages (November 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Bile salt-induced activation of the Src-kinase family member Yes. Hepatocytes were cultured for 24 hours and then exposed to TLCS, TCDC, or TC (100 μmol/L, each); GCDC (50 μmol/L); or sodium orthovanadate (1 mmol/L) for the time periods indicated. When indicated, N-acetylcysteine (NAC; 30 mmol/L), SU6656 (10 μmol/L), PP-2 (10 μmol/L), herbimycin A (1 μmol/L), emodin (10 μmol/L), DB-cAMP (100 μmol/L), H89 (5 μmol/L), AG1478 (5 μmol/L), genistein (100 μmol/L), JNK-inhibitor (L-JNKI1; 5 μmol/L), Gö6850 (10 μmol/L), trolox (20 μmol/L), melatonin (100 μmol/L), or epicatechin (10 μmol/L) were preincubated 30 minutes prior to bile acid addition. Activating phosphorylation of Yes-Y418 (∼62 kilodaltons), Fyn-Y418 (∼59 kilodaltons) was detected after Yes- or Fyn-immunoprecipitation, respectively, and subsequent Western blotting using an anti-phospho-Src familiy-Y418 specific antibody. Total Yes and Fyn, respectively, served as loading controls. c-Src activation was measured using a c-Src-Y418 (∼60 kilodaltons) phosphospecific antibody. c-Src- and Lck-phosphorylation was also detected using phosphospecific antibodies against c-Src-Y529 (∼60 kilodaltons) and Lck-Y505 (∼56 kilodaltons), respectively. Total Src and Lck, respectively, served as loading controls. Representative immunoblots from at least 3 independent experiments are shown. (A) TLCS, TCDC, and GCDC, but not TC, induce within 1 minute Yes activation. This is mimicked by the tyrosine phosphatase inhibitor vanadate. (B–D) None of these bile salts activates Fyn or c-Src. In addition, no change in Lck-Y505 phosphorylation occurs upon TLCS exposure. (E) Concentration dependence of TLCS-induced Yes activation. Hepatocytes were exposed for 1 minute to TLCS at the concentrations indicated. TLCS-induced Yes activation was quantified by densitometric analysis of Yes-Y418 phosphorylation and normalized for total Yes expression in the latter samples. (F) Inhibitor profile. Hepatocytes were exposed to 100 μmol/L TLCS for 1 minute. TLCS-induced Yes activation was sensitive to N-acetylcysteine (NAC), SU6656, and genistein, whereas cAMP, AG1478, Gö6850, PP-2, herbimycin A, emodin, and inhibition of JNKs were ineffective. In addition, TLCS (100 μmol/L, 1 minute)-induced Yes activation was sensitive to antioxidants such as NAC, trolox, melatonin, and epicatechin.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 1
Bile salt-induced activation of the Src-kinase family member Yes. Hepatocytes were cultured for 24 hours and then exposed to TLCS, TCDC, or TC (100 μmol/L, each); GCDC (50 μmol/L); or sodium orthovanadate (1 mmol/L) for the time periods indicated. When indicated, N-acetylcysteine (NAC; 30 mmol/L), SU6656 (10 μmol/L), PP-2 (10 μmol/L), herbimycin A (1 μmol/L), emodin (10 μmol/L), DB-cAMP (100 μmol/L), H89 (5 μmol/L), AG1478 (5 μmol/L), genistein (100 μmol/L), JNK-inhibitor (L-JNKI1; 5 μmol/L), Gö6850 (10 μmol/L), trolox (20 μmol/L), melatonin (100 μmol/L), or epicatechin (10 μmol/L) were preincubated 30 minutes prior to bile acid addition. Activating phosphorylation of Yes-Y418 (∼62 kilodaltons), Fyn-Y418 (∼59 kilodaltons) was detected after Yes- or Fyn-immunoprecipitation, respectively, and subsequent Western blotting using an anti-phospho-Src familiy-Y418 specific antibody. Total Yes and Fyn, respectively, served as loading controls. c-Src activation was measured using a c-Src-Y418 (∼60 kilodaltons) phosphospecific antibody. c-Src- and Lck-phosphorylation was also detected using phosphospecific antibodies against c-Src-Y529 (∼60 kilodaltons) and Lck-Y505 (∼56 kilodaltons), respectively. Total Src and Lck, respectively, served as loading controls. Representative immunoblots from at least 3 independent experiments are shown. (A) TLCS, TCDC, and GCDC, but not TC, induce within 1 minute Yes activation. This is mimicked by the tyrosine phosphatase inhibitor vanadate. (B–D) None of these bile salts activates Fyn or c-Src. In