Several studies have highlighted the role of the transcription factor p53 in antiviral responses (7
); however, the molecular mechanism remains to be clarified. Because the initiation of antiviral response is mediated, in part, by TLRs, which are important receptors that recognize microbial pathogens, we focused on the expression of TLRs and their possible regulation by p53. A principal finding in this study is that p53 positively affects the expression of TLR3 in epithelial cells (HCT116, HepG2, and A549). The present study is the first report on the transcriptional control of a TLR family member by p53 in epithelial cells and in some mouse tissues (liver and intestine). We have presented the following lines of evidence that TLR3 is a bona fide target of p53 (32
) in epithelial cells: (i) a p53 response element (RE) is present in the TLR3 promoter; (ii) TLR3 mRNA and protein levels are upregulated in HCT116 p53+/+
cells but not in p53−/−
cells; (iii) the TLR3 promoter containing the p53 RE could activate the luciferase reporter, but this activity was compromised when the RE was absent or mutated; and (iv) endogenous p53 binds to the RE in the TLR3 promoter region, as determined by the ChIP assay.
Importantly, the regulation of TLR3 expression by p53 impacts the TLR3 signaling pathways and the subsequent induction of cytokines downstream of TLR3 in response to poly(I-C) (Fig. and ). Response to poly(I-C) and dsRNA could also be mediated by the cytoplasmic viral sensors, the RNA helicases retinoic inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) (13
). It was recently reported by Hirata et al. that the recognition of poly(I-C) in intestinal epithelial cells is mediated by RIG-I but not by TLR3 (17
). The different cell lines used may account for the conflicting findings: whereas, we used the HCT116 cell line, which expresses wild-type p53, Hirata et al. utilized cell lines with mutated p53 (17
). We observed that HCT116 p53+/+
cells showed the same expression levels of RIG-I and MDA-5, which indicated that the regulation of their expression might not be dependent on p53 (data not shown). In our study, the specific knockdown of TLR3 by siRNA in HCT116 p53+/+
cells drastically reduced the IFN-β and IL-8 mRNA induction by poly(I-C) (Fig. ), which demonstrated the essential role of TLR3 in the cell's response to poly(I-C). Several studies have confirmed the importance of TLR3, RIG-I, or MDA-5 in poly(I-C) recognition (19
), and the question of whether RIG-I or TLR3 predominantly recognizes poly(I-C) in cells remains unresolved. Based on our results that p53 affects the expression of TLR3 but not that of MDA-5 or RIG-I, it is tempting to speculate that the activation of these pattern recognition receptors by poly(I-C) may, in part, depend on the p53 status of cells or tissues.
Although we did not observe a difference in the responses of the IL-8 promoter activity to other TLR ligands (PGN, LPS, CpG, and R-848) between p53+/+
cells (Fig. ), we could not rule out the possibility that p53 affects the steady-state level of other TLRs and the induction of cytokines other than IL-8, especially in light of studies proving that p53 is necessary in mounting a response against a variety of viruses, some of which may not be recognized by TLR3 (25
). Interestingly, our preliminary investigations showed that the mRNA expression of TLR7 and TLR8 was downregulated in HCT116 p53−/−
cells (data not shown), which hints at the possibility that p53 may also regulate the basal transcription of TLR7/8. Further investigations may clarify this issue.
It is noteworthy that hepatitis C virus (HCV) can induce anomalies in p53 function (3
) and that TLR3 expression is downregulated in chronic HCV infection through an unknown mechanism (4
). Although HCV is a single-stranded virus, which is susceptible to detection by TLR7/8, its genome also encodes regions of extensive secondary dsRNA structure that could be engaged by dsRNA-sensing receptor such as TLR3 (12
). Based on these observations, it may be likely that certain viruses, which can induce downregulation of p53, may also in part circumvent TLR3 antiviral functions (and cause persistent infection) due to TLR3 being a molecular target of p53.
It has been reported that primary induction of IFNs by virus infection transcriptionally activates p53 to trigger apoptosis in infected cells (29
). While these previous studies clearly indicate that host protection provided by p53 is dependent on its role as inducer of apoptosis (5
), our present results suggest a novel mechanism of p53's function in antiviral signaling which is through its transactivation of TLR3 expression (and possibly that of other TLRs as well). Considering together the previous and current findings, it is likely that a positive feedback loop may exist between p53, TLR3, and IFN-β for antiviral host defense.
In conclusion, our results here first demonstrate a molecular link between p53 and a virus-sensing molecule.