Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage.

Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage.

Programmed cell death 6, a novel p53-responsive gene, targets to the nucleus in the apoptotic response to DNA damage.

The cellular response to genotoxic stress is multifaceted in nature. Following DNA damage, the tumor suppressor gene p53 activates and plays critical roles in cell cycle arrest, activation of DNA repair and in the event of irreparable damage, induction of apoptosis. The breakdown of apoptosis causes the accumulation of mutant cells. The elucidation of the mechanism for the p53-dependent apoptosis will be crucial in applying the strategy for cancer patients. However, the mechanism of p53-dependent apoptosis remains largely unclear. Here, we carried out ChIP followed by massively parallel DNA sequencing assay (ChIP-seq) to uncover mechanisms of apoptosis. Using ChIP-seq, we identified PDCD6 as a novel p53-responsive gene. We determined putative p53-binding sites that are important for p53 regulation in response to DNA damage in the promoter region of PDCD6. Knockdown of PDCD6 suppressed p53-dependent apoptosis. We also observed that cytochrome c release and the cleavage of PARP by caspase-3 were suppressed by depletion of PDCD6. We further observed that PDCD6 localizes in the nucleus in response to DNA damage. We identified the nuclear localization signal of PDCD6 and, importantly, the nuclear accumulation of PDCD6 significantly induced apoptosis after genotoxic stress. Therefore, we conclude that a novel p53-responsive gene PDCD6 is accumulated in the nucleus and induces apoptosis in response to DNA damage.

Identification of Evi-1 as a novel effector of PKCδ in the apoptotic response to DNA damage.

Protein kinase C delta (PKCδ), a PKC family isoform, regulates diverse signal transduction pathways during DNA damage to induce apoptosis. To explore the apoptosis mechanism that PKCδ modulates, we sought to uncover transcription factor targets of PKCδ by devising a screening strategy that utilizes ChIP-cloning and microarray analysis. Transcription factor candidates were generated with the application of public access data-mining tools and this resulted in the identification of Evi-1 as a novel PKCδ-mediated DNA damage responsive molecule. The results demonstrated that PKCδ is constitutively associated with Evi-1. PKCδ regulated Evi-1 to activate PLZF transcription upon genotoxic stress. Furthermore, both Evi-1 and PLZF were associated with DNA damage-stimulated apoptosis. Taken together, we have discovered a novel regulation of Evi-1, which transactivates PLZF, by PKCδ to induce cell death in response to genotoxic stress.

D4S234E, a novel p53-responsive gene, induces apoptosis in response to DNA damage.

Understanding intracellular transduction pathways on apoptosis is indispensable for clinical application to develop effective cancer therapies. Transcription factor p53 is activated in response to genotoxic damage and plays crucial roles in apoptosis. p53 regulates the expression of numerous apoptosis-related genes to induce cell death. Here, through genome-wide comprehensive gene expression profile, we identified D4S234E as a novel p53-responsive gene. We determined the p53-binding region in the D4S234E promoter, which is important for p53 regulation in response to DNA damage. Inhibition of D4S234E expression by RNA interference suppressed apoptosis. Furthermore, we observed that D4S234E partially localizes in the endoplasmic reticulum (ER). We found a functional ER retention signal in D4S234E and, more importantly, ER targeting is essential for D4S234E-mediated apoptosis. Finally, depletion of D4S234E diminished genotoxic stress-induced reduction of Bcl-2 and augmentation of CHOP. We thus concluded that a novel p53-responsive gene D4S234E is accumulated in the ER and induces apoptosis in response to DNA damage.

DNA damage signalling recruits RREB-1 to the p53 tumour suppressor promoter.

Transcriptional regulation of the p53 tumour suppressor gene plays an important role in the control of the expression of various target genes involved in the DNA damage response. However, the molecular basis of this regulation remains obscure. In the present study we demonstrate that RREB-1 (Ras-responsive-element-binding protein-1) efficiently binds to the p53 promoter via the p53 core promoter element and transactivates p53 expression. Silencing of RREB-1 significantly reduces p53 expression at both the mRNA and the protein levels. Notably, disruption of RREB-1-mediated p53 transcription suppresses the expression of the p53 target genes. We also show that, upon exposure to genotoxic stress, RREB-1 controls apoptosis in a p53-dependent manner. These findings provide evidence that RREB-1 participates in modulating p53 transcription in response to DNA damage.

Protein kinase Cdelta activates RelA/p65 and nuclear factor-kappaB signaling in response to tumor necrosis factor-alpha.

Nuclear factor-kappaB (NF-kappaB) is tightly modulated by IkappaB kinases and IkappaBalpha in the cytoplasm. On stimulation, NF-kappaB translocates into the nucleus to initiate transcription; however, regulation of its transcriptional activity remains obscure. Here, we show that protein kinase C (PKC) delta controls the main subunit of NF-kappaB, RelA/p65. On exposure to tumor necrosis factor-alpha (TNF-alpha), the expression of RelA/p65 target genes such as IkappaBalpha, RelB, and p100/p52 is up-regulated in a PKCdelta-dependent manner. The results also show that PKCdelta is targeted to the nucleus and forms a complex with RelA/p65 following TNF-alpha exposure. Importantly, kinase activity of PKCdelta is required for RelA/p65 transactivation. In concert with these results, PKCdelta activates RelA/p65 for its occupancy to target-gene promoters, including IkappaBalpha and p100/p52. Moreover, functional analyses show that inhibition of PKCdelta is associated with substantial attenuation of NF-kappaB activity in response to TNF-alpha. These findings provide evidence that PKCdelta orchestrates RelA/p65 transactivation, a requisite for NF-kappaB signaling pathway in the nucleus.

Protein kinase C delta induces transcription of the TP53 tumor suppressor gene by controlling death-promoting factor Btf in the apoptotic response to DNA damage.

Expression of the TP53 tumor suppressor is tightly controlled for its ability to function as a critical regulator of cell growth, proliferation, and death in response to DNA damage. However, little is known about the mechanisms and contributions of the transcriptional regulation of TP53. Here we report that protein kinase C delta (PKCdelta), a ubiquitously expressed member of the novel subfamily of PKC isoforms, transactivates TP53 expression at the transcriptional level. Reporter assays demonstrated that PKCdelta induces the promoter activity of TP53 through the TP53 core promoter element (CPE-TP53) and that such induction is enhanced in response to DNA damage. The results also demonstrate that, upon exposure to genotoxic stress, PKCdelta activates and interacts with the death-promoting transcription factor Btf to co-occupy CPE-TP53. Inhibition of PKCdelta activity decreases the affinity of Btf for CPE-TP53, thereby reducing TP53 expression at both the mRNA and the protein levels. In concert with these results, we show that disruption of Btf-mediated TP53 gene transcription by RNA interference leads to suppression of TP53-mediated apoptosis following genotoxic stress. These findings provide evidence that activation of TP53 gene transcription by PKCdelta triggers TP53-dependent apoptosis in response to DNA damage.