Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
Chin Med J (Engl). 2012 Nov;125(21):3851-5.
Hedgehog (Hh) signaling plays an important role in both embryonic development and postnatal tissue homeostasis. Aberrant Hh activation results in a large variety of cancers. This study was designed to discover novel modulators in Hh signaling pathway.
We performed yeast-two-hybrid screening and immunoprecipitation to identify the interaction of Nedd4 and Smo. To verify whether Nedd4 is involved in the regulation of Hh signaling, we monitored the activation of Gli-luciferase reporter by overexpressing Nedd4 together with Gli-luciferase reporter. In order to examine the role of endogenous Nedd4 in regulating Hh signaling, we used a short hairpin RNA (shRNA) interference strategy to silence the Nedd4 expression, and then perform dual-luciferase reporter assay. Statistical comparisons were performed by Student's t tests.
We showed that Nedd4 binds to Smo in the transfected HEK293 cells. Overexpression of Nedd4 alone did not significantly activate the Gli reporter compared to pcDNA3 control (Nedd4 group: dimethyl sulfoxide (DMSO), relative luciferase unit (RLU) 1.87 ± 0.41). However, Smo agonist (SAG)-stimulated activation of Gli-luciferase reporter was markedly potentiated in Nedd4 transfected cells (Nedd4 group: SAG, RLU 13.49 ± 1.04, P < 0.05), indicating that overexpression of Nedd4 increases Gli luciferase reporter activity and Nedd4-induced activation of Hh signaling is activity dependent. In Nedd4 knockdown NIH 3T3 cells, the luciferase reporter activity was measured basally and after SAG treatment. In scrambled cells, compared to DMSO, SAG could activate reporter activity by (4.16 ± 0.84)-fold. In Nedd4 knockdown cells, the luciferase reporter activation by SAG was significantly inhibited (SAG, RLU 1.72 ± 0.24, P < 0.05); knockdown of Nedd4 did not change the basal activity of luciferase activity (DMSO, RLU 0.86 ± 0.11), suggesting that the loss of Nedd4 expression diminishes Gli-dependent activity in the Hh pathway and the regulation of Nedd4 in the Hh signaling pathway is activity-dependent.
Nedd4 positively regulates the Hh pathway and provides a potential target for inhibiting Hh signaling in cancer therapy.
Hedgehog（Hh）信号通路在胚胎发育和出生后组织稳态中都发挥着重要作用。异常的 Hh 激活会导致多种癌症。本研究旨在发现 Hh 信号通路中的新型调节剂。
我们进行了酵母双杂交筛选和免疫沉淀，以鉴定 Nedd4 和 Smo 的相互作用。为了验证 Nedd4 是否参与 Hh 信号的调节，我们通过共转染 Nedd4 和 Gli-luciferase 报告基因来监测 Gli-luciferase 报告基因的激活。为了研究内源性 Nedd4 在调节 Hh 信号中的作用，我们使用短发夹 RNA（shRNA）干扰策略沉默 Nedd4 的表达，然后进行双荧光素酶报告基因检测。统计比较采用 Student's t 检验。
我们表明，Nedd4 在转染的 HEK293 细胞中与 Smo 结合。与 pcDNA3 对照相比，单独过表达 Nedd4 对 Gli 报告基因的激活作用并不明显（Nedd4 组：二甲基亚砜（DMSO），相对荧光素酶单位（RLU）为 1.87±0.41）。然而，Smo 激动剂（SAG）刺激的 Gli-luciferase 报告基因的激活在 Nedd4 转染的细胞中明显增强（Nedd4 组：SAG，RLU 为 13.49±1.04，P<0.05），表明过表达 Nedd4 增加了 Gli 荧光素酶报告基因的活性，并且 Nedd4 诱导的 Hh 信号激活是活性依赖性的。在 Nedd4 敲低的 NIH 3T3 细胞中，在基础状态和 SAG 处理后测量荧光素酶报告基因的活性。在对照细胞中，与 DMSO 相比，SAG 可使报告基因活性激活（4.16±0.84）倍。在 Nedd4 敲低的细胞中，SAG 对荧光素酶报告基因的激活明显受到抑制（SAG，RLU 为 1.72±0.24，P<0.05）；Nedd4 敲低不改变荧光素酶活性的基础活性（DMSO，RLU 为 0.86±0.11），表明 Nedd4 表达的缺失降低了 Hh 通路中的 Gli 依赖性活性，并且 Nedd4 在 Hh 信号通路中的调节是活性依赖性的。
Nedd4 正向调节 Hh 通路，为癌症治疗中抑制 Hh 信号提供了一个潜在的靶点。
