Prostaglandin E2 modifies SMAD2 and promotes SMAD2–SMAD4 complex formation

We report that PGE₂ promotes Smad2–Smad4 complex formation and this phenomenon could be blocked by DIDS, an anion transporter inhibitor. Our data suggest that PGE₂ had no effects on Smad2 phosphorylation, suggesting that PGE₂-mediated Smad2–Smad4 complex formation is independent of TGF-β signaling and that PGE₂ induced Smad2 modification which is different from TGF-β-mediated phosphorylation. We demonstrate that in primary human glomerular mesangial cells PGE₂ caused modification of Smad2 as detected by Smad2N antibody, raised against a peptide near the N-terminus of Smad2. We hypothesize that Smad2 protein is post-translationaly modified by PGE₂. Direct evidence of Smad2 modification by PGE₂ was achieved by avidin pulldown assay which showed that endogenous Smad2 and recombinant Smad2 protein were attached by biotin-labeled PGE₂. Taken together, our results provided evidence that post-translational modification of Smad2 could be a mechanism for the action of PGE₂ in the pathogenesis of human pathologies.     

用酵母双杂交系统研究SMAD3和SMAD4的相互作用

利用酵母双杂交试验,鉴定了细胞内信号传导蛋白SMAD3和SMAD4的相互作用。通过SMAD3和SMAD4各突变体的同源和异源相互作用的双杂交反应,确定SMAD4介导信号传递的功能区在中间连接区,SMAD3的功能区在C末端。     

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

SMAD介导的TGF—β信号转导与大肠癌

转化生长因子β（TGF－β）家族成员与各自的膜受体（Ⅰ型及Ⅱ型受体）结合后,通路限制性SMADs被磷酸化,并与共介导Smad4形成杂聚体而转位至细胞核,从而调节一些靶基因的转录而对TGF－β产生反应,抑制性SMADs对此过程有负性调节作用,由SMADs介导的TGF－β信号转导通路的异常在大肠癌的发生发展中发挥重要作用,主要包括膜受体,通路限制性SMADs和共介导Smad4的改变,而抑制SMADs的改变甚为少见。     

SMAD3与细胞周期蛋折CyclinB的相互作用

ＳＡＭＤｓ是ＴＧＦβ家族的细胞内信号介导。为了研究ＳＭＡＤ３的信号传递过程,我们以ＳＡＭＤ３为诱饵蛋白用酵母双杂交系统筛选与ＳＡＭＤ３相互作用的蛋白,发现Ｃｙｃｌｉｎ Ｂ可以与ＳＭＡＤ３发生相互作用,并在ＣＯＳ７细胞中证实了该相互作用。提示ＴＧＦβ可能通过ＳＭＡＤｓ与细胞周期素的相互作用来调节细胞周期的变化。     

SMAD3抑制小鼠胚胎成纤维细胞表达MMP-2

SMAD3是TGF-β信号转导通路中重要的受体激活型SMADs之一。Smad3基因缺失可以引起小鼠创伤愈合速度加快。检测Smad3不同基因型小鼠皮肤创伤局部MMP-2时,发现Smad3缺失小鼠创面MMP-2出现的时间早于野生型和杂合性小鼠。Smad3突变小鼠血清中MMP-2的活性亦显著高于野生型和杂合性小鼠。分离不同Smad3基因型小鼠胚胎成纤维细胞并检测MMP-2的表达,结果显示：Smad3基因缺失小鼠成纤维细胞中MMP-2的表达与活性显著高于野生型细胞;TGF-β1可以提高野生型成纤维细胞MMP-2的活性;Smad3基因缺失细胞暂时恢复SMAD3表达后MMP-2活性下降,阻断野生型细胞表达SMAD3导致MMP-2活性上升。结果表明,SMAD3抑制MMP-2在小鼠胚胎成纤维细胞的表达。     

SMAD蛋白家族:一类新的细胞内信号传导蛋白

SMADs是新近发观的一族细胞内信号传导蛋白,包括8个成员,即SMAD1～8。SMAD1、2、3、5和8是一类,它们被TGF-β受体或BMP受体激活而磷酸化,称为受体调节SMAD,传导TGF-β或BMP的信号。SMAD6和7是另一类,它们抑制受体调节SMAD传导信号。SMAD4是第2类,它是受体调节SMAD传导信号的伴侣。受体调节SMAD传导信号必须先与SMAD4结合形成异源复合物,才能进到核中,调节转录活动。本文简要介绍了各成员的特性及作用。     

