Deacetylation of p53 induces autophagy by suppressing Bmf expression

Abstract

Interferon γ (IFN-γ)-induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-γ on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-γ down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death-stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-γ did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-γ-induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53's proline-rich domain. Suppression of Bmf facilitated IFN-γ-induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf(-/-) but not in bmf(+/+) cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy.

Figure 1.

IFN-γ down-regulates expression of proapoptotic Bmf isoforms. (A–E) Bmf mRNA levels in AALEB cells (A), HAECs (B), wild-type MAECs (C), Bik^−/− MAECs (D), or STAT1^−/− MAECs (E) 48 h after treatment with 50 ng/ml IFN-γ as quantified by qRT-PCR. The relative standard curve method was used for analysis of unknown samples, and data are presented as fold change after averaging the ΔCt values for the nontreated (NT) samples. (F) Bmf_S, Bmf_CUG, and Bmf_L mRNA levels in IFN-γ–treated or untreated MAECs as analyzed by RT-PCR with GAPDH levels as controls. (G) Western blot of protein lysates extracted from homogenized lung or thymus tissue from bmf^−/− and bmf^+/+ mice and analyzed for Bmf expression. (H) Bmf protein levels in protein lysates prepared from IFN-γ–treated or nontreated MAECs. (I) Bmf protein levels in protein lysates from MAECs treated with 300 nM TSA, infected with 100 MOI of Ad-Bmf_S, Ad-Bmf_CUG, or nontreated controls, and the percentage of viable cells. Cells were harvested and quantified using trypan blue exclusion. Data presented are means ± SEM for three independent experiments. *, P < 0.05.

Figure 2.

p53 mediates IFN-γ–induced Bmf suppression. (A) Bmf expression levels in p53-sufficient and -deficient cell lines evaluated by qRT-PCR. Bmf mRNA levels relative to p53-sufficient A549 are shown for AALEB cells and the p53-deficient SAOS-2 and Calu-6 cells. Data presented are representative of four independent experiments. (B) Western blot analysis of p53 in Calu-6 cells infected with an adenoviral expression vector for p53 and the relative Bmf mRNA levels quantified by qRT-PCR in these cell lines when exposed to 10 mJ UV radiation compared with the respective nontreated controls. (C) p53 protein levels in MAEC 6 h after exposure to 10 mJ UV radiation and Bmf mRNA levels quantified by qRT-PCR in MAECs exposed to10 mJ UV and nontreated (NT) controls. (D) Bmf mRNA levels in AALEB cells 6, 12, and 24 h after exposure to 10 mJ UV radiation compared with nontreated controls. Data presented are means ± SEM for three independent experiments. *, P < 0.05.

Figure 3.

p53 suppresses IFN-γ– and HDACi-induced Bmf expression. (A) Bmf mRNA in IFN-γ–treated and nontreated (NT) p53^−/− MAECs. (B) Bmf protein levels in protein lysates prepared from p53^−/− MAECs treated with nothing or IFN-γ for 48 h. (C) Bmf mRNA levels in AALEB cells treated with 5 mM sodium butyrate, 300 nM TSA, and 5 µM MS-275 for 18 h compared with nontreated controls. (D) Bmf mRNA expression in p53^−/− and p53^+/+ MAECs treated with 300 nM TSA for 18 h. Error bars indicate ±SEM (n = 3 independent experiments). *, P < 0.05; statistically significant difference from controls.

Figure 4.

p53 is enriched in the nucleus and interacts with the Bmf promoter. (A) ChIP assays on p53^+/+ or p53^−/− HCT116 cells using a polyclonal antibody to HDAC1 or rabbit IgG₁ as a control. DNA identification was performed using PCR with primers specific for Bmf promoter regions at −97 or −560 regions. (B) ChIP assay on AALEB cells nontreated (NT) or treated with IFN-γ for 48 h using a monoclonal antibody to p53 or mouse IgG₁ as a control using primers specific for Bmf promoter regions at −97 or −560 regions. Densitometry of PCR products from six independent experiments for DNA pulled down with p53 normalized to IgG₁ and IFN-γ–treated cells normalized to the nontreated values. IN, input. (C) Increased p53 protein levels in the nuclear fractions of AALEB cells 48 h after treatment with IFN-γ compared with nontreated controls. Nuclear and cytosolic extracts were analyzed for p53, lamin, and actin. Panel is representative of three independent experiments. (D) Representative photomicrographs of nontreated and IFN-γ–treated AALEB cells immunostained with the anti-p53 antibody and quantification of p53-positive nuclei (n = 4/group). Bar, 15 µm. (E) Bmf mRNA levels in MAECs isolated from p53^ΔP, p53^AXXA, and p53^wt littermates 48 h after IFN-γ treatment compared with nontreated MAECs (n = 3 independent experiments). WT, wild type. Error bars indicate ±SEM. *, P < 0.05.

Figure 5.

