Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation.

The p53-regulated stress-inducible gene GADD45 has been shown to participate in cellular response to DNA damage, including cell cycle checkpoint, apoptosis, and DNA repair. However, the regulation of GADD45 expression is complex and may involve both p53-dependent and -independent pathways. Recent findings have demonstrated that the p53-independent induction of GADD45 is mainly regulated by the transcription factors Oct-1 and NF-YA, which directly bind to their consensus motifs located at the GADD45 promoter region. Here, we report that mitogen-activated protein (MAP) kinases are involved in the induction of the GADD45 promoter after DNA damage. Inhibition of JNK1 and ERK kinase activities either by expression of the dominant negative mutant JNK1 or by treatment with a selective chemical inhibitor of ERK (PD098059) substantially abrogates the UV induction of the GADD45 promoter. In contrast, a p38 kinase inhibitor (SB203580) has little effect on GADD45 induction by UV. In addition, the GADD45 promoter is strongly activated following expression of JNK1; Raf-1, which is an upstream activator of the ERK pathway; or MEK1, an upstream activator of both the ERK and the JNK pathways. Activation of the GADD45 promoter by MAP kinases does not require normal p53 function. Interestingly, the MAP kinase-regulatory effect appears to be mediated via OCT-1 and CAAT motifs since disruption of these sites abrogates activation of the GADD45 promoter by MAP kinases. Therefore, these findings indicate that the MAP kinase pathways are involved in the regulation of the p53-independent induction of the GADD45 promoter, probably via interaction with transcription factors that directly bind to OCT-1 and CAAT motifs.

Transcription factors Oct-1 and NF-YA regulate the p53-independent induction of the GADD45 following DNA damage.

The p53-regulated GADD45 gene is one of the important players in cellular response to DNA damage, and probably involved in the control of cell cycle checkpoint, apoptosis and DNA repair. There are both the p53-dependent and -independent pathways that regulate GADD45 induction. Following ionizing radiation, induction of the GADD45 gene is regulated by p53 through the p53-binding motif located in the third intron of the GADD45 gene. In contrast, GADD45 induction by methyl methanesulfonate (MMS), UV radiation (UV), and medium starvation is independent of p53 status although p53 may contribute to these responses. However, the regulatory elements that control the p53-independent induction of GADD45 remain uncertain. In this report, we have performed detailed analyses to characterize the responsive components that are required for the induction of the GADD45 promoter. We have found that the region between -107 and -62 of the GADD45 promoter is crucial for the induction. Sequence analysis indicates that there are two OCT-1 sites and one CAAT box located in this region. Site-directed mutations of both OCT-1 and CAAT motifs substantially abrogate the induction of the GADD45 promoter by DNA damage. In addition, both Oct-1 protein (binding to OCT-1 site) and NF-YA protein (binding to CAAT box) are induced after cell exposure to DNA damaging agents. Moreover, the Electrophoretic Mobility Shift Assay (EMSA) has demonstrated the direct bindings of Oct-1 and NF-YA proteins to their consensus sequences in the GADD45 promoter. Therefore, these results have presented the novel observation that transcription factors Oct-1 and NF-YA participate in the cellular response to DNA damage and are involved in the regulation of stress-inducible genes.