Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA-binding protein HuR

Abstract

We have reported previously that apigenin, a naturally occurring nonmutagenic flavonoid, increased wild-type p53 protein expression in the mouse keratinocyte 308 cell line by a mechanism involving p53 protein stabilization. Here we further demonstrated that the increase in p53 protein level induced by apigenin treatment of 308 keratinoyctes was not the result of enhanced transcription, mRNA stabilization or cytoplasmic export of p53 mRNA. Instead, biosynthetic labeling showed that apigenin increased nascent p53 protein synthesis by enhancing p53 translation. The AU-rich element (ARE) within the 3'-untranslated region (UTR) of p53 mRNA was found to be responsible for apigenin's ability to increase p53 translation, as demonstrated in studies wherein the 3'-UTR of p53 mRNA containing the ARE was fused downstream of a luciferase reporter gene. Furthermore, apigenin treatment increased the level of association of the RNA binding protein HuR with endogenous p53 mRNA. Apigenin treatment also augmented HuR translocation into the cytoplasm. Overexpression of HuR enhanced apigenin-induced p53 protein expression in 308 keratinocytes, whereas siRNA-mediated HuR reduction suppressed apigenin-induced p53 protein expression and de novo translation of p53. Moreover, apigenin treatment of cells induced p16 protein expression, which in turn was correlated with cytoplasmic localization of HuR induced by apigenin. Overall, these findings indicate that, in addition to modulating p53 protein stability, one of the mechanisms by which apigenin induces p53 protein expression is enhancement of translation through the RNA binding protein HuR.

Figure 1. Apigenin-induced expression of p53 and its downstream target genes in 308 mouse keratinocytes

(A) Western blot analysis showing dose-dependent induction of p53 protein at 8 h after apigenin treatment. (B) Time-dependent induction of p53 protein expression by apigenin. Cells were treated with 50 μM apigenin, harvested at the indicated time points, and analyzed by Western blot. (C) Apigenin-mediated expression of p21, Bax and PUMA. Cells were treated with 50 μM apigenin, harvested at the indicated time points, and subjected to Western blot analysis.

Figure 2. Effects of apigenin on p53 mRNA

(A) Real-time PCR analysis of steady-state p53 mRNA levels. Cells were treated with 50 μM apigenin, and total RNA was harvested at the indicated times. TaqMan real-time quantitative PCR was carried out as described in Materials and Methods (* = significantly different from 0 h, P< 0.001). (B) Apigenin treatment does not change the stability of p53 mRNA. Cells were pre-treated with medium containing 50 μM apigenin or DMSO vehicle for 2 h, 10 μM actinomycin D was added and incubated for 0, 3, 6, 9 and 12 h. Total RNA was isolated, reverse transcribed and analyzed for p53 mRNA using TaqMan real-time PCR. Amount of p53 mRNA is expressed as a percentage of levels measured at the 0 h time point. (C) Total, cytoplasmic, and nuclear fractions were prepared for real-time PCR analysis of p53 mRNA at 2 h after apigenin (50 μM) treatment. All the graphs represent the results of three independent experiments.

Figure 3. Apigenin induces p53 protein phosphorylation and enhances nascent p53 protein synthesis

(A) Cells were exposed to 50 μM apigenin, harvested at the indicated time points, and Western blot analysis was carried out to detect the levels of phosphorylated p53 protein (Ser-15) and total p53 protein. (B) Control cells (C) and cells that had been treated with 50 μM apigenin for 4 h (A) were incubated with L-[³⁵S]methionine and L-[³⁵S]cysteine for 15 min, followed by IP using either normal IgG or anti-p53 antibody. Samples were resolved by SDS-PAGE and the level of nascent p53 protein was measured by autoradiography (upper panel). Analysis of whole-cell extracts (WCE) showed that equal amount of [³⁵S]methionine/cysteine was incorporated into the cells and that protein translation in general was not affected by apigenin treatment (lower panel).

Figure 4. The ARE of p53 mRNA is important for translation efficiency

(A) Structure of luciferase reporter gene constructs. Various regions of the 3′-UTR (gray bars) of p53 were fused to reporter gene luciferase (black bars) to generate the constructs containing the full-length 3′-UTR (nt 1302-1743), the ARE-containing region (nt 1605-1743), the ARE-containing region deleted from the full-length 3′-UTR (nt 1302-1624) or luciferase control (pGL3c) without the 3′-UTR. The locations of AUUUA sequence and AU-rich region are indicated by asterisks and white bar, respectively. (B) Effects of various regions of the 3′-UTR of the p53 gene on luciferase activity when fused to the 3′ end of the reporter gene. Cells were co-transfected with the different reporter gene constructs and Renilla luciferase plasmid. At 24 h after transfection, cells were treated with 50 μM apigenin for another 8 or 24 h. Relative luciferase activity for each construct was represented as the ratio between apigenin-treated versus untreated culture. All values were normalized to Renilla luciferase activity and results are the means ± S.E. for three independent experiments, each performed in duplicate (* = significantly different from pGL3c, P < 0.001). (C) Effects of various regions from the 3′-UTR of the p53 gene on the luciferase reporter gene message levels. Cells were transfected as described in (B) and treated with apigenin for 24 h. Total RNA was extracted and assayed for luciferase and GAPDH mRNAs by using real-time PCR as described in Materials and Methods. Luciferase message levels were normalized to GAPDH mRNA and expressed relative to untreated cells (* = significantly different from pGL3c, P < 0.001).

