R-spondin：一个新的Wnt信号通路相关蛋白家族的研究进展

R-spondin（Rspo）是近年来新发现的蛋白家族,包括4个成员（Rspo1～4）。已报道Rspo蛋白家族所有成员均为分泌性蛋白,均有两个富含半胱氨酸的furin-like结构域、1个TSP1结构域和富含碱性氨基酸的C端区域。Rspos通过激活并协同Wnt/β-catenin信号通路参与对细胞增殖和分化的调控,影响骨骼、肌肉、血管等组织的发育以及肢体和性腺的形成,并在多种疾病的发生过程中起重要作用。该文结合最新研究进展,就Rspo家族蛋白的结构、主要功能及其对经典Wnt信号通路的调控机理做一综述。     

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function

R-spondins (Rspos) potentiate Wnt/β-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein, demonstrating for the first time specific amino acid residues in Rspos that are critical for β-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide-bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a β-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.     

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.     

Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb

The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate β-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(−/−) mice. Consistent with the ligand role of R-spondins in the Wnt/β-catenin signaling pathway, expression of Axin2 and Sp8, targets for β-catenin signaling, within AER was greatly reduced in Rspo2(−/−) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(−/−) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/β-catenin signaling.     

Rspo1调控斑马鱼胚胎汇聚延伸运动的研究

Rspo1 (R-spondin 1)是分泌型Rspos (R-spondins)蛋白家族的成员,在雌性发育、血管生成和癌症等多个方面具有调控作用。为了研究Rspo1在早期胚胎发育中的功能,以斑马鱼(Danio rerio)作为模式生物,利用反转录PCR及原位杂交技术检测rspo1基因的时空表达模式;通过显微注射rspo1 mRNA或rspo1反义寡核苷酸(Morpholino, MO)对rspo1进行过表达或敲降;通过形态观察及原位杂交技术检测胚胎汇聚延伸(Convergence and extension, CE)运动是否正常;利用荧光素酶活性检测实验测定Wnt/PCP信号通路活性水平;通过蛋白印迹法检测表征Wnt/PCP信号通路活性的磷酸化JNK (Jun N-terminal kinase)蛋白的水平。结果显示:rspo1为母源基因,在12hpf前胚胎中呈全身性表达, rspo1的过表达或敲降均影响胚胎的CE运动;过表达rspo1降低Wnt/PCP信号通路报告质粒的活性,而敲降rspo1则增加其活性,与之相一致, rspo1敲降的胚胎中磷酸化JNK的水平显著升高;此外, rsp...     

Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

Ablation of PKM2 ameliorated ER stress-induced apoptosis and associated inflammation response in IL-1β-treated chondrocytes via blocking Rspo2-mediated Wnt/β-catenin signaling

Endoplasmic reticulum (ER) stress and the related apoptosis and inflammation damage play key roles in osteoarthritis development. The aim of the present work was to investigate the exact role and potential underlying mechanism of pyruvate kinase M2 (PKM2) in rat chondrocytes exposed to interleukin-Iβ (IL-1β). We observed that IL-1β stimulation resulted in an apparent enhancement in PKM2 expression. Additionally, loss of PKM2 evidently ascended cell viability in response to IL-1β exposure. Simultaneously, elimination of PKM2 manifestly repressed IL-1β-stimulated chondrocyte apoptosis, concomitant with attenuated in the proapoptotic protein markers Bax and cleaved caspase-3, and elevated the antiapoptotic protein Bcl-2. In the meanwhile, knockdown of PKM2 ameliorated ER stress in IL-1β-treated chondrocytes, as evidenced by reduced expression of the ER stress-associated proteins GRP78, CHOP, and cleaved caspase-12. Furthermore, PKM2 silencing protected chondrocytes against IL-1β-triggered inflammatory response, as reflected by the downregulated release of proinflammatory mediators, including tumor necrosis factor-α, IL-6, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2, as well as decreased nitric oxide generation. More important, abrogating PKM2 expression caused a marked decline in Rspo2 expression, and subsequently blocked Wnt/β-catenin signaling. Mechanistically, the Wnt/β-catenin signaling activator Licl effectively impeded the beneficial effects of PKM2 ablation on IL-1β-stimulated apoptosis and inflammatory response. These findings collectively implicated that PKM2 inhibition protected against ER stress-mediated cell apoptosis and inflammatory injury in rat chondrocytes stimulated with IL-1β by inactivating Rspo2-mediated Wnt/β-catenin pathway, and may represented a novel therapeutic target for osteoarthritis.     

Structure-based discovery of a novel inhibitor targeting the β-catenin/Tcf4 interaction

Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.     

Activation of Wnt/β-catenin protein signaling induces mitochondria-mediated apoptosis in hematopoietic progenitor cells

The canonical Wnt/β-catenin signaling is activated during development, tumorigenesis, and in adult homeostasis, yet its role in maintenance of hematopoietic stem/progenitor cells is not firmly established. Here, we demonstrate that conditional expression of an active form of β-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/β-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨ(m)), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated β-catenin in HPCs is associated with down-regulation of Bcl2 and expression of Casp3. Bone marrow transplantation assays reveal that enhanced cell survival by a Bcl2 transgene re-establishes the reconstitution capacity of HSCs/HPCs that express activated β-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/β-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.     

Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin

Background

Canonical Wnt signals, transduced by stabilized β-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/β-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here.

Results

Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3β but upstream of β-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b.

Conclusions

We conclude that Gtpbp2 is required for canonical Wnt/β-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the β-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant β-catenin signaling in cancer and other Wnt-related diseases.