Chen Dong-bao, Davis John S
The Women's Research Institute, Department of Obstetrics and Gynecology, University of Kansas School of Medicine-Wichita, 1010 North Kansas, Wichita 67214, USA.
Mol Cell Endocrinol. 2003 Feb 28;200(1-2):141-54. doi: 10.1016/s0303-7207(02)00379-9.
Epidermal growth factor (EGF) modulates the actions of gonadotropins in the corpus luteum. The membrane-associated EGF receptors undergo rapid tyrosine phosphorylation and internalization upon ligand binding in ovarian cells, including luteal cells. However, little is known about the post-receptor signaling events induced by EGF that lead to the transcriptional regulation of EGF-responsive genes in the ovary. The present study was designed to examine in bovine luteal cells (1) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling cascade (Raf/MEK/ERK) by EGF; (2) mRNA expression of AP-1 transcription factors, i.e. c-fos and c-jun, in response to EGF; and (3) the role of ERK in EGF-induced expression of c-fos and c-jun mRNA. Raf-1 and B-Raf, but not A-Raf, were activated by EGF (10 ng/ml) and the pharmacological protein kinase C (PKC) activator phorbol myristate acetate (PMA, 20 nM). Activation of Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1) which subsequently activated ERKs. Treatment with EGF-induced the phosphorylation of both ERK2 and ERK1 in a time and concentration dependent manner. Additionally, activated ERK was found in the nucleus of the cells following treatment with EGF (10 ng/ml) and PMA (PMA, 20 nM) for 5 min. Depletion of PKC by chronic PMA treatment (2.5 microM, 24 h) only partially inhibited the stimulatory effects of EGF on Raf-1, ERK2 and ERK1. These data demonstrate that PKC-dependent and independent-mechanisms are involved in EGF activation of the Raf/MEK/ERK signaling cascade in bovine luteal cells. EGF rapidly and transiently stimulated the expression of c-fos and c-jun mRNA in bovine luteal cells. Maximal induction of c-fos and c-jun mRNA by EGF occurred within 30 min of treatment with 10 ng/ml EGF. Treatment with the MEK1 inhibitor PD098059 (50 microM) abolished EGF-induced ERK activation. However, blocking EGF-induced ERK activation by pretreatment with PD098059 only partially attenuated EGF-induced c-fos and c-jun mRNA expression. Thus, additional pathways are implicated in the regulation of c-fos and c-jun mRNA expression by EGF in bovine luteal cells.
表皮生长因子（EGF）可调节黄体中促性腺激素的作用。膜相关的EGF受体在与配体结合后，包括黄体细胞在内的卵巢细胞中会迅速发生酪氨酸磷酸化并内化。然而，关于EGF诱导的受体后信号转导事件如何导致卵巢中EGF反应性基因的转录调控，我们所知甚少。本研究旨在检测牛黄体细胞中：（1）EGF对细胞外信号调节激酶（ERK）丝裂原活化蛋白激酶（MAPK）信号级联（Raf/MEK/ERK）的激活作用；（2）EGF作用下AP-1转录因子即c-fos和c-jun的mRNA表达；（3）ERK在EGF诱导的c-fos和c-jun mRNA表达中的作用。EGF（10 ng/ml）和药理蛋白激酶C（PKC）激活剂佛波酯肉豆蔻酸酯乙酸酯（PMA，20 nM）可激活Raf-1和B-Raf，但不能激活A-Raf。Raf的激活导致丝裂原活化蛋白激酶激酶（MEK1）的磷酸化和激活，进而激活ERK。EGF处理以时间和浓度依赖的方式诱导ERK2和ERK1的磷酸化。此外，在用EGF（10 ng/ml）和PMA（20 nM）处理5分钟后，在细胞核中发现了活化的ERK。通过慢性PMA处理（2.5 microM，24小时）耗尽PKC，仅部分抑制了EGF对Raf-1、ERK2和ERK1的刺激作用。这些数据表明，PKC依赖性和非依赖性机制均参与了牛黄体细胞中EGF对Raf/MEK/ERK信号级联的激活。EGF可快速短暂地刺激牛黄体细胞中c-fos和c-jun mRNA的表达。用10 ng/ml EGF处理30分钟内，EGF对c-fos和c-jun mRNA的诱导作用达到最大值。用MEK1抑制剂PD098059（50 microM）处理可消除EGF诱导的ERK激活。然而，用PD098059预处理阻断EGF诱导的ERK激活，仅部分减弱了EGF诱导的c-fos和c-jun mRNA表达。因此，在牛黄体细胞中，EGF对c-fos和c-jun mRNA表达的调控还涉及其他途径。