Figure 5. Apigenin-dependent binding of HuR to endogenous p53 mRNA

Cytoplasmic extracts were prepared from control and apigenin-treated cells, then protein-RNA complexes were immunoprecipitated using either anti-HuR antibody or normal IgG, followed by RT-PCR analysis to detect endogenous p53 mRNA and β-Actin mRNA (M, DNA marker; C, control; A, 50 μM apigenin for 4 h).

Figure 6. Effect of apigenin on the subcellular localization of HuR

(A) Whole cell, cytoplasmic and nuclear lysates were prepared and subjected to Western blot analysis for HuR. Expression of the cytoplasmic marker α-Tubulin and the nuclear marker hnRNP A0 were also detected (C, control; A, 50 μM apigenin for 4 h). (B) The subcellular localization of HuR was monitored by immunofluorescence. The slides were also stained with diamidinophenylindole (DAPI) for visualization of nuclei. (C) Western blot showing subcelluar localization of HuR in cytosol and polysomal fractions (C, control; A, 50 μM apigenin for 4 h). (D) RNA was extracted from polysomal fraction (untreated control or 50 μM apigenin for 4 h), and the levels of p53 mRNA was measured by real-time RT-PCR (*, P< 0.01).

Figure 6. Effect of apigenin on the subcellular localization of HuR

(A) Whole cell, cytoplasmic and nuclear lysates were prepared and subjected to Western blot analysis for HuR. Expression of the cytoplasmic marker α-Tubulin and the nuclear marker hnRNP A0 were also detected (C, control; A, 50 μM apigenin for 4 h). (B) The subcellular localization of HuR was monitored by immunofluorescence. The slides were also stained with diamidinophenylindole (DAPI) for visualization of nuclei. (C) Western blot showing subcelluar localization of HuR in cytosol and polysomal fractions (C, control; A, 50 μM apigenin for 4 h). (D) RNA was extracted from polysomal fraction (untreated control or 50 μM apigenin for 4 h), and the levels of p53 mRNA was measured by real-time RT-PCR (*, P< 0.01).

Figure 7. Level of expression of HuR affects apigenin-induced p53 protein expression

(A) Representative Western blot of HuR and p53 expression levels in 308 cells that had been transfected either with an empty control plasmid or an HuR-expression plasmid (C, control; A, 50 μM apigenin for 8 h). (B) Quantitative analysis of the effect of HuR over-expression on apigenin-induced p53 expression. p53 protein bands were scanned by densitometry and normalized to their corresponding Actin bands, graph represents the results of three independent experiments (*, P< 0.05). (C) Representative Western blot of HuR and p53 expression levels in 308 cells that had been transfected with either HuR siRNA duplex or control non-targeting siRNA duplex, and exposed to 50 μM apigenin for another 8 h. (D) The effect of HuR knockdown on apigenin-induced p53 expression was measured as described in (B) (*, P< 0.001). (E) Cells were transfected with siRNA, treated with 50 μM apigenin for 4 h, and followed by a brief incubation (15 min) with L-[³⁵S]methionine and L-[³⁵S]cysteine. Upper panel: cell lysates were immunoprecipitated by using either normal IgG or anti-p53 antibody, then resolved by SDS-PAGE and the level of nascent p53 protein was measured by autoradiography; lower panel: analysis of whole-cell extracts (WCE) showed that equal amount of [³⁵S]methionine/cysteine was incorporated into the cells and that protein translation in general was not affected by apigenin treatment.

Figure 7. Level of expression of HuR affects apigenin-induced p53 protein expression

(A) Representative Western blot of HuR and p53 expression levels in 308 cells that had been transfected either with an empty control plasmid or an HuR-expression plasmid (C, control; A, 50 μM apigenin for 8 h). (B) Quantitative analysis of the effect of HuR over-expression on apigenin-induced p53 expression. p53 protein bands were scanned by densitometry and normalized to their corresponding Actin bands, graph represents the results of three independent experiments (*, P< 0.05). (C) Representative Western blot of HuR and p53 expression levels in 308 cells that had been transfected with either HuR siRNA duplex or control non-targeting siRNA duplex, and exposed to 50 μM apigenin for another 8 h. (D) The effect of HuR knockdown on apigenin-induced p53 expression was measured as described in (B) (*, P< 0.001). (E) Cells were transfected with siRNA, treated with 50 μM apigenin for 4 h, and followed by a brief incubation (15 min) with L-[³⁵S]methionine and L-[³⁵S]cysteine. Upper panel: cell lysates were immunoprecipitated by using either normal IgG or anti-p53 antibody, then resolved by SDS-PAGE and the level of nascent p53 protein was measured by autoradiography; lower panel: analysis of whole-cell extracts (WCE) showed that equal amount of [³⁵S]methionine/cysteine was incorporated into the cells and that protein translation in general was not affected by apigenin treatment.

Figure 8. Knockdown of p16 expression correlates with reduction in apigenin-induced cytoplasmic distribution of HuR

(A) Time-dependent induction of p16 protein expression by apigenin. Cells were treated with 50 μM apigenin, harvested at the indicated time points, and analyzed by Western blot. (B) Cells were transfected with either p16 siRNA duplex or control non-targeting siRNA duplex, and whole cell lysates were used to examine p16 protein level. (C) After p16 knockdown by siRNA, cytoplasmic and nuclear lysates were separated and subjected to Western blot analysis for HuR, α-Tubulin and hnRNP A0 (C, control; A, 50 μM apigenin for 4 h).