Davoodi Homa, Hashemi Seyed Reza, Seow Heng Fong
Golestan University of Medical Sciences, Gorgan, Iran.
Iran J Allergy Asthma Immunol. 2012 Jun;11(2):121-32.
Toll-Like Receptor 4 (TLR4), considered one of the most important TLR, recognizes lipopolysaccharide of Gram-negative bacteria. Recognition of ligands by TLRs induces signaling pathways resulting in activation of transcriptional factors such as NF-κB which are involved in the expression of inflammatory cytokines and chemokines. To prevent an inappropriate immune response, a complex network of molecules negatively regulates TLRs and their associated signaling pathways. Two cosegregating single nucleotide polymorphisms of the human TLR4 gene, namely Asp299Gly and Thr399Ile, have been associated with hyporesponsiveness to inhaled LPS. The purpose of this study was to determine the impact of TLR4 gene variant on NF-κB activity in colorectal cancer cell line. HCT116 cells were transfected with wild-type and mutants Flag-CMV1-TLR4 expression vectors. Western blot analysis was performed to evaluate selected molecules involved in TLR4 signaling. NF-κB activity was assessed by dual-luciferase reporter assay and cytokine profiles were evaluated byELISA and Cytometric Bead Array method. Results showed that the activity of pNF-κB was higher in cells harboring TLR4 D299G compared to the other cells. However, the activity of pAKT, pERK1 and pIRAK was higher in wild-type. The results of cytokine measurements showed about four fold higher level of IL-8 in cells with wild-type TLR4. This study suggest that TLR4 Asp299Gly gene variant has an impact on TLR4 signaling and potentially on intestinal homeostasis due to impaired control signals at the epithelial cell level which may lead to chronic intestinal inflammation and interrupted intestinal homeostasis and may eventually lead to colorectal cancer.
Toll样受体4（TLR4）被认为是最重要的TLR之一，可识别革兰氏阴性菌的脂多糖。TLR对配体的识别会诱导信号通路，导致转录因子如NF-κB的激活，这些转录因子参与炎症细胞因子和趋化因子的表达。为防止不适当的免疫反应，一个复杂的分子网络会对TLR及其相关信号通路进行负调控。人类TLR4基因的两个共分离单核苷酸多态性，即Asp299Gly和Thr399Ile，与吸入LPS的低反应性有关。本研究的目的是确定TLR4基因变异对结肠癌细胞系中NF-κB活性的影响。用野生型和突变型Flag-CMV1-TLR4表达载体转染HCT116细胞。进行蛋白质免疫印迹分析以评估参与TLR4信号传导的选定分子。通过双荧光素酶报告基因测定评估NF-κB活性，并通过酶联免疫吸附测定和细胞计数微球阵列方法评估细胞因子谱。结果显示，与其他细胞相比，携带TLR4 D299G的细胞中pNF-κB的活性更高。然而，野生型细胞中pAKT、pERK1和pIRAK的活性更高。细胞因子测量结果显示，野生型TLR4细胞中IL-8水平高出约四倍。本研究表明，TLR4 Asp299Gly基因变异对TLR4信号传导有影响，并且可能由于上皮细胞水平的控制信号受损而影响肠道稳态，这可能导致慢性肠道炎症和肠道稳态中断，并最终可能导致结直肠癌。
