Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway.
Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.
Fabregat
I
I
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia (Centro Mixto CSIC/UCM), Universidad Complutense de Madrid, Spain. isabelf@eucmax.sim.ucm.es
Herrera
B
B
Fernández
M
M
Alvarez
A M
AM
Sánchez
A
A
Roncero
C
C
Ventura
J J
JJ
Valverde
A M
AM
Benito
M
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000502:physiology*; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000196:embryology*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; C494813:Bcl2l1 protein, rat; D003574:Cytochrome c Group; D019253:Proto-Oncogene Proteins c-bcl-2; D016212:Transforming Growth Factor beta; D051020:bcl-X Protein; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases