FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal - PubMed (original) (raw)

FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal

Han You et al. J Exp Med. 2006.

Erratum in

Abstract

Puma is an essential mediator of p53-dependent and -independent apoptosis in vivo. In response to genotoxic stress, Puma is induced in a p53-dependent manner. However, the transcription factor driving Puma up-regulation in response to p53-independent apoptotic stimuli has yet to be identified. Here, we show that FOXO3a up-regulates Puma expression in response to cytokine or growth factor deprivation. Importantly, dysregulated Akt signaling in lymphoid cells attenuated Puma induction upon cytokine withdrawal. Our results suggest that Puma, together with another BH3 only member, Bim, function as FOXO3a downstream targets to mediate a stress response when PI3K/Akt signaling is down-regulated.

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Figures

Figure 1.

Figure 1.

PI3K/Akt/FOXO3a are involved in cytokine withdrawal-induced apoptosis in activated T cells. (A) Resistance of Pten-deficient activated T cells to apoptosis induced by IL-2 withdrawal. (left) Western blot showing levels of indicated proteins in untreated WT and Pten-deficient T cells. (right) Activated T cells were cultured in IL-2–free medium for 24 h and numbers of viable cells were determined by flow cytometric analysis. Data shown are means ± SD from three independent experiments. (B) FOXO3a phosphorylation status in IL-2–deprived activated T cells derived from p53−/− mice. Phosphorylated FOXO3a (Thr 32) (FOXO3a-P) and total FOXO3a (FOXO3a-T) were detected by Western blotting.

Figure 2.

Figure 2.

Puma is a FOXO3a transcriptional target gene. (A) p53+/+ and p53−/− FOXO3a TM-ER MEFs were exposed to 4-OHT (0.5 μM) for 6 h and lysates were subjected to Western blotting using antibodies directed against the indicated proteins. (B) Induction of Puma expression by FOXO3a-TM-ER. Levels of Puma transcripts or Puma protein in WT TM-ER and p53−/− TM-ER MEFs either left untreated (−) or treated with 4-OHT (+) for 8 h were assessed by RT-PCR or Western blotting (top) and QRT-PCR (bottom). HPRT and TBP, normalization control. Cell extracts from puma−/− MEFs were used as a negative control. QRT-PCR data are means ± SD from four independent experiments. (C) Identification of a conserved FHRE site in the human and mouse Puma promoters. p53BE, p53-binding element. (D) FOXO3a-TM activates a luciferase reporter gene driven by the Puma promoter. p53−/− MEFs were cotransfected with constructs as indicated. Luciferase assays were performed 24 h after transfection. Data shown are means ± SD from five independent experiments conducted in triplicates each time. (E) Quantification of FOXO3a association with the Puma promoter. QRT-PCR assays were conducted after chromatin IP using samples from cells that were either left untreated (con) or treated with 4-OHT. Numbers on the y-axis represent the levels of FOXO3a association with the Puma promoter region after normalizing to Ct values from input samples. Data shown are means ± SD from three independent experiments.

Figure 3.

Figure 3.

FOXO3a-dependent regulation of Puma expression in lymphoid cells upon removing of IL-2. (A) Pten loss impairs Puma and bim up-regulation induced by IL-2 deprivation. RNA extracted from Pten +/+ Lck-Cre or Pten flox/flox Lck-Cre activated T cells that were either left untreated or deprived of IL-2 for 10 h was subjected to QRT-PCR. Data represent the mean and error of four independent experiments. (B) Induction of Puma and Bim protein levels was attenuated in the absence of Pten. Activated T cells were deprived of IL-2 for 24 h and cell lysates were subjected to Western blotting with antibodies as indicated. Phosphorylated FOXO3a and total FOXO3a levels were determined by immunoprecipitation and Western blotting. (C) Synergistic cooperation between Puma and Bim in mediating cytokine withdrawal-induced cell death in lymphocytes. T cells were isolated from WT, puma −/−, bim −/−, or DKO mice and expanded in the presence of IL-2 and mitogen. Activated T cells were deprived of IL-2 for the indicated times, cell viability was determined by PI staining, and FACS analysis. p-values (Student's t test) were determined by comparing indicated KO T cells to WT T cells (green line) or DKO T cells to bim −/− T cells (red line). Data shown are means ± SD from three independent experiments. (D) Induction of Puma and bim mRNA (top) and protein levels (bottom) in activated T cells. p53+/+ and p53−/− activated T cells were subjected to IL-2 withdrawal for 10 h. QRT-PCR was performed to detect Puma (top left) and bim (top right) expression levels. Fold induction was determined after normalization to TBP. Results shown are representative of three independent experiments conducted in duplicates each time. (bottom) phosphorylated FOXO3a (Thr 32) (FOXO3a-P) and total FOXO3a (FOXO3a-T) were determined by immunoprecipitation followed by Western blotting. Puma, p53, phosphorylated Akt (Ser473) (Akt-P), and total Akt (Akt-T) were detected by Western blotting. (E) FOXO3a binds to the Puma and bim promoters. Activated T cells generated from p53−/− mice were cultured in IL-2–free medium for 8 h and ChIP assays were conducted as described in Materials and methods.

Figure 4.

Figure 4.

Direct role of FOXO3a in transcriptional regulating Puma. (A and B) FOXO3a is required for induction of Puma upon serum withdrawal. (left) Western blots show that FOXO3a shRNA successfully blocked FOXO3a expression in p53QSA135V cells (A) or 293T cells (B). Protein levels of Puma, Akt-T/P, and tubulin before and after serum starvation (24 h) were detected by Western blotting. (right) RT-PCR shows that Puma transcripts were elevated in serum-starved p53QSA135V cells (A) or 293T cells (B) expressing control shRNA (con). Ablation of endogenous FOXO3a by FOXO3a shRNA inhibited this elevation in Puma expression in both cell types. SS, serum starvation.

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