p53 positively regulates the expression of TLR3 in epithelial cells. (A) HCT116 p53^+/+ and p53^−/− cells were transfected with IL-8 promoter plasmid and treated with 10 μg/ml each of the indicated TLR agonists for 6 h. IL-8 promoter activity was measured 48 h after transfection of IL-8 promoter. The data shown are means ± standard errors from triplicate platings and represent three independent experiments. **, P < 0.001 versus control (Con; phosphate-buffered saline [HCT116 p53^+/+]) assessed by ANOVA with Dunnett's procedure. (B) TLR3 mRNA in HCT116 p53^+/+ and p53^−/− cells was determined by real-time quantitative PCR. The TLR3 mRNA level was normalized to GAPDH (internal control). The data shown are means ± standard deviations from triplicate determinations from three independent experiments. *, P < 0.05 assessed by Student's t test. (C) TLR3 mRNA was examined in HCT116 p53^+/+ and in HCT116 p53^−/− cells untransfected or transfected with increasing amounts of p53 expression plasmid. (D) TLR3 mRNA was determined in HCT116 p53^+/+ and p53^−/− cells treated for 24 h with 4 or 8 μM 5-FU. For panels C and D, p21 served as a positive control. GAPDH was used as an internal control for RT-PCR analyses. (E) HCT116 p53^+/+ cells were transfected with p53 siRNA (si-p53) or control siRNA duplex (si-GL2), and total RNA from these cells was isolated for analysis of TLR3 and p53 mRNA by RT-PCR. (F) The TLR3 protein level was determined in lysates of HCT116 p53^+/+ and p53^−/− cells by immunoprecipitation (IP) and Western blotting using anti-TLR3 antibody. Hsc70 was used as internal control. con-IgG, control IgG. (G) A549 cells were treated with 4 or 8 μM 5-FU for 24 h. (H) si-GL2 or the indicated amount of p53 siRNA duplex was transfected in A549 cells. Twenty-four hours later, RNA was extracted. (I to K) HepG2 (I), Caco2 (J), and Calu-3 (K) cells were treated with 400 or 800 μM 5-FU for 8 h. For panels G to K, total RNA was extracted and analyzed by RT-PCR for the expression of TLR3, p21 (positive control), or p53. GAPDH served as the internal control.

Activation of TLR3 signaling pathway by poly(I-C) is impaired in HCT116 p53^−/− cells. (A to C) Cytoplasmic lysates (A and C) or nuclear extracts (B) from HCT116 p53^+/+ and p53^−/− cells unstimulated or stimulated for 3 h with poly(I-C) at the indicated concentration (A and C) or 5 μg/ml (B) were analyzed by Western blotting for phosphorylated IκB-α (p-IκB-α) and basal IκB-α (A), p65 (B), phosphorylated IRF-3 (p-IRF-3), and basal IRF-3 (C) expression. Hsc70 was used as loading control for panels A and C. γ-Tubulin was used as the loading control for panel B.

The TLR3 mRNA level is downregulated in tissues of p53^−/− mice. (A to C) Total RNA isolated from liver (A), intestine (B), and spleen (C) of p53^+/+, p53^+/−, and p53^−/− mice was analyzed for the expression of TLR3 by real-time quantitative RT-PCR. TLR3 mRNA levels were normalized to HPRT (internal control). The results represent means ± standard deviations (n = 3). * and **, P < 0.01 and P < 0.001, respectively, against p53 wild-type mice (+/+), as assessed by ANOVA with Dunnett's test.

