Bishnupuri Kumar S, Luo Qizhi, Murmu Nabendu, Houchen Courtney W, Anant Shrikant, Dieckgraefe Brian K
Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Gastroenterology. 2006 Jan;130(1):137-49. doi: 10.1053/j.gastro.2005.10.001.
BACKGROUND & AIMS: Reg IV, a secreted protein and member of the Reg multigene family, is up-regulated in malignancies of the human gastrointestinal tract, including colorectal carcinoma (CRC). However, in vitro signal transduction pathway(s) utilized by Reg IV are not yet known.
To determine the signaling pathway(s) responsive to Reg IV, we examined the effects of purified recombinant human Reg IV (rhR4) on HCT116 and HT29 colon adenocarcinoma cells.
Addition of rhR4 to cultures led to a dose-dependent increase in cell number similar to that observed after treatment with epidermal growth factor (EGF). In addition, rhR4 treatment resulted in rapid phosphorylation of EGF receptor at Tyr992 and Tyr1068 and Akt at Thr308 and Ser473. Using luciferase reporter gene assays, we demonstrated that Reg IV signaling through EGF receptor and Akt results in increased activator protein-1 (AP-1) transcription factor activity. Real-time reverse-transcription polymerase chain reaction and Western blot analyses revealed quantitative increases in c-Jun, JunB, JunD, and FosB expression associated with increased AP-1 activity. Electrophoretic mobility shift assay further revealed significant increases in AP-1 binding activity in rhR4-treated cells, with increased supershift in the presence of antibodies to JunB, JunD, and FosB. Furthermore, rhR4 treatments led to the increased expression of Bcl-2, Bcl-XL, survivin, and matrilysin, genes associated with a poor prognosis in advanced CRC.
Reg IV is a potent activator of the EGF receptor/Akt/AP-1 signaling pathway in CRC. Disruption of Reg signaling may have utility as a therapeutic intervention for human gastrointestinal adenocarcinomas.
Reg IV是一种分泌蛋白，属于Reg多基因家族成员，在包括结直肠癌（CRC）在内的人类胃肠道恶性肿瘤中表达上调。然而，Reg IV在体外利用的信号转导途径尚不清楚。
为了确定对Reg IV有反应的信号通路，我们检测了纯化的重组人Reg IV（rhR4）对HCT116和HT29结肠腺癌细胞的影响。
向培养物中添加rhR4导致细胞数量呈剂量依赖性增加，类似于用表皮生长因子（EGF）处理后观察到的情况。此外，rhR4处理导致EGF受体在Tyr992和Tyr1068以及Akt在Thr308和Ser473处快速磷酸化。使用荧光素酶报告基因测定法，我们证明通过EGF受体和Akt的Reg IV信号传导导致激活蛋白-1（AP-1）转录因子活性增加。实时逆转录聚合酶链反应和蛋白质印迹分析显示，与AP-1活性增加相关的c-Jun、JunB、JunD和FosB表达定量增加。电泳迁移率变动分析进一步显示，rhR4处理的细胞中AP-1结合活性显著增加，在存在针对JunB、JunD和FosB的抗体时超迁移增加。此外，rhR4处理导致Bcl-2、Bcl-XL、survivin和基质溶素的表达增加，这些基因与晚期CRC的不良预后相关。
Reg IV是CRC中EGF受体/Akt/AP-1信号通路的有效激活剂。破坏Reg信号传导可能作为人类胃肠道腺癌的一种治疗干预手段。
