cAMP-guanine exchange factor protection from bile acid-induced hepatocyte apoptosis involves glycogen synthase kinase regulation of c-Jun NH₂-terminal kinase

Abstract

Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (cAMP-GEF)/phosphoinositide-3-kinase (PI3K)/Akt pathway. The aim of these studies was to identify the downstream substrate in this pathway and to determine at what level in the apoptotic cascade cytoprotection occurs. Since inhibitory phosphorylation of glycogen synthase kinase-3 (GSK) occurs downstream of PI3K/Akt and this phosphorylation has been implicated in cell survival, we conducted studies to determine whether GSK was downstream in cAMP-GEF/PI3K/Akt-mediated cytoprotection. Our results show that treatment of hepatocytes with the cAMP-GEF-specific analog, 4-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP, results in PI3K-dependent phosphorylation of GSK. Direct chemical inhibition of GSK in rat hepatocytes or human HUH7-NTCP cells with several structurally and functionally distinct inhibitors including bromoindirubin-3'-oxime (BIO), maleimides (SB216763, SB415286), thiadiazolidine derivatives, and LiCl attenuates apoptosis induced by glycochenodeoxycholate (GCDC). In addition, genetic silencing of the GSK β isoform with small interfering RNA attenuates GCDC apoptosis in HUH7-NTCP cells. Adenoviral inhibition of the Rap1 blocks both cAMP-GEF-mediated cytoprotection against GCDC-induced apoptosis and Akt/GSK3β phosphorylation. GCDC-induced phosphorylation of the proapoptotic kinase, c-Jun NH(2)-terminal kinase (JNK) is inhibited by GSK inhibition or cAMP-GEF activation. GCDC-induced apoptosis is accompanied by phosphorylation of the endoplasmic reticulum stress markers pIEF2α and IRE-1, and pretreatment with the cAMP-GEF analog or GSK inhibitors prevents this phosphorylation. Collectively, our results support the presence of a cAMP/cAMP-GEF/Rap1/PI3K/Akt/GSKβ survival pathway in hepatocytes that inhibits bile acid-induced JNK phosphorylation.

Phosphodiesterase 4 Inhibition as a Therapeutic Target for Alcoholic Liver Disease: From Bedside to Bench

“…Indeed, rolipram‐treated mice with adequate cAMP levels had much lower ER stress protein expression including a critical apoptotic protein, CHOP. Our in vitro findings further established that the cAMP pathway is a negative regulator of ER stress and TNFα‐induced toxicity induced by ethanol, which is in agreement with previous studies examining the role of cAMP signaling in TNFα and bile acid–induced toxicity …”

Role of cAMP and phosphodiesterase signaling in liver health and disease

“…Specifically, it has been demonstrated that increased cAMP, and specifically EPAC signaling, protects hepatocytes from bile acid induced apoptosis via PI3/Akt pathway. Moreover, this protection involves glycogen synthase kinase 3 (GSK3)-mediated inhibition of pro-apoptotic kinase, c-Jun NH2-terminal kinase (JNK) and ER stress [114]. EPAC activation also protects hepatocytes from bile acid induced mitochondrial apoptosis [113, 114].…”

“…Moreover, this protection involves glycogen synthase kinase 3 (GSK3)-mediated inhibition of pro-apoptotic kinase, c-Jun NH2-terminal kinase (JNK) and ER stress [114]. EPAC activation also protects hepatocytes from bile acid induced mitochondrial apoptosis [113, 114]. Another pathway of cAMP-mediated protection involves PKA-mediated phosphorylation of CD95 (FasR) and preventing formation of deathinducing signal complex (DISC), activation of caspases and apoptosis [115, 116].…”

Rolipram Attenuates Bile Duct Ligation–Induced Liver Injury in Rats: A Potential Pathogenic Role of PDE4

“…Hence, in addition to its anti-inflammatory effects, PDE4 inhibition, which leads to increased cellular cAMP levels, could also attenuate hepatocyte death. Indeed, cAMP has been shown to protect hepatocytes from bile acid-induced death by affecting protein kinase A (PKA) and cAMP-guanine exchange factor-mediated signaling (Webster et al, 2002;Cullen et al, 2004;Reinehr and Haussinger, 2004;Johnston et al, 2011). Furthermore, along with inhibiting TGF-b and its receptor TbR1, PDE4 inhibition also reduced phosphorylation of Smad3 (Fig.…”