Death receptor-4 (DR4) expression is regulated by transcription factor NF-κB in response to etoposide treatment

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Abstract
Tumour necrosis factor related apoptosis inducing ligand (TRAIL) binds to death receptor 4 (DR4) activating the apoptotic signalling pathway. DNA damaging agents (genotoxins) such as etoposide increase DR4 expression and when combined with TRAIL induce a synergistic apoptotic response. The mechanism for up-regulation of DR4 expression following genotoxin treatment is not well understood. Herein, we determined that transcription factor NF-κB plays a role in genotoxin induced DR4 expression. Increased expression of DR4 following etoposide treatment is blocked by inhibition of the NF-κB pathway. Moreover, expression of the p65 subunit of NF-κB is sufficient to increase DR4 protein levels. Indeed, knockdown of p65 by RNA interference blocked etoposide up-regulation of DR4. We further identified a functional NF-κB binding site located in the DR4 promoter. Mutation of this site abrogates the induction of luciferase activity after p65 over-expression. Furthermore, electromobility shift assays and chromatin immunoprecipitaton suggest that NF-κB binds to this site upon etoposide treatment. MEK kinase 1 (MEKK1) is a serine threonine kinase that is activated following etoposide treatment and activates NF-κB. Expression of the kinase inactive MEKK1 (MEKK1-KM) abrogates the up-regulation of DR4 after etoposide treatment. Taken together, NF-κB plays a role in up-regulation of DR4 following etoposide treatment.