1. Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95 
Roland Reinehr, Dieter Häussinger 
Gastroenterology 
Volume 126, Issue 1, Pages (January 2004)
DOI: /j.gastro

2. Figure 1
Bile salt-induced CD95 phosphorylations. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L), GCDC (100 μmol/L), and/or DB-cAMP (100 μmol/L) for the time periods indicated. Samples were collected, and CD95 was immunoprecipitated as described in the Materials and Methods section and then CD95 was detected for phosphorylation on serine/threonine/tyrosine residues by Western blotting. Total CD95 served as a loading control. (A) TLCS induces CD95-Tyr phosphorylation within 30 minutes, which was maximal at about 3 hours and slowly decreased thereafter. After about 5 hours, a CD95-Ser/Thr phosphorylation becomes detectable. (B) DB-cAMP induces CD95-Ser/Thr phosphorylation within 10 minutes but no CD95-Tyr phosphorylation. (C) Simultaneous addition of TLCS and DB-cAMP prevents CD95-Tyr phosphorylation and induces CD95-serine/threonine phosphorylation with kinetics similar to that induced by DB-cAMP alone. (D) DB-cAMP inhibits TLCS-induced CD95-tyrosine phosphorylation in a dose-dependent way when incubated together with TLCS for 60 minutes. One micromolar DB-cAMP was sufficient to induce CD95-Ser/Thr phosphorylation and to inhibit CD95-Tyr phosphorylation. (E) DB-cAMP inhibits GCDC-induced CD95-tyrosine phosphorylation in a dose-dependent way when incubated together with GCDC for 60 minutes. One micromolar DB-cAMP was sufficient to induce CD95-Ser/Thr phosphorylation and to inhibit CD95-Tyr phosphorylation.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Effect of cAMP on bile salt-induced EGF-R- and JNK-activation. Hepatocytes were cultured for 24 hours and then exposed to control medium, TLCS (100 μmol/L), and/or DB-cAMP (100 μmol/L) for the time periods indicated. The PKA-inhibitor H89 (5 μmol/L) was preincubated 30 minutes prior to the bile salt addition. EGF-R phosphorylation and JNK phosphorylation were detected by immunoblotting. Representative blots from 3 independent experiments are shown. (A) TLCS induces within 1 minute a marked EGF-R phosphorylation, which was abolished when incubated together with DB-cAMP in a H89-sensitive way, suggesting that cAMP prevents TLCS-induced EGF-R activation in a PKA-dependent manner. (B) TLCS, but not DB-cAMP, induces JNK-1 protein phosphorylation within 15 minutes, which lasts for up to 6 hours. cAMP has no effect on the TLCS-induced JNK-1 activation during the first hour, whereas JNK-1 phosphorylation after 3 hours was inhibited. Similar data were obtained for JNK-2 protein phosphorylation (not shown).
