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

2011 ◽  
Vol 301 (2) ◽  
pp. G385-G400 ◽  
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.

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

2009 ◽  
Vol 296 (4) ◽  
pp. G764-G774 ◽  
Author(s):  
Anna Gates ◽  
Simon Hohenester ◽  
M. Sawkat Anwer ◽  
Cynthia R. L. Webster
Keyword(s):  
Bile Acid ◽  
Signaling Pathway ◽  
Rat Hepatocytes ◽  
Cytoprotective Effect ◽  
Src Tyrosine Kinase ◽  
Akt Phosphorylation ◽  
Egfr Inhibition ◽  
Phosphoinositide 3 Kinase ◽  
Induced Apoptosis ◽  
Α Subunits

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.

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

2002 ◽  
Vol 283 (3) ◽  
pp. G727-G738 ◽  
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.

scholarly journals Discovery and Optimization of Novel Pyridines as Highly Potent and Selective Glycogen Synthase Kinase 3 Inhibitors

2019 ◽  
Author(s):  
Savithri Ramurthy ◽  
Keith B. Pfister ◽  
Rustum S. Boyce ◽  
Sean P. Brown ◽  
Abran Q. Costales ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Insulin Dependent Diabetes Mellitus ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Pharmacological Intervention ◽  
Dependent Diabetes Mellitus ◽  
Energy Regulation ◽  
Structural Determinants ◽  
Disease States

<div>Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-beta in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.</div><div><br></div>

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

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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