Todisco A, Takeuchi Y, Urumov A, Yamada J, Stepan V M, Yamada T
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109, USA.
Am J Physiol. 1997 Oct;273(4):G891-8. doi: 10.1152/ajpgi.1997.273.4.G891.
We have previously observed that gastrin has a cholecystokinin B (CCK-B) receptor-mediated growth-promoting effect on the AR42J rat pancreatic acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose dependently induced luciferase activity in AR42J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element (SRE) of the c-fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the specific CCK-A receptor antagonist L-364,718 or by pertussis toxin, indicating that gastrin targets the SRE via specific CCK-B receptors through a mechanism independent of Gi. Inhibition of protein kinase C (PKC) either by prolonged (24 h) exposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (100 nM) or by incubation with the selective inhibitor GF-109203X (3.5 microM) resulted in an 80% reduction in luciferase activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 microM). We measured ERK2 activity in AR42J cells via in-gel kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2 enzyme activity in a dose-dependent manner. Addition of GF-109203X and PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin induction of ERK2 activity also resulted in a threefold increase in the transcriptional activity of Elk-1, a factor known to bind to the c-fos SRE and to be phosphorylated and activated by ERK2. PD-98059 blocked the growth-promoting effect of gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced c-fos gene expression via PKC-dependent and -independent pathways.
我们先前观察到胃泌素对AR42J大鼠胰腺腺泡细胞系具有胆囊收缩素B（CCK - B）受体介导的促生长作用，且这一作用与早期反应基因c - fos表达的诱导相平行。我们进行这些实验以阐明c - fos诱导的机制以及该作用与胃泌素营养作用的联系。胃泌素（0.1 - 10 nM）以剂量依赖的方式诱导用由与c - fos启动子的血清反应元件（SRE）偶联的荧光素酶报告基因构建体转染的AR42J细胞中的荧光素酶活性。这种作用被特异性CCK - B受体拮抗剂D2阻断，但不被特异性CCK - A受体拮抗剂L - 364,718或百日咳毒素阻断，表明胃泌素通过一种不依赖Gi的机制经由特异性CCK - B受体靶向SRE。通过将细胞长时间（24小时）暴露于佛波酯12 - O - 十四酰佛波醇13 - 乙酸酯（100 nM）或与选择性抑制剂GF - 109203X（3.5 microM）孵育来抑制蛋白激酶C（PKC），导致荧光素酶活性降低80％。在存在特异性细胞外信号调节激酶（ERK）激酶（MEK）抑制剂PD - 98059（50 microM）的情况下观察到类似结果。我们通过凝胶内激酶测定法测量AR42J细胞中的ERK2活性，并观察到胃泌素（1 pM - 100 nM）以剂量依赖的方式诱导ERK2酶活性。单独或联合添加GF - 109203X和PD - 98059分别对胃泌素诱导的ERK2活性产生部分和完全抑制。胃泌素诱导ERK2活性还导致Elk - 1的转录活性增加三倍，Elk - 1是一种已知与c - fos SRE结合并被ERK2磷酸化和激活的因子。PD - 98059阻断了胃泌素对AR42J细胞的促生长作用，表明这种作用取决于MEK的激活。我们的数据使我们得出结论，胃泌素的营养作用是通过ERK2诱导的c - fos基因表达经由PKC依赖性和非依赖性途径介导的。
