Medicine: Mitogenic influence of human R-spondin1 on the intestinal epithelium

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


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 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.
    Journal of Structural Biology 06/2015; 191(2). DOI:10.1016/j.jsb.2015.05.008 · 3.23 Impact Factor
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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.
    Genes & Development 09/2014; 28(20). DOI:10.1101/gad.245142.114 · 10.80 Impact Factor
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    • "The last decade of research into the intestinal epithelial stem cell niche has been marked by exciting new insights which have resulted in numerous experimental systems of long-term in vitro epithelial growth. This work has been facilitated by the identification of critical growth factors, the use of three-dimensional culture techniques that provide a near-physiologic extracellular environment, and better characterization of supportive cells within the niche [1]–[4]. Though these culture systems have proven essential for advancement of in vitro research in the field, few studies have sought to further develop their clinical applicability. "
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    ABSTRACT: Background: We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. Methods: Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. Results: Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. Conclusion: Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.
    PLoS ONE 09/2014; 9(9):e107814. DOI:10.1371/journal.pone.0107814 · 3.23 Impact Factor
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