IFN-γ causes HDAC1–p53 interaction and deacetylates p53. (A) HDAC1 interacts with p53. Nuclear lysates were prepared from AALEB cells after treatment with IFN-γ for 48 h and were immunoprecipitated (IP) using a p53-specific monoclonal antibody. The nuclear lysates (input) and immunoprecipitates were resolved by SDS-PAGE and analyzed using Western blotting with antibodies to HDAC1, total p53, and acetyl-p53. Panel is representative of three independent experiments. (B) Nuclear extracts prepared from IFN-γ–treated and nontreated MAECs and probed for total p53, acetyl-p53, and lamin. (C) Bmf mRNA levels in IFN-γ–, TSA-, or IFN-γ + TSA–treated AALEB cells compared with nontreated (NT) controls. (D) Representative photomicrographs IFN-γ–, TSA-, or IFN-γ/TSA-treated or nontreated AALEB cells immunostained for p53 (shown in red) and acetyl-p53 (shown in green). Nuclei were stained with DAPI (shown in blue), and merged images are shown in the right-most column. Bars, 10 µm. (E) Quantification of the percentage of cells positive for p53 and acetyl-p53 (Ac-p53) in wild-type (WT) and AXXA MEFs either nontreated or treated with IFN-γ, TSA, or IFN-γ/TSA. Error bars show group means ± SEM (n = 4/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

IFN-γ deacetylates p53 but does not affect IFN-γ–induced cell death. (A) Nuclear extracts from MAECs 0.5 h after IFN-γ treatment or nontreated (NT) controls that were probed for total p53, acetyl-p53, and lamin. (B) Bmf mRNA levels in p53^+/+ HCT116 cells 0.5 h after IFN-γ treatment or nontreated controls. *, P < 0.05. (C) Bmf mRNA in p53^−/− and p53^+/+ MAECs treated with IFN-γ for 0.5 h (n = 3 independent experiments). (D) Quantification of viable p53^−/− and p53^+/+ MAECs treated with IFN-γ for 48 h as assessed by trypan blue exclusion. (E) Quantification of viable AALEB cells transfected with an empty vector plasmid (shRNA control [CTR]) or one expressing p53 shRNA were treated with IFN-γ for 48 h. (F) Bcl-x_L and Bcl-2 protein levels in protein lysates prepared from p53^−/− and p53^+/+ MAECs treated with nothing or IFN-γ for 48 h. Error bars indicate ±SEM.

Figure 7.

IFN-γ suppresses Bmf expression to induce autophagy. (A and B) IFN-γ increases protein levels of Beclin-1 and LC3B in AALEB cells (A) and MAECs (B) compared with nontreated (NT) controls as detected by Western blot analysis. (C) AALEB cells treated with IFN-γ for 48 h and infected with 50 MOI of Ad-Bmf_CUG or Ad-GFP. Bmf expression inhibits IFN-γ–induced Beclin-1 and LC3B protein levels. (D) Western blot of lung and thymus tissues from bmf^+/+ (wild type [WT]) and bmf^−/− (knockout [KO]) mice probed for Beclin-1 and LC3B proteins. (E) Western blot analysis of protein extracts from bmf^+/+ (wild type) and bmf^−/− (knockout) MAECs and MEFs probed for Beclin-1, LC3B, and β-actin. (F) Representative micrographs of bmf^+/+ and bmf^−/− MAECs and MEFs expressing mCherry-LC3B and cells with punctuate LC3B were quantified from >50 bmf^+/+ and bmf^−/− MAECs. (G) Representative electron micrographs of bmf^+/+ and bmf^−/− MAECs that were cultured in 6-well dishes. Thin sections of cells were analyzed by scanning electron microscopy. The arrowhead denotes an autophagic vesicle, and the arrow denotes a mitochondrion surrounded by a double membrane. Quantification of autophagic vesicles per 100 µm² in >30 each bmf^+/+ and bmf^−/− MAECs. (H) Quantification of viable bmf^+/+ and bmf^−/− MEFs maintained in starvation media for 4 or 24 h relative to cell grown in regular media (n = 3 independent experiments). (I) Quantification of viable bmf^+/+ and bmf^−/− MEFs 24 h after treatment with the mTOR inhibitor, pp242, at 2.5 µM relative to nontreated control cells (n = 3 independent experiments). (J) Knockdown of Bmf mRNA using shBmf in p53^−/− HCT116 cells reduced Bmf mRNA levels. Western blot analysis of shRNA control (shCtr)– and shBmf-transfected p53^−/− HCT116 cells probed with Beclin-1, LC3B, and β-actin antibodies. The Western blot is representative of three experiments using three different shBmf constructs. (K) Western blot of IFN-γ–treated p53^+/+ and p53^−/− HCT116 cells probed with Beclin-1, LC3B, and β-actin antibodies. (L) Immunoprecipitation of protein extracts from IFN-γ–treated and nontreated bmf^+/+ and nontreated bmf^−/− MEFs using anti–Beclin-1 and Western blot analysis of input and immunoprecipitates (IP) with Bcl-2, Beclin-1, Bmf, and β-actin antibodies. Results are representative of four independent immunoprecipitations. Error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.