Mechanism of anthracycline-mediated down-regulation of GATA4 in the heart

Abstract

Aims: Anthracyclines such as daunorubicin (DNR) and doxorubicin are effective cancer chemotherapeutic agents, but can induce cardiotoxicity. GATA4 has been shown to serve as a survival factor of cardiac muscle cells, and anthracyclines promote apoptosis in part by down-regulating GATA4. The present study investigated the mechanism of anthracycline action to down-regulate GATA4.

Methods and results: DNR inhibited the transcriptional activity exhibited by the 250 bp conserved region immediately upstream from the transcriptional start site of the Gata4 gene. Mapping this region identified that the CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) binding to the CCAAT box was inhibited by DNR in HL-1 cardiac muscle cells and in perfused isolated mouse hearts. The DNR action on the Gata4 promoter was found to be dependent on p53, since DNR promoted nuclear binding of p53 to CBF/NF-Y and pifithrin-α (a p53 inhibitor) attenuated DNR down-regulation of GATA4.

Conclusion: Anthracycline down-regulation of GATA4 is mediated by the inhibition of Gata4 gene transcription via a novel mechanism that involves the p53-dependent inhibition of CBF/NF-Y binding to the CCAAT box within the Gata4 promoter.

Figure 1

Effects of DNR on gene transcription controlled by the proximal Gata4 promoter region. (A) HL-1 cells were co-transfected with firefly luciferase vector controlled by the proximal 1000 bp region of the mouse Gata4 promoter [Gata4 (1000)-Luc] and Renilla luciferase vector controlled by the TK promoter (TK-Luc). Seven hours after transfection, cells were treated with DNR (2 µM) for 17 h and then relative luciferase units (RLU) were measured. Values represent means ± S.E. (n = 9). (B) HL-1 cells were transfected with luciferase construct controlled by proximal 1000, 500, or 250 bp region of the Gata4 promoter then treated with DNR (2 µM) for 17 h. Values represent means ± S.E. (n = 9–12). (*) denotes values significantly different from the control value at P < 0.05.

Figure 2

Effects of DNR on DNA-binding activities towards various segments of proximal 250 bp Gata4 promoter. (A) Schematic of various probes containing sequences from the 250 bp Gata4 promoter region. (B) Basal DNA-binding activities of ³²P-labelled Probes #1–7. (C) Effects of DNR (2 µM) on DNA-binding activities of nuclear extracts from control and DNR-treated HL-1 cells towards ³²P-labelled Probes #1–7. The arrow indicates the band that was affected by DNR. (D) The line graph represents means ± S.E. of the intensity of the Probe #2 binding band affected by DNR as determined by densitometry (n = 4). (*) denotes values significantly different from the control value at P < 0.05.

Figure 3

Effects of DNR on perfused isolated mouse hearts. (A) Mouse hearts were subjected to Langendorff perfusion with or without 2 µM of DNR for 2 h. Nuclear extracts (5 µg) were subjected to EMSA using the ³²P-labelled Probes #2 and #3 of the Gata4 promoter region. Values in the bar graph represent means ± S.E. (n = 4). (B) Sequences of Probe #2 and Center Probe. (C) Mouse heart nuclear extracts were subjected to EMSA using ³²P-labelled Center Probe. Values in the bar graph represent means ± S.E. of fold change binding for Bands a and b. (*) denotes values significantly different from the untreated control at P < 0.05.

Figure 4

Effects of DNR on Gata4 promoter activity. HL-1 cells were transfected with the luciferase construct controlled by the 250 bp proximal region of the wild-type (WT) Gata4 promoter, or mutant (Mut) Gata4 promoter at CCAAT box, then treated with DNR (2 µM) for 17 h. Values represent means ± S.E. of the ratio of 250 bp Gata4 promoter-controlled firefly luciferase activity to TK promoter-controlled Renilla luciferase activity (n = 6). (*) denotes values significantly different from the wild-type Gata4 promoter value at P < 0.05.

Figure 5

Effects of DNR on p53. (A) Nuclear extracts from HL-1 cells untreated or treated with DNR were immunoprecipitated with rabbit anti-CBF-B IgG or with normal rabbit IgG (as control), then immunoblotted with mouse anti-p53 IgG. Values in the bar graph represent means ± S.E. (n = 5). (B) HL-1 cells were treated with DNR (2 µM) for 4 h. p53 protein levels in nuclear extracts were measured by western blot. Values in the bar graph represent means ± S.E. (n = 4). (C) p53 protein levels in total cell lysates were measured by western blot. Values in the bar graph represent means ± S.E. (n = 4). (*) denotes values significantly different from the untreated control at P < 0.05.

Figure 6

Effects of p53 inhibitor. (A) HL-1 cells were treated with DNR (2 µM) and pifithrin-α (Pif; 10 µM) for 24 h. Nuclear extracts were subjected to EMSA using ³²P-labelled Probe #2 of the Gata4 promoter. Bar graphs represent means ± S.E. (n = 4). (*) denotes values significantly different from each other at P < 0.05. CBF-B Ab indicates the supershift experiment with CBF-B antibody. (B) Nuclear extracts were subjected to western blotting with CBF-B antibody. D + P, DNR + Pif. (C) Total RNA was isolated, and levels of Gata4 mRNA and 28S rRNA were monitored by reverse transcription PCR. The bar graph represents means ± S.E. of the intensity of Gata4 mRNA levels determined by densitometry (n = 4). (*) denotes values significantly different from each other at P < 0.05. (D) Nuclear extracts were subjected to EMSA using the ³²P-labelled double-stranded oligonucleotide containing GATA consensus elements. GATA4 Ab indicates supershifting with the GATA4 antibody.