Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms Dirk Graf, Roland Reinehr, Anna Kordelia Kurz, Richard Fischer, and Dieter H€ aussinger * Department for Gastroenterology, Hepatology and Infectiology, Heinrich Heine University D€ usseldorf, Moorenstrasse 5, D-40225 D€ usseldorf, Germany Received 12 March 2003, and in revised form 30 April 2003 Abstract The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 lmol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38 MAPK , and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase acti- vation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Apoptosis; Bile acids; CD95 trafficking; TRAIL-2; PKA/MAP kinases In cholestatic disorders, hepatocellular damage is augmented by the retention of hydrophobic bile acids in hepatocytes, which may induce apoptosis [1–6], as dem- onstrated in isolated rat hepatocytes, hepatoma cell lines [1,2,6],andtheliverinvivo[7].Differentmechanismshave been proposed to mediate bile acid-induced apoptosis, including ligand-independent CD95 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) 1 re- ceptor-2 activation [8–12], protein kinase C (PKC) [13], c-Jun N-terminal kinase (JNK) [11], extracellular signal- regulated kinase (ERK) [14], cathepsin B [15], and oxi- dativestress[4–6,16].cAMPwasrecentlyshowntoinhibit bile acid-induced apoptosis in isolated hepatocytes [17,18] and to modulate the apoptotic program in other cell types [19]. The mechanisms underlying the protective effect of cAMP on bile acid-induced apoptosis are in- completely understood, although it was suggested that inhibition of hepatocyte apoptosis by cAMP is mediated by activation of PKA and phosphatidylinositol-3-kinase (PI3-kinase) [17,18]. Here, we demonstrate that cAMP prevents bile acid-induced apoptosis due to multiple ef- fects on apoptotic pathways involving PKA-dependent and -independent mechanisms. Materials and methods Materials The antibodies against CD95-receptor and c-Jun N-terminal kinase (JNK)-1 were from Santa Cruz Archives of Biochemistry and Biophysics 415 (2003) 34–42 www.elsevier.com/locate/yabbi ABB * Corresponding author. Fax: +49-211-811-8838. E-mail address: haeussin@uni-duesseldorf.de (D. H€ aussinger). 1 Abbreviations used: cAMP, cyclic AMP; TLCS, taurolithocholate 3-sulfate; MAP, mitogen activated protein; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PARP, poly(ADP) ribose polymerase; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; JNK-1, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; PI3-kinase, phosphatidylinositol-3-kinase; GCDC, glycochenodeoxycholate; TBST, Tris-buffered saline; PI, propidium iodide; PBS, phosphate-buffered saline; TUDC, taur- oursodeoxycholate; MKP-1, MAP-kinase-phosphatase 1. 0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00224-8