RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis

WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupts myogenic differentiation and significantly diminishes the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the transforming growth factor β (TGF-β) antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular cross talk between WNT/β-catenin and TGF-β signaling pivotal in myogenic differentiation.     

RSPO2 enhances cell invasion and migration via the WNT/β-catenin pathway in human gastric cancer

R-spondins comprise a group of secreted WNT agonists. R-spondin2 (RSPO2) plays a crucial role in the activation of the WNT/β-catenin pathway and oncogenesis, though its specific role in human gastric cancer (GC) remains unclear. In the current study, RSPO2 expression levels were upregulated in cancer specimens and cell lines (AGS and BGC-823). Inhibition of RSPO2 expression levels had distinct effects on cell invasion, migration, and epithelial-mesenchymal transition (EMT) in AGS and BGC-823 cells in vitro. Furthermore, RSPO2 positively correlated with leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), the receptor of RSPO2. Silencing RSPO2 reduced the expression of LGR5 and WNT/β-catenin effector molecule β-catenin together with downstream targets TCF-4 and Cyclin-D1. These observations demonstrate that upregulation of RSPO2 in GC specimens and cell lines is closely related to tumor invasion and migration and that RSPO2 promotes EMT in gastric cancer cells by activating WNT/β-catenin signaling.     

The R-spondin family of proteins: Emerging regulators of WNT signaling

Recently, the R-spondin (RSPO) family of proteins has emerged as important regulators of WNT signaling. Considering the wide spectrum of WNT signaling functions in normal biological processes and disease conditions, there has been a significantly growing interest in understanding the functional roles of RSPOs in multiple biological processes and determining the molecular mechanisms by which RSPOs regulate the WNT signaling pathway. Recent advances in the RSPO research field revealed some of the in vivo functions of RSPOs and provided new information regarding the mechanistic roles of RSPO activity in regulation of WNT signaling. Herein, we review recent progress in RSPO research with an emphasis on signaling mechanisms and biological functions.     

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...     

Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6-9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination.     

Cellular signaling and biological functions of R-spondins

R-spondins (RSPOs) are a family of cysteine-rich secreted proteins containing a single thrombospondin type I repeat (TSR) domain. A vast amount of information regarding cellular signaling and biological functions of RSPOs has emerged over the last several years, especially with respect to their roles in the activation of the WNT signaling pathway. The identification of several classes of RSPO receptors may indicate that this family of proteins can affect several signaling cascades. Herein, we summarize the current understanding of RSPO signaling and its biological functions, and discuss its potential therapeutic implications to human diseases.     

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。     

Wnt/β-catenin-mediated heat exposure inhibits intestinal epithelial cell proliferation and stem cell expansion through endoplasmic reticulum stress

Heat stress induced by continuous high ambient temperatures or strenuous exercise in humans and animals leads to intestinal epithelial damage through the induction of intracellular stress response. However, the precise mechanisms involved in the regulation of intestinal epithelial cell injury, especially intestinal stem cells (ISCs), remain unclear. Thereby, in vitro a confluent monolayer of IPEC-J2 cells was exposed to the high temperatures (39, 40, and 41°C), the IPEC-J2 cell proliferation, apoptosis, differentiation, and barrier were determined, as well as the expression of GRP78, which is a marker protein of endoplasmic reticulum stress (ERS). The Wnt/β-catenin pathway-mediated regenerative response was validated using R-spondin 1 (Rspo1). And ex-vivo, three-dimensional cultured enteroids were developed from piglet jejunal crypt and employed to assess the ISC activity under heat exposure. The results showed that exposure to 41°C for 72 hr, rather than 39°C and 40°C, decreased IPEC-J2 cell viability, inhibited cell proliferation and differentiation, induced ERS and cell apoptosis, damaged barrier function and restricted the Wnt/β-catenin pathway. Nevertheless, Wnt/β-catenin reactivation via Rspo1 protects the intestinal epithelium from heat exposure-induced injury. Furthermore, exposure to 41°C for 24 hr reduced ISC activity, stimulated crypt-cell apoptosis, upregulated the expression of GRP78 and caspase-3, and downregulated the expression of β-catenin, Lgr5, Bmi1, Ki67, KRT20, ZO-1, occludin, and claudin-1. Taken together, we conclude that heat exposure induces ERS and downregulates the Wnt/β-catenin signaling pathway to disrupt epithelial integrity by inhibiting the intestinal epithelial cell proliferation and stem cell expansion.     

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ(1)

Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.     

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.     

Craniofacial malformation in R-spondin2 knockout mice

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/β-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/β-catenin signaling, Rspo2, we generated and characterized Rspo2^−/− mice. We found CLP with mild facial skeletal defects in Rspo2^−/− mice. Additionally, Rspo2^−/− mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/β-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2^−/− mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/β-catenin signaling.     

A cyclic AMP-dependent pathway regulates the expression of acetylcholinesterase during myogenic differentiation of C2C12 cells

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.