Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95

doi:10.1053/j.gastro.2003.09.044

Gastroenterology

Volume 126, Issue 1, January 2004, Pages 249-262

https://doi.org/10.1053/j.gastro.2003.09.044 Get rights and content

Abstract

$Background & Aims$ : Cyclic AMP (cAMP) inhibits bile salt-induced hepatocyte apoptosis; the underlying mechanisms are unclear. $Methods$ : The effects of cAMP on taurolithocholate-3-sulfate-(TLCS)- or glycochenodesoxycholate (GCDC)-induced CD95 (Fas/APO-1) activation and apoptosis were studied in 24-hour cultured rat hepatocytes and in perfused rat liver. $Results$ : TLCS induced a rapid oxidative stress response, c-Jun-N-terminal kinase (JNK) and epidermal growth factor (EGF) receptor (EGF-R) activation, subsequent EGF-R/CD95 association and CD95 tyrosine phosphorylation, CD95 membrane targeting, death-inducing signal complex (DISC) formation and hepatocyte apoptosis. None of these responses was triggered by cAMP; however, cAMP induced H89-sensitive serine/threonine phosphorylation of CD95. Similar data were obtained with GCDC, another proapoptotic bile acid. cAMP did not prevent the TLCS-induced oxidative stress response, JNK activation and EGF-R/CD95 association, however abolished EGF-R activation and subsequent CD95 tyrosine phosphorylation, CD95 membrane trafficking, and DISC formation in a H89-sensitive way. Also in presence of TLCS, cAMP induced rapid Ser/Thr phosphorylation of CD95 within 10 min. The effects of cAMP on the various steps of CD95 activation were also found in the intact perfused rat liver. Evidence is given that a cAMP-induced Ser/Thr phosphorylation favors internalization of CD95. $Conclusions$ : Inhibition of bile salt-induced apoptosis by cAMP involves both PKA-dependent Ser/Thr phosphorylation of the CD95 and inhibition of EGF-R activation, which results in an inhibition of CD95 tyrosine phosphorylation, CD95 membrane targeting, and DISC formation. CD95 regulation involves complex phosphorylations with CD95-tyrosine phosphorylation favoring CD95 membrane trafficking and DISC formation, whereas CD95 Ser/Thr phosphorylation inhibits these processes.

Section snippets

Materials

The materials used were purchased as follows: collagenases from Boehringer (Mannheim, Germany); William’s E medium and fetal calf serum from Gibco Life Technologies (Gaithersburg, MD); taurolithocholate-3-sulfate (TLCS) and glycochenodeoxycholate (GCDC) from Sigma-Aldrich (Deisenhofen, Germany); penicillin/streptomycin from Biochrom (Berlin, Germany); carboxy-2,7-dichlorofluorescin diacetate (carboxy-H₂-DCFDA)29 from Molecular Probes (Eugene, OR); H89 from Tocris Cookson Ltd. (Bristol, United

Effects of cAMP and TLCS on CD95 phosphorylation pattern

Because cAMP was shown to inhibit TLCS-induced CD95 membrane targeting, caspase activation, and hepatocyte apoptosis,24 its effect on CD95 phosphorylation was studied. In line with recent data18 and as shown in Figure 1A, TLCS (100 μmol/L) induced within 30 minutes CD95 tyrosine phosphorylation, which persisted for more than 12 hours. No Ser/Thr phosphorylation of CD95 was observed during the first 3 hours of TLCS addition; however, thereafter, Ser/Thr phosphorylation of CD95 started to

Mechanism of cAMP-induced inhibition of CD95 activation by hydrophobic bile acids

Recent data indicated that CD95 activation in hepatocytes is a complex process,12, 18 which is not only triggered by hydrophobic bile acids but also by other proapoptotic stimuli, such as CD95 ligand and hyperosmolarity.28 This process of CD95 activation involves an almost instantaneous oxidative stress response, which triggers a tyrosine kinase-dependent EGF-R activation and JNK activation. This JNK signal together with a PKC signal is required for a CD95/EGF-R association with subsequent CD95

Acknowledgements

The authors thank C. Holneicher and N. Schumacher for expert help in liver perfusion and S. Becker for excellent technical assistance.

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^☆: Supported by Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575 “Experimentelle Hepatologie” (Düsseldorf).

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Basic-liver, pancreas, and biliary tractInhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95☆

Abstract

Section snippets

Materials

Effects of cAMP and TLCS on CD95 phosphorylation pattern

Mechanism of cAMP-induced inhibition of CD95 activation by hydrophobic bile acids

Acknowledgements

Gastroenterology

Hepatology

Gastroenterology

J Hepatol

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Hepatology

Gastroenterology

Gastroenterology

Arch Biochem Biophys

J Biol Chem

J Biol Chem

J Biol Chem

Hepatology

Gastroenterology

J Biol Chem

JBC

J Biol Chem

Basic-liver, pancreas, and biliary tract
Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95☆