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
cAMP does not prevent TLCS-induced EGF-R/CD95-association. Hepatocytes were cultured for 24 hours and then exposed to TLCS (100 μmol/L) and/or DB-cAMP (100 μmol/L) for the time periods indicated. CD95 was immunoprecipitated and EGF-R-association was detected by immunoblotting. Representative blots from 3 independent experiments are shown. In line with previous data,18 TLCS induced CD95/EGF-R association (A), whereas DB-cAMP had no effect (B), DB-cAMP did not alter the TLCS-induced CD95/EGF-R association (C), indicating that inhibition of TLCS-induced CD95-tyrosine phosphorylation by cAMP does not occur at the level of CD95/EGF-R association.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Effects of cAMP and H89 on TLCS- and GCDC-induced activation of the CD95 system. Hepatocytes were cultured for 24 hours and then exposed to control medium, TLCS (100 μmol/L), GCDC (100 μmol/L), or DB-cAMP (100 μmol/L). If indicated, the PKA-specific inhibitor H89 (5 μmol/L) or the PI-3-kinase-specific inhibitors LY (10 μmol/L) or wortmannin (100 nmol/L) were preincubated for 30 minutes. Samples were collected and either underwent Western blotting for EGF-R phosphorylation (1) or immunoprecipitation for CD95 with subsequent Western blotting for EGF-R (2), phospho-serine/threonine/tyrosine (3), or DISC-formation as indicated by caspase 8 and FADD immunoblotting (4). Total CD95 served as loading control. Representative blots of 3 independent experiments are shown. (1) Bile salt-induced EGF-R phosphorylation was detected after 10 minutes of bile salt addition and was inhibited by DB-cAMP in a H89-sensitive but LY and wortmannin-insensitive way. (2) EGF-R/CD95 association induced by TLCS or GCDC was measured after 60 minutes of bile salt addition and was not affected by DB-cAMP. (3) CD95 phosphorylations were detected after 60-minute incubation of bile salts and/or DB-cAMP. Whereas TLCS and GCDC induced CD95-Tyr phosphorylation, DB-cAMP induced phosphorylation of CD95 on Ser/Thr residues. When DB-cAMP was incubated together with TLCS or GCDC, bile salt-induced CD95-Tyr phosphorylation was inhibited, whereas CD95-Ser/Thr phosphorylation still occurred in a PKA-inhibition-sensitive way. (4) DISC-formation was measured 3 hours after bile salt addition. TLCS and GCDC induced caspase 8 and FADD association with CD95, which was abolished in a H89-sensitive way when TLCS or GCDC were incubated together with DB-cAMP. Inhibition of PI-3 kinase with either wortmannin or LY did not alter DB-cAMP-mediated effects on TLCS-induced activation of the CD95-system.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
cAMP abolishes TLCS-induced apoptosis in a H89-sensitive way. Hepatocytes were cultured for 24 hours and then exposed to control medium (•) or TLCS (100 μmol/L, ○) for the given time periods. When indicated, H89 (5 μmol/L) was preincubated for 30 minutes prior to the TLCS addition (▴). DB-cAMP (100 μmol/L) was either added together with TLCS (0′, □/+H89, ♢) or after 150 minutes of TLCS incubation (150′, ■/+H89, ▵). The percentage of TUNEL-positive hepatocytes was determined as described in the Materials and Methods section. TLCS induced a time-dependent increase in apoptotic cells, which was enhanced by the inhibition of PKA and inhibited by DB-cAMP (in a H89 sensitive way), especially if given simultaneously with TLCS. The antiapoptotic effect of cAMP was strongly blunted when cAMP was added 150 minutes after TLCS.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 9
Enrichment of Tyr-phosphorylated CD95 in the membrane and Ser/Thr-phosphorylated CD95 in the cytosolic fraction of rat hepatocytes. Twenty-four-hour cultured hepatocytes were exposed to control medium, TLCS (100 μmol/L), and DB-cAMP (100 μmol/L) for the given time periods. When indicated, H89 (5 μmol/L) was added 30 minutes prior to the bile salt addition. DB-cAMP was either added together with TLCS (cAMP 0′) or 150 minutes after TLCS (cAMP 150′). Samples were collected, and membrane and cytosolic fractions were separated by ultracentrifugation as described in the Materials and Methods section. CD95 was immunoprecipitated first and then CD95 phosphorylations were detected by immunoblotting. CD95 served as a loading control. A