addition, no change in Lck-Y505 phosphorylation occurs upon TLCS exposure. (E) Concentration dependence of TLCS-induced Yes activation. Hepatocytes were exposed for 1 minute to TLCS at the concentrations indicated. TLCS-induced Yes activation was quantified by densitometric analysis of Yes-Y418 phosphorylation and normalized for total Yes expression in the latter samples. (F) Inhibitor profile. Hepatocytes were exposed to 100 μmol/L TLCS for 1 minute. TLCS-induced Yes activation was sensitive to N-acetylcysteine (NAC), SU6656, and genistein, whereas cAMP, AG1478, Gö6850, PP-2, herbimycin A, emodin, and inhibition of JNKs were ineffective. In addition, TLCS (100 μmol/L, 1 minute)-induced Yes activation was sensitive to antioxidants such as NAC, trolox, melatonin, and epicatechin.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 2
Bile salt-induced activation of c-Jun-N-terminal kinase (JNK) is not mediated by Src familiy kinases. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L) for the time periods indicated. When indicated, SU6656 (10 μmol/L), PP-2 (10 μmol/L), herbimycin A (1 μmol/L), or emodin (10 μmol/L) were preincubated for 30 minutes. Phospho-JNK-1/-2 (∼46/54 kilodaltons), as markers of JNK-activation, were detected by Western blotting. Total JNK-1/-2 (∼46/54 kilodaltons) served as loading control. Representative blots from 3 independent experiments are shown. TLCS exposure led, within 15 minutes, to JNK-activation, which was not affected by SU6656, PP-2, herbimycin A, or emodin.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 3
Bile salt-induced EGFR/Yes association. Hepatocytes were cultured for 24 hours and then stimulated as indicated below. (A) Yes was immunoprecipitated as described in the Materials and Methods section, and its association with the epidermal growth factor receptor (EGFR; ∼170 kilodaltons) was detected by Western blotting. Total Yes (∼62 kilodaltons) served as loading control. In addition, phospho-JNK-1/-2 (∼46/54 kilodaltons), as markers of JNK-activation, were detected in Western blots with total JNK-1/-2 (∼46/54 kilodaltons) serving as a loading control. When indicated, SU6656 (10 μmol/L) or DB-cAMP (100 μmol/L) were preincubated for 30 minutes. TLCS (100 μmol/L) induced within 1 minute an association of the EGFR with Yes, which was sensitive to SU6656, indicating that EGFR/Yes association requires functionally active Yes kinase. Also, cAMP inhibited EGFR/Yes association in a H89-sensitive way (see also Figure 3 B), suggestive for a PKA-mediated inhibition of EGFR/Yes association. Neither SU6656 nor cAMP affected TLCS-induced JNK activation. (B) Hepatocytes were exposed to TLCS (100 μmol/L). When indicated, cells were preincubated for 30 minutes with NAC (30 mmol/L), SU6656 (10 μmol/L), PP-2 (10 μmol/L), herbimycin A (1 μmol/L), emodin (10 μmol/L), DB-cAMP (100 μmol/L), or AG1478 (5 μmol/L) or genistein (100 μmol/L), L-JNKI1 (5 μmol/L), or Gö6850 (10 μmol/L). If indicated, H89 (5 μmol/L) or PKI (5 μmol/L) were incubated 30 minutes before cAMP addition to inhibit PKA activity. Unless indicated otherwise, samples were examined 1 minute after bile salt exposure to detect EGFR/Yes association and EGFR-activation. Bile salt-induced Yes/EGFR association was sensitive to NAC, SU6656, cAMP, and genistein. All maneuvers that prevented Yes activation (see Figure 1F) and/or Yes/EGFR association also prevented EGFR activation. (C) After bile salt exposure, Yes or EGFR, respectively, were immunoprecipitated as described in the Materials and Methods section to detect an association with the docking protein c-Cbl (∼120 kilodaltons) by Western blot. EGF (50 ng/mL, 60 minutes) served as a positive control. TLCS neither induced a Yes/Cbl nor EGFR/Cbl association, indicating that Cbl may not be involved in Yes/EGFR interaction. However, in line with the literature, EGFR/Cbl association was found after stimulation with EGF. 43 (D) Hepatocytes were exposed to cAMP (100 mmol/L) for 30 minutes. When