Submitted on December 12, 2003
Accepted on March 20, 2004
Activation of cAMP-guanine exchange factor (cAMP-GEF) confers protein kinase A independent protection from hepatocyte apoptosis
Kimberly Cullen1, John McCool1, M Sawkat Anwer2, and Cynthia RL Webster1*
1 Department of Clinical Science, Tufts University School of Veterinary Medicine, North Grafton, MA, USA
2 Department of Biomedical Science, Tufts University School of Veterinary Medicine, North Grafton, MA, USA
* To whom correspondence should be addressed. E-mail: cynthia.leveille-webster{at}tufts.edu.
Cyclic AMP has previously been shown to promote cell survival in a variety of
cell types, but the downstream signaling pathway(s) of this anti-apoptotic effect is
unclear. Thus, the role of cAMP signaling through protein kinase A (PKA) and cAMP
regulated guanine nucleotide exchange factors (cAMP-GEF's) in cAMP's anti-apoptotic
action was investigated in the present study. Cyclic AMP's protective effect against bile
acid, Fas ligand and tumor necrosis factor-
(TNF-) induced apoptosis in rat
hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-4-
chlorophenythio-cAMP (Rp-cAMP). In contrast, a novel cAMP analogue, 8-(4-chloro-phenylthio)-
2'-O-methyladenosine-3'-5'-cyclic monophosphate, (CPT-2-Me-cAMP),
which activated cAMP-GEF's in hepatocytes without activating PKA, protected
hepatocytes against apoptosis induced by bile acids, Fas ligand and TNF-. 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, while CPT-2-Me-cAMP, which did not activate PKA,
induced phosphoinositide-3-kinase (PI3K) dependent activation of Akt. Pretreatment of
hepatocytes with the PI3K inhibitor, Ly 294002, prevented CPT-2-Me-cAMP's
protective effect against bile acid and Fas ligand, but not TNF-, mediated apoptosis.
Glucagon, CPT-cAMP and CPT-2-Me-cAMP all activated Rap1, a downstream effector
of cAMP-GEF. Taken together, these results suggest that a PKA independent
cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEF's
promotes Akt phosphorylation and hepatocyte survival. Thus, a cAMP/cAMP-GEF/Rap/PI3K/Akt signaling pathway may confer protection against bile acid and Fas
induced apoptosis in hepatocytes.
