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.
Pucci
Bruna
B
Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, Rome, Italy.
Indelicato
Manuela
M
Paradisi
Valentina
V
Reali
Valentina
V
Pellegrini
Laura
L
Aventaggiato
Michele
M
Karpinich
Natalie O
NO
Fini
Massimo
M
Russo
Matteo A
MA
Farber
John L
JL
Tafani
Marco
M
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