China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China.
Department of Medical Education & Primary Care, Kyoto Prefectural University of Medicine, Kajiicho 465, Kamikyo-ku, Kyoto City, Kyoto 602-8566, Japan.
Mol Immunol. 2018 Jul;99:19-29. doi: 10.1016/j.molimm.2018.04.001. Epub 2018 Apr 16.
Ultraviolet B (UVB) irradiation exerts multiple effects on skin cells, inducing apoptosis, senescence and carcinogenesis. Toll-like receptor 3, a member of pattern recognition receptors, is reported to initiate inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. It has not been studied, however, whether apoptosis induction in UVB irradiation is attributed to TLR3 activation. Here, we report on the pro-apoptotic role of TLR3 in UVB-irradiated epidermal cells. Poly I:C, an analogue of dsRNA that activates TLR3, was used in combination with sub-lethal UVB (4.8 mJ/cm) irradiation for investigating the effects of TLR3 activation on human immortalized keratinocyte HaCaT cells. Although sub-lethal dose of either Poly I:C or UVB alone did not induce cell death, UVB-Poly I:C co-treatment synergistically induced cell death by activation of caspase-3 and cleavages of ICAD and PARP, with apoptotic features when stained with Annexin V/PI or Hoechst 33342. Treatment with pan-caspase inhibitor, Z-VAD, attenuated UVB-Poly I:C-induced cell death. Silencing TLR3 by siRNA rescued HaCaT cells from UVB-Poly I:C-induced apoptosis. NF-κB, a major downstream component of TLR3 pathway, that usually negatively regulates the classical TLR3 apoptotic pathway, was analyzed by western blotting and immunofluorescence confocal microscopy. The results indicate to our surprise that NF-κB is translocated to nucleus in the cells co-treated with UVB-Poly I:C. The nuclear translocation of NF-κB is attenuated by TLR3 silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-Poly I:C-induced apoptosis. Therefore, we conclude that NF-κB pathway plays a cytotoxic role in UVB-Poly I:C-treated HaCaT cells, mediating TLR3-related apoptosis.
紫外线 B（UVB）照射对皮肤细胞有多种影响，可诱导细胞凋亡、衰老和癌变。模式识别受体中的 Toll 样受体 3 被报道可通过识别从 UVB 照射的细胞中释放的双链 RNA（dsRNA）来引发炎症。然而，尚未研究 UVB 照射诱导的细胞凋亡是否归因于 TLR3 的激活。在这里，我们报告了 TLR3 在 UVB 照射的表皮细胞中诱导凋亡的作用。聚肌苷酸：聚胞苷酸（poly I:C）是一种激活 TLR3 的 dsRNA 类似物，与亚致死剂量的 UVB（4.8 mJ/cm）照射联合使用，用于研究 TLR3 激活对人永生化角质形成细胞 HaCaT 细胞的影响。尽管单独使用亚致死剂量的 Poly I:C 或 UVB 均不会诱导细胞死亡，但 UVB-Poly I:C 共同处理通过激活 caspase-3 和切割 ICAD 和 PARP 协同诱导细胞死亡，并用 Annexin V/PI 或 Hoechst 33342 染色具有凋亡特征。用泛半胱天冬酶抑制剂 Z-VAD 处理可减轻 UVB-Poly I:C 诱导的细胞死亡。用 siRNA 沉默 TLR3 可使 HaCaT 细胞免于 UVB-Poly I:C 诱导的凋亡。TLR3 途径的主要下游成分 NF-κB，通常负调控经典 TLR3 凋亡途径，通过 Western 印迹和免疫荧光共聚焦显微镜进行分析。结果令人惊讶地表明，在共同用 UVB-Poly I:C 处理的细胞中，NF-κB 被转位到核内。TLR3 沉默可减弱 NF-κB 的核转位。用 NF-κB 途径抑制剂 BAY 处理可阻断 UVB-Poly I:C 诱导的细胞凋亡。因此，我们得出结论，NF-κB 途径在 UVB-Poly I:C 处理的 HaCaT 细胞中发挥细胞毒性作用，介导 TLR3 相关的凋亡。
