Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element
Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element
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DOI：
10.1042/bj20031793
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发表时间：
2004-07-15
影响因子：
4.1
通讯作者：
Dockray, GJ
中科院分区：
文献类型：
--
作者：
Ashcroft, FJ;Varro, A;Dockray, GJ
许多组织中的应激、炎症和损伤会导致分泌型凝集素样蛋白 Reg 家族成员的表达增加。在胃中，Reg 位于肠嗜铬样细胞中，其表达受到胃激素胃泌素的刺激。我们研究了胃泌素刺激 Reg-1 表达的机制。将荧光素酶报告载体中大鼠 Reg-1 启动子 2.1 至 0.1 kb 的缺失突变瞬时转染至胃癌 AGS-G(R) 细胞中。所有测试的启动子片段均显示响应于胃泌素(1nM)的荧光素酶表达的类似相对增加。最小（104 bp）构建体对胃泌素的反应是基础的 4.2 +/- 0.4 倍。 Ro-32-0432 减少了这些响应。一种蛋白激酶 C 抑制剂，通过 C3 转移酶、肉毒杆菌毒素和 Rho 家族 GTP 酶 RhoA 的选择性抑制剂，以及通过与 RhoA 的显性失活形式共转染。与 RhoA 组成型活性形式的共转染刺激表达比基础表达高 11.6 +/- 1.7 倍。通过 104 bp 构建体的突变鉴定出对胃泌素、PKC（蛋白激酶 Q 和 L63RhoA（人 RhoA 蛋白的组成型活性形式，在第 63 位含有谷氨酰胺到亮氨酸取代）作出反应所需的富含 C 的元件 (C-79CCCTCCC-72)。使用对照和对照的核提取物进行 EMSA（电泳迁移率变化测定）。 胃泌素刺激的 AGS-GR 细胞和跨度 86 至 64 bp 的探针揭示了多种结合蛋白。胃泌素对结合模式没有影响。 Supershift 测定表明转录因子 Sp1 和 Sp3 结合富含 C 的序列。我们得出结论，胃泌素通过激活 PKC 和 RhoA 刺激 Reg 表达，富含 C 的区域（- 79 至 - 72）对于该反应至关重要 Sp 家族转录因子与启动子的该区域结合。
The expression of members of the Reg family of secreted lectin-like proteins is increased in response to stress, inflammation and damage in many tissues. In the stomach, Reg is located in enterochromaffin-like cells, where its expression is stimulated by the gastric hormone gastrin. We have examined the mechanisms by which gastrin stimulates expression of Reg-1. Deletional mutations of 2.1 to 0.1 kb of the rat Reg-1 promoter in a luciferase reporter vector were transiently transfected into gastric cancer AGS-G(R) cells. All promoter fragments tested showed similar relative increases in luciferase expression in response to gastrin (I nM). The response to gastrin of the smallest (104 bp) construct was 4.2 +/- 0.4-fold over basal. These responses were reduced by Ro-32-0432. a protein kinase C inhibitor, by C3-transferase, a Clostridium botulinum toxin and a selective inhibitor of the Rho family GTPase RhoA, and by co-transfection with a dominant negative for-m of RhoA. Co-transfection with a constitutively active form of RhoA stimulated expression 11.6 +/- 1.7-fold over basal. Mutations through the 104 bp construct identified a C-rich element (C-79CCCTCCC-72) required for responses to gastrin, PKC (protein kinase Q and L63RhoA (the constitutively active form of human RhoA protein containing a glutamine-to-leucine substitution at position 63). EMSAs (electrophoretic-mobility-shift assays) using nuclear extracts of control and gastrin-stimulated AGS-GR cells and a probe spanning - 86 to - 64 bp revealed multiple binding proteins. There was no effect of gastrin on the pattern of binding. Supershift assays indicated that transcription factors Sp1 and Sp3 bound the C-rich sequence. We conclude that gastrin stimulates Reg expression via activation of PKC and RhoA, that a C-rich region (- 79 to - 72) is critical for the response and that Sp-family transcription factors bind to this region of the promoter.