high efficacy of separation is shown by the exclusive detection of the membrane marker annexin II in the membrane fraction and the cytosolic enzyme GAPDH in the cytosolic fraction only. CD95 membrane trafficking was detected using CD95 immunoblotting in the latter fractions. Annexin II and GAPDH served as a loading control. (A) TLCS induced within 3 hours an enrichment of Tyr-phosphorylated CD95 in the membrane fraction, which was abolished in the presence of DB-cAMP in a H89-sensitive manner. Simultaneously, cAMP induced a CD95-Ser/Thr phosphorylation, which was only detectable in the cytosolic fraction. Prolonged exposure (12 hours) to TLCS also induced CD95-Ser/Thr phosphorylation, which was cytosolic. When cAMP was added 150 minutes after TLCS, Ser/Thr phosphorylation of CD95 occurred in both the cytosolic and membrane fraction. The findings suggest that CD95-Ser/Thr phosphorylation may provide a signal for its internalization and/or prevention of its externalization. (B) TLCS induced a CD95 enrichment in the membrane fraction, indicating a TLCS-induced CD95 membrane trafficking. This CD95 membrane trafficking was abolished by addition of DB-cAMP in a H89-sensitive way.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 6
cAMP generation and PKA activation in response to adenosin and forskolin in 24-hour cultured rat hepatocytes. (A) Hepatocytes were cultured for 24 hours and then exposed to control medium (○), adenosine (100 μmol/L, ■), or forskolin (100 μmol/L, ♦) for the time periods indicated. Measurement of intracellular cAMP was performed as described in the Materials and Methods section. All agonists led to a significant cAMP-generation within 30 seconds (P < 0.05; n = 3). Basal cAMP level was 21 ± 1 fmol cAMP/μg protein (n = 21). (B) After 24 hours of culture, hepatocytes were exposed to control medium (○), adenosine (100 μmol/L, ■), forskolin (100 μmol/L, ♦), or DB-cAMP (100 μmol/L, ▴) for up to 180 minutes. PKA activity was measured as described in the Materials and Methods section and was elevated significantly for all agonists used (P < 0.05; n = 3).
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 7
Inhibition of the TLCS-induced CD95 activation by adenosine and forskolin. Hepatocytes were cultured for 24 hours and then exposed to control medium, TLCS (100 μmol/L), adenosine (100 μmol/L), or forskolin (100 μmol/L). When indicated, the PKA inhibitor H89 (5 μmol/L) was preincubated for 30 minutes. Samples were collected and either underwent Western blotting for EGF-R phosphorylation (1) or immunoprecipitation for CD95 with subsequent Western blotting for EGF-R (2), phospho-serine/threonine/tyrosine (3), or DISC formation as indicated by caspase 8 and FADD immunoblotting (4). Total CD95 served as loading control. Representative blots of 3 independent experiments are shown. (1) TLCS-induced EGF-R phosphorylation was detected after 10 minutes of bile salt exposition and was sensitive to adenosine or forskolin addition in a H89-sensitive way. (2) EGF-R/CD95 association induced by TLCS was measured after 60 minutes of bile salt addition and was not affected by adenosine or forskolin. (3) CD95 phosphorylations were detected after 60-minute incubation of TLCS, adenosine, or forskolin. Whereas TLCS induced CD95-Tyr phosphorylation, adenosine and forskolin induced phosphorylation of CD95 on Ser/Thr residues. When the latter compounds were incubated together with TLCS, TLCS-induced CD95-Tyr phosphorylation was blunted, and CD95-Ser/Thr phosphorylation still occurred in a H89 sensitive way. (4) DISC formation was measured 3 hours after bile salt addition. TLCS-induced caspase 8 and FADD association with CD95 was abolished by adenosine or forskolin in a H89-sensitive manner.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 8
Effect of TLCS on cAMP levels and PKA activity. Hepatocytes were cultured for 24 hours and then exposed to control medium (○) or TLCS (100 μmol/L, •). cAMP-levels (A) and PKA activity (B) were measured as described in the Materials and Methods section. TLCS increased cAMP levels and PKA activity (∗indicates P < 0.05; n = 3) after 6–7 hours of TLCS exposure. This suggests that the late TLCS-induced CD95-Ser/Thr phosphorylation is caused by TLCS-induced cAMP generation and PKA activation.
