ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells.
Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death.
Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, Rome, Italy.
Bruna
B
Pucci
Manuela
M
Indelicato
Valentina
V
Paradisi
Valentina
V
Reali
Laura
L
Pellegrini
Michele
M
Aventaggiato
Natalie O
NO
Karpinich
Massimo
M
Fini
Matteo A
MA
Russo
John L
JL
Farber
Marco
M
Tafani
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053181:Caspase 8 / Q000378:metabolism; D003513:Cycloheximide / Q000378:metabolism / Q000494:pharmacology; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D015398:Signal Transduction / Q000502:physiology; D048069:Tumor Necrosis Factors / Q000378:metabolism* / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D051025:bcl-Associated Death Protein / Q000378:metabolism*
C494826:BAX protein, human; D048069:Tumor Necrosis Factors; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D003513:Cycloheximide; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D053181:Caspase 8