Transcription Factor NF-κB Differentially Regulates Death Receptor 5 Expression Involving Histone Deacetylase 1

Abstract

The transcription factor nuclear factor B (NF-B) regulates the expression of both antiapoptotic and proapoptotic genes. Death receptor 5 (DR5, TRAIL-R2) is a proapoptotic protein considered to be a potential target for cancer therapy, and its expression is mediated by NF-B. The mechanism of NF-B-induced DR5 expression is, however, unknown. Herein, we determined that etoposide-induced DR5 expression requires the first intronic region of the DR5 gene. Mutation of a putative NF-B binding site in this intron eliminates DR5 promoter activity, as do mutations in the p53 binding site in this region. Reduction in p53 expression also blocks p65 binding to the intronic region of the DR5 gene, indicating cooperation between p53 and p65 in DR5 expression. In contrast, the antiapoptotic stimulus, epidermal growth factor (EGF), fails to increase DR5 expression but effectively activates NF-B and induces p65 binding to the DR5 gene. EGF, however, induces the association of histone deacetylase 1 (HDAC1) with the DR5 gene, whereas etoposide treatment fails to induce this association. Indeed, HDAC inhibitors activate NF-B and p53 and upregulate DR5 expression. Blockage of DR5 activation decreased HDAC inhibitor-induced apoptosis, and a combination of HDAC inhibitors and TRAIL increased apoptosis. This provides a mechanism for regulating NF-B-mediated DR5 expression and could explain the differential roles NF-B plays in regulating apoptosis.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family. It is capable of inducing apoptosis in transformed cancer cells but not in normal cells (14,53). TRAIL binds to two receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5, TRAIL-R2) (33,40,41,52,57). Upon binding, an adaptor protein called Fas-associated death domain (FADD) is recruited to the death receptors, forming a death-inducing signaling complex (46). Fas-associated death domain constitutively associates with procaspase 8 and, upon recruitment to death receptors, caspase 8 is activated. This leads to further caspase activation and release of mitochondrial proteins and, ultimately, to apoptosis (3, 38).Chemotherapeutic drugs, including etoposide and doxorubicin in combination with TRAIL, give a synergistic apoptotic response in cancer cells (11,21,43). This synergy is due partially to the ability of chemotherapeutic drugs and TRAIL to induce DR4 and DR5 expression. Up-regulation of DR5 in lung cancer cell lines is p53 dependent, and the p53-responsive element responsible for DR5 expression has been identified in the first intron region of the DR5 gene (47). Indeed, primary chronic lymphocytic leukemia cells lacking functional p53 fail to up-regulate DR5 following genotoxin treatment (20). Besides p53 involvement, blockage of the transcription factor NF-B effectively inhibits etoposide and TRAIL-induced DR5 expression and synergistic apoptotic response (11,42). The mechanism for NF-B up-regulation of DR5 is currently unknown, but a putative NF-B binding site has been i...

Dual role of TMS1/ASC in death receptor signaling

“…Thus, whereas p53 may have some impact on expression of TMS1, it may be indirect. Alternatively, the regulation of TMS1 by genotoxins may involve transcriptional crosstalk between p53 and NF-kB (Webster and Perkins, 1999) as has been described for other pro-apoptotic NF-kB target genes, such as DR5 (Shetty et al, 2005).…”

DAPK2 is a novel modulator of TRAIL-induced apoptosis

“…DR5 is transcriptionally regulated by the tumour suppressor p53 and by NF-kB. 19 Upon silencing DAPK2, there was no change in p53 mRNA levels and both prostate cancer PC3 cells, which are p53 null, and bladder cancer T24 cells, which have an inactivating mutation in p53, can be sensitised to TRAIL by downregulating Figure S6). These data suggested that p53 was not necessary to sensitise TRAIL-resistant cells to TRAIL-induced apoptosis and we thus focused on NF-kB.…”

“…DR expression can be induced by several chemicals, which can cause either their mRNA stabilisation 23 or transcriptional upregulation by various transcription factors. 19,24 DR induction leads to an increase in the number of receptor molecules on the cell surface, thus overcoming the threshold required to sensitise cells to TRAIL cytotoxicity. As U2OS cells do not express DR4 and silencing DAPK2 leads to the upregulation of DR5 in both U2OS and A549 cells (Figure 2), we focused on the regulation of DR5 induction/expression upon DAPK2 depletion.…”

“…DR5 is regulated by a variety of transcription factors such as p53 and NF-kB. 19 The fact that DAPK2 silencing can also sensitise p53-mutated (mt) T24 colon cancer cells and p53-null prostate cancer PC3 cells rules out the absolute requirement for p53 (Supplementary Figure S6). In contrast, activation of NF-kB ( Figure 5) is absolutely essential (Figure 6 and Supplementary Figures S7 and S8).…”

“…For example, there are reports suggesting an antiapoptotic role for NF-kB, involving the upregulation of DcR1 25 or of antiapoptotic BCL-X L , 26 whereas other reports suggest a proapoptotic role due to the induction of DR4 or DR5. 19,24 Interestingly, binding of TRAIL to DcR2 has been shown to activate NF-kB, which then initiates a negative feedback loop protecting cells from TRAIL-induced apoptosis. 27 Using an NF-kB reporter plasmid, we were able to show that NF-kB is transcriptionally active in both U2OS and A549 cells following DAPK2 knockdown (Figures 5g and h).…”