Department of Ultrasound, Shengjing Hospital of China Medical University, 110004, Shenyang, China,
Inflammation. 2013 Oct;36(5):1064-74. doi: 10.1007/s10753-013-9638-7.
The role of Toll-like receptor 4 (TLR4) in immune cells is well characterized, but its biological properties in bladder epithelial cells (BECs), especially reciprocal crosstalk between mitogen-activated protein (MAP) kinase pathway and signal transducer and activator of transcription (STAT)3-mediated signal transduction elicited by TLR4 have not been demonstrated so far. The present studies were to demonstrate the signal transduction and inflammatory cytokine response elicited through activation of TLR4 in BECs with a special focus on the crosstalk between the MAPK-pathway and STAT3-mediated signals and its regulatory relevance for the inflammatory response towards lipopolysaccharide (LPS). We selected human bladder cancer T24 cell line in the present study and examined its expression of TLR4 by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression of p38, extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and STAT3 were performed by RT-PCR, quantitative PCR, and Western blotting. Signal transduction was analyzed by Western blotting. Interleukin-6 (IL-6) and interleukin-10 (IL-10) secretion in culture supernatants were tested by human enzyme-linked immunosorbent assay (ELISA) kit. BECs of rat infection in vivo model and patients with cystitis glandularis (CG) were analyzed as described above. Our study demonstrated that TLR4 was significantly upregulated following LPS treatment, with the maximum mRNA expression occurring at 4 h after stimulation. Activation of TLR4 signaling by LPS resulted in phosphorylation of MAPK and STAT pathways and upregulation of IL-10 in dose- and time-dependent manners in T24 cells. Pretreatment of cells with SB203580 (inhibitor of p38) and SP600125 (inhibitor of JNK) attenuated LPS-induced IL-10 expression, whereas it markedly inhibited the STAT3 expression. IL-10 mRNA expression was increased in inflamed lesions compared to noninflamed tissue in rats and patients with CG disease. Our results demonstrate that activation of TLR4 signaling in BECs induces IL-10 expression via activation of p38 and JNK, and the activation of STAT-3 was upregulated. Our data indicated that the reciprocal crosstalk between the MAPK pathway and STAT3-mediated signal transduction forms a critical axis successively activated by LPS in BECs.
Toll 样受体 4（TLR4）在免疫细胞中的作用已得到充分阐明，但在膀胱上皮细胞（BEC）中，其生物学特性，特别是丝裂原活化蛋白（MAP）激酶途径和信号转导及转录激活因子（STAT）3 介导的信号转导之间的相互交流，迄今为止尚未得到证实。本研究旨在通过激活 BEC 中的 TLR4 来证明信号转导和炎症细胞因子反应，特别关注 MAPK 途径和 STAT3 介导的信号之间的串扰及其对脂多糖（LPS）炎症反应的调节相关性。我们在本研究中选择人膀胱癌 T24 细胞系，并通过逆转录聚合酶链反应（RT-PCR）和免疫组织化学检查 TLR4 的表达。通过 RT-PCR、定量 PCR 和 Western 印迹检查 p38、细胞外信号调节激酶（ERK）、c-Jun NH2-末端激酶（JNK）和 STAT3 的表达。通过 Western 印迹分析信号转导。通过人酶联免疫吸附试验（ELISA）试剂盒检测培养上清液中白细胞介素-6（IL-6）和白细胞介素-10（IL-10）的分泌。如上所述，分析了大鼠感染体内模型和腺性膀胱炎（CG）患者的 BEC。我们的研究表明，LPS 处理后 TLR4 显著上调，刺激后 4 小时最大 mRNA 表达。TLR4 信号的激活导致 MAPK 和 STAT 途径的磷酸化，并以剂量和时间依赖性方式上调 T24 细胞中的 IL-10。用 SB203580（p38 抑制剂）和 SP600125（JNK 抑制剂）预处理细胞可抑制 LPS 诱导的 IL-10 表达，但明显抑制 STAT3 表达。与非炎症组织相比，炎症病变中 IL-10 mRNA 表达增加。在大鼠和 CG 疾病患者中。我们的结果表明，BEC 中 TLR4 信号的激活通过激活 p38 和 JNK 诱导 IL-10 表达，并且 STAT-3 的激活上调。我们的数据表明，MAPK 途径和 STAT3 介导的信号转导之间的相互串扰形成了 LPS 在 BEC 中依次激活的关键轴。
