cAMP-GEF cytoprotection by Src tyrosine kinase activation of phosphoinositide-3-kinase p110 β/α in rat hepatocytes

Cyclic AMP protects against hepatocyte apoptosis by a protein kinase A-independent cAMP-GEF/phosphoinositide-3-kinase (PI3K)/Akt signaling pathway. However, the signaling pathway coupling cAMP-GEF with PI3K is unknown. The aim of this study was to investigate the role of Src tyrosine kinases (Src-TYK) and PI3K-p110 isoforms in this pathway. Studies were done in rat hepatocytes using the hydrophobic bile acid glycochenodeoxycholic acid (GCDC) to induce apoptosis. cAMP-binding guanine nucleotide exchange factors (cAMP-GEFs) were selectively activated by using 4-(4-chloro-phenylthio)-2′- O-methyladenosine-3′-5′-cyclic monophosphate (CPT-2-Me-cAMP), which sequentially phosphorylated Src-TYK (within 1 min) followed by Akt (within 5 min). The Src inhibitors PP2 and SU6656 inhibited basal and CPT-2-Me-cAMP-mediated Src and Akt phosphorylation. These inhibitors had no effect on CPT-2-Me-cAMP-mediated activation of Rap GTPases. CPT-2-Me-cAMP induced transient Src dependent autophosphorylation of the epidermal growth factor receptor (EGFR). Inhibition of the EGFR with AG 1478 partially inhibited the ability of CPT-2-Me to phosphorylate Akt. Whereas PP2 completely abolished the protective effect of CPT-2-Me-cAMP in GCDC induced apoptosis, AG 1478 partially inhibited the cytoprotective effect. CPT-2-Me-cAMP treatment resulted in Src-dependent activation of the p110 β and α subunits of PI3K, but only the latter was sensitive to inhibition with AG 1478. In conclusion, activation of cAMP-GEFs results in phosphorylation of Src-TYK and Akt and activation of the p110 β/α subunits of PI3K. Maximal cAMP-GEF-mediated Akt phosphorylation as well as protection from bile acid-induced apoptosis requires activation of Src-TYK and the EGFR. These studies support the existence of two pathways: cAMP-GEF/Rap/Src/PI3Kβ/Akt and cAMP-GEF/Rap/Src/EGFR/PI3Kα/Akt, both of which are necessary for maximal cytoprotective effect of cAMP-GEFs in hepatocytes.

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cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochromecrelease

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00410.2001 ◽

2002 ◽

Vol 283 (3) ◽

pp. G727-G738 ◽

Cited By ~ 40

Author(s):

Cynthia R. L. Webster ◽

Paul Usechak ◽

M. Sawkat Anwer

Keyword(s):

Bile Acid ◽

Cytochrome C ◽

Rat Hepatocytes ◽

Dependent Manner ◽

Cytochrome C Release ◽

Akt Phosphorylation ◽

Apoptotic Effect ◽

Cultured Rat Hepatocytes ◽

Phosphoinositide 3 Kinase ◽

Induced Apoptosis

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00430.2010 ◽

2011 ◽

Vol 301 (2) ◽

pp. G385-G400 ◽

Cited By ~ 12

Author(s):

A. Johnston ◽

K. Ponzetti ◽

M. S. Anwer ◽

C. R. L. Webster

Keyword(s):

Bile Acid ◽

Glycogen Synthase ◽

Rat Hepatocytes ◽

Glycogen Synthase Kinase ◽

Exchange Factor ◽

Stress Markers ◽

Guanine Exchange Factor ◽

Phosphoinositide 3 Kinase ◽

Induced Apoptosis ◽

Interfering Rna

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 NH2-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.

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Phosphoinositide 3-kinase, but not mitogen-activated protein kinase, pathway is involved in hepatocyte growth factor-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes

Hepatology ◽

10.1053/jhep.2001.22756 ◽

2001 ◽

Vol 33 (3) ◽

pp. 608-615 ◽

Cited By ~ 48

Author(s):

C Webster

Keyword(s):

Bile Acid ◽

Growth Factor ◽

Rat Hepatocytes ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Cultured Rat Hepatocytes ◽

Phosphoinositide 3 Kinase ◽

Induced Apoptosis ◽

Hepatocyte Growth ◽

Kinase Pathway

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Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00517.2003 ◽

2004 ◽

Vol 287 (2) ◽

pp. G334-G343 ◽

Cited By ~ 36

Author(s):

Kimberly A. Cullen ◽

John McCool ◽

M. Sawkat Anwer ◽

Cynthia R. L. Webster

Keyword(s):

Bile Acid ◽

Protective Effect ◽

Signaling Pathway ◽

Fas Ligand ◽

Kinase Inhibitor ◽

Pi3 Kinase ◽

Akt Phosphorylation ◽

Tnf Α ◽

Induced Apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-α-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2′- O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-α. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-α-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.