indicated H89 (5 μmol/L) or PKI (5 μmol/L) were preincubated 30 minutes prior to the cAMP addition. TLCS (100 μmol/L) was added for 1 minute after 30 minutes of cAMP preincubation. Yes or EGFR, respectively, was immunoprecipitated as described in the Materials and Methods section, and phosphorylation on serine or threonine sites was detected by Western blotting using phospho-serine (P-Ser) or phospho-threonine (P-Thr) specific antibodies, respectively. Total Yes and total EGFR served as a loading control. Cyclic AMP induced a H89- and PKI-sensitive Ser/Thr-phosphorylation of Yes but not of the EGFR.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 4
TLCS-induced EGFR tyrosine phosphorylation pattern. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L) for the time periods indicated. If indicated, SU6656 (10 μmol/L) or AG1478 (5 μmol/L) were preincubated for 30 minutes to inhibit Yes- or EGFR-tyrosine kinase activity, respectively. EGFR tyrosine phosphorylation at positions Y845, Y1045, and Y1173 was detected by Western blot using phosphospecific antibodies (∼170 kilodaltons). In addition, EGFR was immunoprecipitated, and total EGFR-tyrosine phosphorylation (P-Tyr; ∼170 kilodaltons) was detected as described in the Materials and Methods section. Total EGFR (∼170 kilodaltons) served as a loading control. TLCS induced within 1 minute a SU6656-sensitive EGFR phosphorylation on position Y845, which was then followed by EGFR autophosphorylation on position Y1173 as indictated by its AG1478-sensitivity. This may suggest that Yes-meditated EGFR-Y845 phosphorlyation leads to an activation of EGFR-tyrosine kinase activity. TLCS had little or no effect on EGFR tyrosine phosphorylation on position Y1045, a known target for the Cbl docking peptide.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 5
Bile salt-induced EGFR/Yes association is followed by the formation of the EGFR/CD95 complex. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L) for the time periods indicated. JNK-inhibitor (L-JNKI1; 5 μmol/L) or PKC-inhibitor (Gö6850; 10 μmol/L) were preincubated for 30 minutes. Yes and CD95 were immunoprecipitated as described in the Materials and Methods section. Yes immunoprecipitates were detected for CD95 (∼48 kilodaltons) and EGFR (∼170 kilodaltons) association, whereas immunoprecipitated CD95 samples were detected for Yes (∼62 kilodaltons) or EGFR association (∼170 kilodaltons). Total CD95 (∼48 kilodaltons) and total Yes (∼62 kilodaltons) served as respective loading controls. Representative blots from at least 3 independent experiments are shown. (A) TLCS-induced Yes/EGFR association precedes EGFR/CD95 association. No CD95/Yes association was detectable upon bile salt addition. (B) TLCS-induced EGFR/Yes association is not affected by JNK- or PKC inhibition, whereas subsequent EGFR/CD95 association is sensitive to inhibition of JNK and PKC.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 6
Inhibitor profile of TLCS-induced EGFR/CD95 association and activation of the CD95 system. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L) for 60 minutes (EGFR/CD95 association and CD95-tyrosine phosphorylation) or for 3 hours (DISC formation, i.e., caspase 8 and FADD recruitment to CD95). When indicated, N-acetylcysteine (NAC; 30 mmol/L), SU6656 (10 μmol/L), PP-2 (10 μmol/L), herbimycin A (1 μmol/L), emodin (10 μmol/L), DB-cAMP (100 μmol/L), H89 (5 μmol/L), AG1478 (5 μmol/L), genistein (100 μmol/L), JNK-inhibitor (L-JNKI1; 5 μmol/L), or Gö6850 (10 μmol/L) were preincubated for 30 minutes. CD95 was immunoprecipitated as described in the Materials and Methods section, and immunoprecipitated CD95 samples were detected for EGFR-association (∼170 kilodaltons), for tyrosine phosphorylation (CD95-Tyr-P, ∼48 kilodaltons), and for Fas-associated death domain (FADD, ∼28 kilodaltons) and caspase 8 (Casp8, ∼54/55 kilodaltons) association by Western blotting. Total CD95 (∼48 kilodaltons) served as a loading control. Representative blots from at