Screening of novel signal transducers for BMP using DNA affinity purification

转化生长因子-β (TGF-β)超家族的成员，包括TGF-β、激活素、结蛋白和骨形态发生蛋白（BMPs），是一种多功能细胞因子，可调节多种细胞反应，包括细胞增殖、分化、粘附、迁移和凋亡。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）和细胞内Smad蛋白转导信号。在哺乳动物中已鉴定出8种不同的Smad蛋白，并将其分为三类：受体调节的Smad （r- Smad）、共同伴侣Smad （Co-Smad）和抑制性Smad （I-Smads）。Smad2和Smad3是由TGF-β/激活素/节点受体ALK-4、-5和-7激活的R-Smads，而Smad1、Smad5和Smad8是bmp特异性的R-Smads。Smad4是TGF-β和激活素信号通路与bmp信号通路共享的Co-Smad。哺乳动物中Smad6和Smad7是i - smad；Smad6优先抑制BMP信号，而S…More mad7同时抑制BMP和TGF-β信号。TGF-β在癌症生物学中的作用是复杂的；TGF-β可以抑制或促进肿瘤的生长，这取决于癌症的类型。TGF-β有效抑制上皮细胞、内皮细胞和造血细胞系增殖的能力是其肿瘤抑制作用的核心。然而，随着肿瘤的发展，它们往往对TGF-β介导的生长抑制变得难以抑制，并过度表达TGF-β，从而诱导肿瘤细胞的上皮-间质转化（epithelial-to-mesenchymal transition， EMT），促进免疫抑制、细胞外基质沉积和血管生成。最近有报道称，抑制癌细胞中自分泌TGF-β信号可降低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肿瘤领域药物开发的一个有吸引力的靶点。为了鉴定含有Smad蛋白的转录复合物的新组分，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，并鉴定了作为活化Smad复合物的直接伴侣的共激活子GCN5。GCN5在结构上与PCAF相似，PCAF先前被确定为TGF-β信号通路R-Smads的共激活因子。GCN5的功能类似于PCAF，与TGF-β特异性R-Smads结合，增强TGF-β诱导的转录活性。此外，GCN5而非PCAF与R-Smads相互作用，参与BMP信号通路，并增强BMP诱导的转录活性，这表明GCN5和PCAF在体内具有不同的生理功能。此外，通过RNA干扰使GCN5基因沉默可抑制TGF-β诱导的转录活性。少

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