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
CD95 internalization is induced by a PKA-mediated CD95-Ser/Thr phosphorylation. Twenty-four-hour cultured hepatocytes were exposed to TLCS (100 μmol/L). When indicated, H89 (5 μmol/L) was added 30 minutes prior to the TLCS addition, or DB-cAMP (100 μmol/L) was incubated after 150 minutes of TLCS exposure. CD95 was then immunostained in nonpermeabilized hepatocytes to detect CD95 membrane localization (A), or samples were taken for CD95 immunoprecipitation and subsequent detection of CD95 phosphorylation grade using immunoblotting technique (B and C). (A) TLCS (○) induced a CD95 membrane trafficking, which was maximal at about 3 hours and declined thereafter. This decline was prevented by H89 preincubation (▴). On the other hand, when DB-cAMP was added after 150 minutes, i.e., a time point when TLCS-induced CD95 membrane trafficking was maximal, CD95-internalization was accelerated (■). ∗Indicates significant difference from TLCS-induced CD95 membrane trafficking (P < 0.05). (B) When H89 was incubated together with TLCS, “late” CD95-Ser/Thr phosphorylation was abolished (for TLCS alone see Figure 1A). (C) When DB-cAMP was added after a 150-minute TLCS-preincubation, CD95-Ser/Thr phosphorylation was induced on top of CD95 Tyr phosphorylation (see Figure 1). The data suggest that CD95-Ser/Thr phosphorylation may provide a signal for CD95 internalization, whereas CD95-Tyr phosphorylation is a prerequiste for CD95 membrane trafficking.
Gastroenterology  , DOI: ( /j.gastro )

12. Figure 11
Inhibition of TLCS-induced activation of the CD95 system by cAMP in perfused rat liver. Rat livers were perfused as described in the Materials and Methods section for 60 minutes with TLCS (10 μmol/L), DB-cAMP (100 μmol/L), or DMSO as control. Liver samples were taken at the time points indicated (t = 0 minutes immediately prior to TLCS infusion). Representative blots from 3 independent experiments for each condition are shown. (A) EGF-R-tyrosine phosphorylation (P-EGF-R), a marker for EGF-R activation, was detected by Western blotting. Total EGF-R served as loading control. CD95 was immunoprecipitated as described in the Materials and Methods section and assessed for Ser/Thr/Tyr phosphorylation. Caspase 8 and FADD association with CD95 (DISC formation) was detected using immunoblotting technique. Total CD95 served as loading control. (B) CD95 membrane trafficking was detected by Western blotting of total CD95 recovered in membrane and cytosolic fractions (see Materials and Methods section). Annexin II and GAPDH served as loading controls for the latter fractions.
Gastroenterology  , DOI: ( /j.gastro )

13. Figure 12
Regulation of the CD95 system by proapoptotic bile salts and cAMP. Proapoptotic bile acids such as TLCS, TCDC, or GCDC generate oxidative stress in rat hepatocytes, which triggers EGF-R-tyrosine phosphorylation and JNK-activation. This JNK signal may converge with a PKC-signal, to allow association of activated EGF-R with CD95, which is a substrate for EGF-R-tyrosine kinase activity. Following EGF-R-mediated CD95-Tyr phosphorylation, CD95 membrane trafficking and DISC formation (FADD and caspase 8/CD95 association) and apoptosis are induced. ∗TCDC only induces DISC formation and apoptosis after inhibition of a PI-3-kinase survival pathway.5 cAMP inhibits bile salt-induced apoptosis through a PKA-dependent (1) CD95-Ser/Thr phosphorylation, which may provide a signal for CD95-internalization, and (2) inhibition of CD95 Tyr phosphorylation, which is a prerequisite for CD95 membrane trafficking and DISC formation. Inhibition of CD95 Tyr phosphorylation by cAMP is due to a PKA-dependent inhibition of bile salt-induced EGF-R phosphorylation. A late activation of PKA by hydrophobic bile acids may be involved in the termination of their apoptotic action (dashed line).
Gastroenterology  , DOI: ( /j.gastro )