least 3 independent experiments are shown. In line with previous studies, 18 TLCS induced an EGFR/CD95 association and subsequent CD95-Tyr-P within 60 minutes, being followed by DISC formation. EGFR/CD95 association was sensitive to JNK and PKC inhibition, whereas Src family kinase inhibitors were ineffective. cAMP and Yes inhibition by SU6656 did not affect CD95/EGFR activation but prevented CD95 tyrosine phosphorylation. All maneuvers inhibiting TLCS-induced EGFR/CD95 association prevented CD95 tyrosine phosphorylation, as did inhibition of EGFR tyrosine kinase activity by AG1478 or genistein. DISC formation only occurred under conditions allowing for CD95 tyrosine phosphorylation.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 7
Role of Yes in bile salt-induced activation of the CD95 system in the perfused rat liver. Rat livers were perfused as described in the Materials and Methods section for 120 minutes with TLCS (10 μmol/L), together with SU6656 (1 μmol/L), PP-2 (250 nmol/L), or DMSO as control. Liver samples were taken at the time points indicated (t = 0 minutes; immediately before TLCS infusion). Representative blots from 3 independent experiments for each condition are shown. (A) Yes activation and subsequent Yes/EGFR association and EGFR phosphorylation were detected by Yes or EGFR immunoprecipitation, respectively, and subsequent Western blotting as described above. Within 5 minutes TLCS perfusion led to an activation of the Src family kinase Yes, followed by an EGFR/Yes association and subsequent EGFR phosphorylation. These events were sensitive to SU6656, whereas PP-2 was ineffective, suggestive for a Yes-mediated EGFR transactivation by TLCS in the perfused liver. (B) EGFR/CD95 association, CD95-Tyr-P, and DISC formation were detected by CD95 immunoprecipitation and subsequent Western blotting as described above. Furthermore, total CD95 was detected in membrane and cytosolic fractions obtained by ultracentrifugation to investigate TLCS-induced CD95 membrane trafficking. In line with previous studies, TLCS induced within 30 minutes EGFR/CD95 association, subsequent CD95-Tyr-P, and membrane translocation. Within 60 minutes recruitment of FADD and caspase 8 to the CD95, i.e., DISC formation, became detectable. CD95 tyrosine phosphorylation and DISC formation were sensitive to Yes inhibition by SU6656, but not PP-2.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 8
Yes and EGFR knockdown in cultured primary rat hepatocytes. Yes (A) and EGFR protein (B) were knocked down as described in the Materials and Methods section by use of antisense oligonucleotides. Nonsense oligonucleotides served as a control. Hepatocytes were cultured in cell culture medium (untreated control) or treated with nonsense, Yes- or EGFR-antisense oligonucleotides, respectively, for the time periods indicated. Total Yes (∼62 kilodaltons) or EGFR (∼170 kilodaltons) were detected by Western blotting. Total CD95 amount (∼48 kilodaltons) served as a loading control and to confirm specificity of Yes- or EGFR-antisense oligonucleotides, respectively. Yes antisense (A) and EGFR antisense (B) led within 4 days to a marked decrease in total Yes or EGFR expression, respectively, whereas CD95 protein expression remained unaffected.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 9
Ntcp and Oatp 1, 2, and 4 expression in primary rat hepatocytes during culture. (A) Hepatocytes were cultured for the time periods indicated. Expression of Ntcp (∼50 kilodaltons), Oatp 1 (∼80 kilodaltons), 2 (∼92 kilodaltons), and 4 (∼90 kilodaltons) were detected by Western blotting. GAPDH (∼38 kilodaltons) served as a loading control. Representative blots of 3 independent experiments are shown. There was a strong culture-time-dependent down-regulation of Ntcp and Oatp 1, whereas expression of Oatp 2 and 4 was largely unaffected. (B) In 4-day cultured rat hepatocytes, Oatp 2 and 4, but not Oatp 1, exhibit membrane localization as visualized by immunocytochemistry.
