Runt-related transcription factor 2 (RUNX2) inhibits p53-dependent apoptosis through the collaboration with HDAC6 in response to DNA damage

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.

Figure 1

ADR-mediated induction of RUNX2. (a and b) Expression level of RUNX2 in response to ADR. U2OS cells were exposed to the indicated concentrations of ADR. Twenty-four hours after ADR treatment, cell lysates and total RNA were prepared and processed for immunoblotting (a) and RT-PCR (b), respectively. Actin was used as a loading control and GAPDH served as an internal control

Figure 2

RUNX2 associates with p53 in response to ADR. (a) Indirect immunostaining experiments. U2OS cells were treated with 1.0 μM of ADR or left untreated. Twenty-four hours after ADR treatment, cells were simultaneously incubated with monoclonal anti-p53 and polyclonal anti-RUNX2 antibodies followed by the incubation with rhodamine-conjugated anti-mouse IgG (red) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Merged images (yellow) indicate the colocalization of RUNX2 with p53 in cell nucleus. (b and c) Co-immunoprecipitation experiments. U2OS cells were exposed to 0.5 μM of ADR (b) or left untreated (c). Twenty-four hours after ADR treatment, cell lysates were prepared and immunoprecipitated with NMS or with monoclonal anti-p53 antibody. The immunoprecipitates were analyzed by immnublotting with monoclonal anti-RUNX2 antibody. The reciprocal experiments and 1/20 of inputs were also shown

Figure 3

p53 as well as RUNX2 is recruited onto p53-target promoters. (a) Co-occupancy of p53 and RUNX2 on p53-target promoters. U2OS cells were transfected with the expression plasmids for GFP or with p53 plus RUNX2. Forty-eight hours after transfection, cells were fixed in formaldehyde and incubated with SDS lysis buffer. The extracted chromatin fragments were immunoprecipitated with monoclonal anti-GFP, monoclonal anti-p53 or with monoclonal anti-RUNX2 antibody and the precipitated genomic DNA was analyzed by PCR using primer sets for p21^WAF1 and BAX promoter regions containing their p53-responsive elements. PCR amplification was also performed before immunoprecipitation for the input control. (b) ADR-mediated recruitment of p53 and RUNX2 onto p53-target promoters. U2OS cells were treated with 0.5 μM of ADR (right panels) or left untreated (left panels). Twenty-four hours after ADR treatment, cells were fixed in formaldehyde and incubated with SDS lysis buffer. Soluble chromatin was subjected to immunoprecipitation with NMS, monoclonal anti-p53 or with monoclonal anti-RUNX2 antibody. Immunoprecipitated genomic DNA was PCR amplified using primer sets that span p53-responsive elements of p21^WAF1 and BAX promoters. Input corresponded to 2.5% of the soluble chromatin that was subjected to immunoprecipitation

Figure 4

Forced expression of RUNX2 reduces the expression levels of various p53-target genes. U2OS cells were transfected with the empty plasmid (2.0 μg) or with the increasing amounts of the expression plasmid encoding RUNX2 (0.5, 1.0 or 2.0 μg). Total amounts of the expression plasmid were kept constant (2.0 μg) with pcDNA3. Forty-eight hours after transfection, cell lysates and total RNA were isolated and subjected to immunoblotting with the indicated antibodies (upper panels) and RT-PCR with the indicated primer sets (lower panels), respectively. Immunoblotting with anti-actin antibody was used as a loading control. Expression levels of an internal control GAPDH were also monitored by RT-PCR

Figure 5

Knockdown of RUNX2 enhances ADR-mediated apoptosis. U2OS cells were transfected with control siRNA or with siRNA against RUNX2. Twenty-four hours after transfection, cells were treated with 0.5 μM of ADR or left untreated. Twelve hours after ADR exposure, images were taken (a) and floating and attached cells were collected, fixed in 70% ethanol, stained with propidium iodide (PI) and subjected to FACS analysis (b). Cells with sub-G1 DNA content were considered dead. Under the same experimental conditions, cell lysates and total RNA were prepared and analyzed by immunoblotting (c) and RT-PCR (d), respectively. Actin and GAPDH were a loading control for immunoblotting and an internal control for RT-PCR, respectively

Figure 6

Knockdown of RUNX2 has a negligible effect on H1299 cells in response to ADR. (a and b) RUNX2 has an undetectable effect on H1299 cells following ADR exposure. H1299 cells were transfected with control siRNA or with siRNA targeting RUNX2. Twenty-four hours after transfection, cells were treated with 0.5 μM of ADR or left untreated. Twenty-four hours after ADR treatment, images were taken (a). Floating and attached cells were then collected and subjected to FACS analysis (b). (c) RUNX2 has a negligible effect on p53-target gene expression in response to ADR. H1299 cells were treated as in (a). Twenty-four hours after ADR treatment, total RNA was prepared and processed for RT-PCR. GAPDH was used as an internal control

Figure 7

Interaction among p53, RUNX2 and HDAC6. Co-immunoprecipitation. U2OS cells were left untreated (a) or treated with 0.5 μM of ADR (b). Twenty-four hours after ADR treatment, cell lysates were prepared and immunoprecipitated with NRS or with polyclonal anti-HDAC6 antibody followed by immunoblotting with the indicated antibodies. Reciprocal immunoprecipitation experiments using NMS or monoclonal anti-RUNX2 and 1/20 of input were also shown

Figure 8

HDAC6 contributes to RUNX2-mediated repression of p53. (a) HDAC6 binds to p53-target gene promoters in the presence of ADR. U2OS cells were exposed to 0.5 μM of ADR (lower panels) or left untreated (upper panels). Twenty-four hours after ADR treatment, cells were fixed in formaldehyde and incubated with SDS lysis buffer. Cross-linked chromatin was immunoprecipitated with the indicated antibodies and collected with protein A-agarose beads. After reversal of the cross-link and digestion with proteinase K, genomic DNA was purified by ethanol precipitation followed by PCR amplification using primer sets that span p53-responsive elements of p21^WAF1 and BAX promoters. (b) Tubacin enhances ADR-mediated upregulation of p53-target gene expression. U2OS cells were exposed to HDAC6 inhibitor tubacin (at a final concentration of 5 μM) or left untreated. Twenty-four hours after tubacin treatment, cells were exposed to 0.5 μM of ADR or left untreated. Twenty-four hours after ADR exposure, total RNA was prepared and subjected to RT-PCR. GAPDH was used as an internal control