p53 binds to and transactivates the TLR3 promoter. (A) HCT116 p53^+/+ and p53^−/− cells were transiently transfected with the indicated TLR3 promoter constructs (0.2 μg). Luciferase activity was determined 48 h after transfection of plasmids and is expressed as activation (fold) over that of the pGL3b vector. Values are means ± standard errors from triplicate platings. The data shown are representative of three independent experiments. ** and ***, P < 0.001 and P < 0.0001, respectively, against the corresponding promoter length in HCT116 p53^−/− cells, as determined by Student's t test. (Right panel) Schematic diagram of the TLR3 promoter constructs containing the p53 binding sites. (B) HCT116 p53^−/− cells were transiently transfected with the indicated TLR3 promoter constructs (0.2 μg) and 0.025 μg p53 plasmid or pcDNA3.1 empty vector (con), and luciferase activity was assayed 48 h posttransfection. Values are means ± standard errors from triplicate platings. Data represent three independent experiments. **, P < 0.001, as determined by Student's t test. (C) HCT116 p53^−/− cells were transiently transfected with pGL3b vector or −2 kb TLR3 promoter (TLR3p), cotransfected with p53 expression plasmid at increasing amounts (6.25, 12.5, and 25 ng), and assayed for luciferase activity. Values are means ± standard errors from triplicate platings. The data shown are representative of three independent experiments. ***, P < 0.0001 versus TLR3p, as assessed by ANOVA with Dunnett's procedure. (D) HCT116 p53^+/+ cells were transiently transfected with the −2 kb TLR3 promoter and cotransfected with si-GL2 or si-p53 (50 or 100 nM) oligonucleotide, and luciferase activity was assayed 48 h posttransfection. Values are means ± standard errors from triplicate platings. The data represent three independent experiments. ** and ***, P < 0.001 and P < 0.0001, respectively, against TLR3p, as determined by ANOVA with Dunnett's test. (E) Wild-type or mutant (mutated p53-binding sites) TLR3 promoter (−2 kb) was transfected into HCT116 p53^+/+ cells, and luciferase activity was assayed 48 h after transfection. Values are means ± standard errors from triplicate platings. Data represent three independent experiments. ***, P < 0.0001 against the wild-type (WT) TLR3 promoter, as determined by ANOVA with Tukey-Kramer's test. n.s., not significant. (Upper panel) Schematic diagram of the −2 kb TLR3 promoter with the indicated position of the mutated p53 binding site. (F) HCT116 p53^−/− cells were transfected with pGL3b and wild-type (WT) or mutant TLR3 promoter and cotransfected with pCDNA3.1 empty vector or p53 plasmid, and luciferase activity was assessed 48 h after transfection. Values are means ± standard errors from triplicate platings. The data represent three independent experiments. * and **, P < 0.01 and P < 0.001, respectively, against pCDNA3.1 vector, as assessed by Student's t test. (G) Consensus site of p53 and the sequence of p53 binding site in the TLR3 promoter at −1929. Underlined bases denote sequence variation from the consensus p53 element. (H) Representative result of the ChIP analysis in HCT116 p53^+/+ cells using p53 antibody or mouse IgG for immunoprecipitation (IP) and primers of the indicated promoter region for PCR.

The response of cytokines IL-8 and IFN-β, which are downstream of TLR3, to poly(I-C) stimulation requires p53. (A) Real-time quantitative PCR analysis of IL-8 and IFN-β mRNA was performed on HCT116 p53^+/+ and p53^−/− cells untreated or treated with 5 μg/ml poly(I-C) for 3 h. mRNA expression was normalized to GAPDH. Values are means ± standard deviations of triplicate measurements. ***, P < 0.0001, as analyzed by ANOVA with Tukey-Kramer's test. (B) The mRNA level of IL-8 and IFN-β was determined in HCT116 p53^+/+ and p53^−/− cells stimulated with 5 μg/ml poly(I-C) for the indicated time. p21 and GAPDH served as the positive control and internal control, respectively. (C and D) Promoter activity of IL-8 (C) and IFN-β (D) was examined in HCT116 p53^+/+ and p53^−/− cells stimulated with poly(I-C) at the indicated concentration for 6 h (upper panels) or with 5 μg/ml poly(I-C) for the indicated time (lower panels). Values are means ± standard errors from triplicate platings. The data shown are representative of two to three independent experiments. (E) The mRNA expression of IL-8 and IFN-β was analyzed in HCT116 p53^+/+ cells unstimulated or stimulated with poly(I-C) and untransfected or transfected with siRNA duplex for TLR3 (si-TLR3) or p53 (si-p53) or with si-GL2 (for control). Expression of TLR3 and p53 was knocked down by the transfection of their respective siRNAs in p53^+/+ cells (lower panels).