Article

Kim, K.A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256-1259

Nuvelo, Inc., 675 Almanor Avenue, Sunnyvale, CA 94085, USA.
Science (Impact Factor: 33.61). 09/2005; 309(5738):1256-9. DOI: 10.1126/science.1112521
Source: PubMed

ABSTRACT

Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic
mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin
domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection
into mice, the protein induced rapid onset of crypt cell proliferation involving β-catenin stabilization, possibly by a process
that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced
intestinal mucositis and may have therapeutic application in gastrointestinal diseases.

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Available from: Y. Tom Tang, Aug 20, 2014
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    • "R-spondins (Rspos), identified in 2004 (Kamata et al., 2004), enhance Wnt signaling with Wnts synergistically (Kazanskaya et al., 2004; Kim et al., 2008; Schuijers and Clevers, 2012). Rspos are growth factors for intestinal crypt stem cells in vivo (Kim, Tomizuka, et al., 2005), and a loss-of-function mutation of Rspo1 develops into a recessive syndrome that is characterized by XX sex reversal (Parma, Radi, et al., 2006). Further, aberrant expression of Rspos promotes tumor malignancy (Carmon et al., 2014; Shinmura et al., 2014; Gong et al., 2015; Ilmer et al., 2015). "
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    ABSTRACT: R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein-protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate that Rspo1 is C-mannosylated at W(153) and W(156) by mass spectrometry. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells, which replaced W(153) and W(156) by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 was regulated by C-mannosylation. Recently, DPY19 was reported to be a C-mannosyltransferase in C. elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W(156) but not W(153) by mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we have identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W(156) and found that DPY19L3-mediated C-mannosylation of Rspo1 at W(156) is required for its secretion.
    Preview · Article · Jan 2016 · Molecular biology of the cell
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    • "R-spondins are important stem cell growth factors with reported roles in tissue regeneration, but also cancer progression (Kim et al., 2005; Zhao et al., 2009; Seshagiri et al., 2012; Papapietro et al., 2013). An understanding of the basic principles behind R-spondin signalling is fundamental to the design of novel therapeutic interventions for cases where the Wnt pathway is deregulated. "
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    ABSTRACT: The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalization of the ternary LGR-Rspo-E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5ecto) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2Fu1-Fu2). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterized by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5-mRspo2Fu1-Fu2-mZNRF3ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jun 2015 · Journal of Structural Biology
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    • "2012). Rspo1 has been implicated in many adult stem cell in vitro expansion systems, such as the intestine, stomach , and liver (Kim et al. 2005; Sato et al. 2009; Barker et al. 2010; Huch et al. 2013). However, it remains unclear in vivo which cells produce Rspo proteins in these organs. "
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    ABSTRACT: Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/β-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.
    Full-text · Article · Sep 2014 · Genes & Development
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