Deletion of the N-terminus of IKKgamma induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway.
The regulatory subunit IKKgamma/NEMO is crucial for skin development and function and although devoid of kinase activity, loss of IKKgamma function completely abolishes the activation of NF-kappaB by all pro-inflammatory cytokines. To inhibit the IkappaB kinase (IKK) complex in keratinocytes, we have used a dominant negative approach by generating stable transfectants of an N-terminal deletion of IKKgamma (IKKgamma-DN97) that uncouples formation of the IKK complex. Expression of this mutant in PB keratinocytes (PB-IKKgamma-DN97) delayed growth kinetics, caused morphological changes and dramatically augmented apoptosis even in the absence of pro-apoptotic stimuli, as determined by cell morphology, TUNEL and caspase-3 cleavage. Moreover, in PB-IKKgamma-DN97 cells, TNF-alpha and IL-1 treatment failed to induce degradation of IkappaBalpha, phosphorylation of p65 on Ser 536 and nuclear translocation which, consequently, reduced kappaB-binding activity. In PB-IKKgamma-DN97 cells, accumulation of IkappaBalpha correlated with a downregulation of AKT activity and an increase of PTEN protein levels whereas pro-apoptotic p53 target genes Bax and Puma were upregulated. These effects were most likely mediated through IKK since coexpression of the wild-type form of IKKgamma in keratinocytes partially reversed apoptosis and reduced PTEN expression. Thus, our data suggest a negative cross-talk mechanism involving PTEN and NF-kappaB, critical for the anti-apoptotic role of NF-kappaB in keratinocytes.
Leis
Hugo
H
Centro de Investigación Príncipe Felipe, Valencia, Av. Autopista del Saler 16, Camino de las Moreras, Valencia, Spain.
Sanchis
Ana
A
Pérez
Paloma
P
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002478:Cells, Cultured; D017353:Gene Deletion; D051550:I-kappa B Kinase / Q000235:genetics / Q000502:physiology*; D015603:Keratinocytes / Q000502:physiology*; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000502:physiology; D051058:Oncogene Protein v-akt / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection
D050505:Mutant Proteins; D051058:Oncogene Protein v-akt; D051550:I-kappa B Kinase; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse