Feng Po-Hao, Hsiung Te-Chih, Kuo Han-Pin, Huang Chien-Da
Department of Thoracic Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taiwan.
Chang Gung Med J. 2010 Jan-Feb;33(1):92-9.
Bradykinin (BK), a G-protein-coupled-receptor (GPCR) agonist via the B2 receptor induces interleukin (IL)-6 expression in airway smooth muscle (ASM) cells by involving the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In some cell species, GPCR agonists have been shown to activate the ERK 1/2 pathway via transactivation of epidermal growth factor (EGF) receptor (EGFR). In this study, we tested whether there is cross-talk between BK and EGF in the regulation of IL-6 gene expression in ASM cells.
ASM cells were treated with BK, EGF, AG-1478 and genistein. IL-6 production was analyzed by enzyme-linked immunosorbent assay (ELISA). Immunoblot study was used for detection of ERK1/2 activation. Transactivation of EGFR phosphorylation was detected by immunoprecipitation.
ELISA showed that EGF (10 ng/ml, 18 hr) increased IL-6 secretion (from 234 +/- 35 to 923 +/- 494 pg/ml, n = 5, p > 0.05), and significantly enhanced BK-induced IL-6 secretion (from 4383 +/- 296 to 8312 +/- 1267 pg/ml, n = 5, p < 0.05) in ASM. Moreover, AG-1478 (2 microM), reduced BK-induced IL-6 secretion by 28% and abrogated the synergic induction of IL-6 induced by BK plus EGF (from 8312 +/- 1267 to 3229 +/- 597 pg/ml, n = 5, p < 0.05). AG-1478 dual effects on IL-6 secretion induced by BK alone or BK plus EGF were also observed in cells treated with genistein, a tyrosine kinase inhibitor, and AG-825, an ErbB-2 inhibitor. Immunoblot analysis demonstrated that AG-1478 had no effect on ERK1/2 activation by BK (1 microM, 10 min). Immunoprecipitation studies showed that BK (1 muM for 2, 5 and 10 min) did not directly transactivate EGFR phosphorylation.
These data show that BK and EGF act in concert to regulate the expression of IL-6 in ASM cells possibly via transcriptional mechanisms involving EGFR-associated key signaling molecules.
缓激肽（BK）作为一种通过B2受体作用的G蛋白偶联受体（GPCR）激动剂，通过细胞外信号调节激酶1/2（ERK1/2）信号通路诱导气道平滑肌（ASM）细胞中白细胞介素（IL）-6的表达。在某些细胞类型中，GPCR激动剂已被证明可通过表皮生长因子（EGF）受体（EGFR）的反式激活来激活ERK 1/2通路。在本研究中，我们测试了在ASM细胞中IL-6基因表达的调控过程中BK与EGF之间是否存在相互作用。
用BK、EGF、AG-1478和染料木黄酮处理ASM细胞。通过酶联免疫吸附测定（ELISA）分析IL-6的产生。免疫印迹研究用于检测ERK1/2的激活。通过免疫沉淀检测EGFR磷酸化的反式激活。
ELISA显示，EGF（10 ng/ml，18小时）增加了IL-6的分泌（从234±35增加到923±494 pg/ml，n = 5，p>0.05），并显著增强了BK诱导的ASM中IL-6的分泌（从4383±296增加到8312±1267 pg/ml，n = 5，p<0.05）。此外，AG-1478（2μM）使BK诱导的IL-6分泌减少了28%，并消除了BK加EGF诱导的IL-6协同诱导作用（从8312±1267减少到3229±597 pg/ml，n = 5，p<0.05）。在用酪氨酸激酶抑制剂染料木黄酮和ErbB-2抑制剂AG-825处理的细胞中，也观察到AG-1478对单独BK或BK加EGF诱导的IL-6分泌的双重作用。免疫印迹分析表明，AG-1478对BK（1μM，10分钟）激活ERK1/2没有影响。免疫沉淀研究表明，BK（1μM，作用2、5和10分钟）不会直接反式激活EGFR磷酸化。
这些数据表明，BK和EGF协同作用，可能通过涉及EGFR相关关键信号分子的转录机制来调节ASM细胞中IL-6的表达。