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Effect of ionomycin on bile-acid-induced apoptosis in rat hepatocytes

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)48048-7 ◽

2000 ◽

Vol 82 ◽

pp. 146

Author(s):

Mayumi Tsuji ◽

Hideto Oyamada ◽

Masako Okazaki ◽

Katsuji Oguchi

Keyword(s):

Bile Acid ◽

Rat Hepatocytes ◽

Induced Apoptosis

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Nodal induces apoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 β-cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00074.2012 ◽

2012 ◽

Vol 303 (1) ◽

pp. E132-E143 ◽

Cited By ~ 16

Author(s):

Fang Zhao ◽

Fengjie Huang ◽

Mengxiong Tang ◽

Xiaoming Li ◽

Nina Zhang ◽

...

Keyword(s):

Signaling Pathway ◽

Cell Apoptosis ◽

Caspase 3 ◽

Biological Effects ◽

Cell Mass ◽

Type I ◽

Β Cells ◽

Β Cell ◽

Akt Phosphorylation ◽

Induced Apoptosis

We demonstrated previously that the activation of ALK7 (activin receptor-like kinase-7), a member of the type I receptor serine/threonine kinases of the TGF-β superfamily, resulted in increased apoptosis and reduced proliferation through suppression of Akt signaling and the activation of Smad2-dependent signaling pathway in pancreatic β-cells. Here, we show that Nodal activates ALK7 signaling and regulates β-cell apoptosis. We detected Nodal expression in the clonal β-cell lines and rodent islet β-cells. Induction of β-cell apoptosis by treatment with high glucose, palmitate, or cytokines significantly increased Nodal expression in clonal INS-1 β-cells and isolated rat islets. The stimuli induced upregulation of Nodal expression levels were associated with elevation of ALK7 protein and enhanced phosphorylated Smad3 protein. Nodal treatment or overexpression of Nodal dose- or time-dependently increased active caspase-3 levels in INS-1 cells. Nodal-induced apoptosis was associated with decreased Akt phosphorylation and reduced expression level of X-linked inhibitor of apoptosis (XIAP). Remarkably, overexpression of XIAP or constitutively active Akt, or ablation of Smad2/3 activity partially blocked Nodal-induced apoptosis. Furthermore, siRNA-mediated ALK7 knockdown significantly attenuated Nodal-induced apoptosis of INS-1 cells. We suggest that Nodal-induced apoptosis in β-cells is mediated through ALK7 signaling involving the activation of Smad2/3-caspase-3 and the suppression of Akt and XIAP pathways and that Nodal may exert its biological effects on the modulation of β-cell survival and β-cell mass in an autocrine fashion.

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951 SPECIES-DEPENDENT BILE ACID-INDUCED APOPTOSIS: A COMPARISON OF PRIMARY HUMAN, MOUSE, AND RAT HEPATOCYTES

Journal of Hepatology ◽

10.1016/s0168-8278(10)60952-x ◽

2010 ◽

Vol 52 ◽

pp. S368

Author(s):

P. Kleiss ◽

M. van Wenum ◽

R. Wimmer ◽

T. Vennegeerts ◽

A.F. Hofmann ◽

...

Keyword(s):

Bile Acid ◽

Rat Hepatocytes ◽

Induced Apoptosis

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Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

Cellular Physiology and Biochemistry ◽

10.1159/000430262 ◽

2015 ◽

Vol 36 (3) ◽

pp. 866-883 ◽

Cited By ~ 10

Author(s):

Annika Sommerfeld ◽

Roland Reinehr ◽

Dieter Häussinger

Keyword(s):

Bile Acid ◽

Protein Kinase ◽

Protein Kinase A ◽

Rat Hepatocytes ◽

Β1 Integrin ◽

Dependent Manner ◽

Mitogen Activated Protein ◽

Induced Apoptosis ◽

Jnk Activation ◽

Kinase A

Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

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