Gastroenterology  , DOI: ( /j.gastro )
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12. Figure 10
Inhibition of TLCS-induced CD95 activation after Yes and EGFR protein knockdown. Yes (A) and EGFR protein (B) were knocked down as described in the Materials and Methods section by use of antisense oligonucleotides. Nonsense oligonucleotides served as a control. Hepatocytes were cultured for 4 days and then exposed to TLCS (500 μmol/L). Yes, EGFR, and CD95 were immunoprecipitated as described in the Materials and Methods section after the time periods indicated below. The immunoprecipitated CD95 samples were detected for EGFR association (∼170 kilodaltons), for tyrosine phosphorylation (CD95-P-Tyr, ∼48 kilodaltons), and for Fas-associated death domain (FADD, ∼28 kilodaltons) and caspase 8 (Casp8, ∼54/55 kilodaltons) association by Western blotting. Immunprecipitated Yes and EGFR were detected for activating phosporylation by Src family-Y418 (P-Yes-Y418, ∼62 kilodaltons) or tyrosine phosphorylation (P-EGFR, ∼170 kilodaltons). Total Yes (∼62 kilodaltons), total EGFR (∼170 kilodaltons), and total CD95 (∼48 kilodaltons) served as loading controls. Total CD95 amount (∼48 kilodaltons) was also detected in membrane and cytosolic fractions obtained by ultracentrifugation as described in the Materials and Methods section. Representative blots from at least 3 independent experiments are shown. (A) In 4-day cultured hepatocytes, Yes knockdown largely prevents TLCS-induced Yes and EGFR phosphorylation (stimulation for 1 minute, lane 1), whereas JNK activation (stimulation for 30 minutes) in response to TLCS was not affected (lane 2). In line with this, Yes knockdown does not affect TLCS-induced EGFR/CD95 association (lane 3) but strongly blunted CD95 tyrosine phosphorylation (lane 3), which otherwise (nonsense transfection) occurs within 60 minutes of TLCS exposure. TLCS induces within 3 hours formation of the death-inducing signaling complex (DISC), as shown by the recruitment of FADD and caspase 8 to CD95. Yes knockdown prevents TLCS-induced DISC formation (lane 4) and CD95 membrane trafficking (lane 5). (B) In 4-day cultured hepatocytes, EGFR knockdown largely prevents TLCS-induced EGFR phosphorylation (stimulation for 1 minute, lane 1) in response to TLCS, whereas TLCS-induced Yes activation was not affected (stimulation for 1 minute, lane 1). Furthermore, EGFR knockdown blunts TLCS-induced EGFR/CD95 association (lane 2) and CD95 tyrosine phosphorylation (lane 2), which otherwise (nonsense transfection) occur within 60 minutes of TLCS exposure. TLCS induces within 3 hours formation of the death-inducing signaling complex (DISC), as shown by the association of CD95 with FADD and caspase 8. EGFR knockdown prevents bile salt-induced DISC formation (lane 3) and CD95 membrane trafficking (lane 4).
Gastroenterology  , DOI: ( /j.gastro )
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13. Figure 11
Putative mechanism of TLCS-induced activation of the CD95-mediated apoptosis. Proapoptotic bile acids produce an almost instantaneous oxidative stress response, which triggers Yes and JNK activation. Activated Yes associates with EGFR, which becomes activated by Yes-tyrosine kinase activity. The Yes/EGFR complex dissociates and activated EGFR associates with CD95 in JNK and PKC-dependent ways. CD95 becomes tyrosine phosphorylated by EGFR tyrosine kinase activity. CD95 tyrosine phosphorylation provides the signal for membrane trafficking and DISC formation. cAMP inhibits bile salt-induced apoptosis by a protein kinase A-dependent inhibition of Yes/EGFR association and EGFR activation. In addition, cAMP induces Ser/Thr phosphorylation of the CD95, which acts as an internalization signal for CD95 (not shown). 20
Gastroenterology  , DOI: ( /j.gastro )
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