Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity

Abstract

Although runt-related transcription factor 2 (RUNX2) is known to be an essential key transcription factor for osteoblast differentiation and bone formation, RUNX2 also plays a pivotal role in the regulation of p53-dependent DNA damage response. In the present study, we report that, in addition to p53, RUNX2 downregulates pro-apoptotic TAp73 during DNA damage-dependent cell death. Upon adriamycin (ADR) exposure, human osteosarcoma-derived U2OS cells underwent cell death in association with an upregulation of TAp73 and various p53/TAp73-target gene products together with RUNX2. Small interfering RNA-mediated silencing of p73 resulted in a marked reduction in ADR-induced p53/TAp73-target gene expression, suggesting that TAp73 is responsible for the ADR-dependent DNA damage response. Immunoprecipitation and transient transfection experiments demonstrated that RUNX2 forms a complex with TAp73 and impairs its transcriptional activity. Notably, knockdown of RUNX2 stimulated ADR-induced cell death accompanied by a massive induction of TAp73 expression, indicating that RUNX2 downregulates TAp73 expression. Consistent with this notion, the overexpression of RUNX2 suppressed ADR-dependent cell death, which was associated with a remarkable downregulation of TAp73 and p53/TAp73-target gene expression. Collectively, our present findings strongly suggest that RUNX2 attenuates the transcriptional activity and ADR-mediated induction of TAp73, and may provide novel insights into understanding the molecular basis behind the development and/or maintenance of chemoresistance. Thus, we propose that the silencing of RUNX2 might be an attractive strategy for improving the chemosensitivity of malignant cancers.

Keywords: DNA damage; RUNX2; TAp73; cell death; p53.

Figure 1

U2OS cells undergo cell death in response to ADR. (A) Phase-contrast micrograph. U2OS cells were treated with the indicated concentrations of ADR or left untreated. Twenty-four hours after treatment, phase-contrast micrographs were taken. Scale bar = 100 μm. (B) Trypan-blue exclusion assay. U2OS cells were treated as described in (A). Twenty-four hours after treatment, cells were trypsinized and stained with 0.4% of trypan blue. Cells restricting trypan-blue entry were considered viable. (C) Annexin V and PI double staining. U2OS cells were treated with 0.5 μm of ADR (filled boxes) or left untreated (gray boxes). Twenty-four hours after treatment, cells were subjected to annexin V and PI double staining.

Figure 2

ADR-mediated induction of TAp73 and RUNX2 in U2OS cells. U2OS cells were exposed to the indicated concentrations of ADR. Twenty-four hours after treatment, whole cell lysates and total RNA were prepared and subjected to (A) immunoblotting and (B) RT-PCR, respectively. Actin and GAPDH were used as a loading and an internal control, respectively.

Figure 3

Knockdown of p73 attenuates ADR-mediated induction of p53/TAp73-target gene expression. U2OS cells were transfected with control siRNA or with siRNA against p73. Twenty-four hours after transfection, cells were treated with 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, whole cell lysates and total RNA were extracted and processed for (A) immunoblotting and (B) RT-PCR, respectively.

Figure 4

Complex formation between TAp73 and RUNX2 in response to ADR. (A) Nuclear co-localization of TAp73 with RUNX2 after ADR exposure. U2OS cells were exposed to 0.5 μm of ADR (lower) or left untreated (upper). Twenty-four hours after treatment, cells were simultaneously incubated with monoclonal anti-RUNX2 (red) and polyclonal anti-p73 (green) antibodies. Cell nuclei were stained with DAPI (blue). Merged images (yellow) indicated the nuclear co-localization of TAp73 with RUNX2. Scale bar = 25 μm (magnification, × 630) (left). Percentages of RUNX2-, p73- or RUNX2/p73-positive cells were indicated (right). (B) Co-immunoprecipitation. An equal amount of whole cell lysates prepared from ADR-treated U2OS cells was immunoprecipitated with normal mouse serum (NMS) or with monoclonal anti-p73 antibody. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies (middle). The reciprocal experiments (right) and 1 : 20 inputs (left) are also shown.

Figure 5

RUNX2 represses the expression of p53/p73-target genes. (A) U2OS cells were transfected with or without the increasing amounts of the expression plasmid for RUNX2 (0.5, 1.0 or 2.0 μg). Forty-eight hours after transfection, whole cell lysates and total RNA were isolated and analyzed by immunoblotting (left) and RT-PCR (right), respectively. (B, C) RUNX2 promotes cell proliferation. U2OS cells were transfected as described in (A). Forty-eight hours after transfection, phase-contrast micrographs were taken. Scale bar = 100 μm (B). U2OS cells were transfected with 2.0 μg of the expression plasmid encoding RUNX2. At the indicated time points, the number of viable cells was measured (C).

Figure 6

RUNX2 inhibits the transcriptional activity of TAp73. H1299 cells were transfected with the indicated combinations of the expression plasmids. Forty-eight hours after transfection, whole cell lysates and total RNA were prepared and subjected to immunoblotting (upper) and RT-PCR (lower), respectively.

Figure 7

siRNA-mediated knockdown of RUNX2 enhances ADR-dependent cell death and expression of TAp73. (A) Knockdown of RUNX2. 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. After 24 h of incubation, cells were stained with anti-RUNX2 antibody (green). Cell nuclei were stained with DAPI (blue). Scale bar = 25 μm. (B) Genomic DNA fragmentation. U2OS cells were transfected as described in (A). Twenty-four hours post-transfection, cells were exposed to 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, attached and floating cells were collected, and their genomic DNA was prepared and analyzed by 1% agarose gel electrophoresis. (C) RT-PCR analysis of the endogenous TAp73. U2OS cells were transfected as described in (A). Transfected cells were treated with 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, total RNA was prepared and subjected to RT-PCR analysis.

Figure 8

Knockdown of RUNX2 in U2OS cells enhances the sensitivity to ADR. U2OS cells were transiently 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 treatment, cells were subjected to TUNEL assay, and number of TUNEL-positive cells (red) was scored. Cell nuclei were stained with DAPI. Scale bar = 50 μm (magnification, × 100).

Figure 9

RUNX2 prohibits ADR-mediated cell death. U2OS cells were transfected with the empty plasmid, the expression plasmid for RUNX2 (upper panels) or with the expression plasmid for p53 (lower panels), followed by additional incubation with or without 0.5 μm of ADR. Twenty-four hours after treatment, attached and floating cells were collected and analyzed by fluorescence-activated cell sorting.

Figure 10

RUNX2 suppresses ADR-mediated induction of TAp73. U2OS cells were transfected as described in Fig.9. Twenty-four hours after transfection, cells were treated with 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, whole cell lysates and total RNA were prepared and analyzed by (A) immunoblotting and (B) RT-PCR, respectively.

Figure 11

RUNX2 inhibits ADR-mediated upregulation of TAp73 in H1299 cells. H1299 cells were transiently transfected with the empty plasmid or with the expression plasmid for RUNX2. Twenty-four hours after transfection, cells were treated with 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, total RNA was prepared and subjected to RT-PCR.