Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.
Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.
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DOI：
10.1016/j.exer.2012.08.010
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发表时间：
2012-10
影响因子：
3.4
通讯作者：
Dartt, Darlene A.
中科院分区：
文献类型：
--
作者：
Hodges, Robin R.;Bair, Jeffrey A.;Carozza, Richard B.;Li, Dayu;Shatos, Marie A.;Dartt, Darlene A.
本研究的目的是确定表皮生长因子（EGF）用于刺激培养的大鼠结膜杯状细胞分泌粘蛋白的信号通路，并比较EGF与已知的促分泌素毒蕈碱胆碱能激动剂所使用的通路。为此，使用RPMI培养基在培养物中生长来自大鼠结膜的杯状细胞。对于免疫荧光实验，使用针对EGF受体（EGFR）和ERK 2以及毒蕈碱受体（M1AchR、M2 AchR和M3 AchR）的抗体，并通过荧光显微镜观察细胞。用Fura 2/AM测定细胞内[Ca 2 +]i。糖缀合物分泌测定培养杯状细胞与抑制剂预孵育后，然后刺激EGF或胆碱能激动剂卡巴胆碱（Cch）。杯状细胞分泌使用酶联凝集素测定与UEA-I或ELISA测定MUC 5AC。在培养的杯状细胞中，EGF以浓度依赖性方式刺激[Ca 2 +]i的增加。EGF刺激的[Ca 2 +]i的增加被EGF受体的抑制剂和细胞外Ca 2+的去除所阻断。针对EGFR和ERK 1/2的抑制剂阻断EGF刺激的粘蛋白分泌。此外，培养的杯状细胞表达M1 AchR、M2 AchR和M3 AchR。Cch刺激的[Ca 2 +]i增加被M1AchRs、基质金属蛋白酶和EGF受体的抑制剂阻断。EGF受体和ERK 1/2的抑制剂也阻断了Cch刺激的粘蛋白分泌。我们的结论是，在结膜杯状细胞，EGF本身增加[Ca 2 +]i和激活ERK 1/2刺激粘蛋白分泌。EGF刺激的分泌依赖于细胞外Ca 2+。这种作用机制类似于胆碱能激动剂，其使用毒蕈碱受体反式激活EGF受体，增加[Ca 2 +]i，并激活ERK 1/2，导致粘蛋白分泌增加。
The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M1AchR, M2AchR, and M3AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca2+] ([Ca2+]i) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca2+]i in a concentration-dependent manner. EGF-stimulated increase in [Ca2+]i was blocked by inhibitors of the EGF receptor and removal of extracellular Ca2+. Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M1AchR, M2AchR, and M3AchRs. Cch-stimulated increase in [Ca2+]i was blocked by inhibitors for the M1AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca2+]i and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca2+. This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca2+]i, and activate ERK 1/2 leading to an increase in mucin secretion.
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DOI：
10.1111/j.1582-4934.2007.00169.x
发表时间：
2008-09-01
影响因子：
5.3
作者：
Lee, H. Y.;Yea, K.;Ryu, S. H.
通讯作者：
Ryu, S. H.
DOI：
10.1097/aci.0b013e32830e6b04
发表时间：
2008-10
影响因子：
2.8
作者：
Mantelli F;Argüeso P
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作者：
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影响因子：
14.2
作者：
Lee, HM;Malm, L;Nadel, JA
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影响因子：
3.4
作者：
Gu, Jian;Chen, Lili;Dartt, Darlene A.
通讯作者：
Dartt, Darlene A.