Negative Feedback Regulation of Wnt4 Signaling by EAF1 and EAF2/U19

Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.     

The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop

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Axin在Wnt信号转导途径中的作用

Wnt信号转导途径是调控细胞形状、运动、黏附、增殖、分化、癌变及机体发育等过程的主要途径之一.Axin(轴蛋白)是一个体轴发育抑制因子,作为构架蛋白在Wnt信号转导途径中起着关键的作用.Axin通过不同的机制调节β连环蛋白的磷酸化和稳定性.它通过与APC、GSK-3β、β连环蛋白和CKIα结合形成复合体促进β连环蛋白的降解,还通过同源二聚化、核质穿梭、自身磷酸化和稳定性的调控来调节β连环蛋白的稳定性.Axin通过Wnt信号转导途径参与了一系列生物学效应的调控,如体轴发育、细胞死亡、神经元的分化等.作为一个新发现的肿瘤抑制因子,axin将为癌症的诊断和治疗提供新的有效的手段.     

HMGA2通过激活Wnt信号通路促进结直肠癌细胞增殖

目的:HMGA2(high-mobility group AT-hook 2)一个染色质蛋白,被报道在多种癌症中都发挥重要作用。本文研究染色体蛋白HMGA2对Wnt信号传递的影响,及对结直肠癌细胞增殖的影响。方法:本文通过q RT-PCR和免疫印迹法检测HMGA2在结直肠癌样本中m RNA和蛋白水平。用荧光素酶报告基因系统研究HMGA2对Wnt信号通路的作用。用细胞增殖实验检测HMGA2对结直肠癌细胞增殖的作用。结果:在我们检测的大部分结直肠癌样本里,HMGA2表达水平升高;HMGA2蛋白可上调Wnt信号通路荧光素酶报告基因TOPflash-luciferase的表达,并呈现剂量依赖的形式。降低HMGA2表达可抑制由Wnt3a、Dvl(Dishevelled)、Li Cl以及βcatenin(S37A)引起的TOPflash-luciferase报告基因表达上调作用。此外,SW480中过量表达HMGA2可以促进细胞增殖。结论:HMGA2在结直肠癌中表达升高,HMGA2在结直肠癌中通过增强Wnt信号来促进结直肠癌细胞的增殖。     

The Self-Limiting Dynamics of TGF-β Signaling In Silico and In Vitro,with Negative Feedback through PPM1A Upregulation

The TGF-β/Smad signaling system decreases its activity through strong negative regulation. Several molecular mechanisms of negative regulation have been published, but the relative impact of each mechanism on the overall system is unknown. In this work, we used computational and experimental methods to assess multiple negative regulatory effects on Smad signaling in HaCaT cells. Previously reported negative regulatory effects were classified by time-scale: degradation of phosphorylated R-Smad and I-Smad-induced receptor degradation were slow-mode effects, and dephosphorylation of R-Smad was a fast-mode effect. We modeled combinations of these effects, but found no combination capable of explaining the observed dynamics of TGF-β/Smad signaling. We then proposed a negative feedback loop with upregulation of the phosphatase PPM1A. The resulting model was able to explain the dynamics of Smad signaling, under both short and long exposures to TGF-β. Consistent with this model, immuno-blots showed PPM1A levels to be significantly increased within 30 min after TGF-β stimulation. Lastly, our model was able to resolve an apparent contradiction in the published literature, concerning the dynamics of phosphorylated R-Smad degradation. We conclude that the dynamics of Smad negative regulation cannot be explained by the negative regulatory effects that had previously been modeled, and we provide evidence for a new negative feedback loop through PPM1A upregulation. This work shows that tight coupling of computational and experiments approaches can yield improved understanding of complex pathways.     

Wnt/Dkk Negative Feedback Regulates Sensory Organ Size in Zebrafish

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The Protein Stability of Axin,a Negative Regulator of Wnt Signaling,Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys⁵⁰⁵ of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.     

经典Wnt信号途径与结直肠肿瘤发生

癌发生的主要原因是控制细胞生长和存活的信号转导途径被异常激活,而这些信号途径在正常胚胎发育中起重要作用.经典的Wnt信号转导途径是其中最重要的通路之一,目前认为其成分主要包括：细胞外因子（Wnt）、跨膜受体（卷曲蛋白Frizzled,Fz）、辅助受体（低密度脂蛋白受体相关蛋白-5和-6,LRP5/6）、β-链蛋白（β-catenin,β-cat）、结肠腺瘤样息肉病（adenomatous polyposis coli,APC）蛋白、T细胞因子（TCF）等一系列蛋白质.多数结直肠癌患者的转化细胞存在APC基因缺失、突变或失活,导致β-cat在核内的累积,从而影响相关基因转录,这是结直肠癌发生的关键因素之一.对Wnt途径的进一步研究将为探索结直肠癌的发病机制以及寻找有效的诊治手段提供新视觉.     

Regulation of Glycogen Synthase Kinase 3β and Downstream Wnt Signaling by Axin

Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3beta (GSK-3beta) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3beta in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3beta in vivo, suggesting that these domains are critical for proper regulation of GSK-3beta activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3beta activity. Based on these data, we propose that the axin complex may directly regulate GSK-3beta enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3beta can mimic the effect of lithium in developing Xenopus embryos.     

Negative Feedback Regulation of Auxin Signaling by ATHB8/ACL5-BUD2 Transcription Module

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the （pro）cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 （ACL5） and BUSHY AND DWARF2 （BUD2） is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper （HD- ZIP） III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein ARABIDOPSIS THALIANA HOMEOBOX8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as LIKE AUXIN RESISTANT2 （LAX2） and LAX3. We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD-ZIP III genes.     

Differential Role of Axin RGS Domain Function in Wnt Signaling during Anteroposterior Patterning and Maternal Axis Formation

Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.     

Wnt基因/Wnt信号通路与乳腺癌

Wnt蛋白是一组调控胚胎形成期间细胞间信号传导的高度保守的分泌信号分子.在过去的几年里,由Wnt蛋白触发的不同信号通路已经得到了详尽的研究.Wnt基因与Wnt信号通路组成分子的突变可引起发育缺陷,异常的Wnt信号传导可导致人类疾病包括肿瘤的发生.许多证据都表明,Wnt信号通路的失调与乳腺癌的发生发展密切相关.micro...