The nuclear factor kappaB subunits RelA/p65 and c-Rel potentiate but are not required for Ras-induced cellular transformation.

Extensive data indicate that oncoproteins, such as oncogenic H-Ras, initiate signal transduction cascades that ultimately lead to the activation of specific transcription factors. We and others have previously demonstrated that Ras activates the inherent transcriptional activation function of the transcription factor nuclear factor kappaB (NF-kappaB). Supportive of the importance of NF-kappaB in transformation, Ras-induced cellular transformation can be suppressed by expression of IkappaBalpha, an inhibitor of NF-kappaB, or by dominant-negative forms of the upstream activator IkappaB kinase (IKK). However, conclusive evidence for a requirement for NF-kappaB subunits in oncogenic transformation has not been reported. Furthermore, there is little understanding of the gene targets controlled by NF-kappaB that might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Ras-induced cellular transformation, these NF-kappaB subunits are not essential for Ras to transform spontaneously immortalized murine fibroblasts. Microarray analysis identified a set of genes induced by Ras that is dependent on NF-kappaB for their expression and that likely play contributory roles in promoting Ras-induced oncogenic transformation.

Oncoprotein suppression of tumor necrosis factor-induced NF kappa B activation is independent of Raf-controlled pathways.

Extensive data indicate that the transcription factor NF kappa B is activated by signals downstream of oncoproteins such as Ras or breakpoint cluster region (BCR)-ABL. Consistent with this, evidence has been presented that NF kappa B activity is required for Ras and BCR-ABL to transform cells. However, it remains unclear whether these oncoproteins activate a full spectrum of NF kappa B-dependent gene expression or whether they may augment or interfere with other stimuli that activate NF kappa B. The data presented here indicate that BCR-ABL expression in 32D myeloid cells or oncogenic Ras expression in murine fibroblasts blocks the ability of tumor necrosis factor (TNF) to activate NF kappa B. This suppression of NF kappa B is manifested by an inhibition of TNF-induced inhibitor of NF kappa B (IKK) activity and NF kappa B DNA binding potential but not by blocking TNF-induced nuclear accumulation of NF kappa B/p65. The inhibition of NF kappa B is not observed in oncogenic Raf-expressing cells and is not fully restored by the suppression of PI3-kinase or MEK pathways. Oncogenic Ras suppresses the ability of TNF to activate the expression of NF kappa B-dependent genes, such as iNOS (inducible nitric oxide synthase) and RANTES (regulated on activation normal T-cell expressed and secreted). These studies suggest that the ability of Ras and BCR-ABL to activate NF kappa B involves an uncharacterized pathway that does not involve classic IKK activity and that suppresses the TNF-induced IKK pathway through a Raf/MEK/Erk-independent mechanism.

A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression.

NF-kappaB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IkappaB kinase complex (IKK-alpha/beta/gamma). IKK-beta and IKK-gamma are critical for cytokine-induced NF-kappaB function, whereas IKK-alpha is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-alpha in NF-kappaB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-alpha after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-alpha was recruited to the promoter regions of NF-kappaB-regulated genes on stimulation with tumour-necrosis factor-alpha. Notably, NF-kappaB-regulated gene expression is suppressed by the loss of IKK-alpha and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification previously associated with positive gene expression. Furthermore, we show that IKK-alpha can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-alpha is an essential regulator of NF-kappaB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-kappaB-regulated gene expression.