Abstract
Runt-related transcription factor 2 (RUNX2) is the best known as an essential protein for osteoblast differentiation. In this study, we have found for the first time that RUNX2 acts as a negative regulator for p53 in response to DNA damage. On DNA damage mediated by adriamycin (ADR) exposure, p53 as well as RUNX2 was induced at protein and mRNA level in human osteosarcoma-derived U2OS cells in association with a significant upregulation of various p53-target genes. Indirect immunostaining and co-immunoprecipitation experiments demonstrated that RUNX2 colocalizes with p53 in cell nucleus and forms a complex with p53 following ADR treatment. Chromatin immunoprecipitation assays revealed that RUNX2/p53 complex is efficiently recruited onto p53-target promoters in response to ADR, suggesting that RUNX2 might be involved in the regulation of transcriptional activation mediated by p53. Indeed, forced expression of RUNX2 resulted in a remarkable downregulation of p53-target genes. Consistent with these observations, knockdown of RUNX2 enhanced ADR-mediated apoptosis and also elevated p53-target gene expression in response to ADR. On the other hand, depletion of RUNX2 in p53-deficient human lung carcinoma-derived H1299 cells had an undetectable effect on p53-target gene expression regardless of ADR treatment, indicating that RUNX2-mediated downregulation of p53-target genes is dependent on p53. Furthermore, RUNX2/p53 complex included histone deacetylase 6 (HDAC6) and HDAC6 was also recruited onto p53-target promoters following ADR exposure. Of note, HDAC6-specific chemical inhibitor tubacin treatment enhanced ADR-mediated upregulation of p53-target gene expression, indicating that deacetylase activity of HDAC6 is required for RUNX2-mediated downregulation of p53-target gene. Taken together, our present findings strongly suggest that RUNX2 inhibits DNA damage-induced transcriptional as well as pro-apoptotic activity of p53 through the functional collaboration with HDAC6 and therefore might be an attractive therapeutic target for cancer treatment.
Highlights
Under normal conditions, p53 is maintained at a quite low level
We have found that RUNX2 interacts with p53 in response to DNA damage and thereby inhibiting p53 in collaboration with histone deacetylase 6 (HDAC6)
To examine the expression pattern of RUNX2 following DNA damage, p53-proficient human osteosarcoma-derived U2OS cells were exposed to ADR, which leads to DNA double-strand breaks
Summary
MDM2 that acts as an E3 ubiquitin protein ligase for p53, binds to NH2-terminal transactivation domain of p53, catalyzes its ubiquitination at COOH-terminal lysine residues and thereby promoting its ubiquitin-dependent proteasomal degradation.[10,11] As MDM2 is a direct p53-target gene product, MDM2 and p53 create a negative-feedback loop in which p53 transactivates MDM2, which in turn downregulates p53.12 In response to cellular stresses such as DNA damage, p53 is quickly induced to accumulate in cell nucleus.[1] DNA damage-mediated sequential post-translational modifications including phosphorylation (Ser-15, Ser-20 and Ser-46). It has been described that ASPP1/ASPP2 interacts with central DNA-binding domain of p53 and enhances its pro-apoptotic activity.[17] Roe et al.[18] demonstrated that VHL binds to p53 and stimulates its pro-apoptotic activity through suppressing MDM2-mediated p53 ubiquitination. Li et al.[23] demonstrated that SIRT1 binds to p53 and inhibits its pro-apoptotic activity through deacetylation of p53 at Lys-382
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