SMAD4在嗜甲醇酵母中的表达及鉴定

Ｓｍａｄ４是新近发现的一种肿瘤抑制基因。在５０％的胰腺癌和３０％的结肠癌中,此基因发生突变或缺失。利用Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ酵母表达系统,以胞外分泌的方法表达了ＳＭＡＤ４蛋白,并对ＳＭＡＤ４蛋白的生化性质进行了分析。结果显示表达蛋白的分子量约６７ｋＤ,经Ｗｅｓｔｅｒｎ印迹鉴定,与ＳＭＡＤ４抗全有特异的结合反应,Ｎ末端１３个氨基酸的序列与Ｓｍａｄ４ ｃＤＮＡ推断的序列完全一致。     

SMAD4错义突变与先天性心脏病易感性的相关性研究

SMAD4在心脏发育过程中发挥重要作用,已有研究显示,Smad4 缺陷小鼠心脏功能异常.为了进一步探究SMAD4基因错义突变与先天性心脏病(congenital heart disease,CHD)发生的相关性,本研究选取了来自中国山东汉族的417例CHD患者样本和213例健康对照样本,并靶向SMAD4的编码区进行深度测序.我们在SMAD4的编码区检测到两个错义突变c.685C＞A(p.L229M)和c.740G＞A(p.G247E),并经Sanger测序验证两者均为杂合突变,SMAD4L229M和SMAD4G247E在不同物种间的保守性都很高.蛋白免疫印迹实验显示,两种突变型对SMAD4的蛋白表达量没有明显影响,双荧光素酶报告基因实验结果表明,与野生型SMAD4相比较,SMAD4L229M和SMAD4G247E对 TGF-β信号通路的激活作用均有所减弱.我们的研究表明,SMAD4的病例特有的错义突变可能通过减弱TGF-β信号通路的活性从而导致先天性心脏病的发生.     

SMAD3 Negatively Regulates Serum Irisin and Skeletal Muscle FNDC5 and Peroxisome Proliferator-activated Receptor γ Coactivator 1-α (PGC-1α) during Exercise

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-β effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3^−/−) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-β, a TGF-β receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3^−/− mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-β suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle.     

The regulation of TGF-β/SMAD signaling by protein deubiquitination

Transforming growth factor-β (TGF-β) members are key cytokines that control embryogenesis and tissue homeostasis via transmembrane TGF-β type II (TβR II) and type I (TβRI) and serine/threonine kinases receptors. Aberrant activation of TGF-β signaling leads to diseases, including cancer. In advanced cancer, the TGF-β/SMAD pathway can act as an oncogenic factor driving tumor cell invasion and metastasis, and thus is considered to be a therapeutic target. The activity of TGF-β/SMAD pathway is known to be regulated by ubiquitination at multiple levels. As ubiquitination is reversible, emerging studies have uncovered key roles for ubiquitin-removals on TGF-β signaling components by deubiquitinating enzymes (DUBs). In this paper, we summarize the latest findings on the DUBs that control the activity of the TGF-β signaling pathway. The regulatory roles of these DUBs as a driving force for cancer progression as well as their underlying working mechanisms are also discussed.     

IFN-γ/STAT通路抑制TGF-β/SMAD信号转导

ＴＧＦ－β和ＩＦＮ－γ对各种细胞功能有相反的作用,但是此拮抗作用的基础不清楚。ＴＧＦ－β信号转导是通过受体丝氨酸激酶磷酸化并激活转录因子Ｓｍａｄ２和Ｓｍａｄ３实现的,而ＩＦＮ－γ受体及其结合的蛋白质酪氨酸激酶Ｊａｋ１能介导Ｓｔａｔ１的磷酸化和活化。最...     

TGF-β家族信号的细胞内传导蛋白:SMAD蛋白家族

SMADs是新近发现的参与TGFβ超家族的信号在细胞内传导的一族蛋白,包括8个成员,分别称SMAD1～8。SMAD1、2、3、5和8属于一类,它们被TGFβ的受体或BMP的受体激活而磷酸化,称为受体调节SMAD,传导TGFβ或BMP的信号。SMAD6和7属于另一类,它们抑制受体调节SMAD的信号传导。SMAD4是第三类,它是受体调节SMAD传导信号的伴侣。受体调节SMAD传导信号必须先与SMAD4结合形成异源复合物,才能进到核中,调节转录活动 。