The Molecular Mechanism Of Apoptosis Induced By TLR3 Through The Up-regulation Of TAP63α

Objective: Toll-like receptors (TLRs) recognize different pathogen associated molecular patterns (PAMPs), which function to prevent host from different pathogen infection by promoting the synthesis and secretion of inflammatory cytokines to induce inflammatory response and innate immunity. Activation of TLRs also induces the expression of immune molecules in immune cells, promotes the maturation of the antigen-presenting cells, and induces adaptive immunity. So TLRs are the bridges linking innate and adaptive immunity. TLR3 is the member in TLR family to recognize the conserved viral molecular double-strain RNA (dsRNA). It has been assumed that TLR3 plays an important role in antiviral immunity. On one hand, the activation of TLR3 by dsRNA induces the activation of immune cells to secrete antiviral cytokines such as IFNα,βto function as antiviral innate immune response. On the other hand, the activation of TLR3 induces the injury of the cells such as vascular endothelial cells, resulting in impairment of the blood vessel barrier and contributing to the spread of viral infection. In our previous studies, we found that both extrinsic and intrinsic death pathways were involved in the apoptosis induced by Poly (I:C) in HUVEC. The purpose of this study is to further analyze the molecular mechanism of apoptosis induced by TLR3, hint the pivotal molecular for apoptosis control in TLR3 signaling pathway, try to prevent the apoptosis by using this molecule as interference target, and in the end provide a solid basis of therapeutic rationality for the selection of new targets to control and prevent the progress of viral infected disease in clinical therapy.Methods : (1) RT-PCR was selected to detect the gene expression in HUVEC; (2) FACS and PI-Annexin V staining were used to test the death level of HUVEC induced by Poly (I:C); (3) The cytokine protein in the supernatant was measured by ELISA; (4) Antibody neutralization experiments were used to verify the involvement of cytokines in apoptosis induced by Poly(I:C) in HUVEC; (5) The activation and the expression of signal molecules involved in TLR3 signaling in HUVEC were assayed by Western-blotting; (6) The down-regulation of gene expression was induced by siRNA.Results : ELISA analysis showed that the treatment with double-stranded RNA analog Poly (I:C) up-regulated TNF-αsecretion in a dose-dependant manner in HUVEC supernatant. Recombinant TNF-αinduced the activation of NF-κB, but did not induce the apoptosis. Moreover the neutralized antibody against TNF-αfailed to inhibit the apoptosis induced by Poly (I:C) in HUVEC, suggesting that TNF-αis not involved in the apoptosis induced by TLR3. The treatment of HUVEC with Poly (I:C) up-regulated the gene expression of TRAIL (TNF-related Apoptosis-inducing Ligand) and its receptors DR4/DR5 in a dose-dependent manner. FACS results showed that neutralized antibody against TRAIL down-regulated the apoptosis induced by Poly (I:C), indicating that TRAIL-DR4/DR5 are involved in the initiation of the extrinsic death pathway induced by TLR3 in HUVEC. In addition, the treatment of HUVEC with Poly (I:C) down-regulated the expression of Bcl-2, up-regulated the expression of Noxa, Puma and P21. All these molecules can be regulated by tumor suppressor P53 or its family members. These results indicated that P53 or its family members may be involved in the apoptosis induced by TLR3 via intrinsic apoptotic pathway. However, TLR3 activation did not regulate the expression of P53, and the P53 inhibitor Pifithrin did not down-regulate the apoptosis induced by TLR3, suggesting that P53 is not involved in the TLR3 induced apoptosis. In addition, Poly (I:C) up-regulated the expression of TAP63α, an isoform of P63 family. Meanwhile the down-regulation of TAP63a by SiRNA, inhibited the down-regulation of Bcl-2, the up-regulation of Noxa, and depressed the activation of caspase 8, caspase 9 and PARP, and more importantly decreased the apoptosis induced by Poly (I:C). These results suggested that TAP63αis a critical regulator for the cell injury induced by TLR3.Conclusion: TNF-αdoes not contribute to the initiation of extrinsic apoptosis pathway. The interaction of TRAIL and DR4/DR5 is involved in the apoptosis induced by TLR3 via extrinsic apoptosis pathway. The activation of TLR3 down-regulates pro-survival molecule Bcl-2 and up-regulates pro-apoptotic molecule Noxa by which the intrinsic apoptosis pathway is initiated. TAP63α, one of P53 family members, is a critical regulator for apoptosis induced by TLR3.