Screening of novel signal transducers for BMP using DNA affinity purification

使用 DNA 亲和纯化筛选新型 BMP 信号转导器

• 批准号: 15591611
• 负责人: IMAMURA Takeshi
• 金额: $ 2.24万
• 依托单位: Japanese Foundation for Cancer Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591611/
• 关键词: BMP; Smad; Gcn5; 蛋白質-DNA相互作用; シグナル伝達

Members of the transforming growth factor-β (TGF-β) superfamily, including TGF-β, activin, nodal, and bone morphogenetic proteins (BMPs), are multifunctional cytokines that regulate a wide range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis. TGF-β and related proteins transduce signals through two distinct serine/threonine kinase receptors, termed type I and type II, and intracellular Smad proteins. Eight different Smad proteins have been identified in mammals, and are classified into three groups : receptor-regulated Smads (R-Smads), common-partner Smad (Co-Smad), and inhibitory Smads (I-Smads). Smad2 and Smad3 are R-Smads activated by TGF-β/activin/nodal receptors ALK-4, -5, and -7, whereas Smad1, Smad5, and Smad8 are BMP-specific R-Smads. Smad4 is the Co-Smad shared by signaling pathways for TGF-β and activin and those for BMPs. Smad6 and Smad7 are I-Smads in mammals ; Smad6 preferentially suppresses BMP signaling, whereas S … More mad7 inhibits both BMP and TGF-β signaling.The roles of TGF-β in cancer biology are complex ; TGF-β can suppress or promote tumor growth depending on the type of cancer. The ability of TGF-β to potently inhibit the proliferation of epithelial, endothelial, and hematopoietic cell lineages is central to its tumor-suppressive effects. However, as tumors evolve, they often become refractory to TGF-β-mediated growth inhibition and overexpress TGF-β, which induces epithelial-to-mesenchymal transition (EMT) of tumor cells and facilitates immunosuppression, extracellular matrix deposition, and angiogenesis. It was recently reported that inhibition of autocrine TGF-β signaling in carcinoma cells reduces cell invasiveness and tumor metastasis, and that these effects of TGF-β are closely associated with the ability of TGF-β to induce EMT and stimulate cell migration. The TGF-β signaling pathway has correspondingly become an attractive target for drug development in the field of oncology.To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a co-activator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a co-activator for R-Smads for TGF-β signaling pathways. GCN5 functions like PCAF, in that it binds to TGF-β-specific R-Smads, and enhances transcriptional activity induced by TGF-β. In addition, GCN5, but not PCAF, interacts with R-Smads for BMP signaling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo. Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-β. Less

转化生长因子-β (TGF-β) 超家族的成员，包括 TGF-β、激活素、节点蛋白和骨形态发生蛋白 (BMP)，是调节多种细胞反应的多功能细胞因子，包括细胞增殖、分化、 TGF-β 和相关蛋白通过两种不同的丝氨酸/苏氨酸激酶受体（称为 I 型和 II 型）以及细胞内 Smad 蛋白传导信号。已在哺乳动物中发现，并分为三组：受体调节的 Smads (R-Smads)、共同伴侣 Smads (Co-Smads) 和抑制性 Smads (I-Smads)，它们是由 R-Smads 激活的。 TGF-β/激活素/节点受体 ALK-4、-5 和 -7，而 Smad1、Smad5 和 Smad8 是 BMP 特异性 R-Smad。在哺乳动物中，Smad4 是 TGF-β 和激活素信号通路共享的 Co-Smad，Smad6 和 Smad7 是 I-Smad；Smad6 优先抑制 BMP 信号传导，而 Smad7 则同时抑制 BMP 和 TGF-β 信号传导。 TGF-β 在癌症生物学中的作用很复杂；TGF-β 可以抑制或促进肿瘤生长，具体取决于癌症的类型。上皮细胞、内皮细胞和造血细胞谱系的增殖是其肿瘤抑制作用的核心，然而，随着肿瘤的发展，它们常常对 TGF-β 介导的生长抑制产生耐药性，并过度表达 TGF-β，从而诱导上皮间质细胞的增殖。最近报道，抑制肿瘤细胞自分泌 TGF-β 信号传导。癌细胞减少细胞侵袭性和肿瘤转移，而TGF-β的这些作用与TGF-β诱导EMT和刺激细胞迁移的能力密切相关，TGF-β信号通路相应地成为药物开发的有吸引力的靶点。为了鉴定含有 Smad 蛋白的转录复合物的新成分，我们使用 Smad 结合 DNA 元件作为诱饵，从人乳腺癌 MCF-7 细胞核提取物中纯化了 DNA 结合蛋白，并鉴定了一种共激活剂GCN5 作为激活的 Smad 复合物的直接伴侣，在结构上与 PCAF 相似，PCAF 之前被认为是 GCN5 信号通路的 R-Smad 的共激活剂，其功能与 PCAF 类似。 -特异性 R-Smads，并增强 TGF-β 诱导的转录活性。此外，GCN5（而非 PCAF）与 BMP 信号通路的 R-Smads 相互作用，并增强。 BMP 诱导的转录活性表明 GCN5 和 PCAF 在体内具有不同的生理功能，此外，通过 RNA 干扰沉默 GCN5 基因会导致 TGF-β 诱导的转录活性受到抑制。

项目成果

Roles for the MH2 domain of Smad7 in the specific inhibition of transforming growth factor-β superfamily signaling

• DOI: 10.1074/jbc.m313977200
• 发表时间: 2004-07-23
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Mochizuki, T;Miyazaki, H;Miyazono, K
• 通讯作者: Miyazono, K

Kahata K, Imamura T (外7名): "Regulation of transforming growth factor-b and bone morphogenetic protein signaling by transcriptional co-activator GCN5"Genes Cells. 9・2. 143-151 (2004)

Kahata K、Imamura T（其他 7 人）：“转录共激活因子 GCN5 对转化生长因子-b 和骨形态发生蛋白信号的调节”Genes Cells 9·2。

Miyazono K, Suzuki H, Imamura T: "Regulation of TGF-b signaling and its roles in progression of tumors."Cancer Sci. 94・3. 230-234 (2003)

Miyazono K、Suzuki H、Imamura T：“TGF-b 信号传导及其在肿瘤进展中的作用”。Cancer Sci. 94・3 (2003)。

SB-431542 and Gleevec inhibit TGF-β induced growth stimulation of human osteosarcoma cells.

SB-431542 和 Gleevec 抑制 TGF-β 诱导的人骨肉瘤细胞的生长刺激。

• 发表时间: 2003
• 期刊: Cancer Res. 63・22
• 作者: Matsuyama S;Imamura T (外9名)
• 通讯作者: Imamura T (外9名)

Regulation of transforming growth factor-β and bone morphogenetic protein signaling by transcriptional co-activator GCN5.

转录共激活因子 GCN5 对转化生长因子-β 和骨形态发生蛋白信号的调节。

• 发表时间: 2004
• 期刊: Genes Cells. 9
• 作者: Kahata K;Hayashi M;Asaka M;Hellman U;Kitagawa H;Yanagisawa J;Kato S;Imamura T;Miyazono M
• 通讯作者: Miyazono M