Differential activities and mechanisms of the four R-spondins in potentiating Wnt/β-catenin signaling

Park, Soo-Hyun; Cui, Jie; Yu, Wangsheng; Wu, Ling; Carmon, Kendra S.; Liu, Qingyun J.

doi:10.1074/jbc.ra118.002743

Cited by 80 publications

(119 citation statements)

References 43 publications

(91 reference statements)

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“…HA-RNF43-G659Vfs41 was then generated by deleting a G in bp 1979-1976 of the RNF43 open reading frame in the HA-RNF43-WT plasmid by site-directed mutagenesis. FLAG-tagged FZD5 was cloned into pIRESpuro3 as described previously 25 . All plasmids were verified by complete DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…2c, mid panel). To test if the mutant was able to bind to RSPO, we used the fusion protein RSPO2-Fu-F109A-IgG1-Fc which binds to ZNRF3/RNF43 with high affinity but not to LGR4 or LGR5 25 . In both WT-and G659Vfs41-transfected cells, RSPO2-Fu-F109A showed strong binding and rapid internalization ( Fig.…”

Section: Rspo-bindingmentioning

confidence: 99%

“…Recombinant full-length human RSPO2 was purchased from R&D Systems. RSPO2-Fu-WT and RSPO2-Fu-F109A were purified as described previously 25 vector Lenti-CRISPR2 as described 24 . The two guide sequences were provided by Dr. Feng Cong as they were used in the generation of double knockout of RNF43 and ZNRF in HEK293 cells 5 .…”

Section: Recombinant Proteins Antibodies and Western Blottingmentioning

confidence: 99%

“…Wnt/β-catenin signaling TOPFlash reporter enzyme assay and immunofluorescence: TOPFlash assay in STF RNF43-ZNRF3 double KO (RZ-DKO) cells transfected with vector, RNF43-WT, or RNF43-G659Vfs41 were carried out as described previously 25 . All TOPFlash experiments were repeated at least three times with duplicates or triplicates in each experiment.…”

Section: Recombinant Proteins Antibodies and Western Blottingmentioning

confidence: 99%

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The most common RNF43 mutant G659Vfs41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

Park

et al. 2019

Preprint

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RNF43 is an E3 ligase that inhibits Wnt signaling by ubiquitinating Wnt receptors for degradation. It is mutated in various cancer types with the most recurrent mutation being the frameshift G659Vfs41 with frequencies of ~5-8% in colon, stomach and endometrial cancers. The mutation has always been assumed to be loss-of-function that would lead to increased Wnt signaling and tumorigenesis, yet no functional characterization has been reported. We analyzed the distribution of the G659Vfs41 mutation and its association with other cancer gene mutations, and found that the mutation occurred nearly exclusively in tumors with low expression of the DNA mismatch repair gene MLH1. Mutant RNF43-G659Vfs41 was no different from wild type RNF43 in expression, localization, R-spondin binding, promotion of Wnt receptor degradation, inhibition of Wnt signaling. We also found that colon tumors with RNF43-G659Vfs41 had low expression of Wnt/β-catenin signaling markers and were frequently mutated in BRAF. A colon cancer cell line with RNF43-G659Vfs41 and BRAF-V600E mutations was uniquely sensitively to activation of Wnt/β-catenin signaling. These findings indicate that RNF43-G659Vfs41 is likely a passenger mutation in MLH1-down regulated tumors, not a loss-of-function driver mutation that facilitates tumorigenesis. Tumors with RNF43-G659Vfs41 and BRAF-V600E may be susceptible to activators of Wnt signaling.

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Section: Methodsmentioning

confidence: 99%

Section: Rspo-bindingmentioning

confidence: 99%

Section: Recombinant Proteins Antibodies and Western Blottingmentioning

confidence: 99%

Section: Recombinant Proteins Antibodies and Western Blottingmentioning

confidence: 99%

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The most common RNF43 mutant G659Vfs41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

Park

et al. 2019

Preprint

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“…3B, S3B). These data combined with the recent discovery that the various R-spondins can act through different mechanisms suggest that these signals likely play distinct roles under homeostatic conditions but are capable of performing somewhat redundant functions (Park et al, 2018).Of Wnt ligands typically associated with non-canonical pathways, Wnt5a and Wnt5bwere the most abundant. Wnt5a was produced primarily by telocytes, while Wnt5b showed disperse expression across regions and cell types, somewhat analogous to the R-spondin family (Fig.…”

mentioning

confidence: 97%

A Holistic Analysis of the Intestinal Stem Cell Niche Network

Pim

et al. 2019

Preprint

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Although many studies into the intestinal stem cell (ISC) niche have been carried out, they have focused on the role of a single cell type or molecular signal. However, no holistic comparisons of the predominant cell types and signals present within the intestinal mucosa have been conducted to date. We utilize bulk RNA sequencing to profile 20 different mucosal cell types covering four major cell categories: epithelial, stromal, endothelial and immune. We further examined the stromal signaling environment using scRNAseq to provide a more comprehensive view of the signaling microenvironment within the intestinal mucosa. We identified the primary signals for the major ISC regulatory pathways and their respective cellular sources. Our analysis suggests that a 'niche network' exists, with no single cell type being responsible for ISC self-renewal, proliferation, or differentiation; rather, each cell type within the network carries out specific functions in a highly cooperative and coordinated manner. KeywordsStem Cell Niche, Intestinal Stem Cells, ISC, RNA-Sequencing As expected, principle component analysis of the various transcriptomes resulted in the cell types clustering according to their categorical distinctions ( Fig. 1G). To briefly summarize, Grem1 CreErt2 + cells adopted a unique position between stromal and epithelial cells. Nestin GFP + and Ncad tdT + cells clustered together, consistent with their overlap and distribution pattern. Intriguingly, Ng2 dsR + and Dmp1 Cre + cells were transcriptomically similar, yet showed differences in several genes important to cellular function ( Fig. S1B). Innate lymphoid type 3 cells (ILC3) were positioned between adaptive T Cells and Innate Immune Dendritic cells and Macrophages (Fig. 1G). Characterization of Genetic Models and Stromal PopulationsBoth Nestin GFP + and Ncad CreErt2 + stromal cells were present from the smooth muscle to the villus tips ( Fig. 2A-B) and possessed somewhat similar distributions and morphologies to the niche cells described within the bone marrow. Though there was considerable overlap and similarities between these genetic markers, several differences were apparent. Ncad CreErt2 + cells were more likely to encircle the vasculature with a flattened morphology, while Nestin GFP + cells frequently laid on top of these cells and possessed long filopodia which connect with one another ( Fig. S2A-E) Ncad CreErt2 + cells were more likely to be present at the lower "ring", while Nestin GFP + were more likely to localize to the isthmus. Ng2 dsR + cells also overlapped with Nestin GFP + cells but were the only cell type examined that exhibited a strong spatial bias within the mucosa, being almost exclusively found within the villi (Fig. 2C,S2B).Unfortunately, their morphological characteristics could not be determined due to the tendency of the dsRed protein to form aggregates, preventing even cytosolic distribution.Dmp1 Cre + cells showed the greatest amount of morphological and positional diversity, likely due to their constitutively active Cre, wh...

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Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

et al. 2023

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Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear.Here, the authors screen all Wnt-related paracrine factors in 1994 obese cases and 2161 controls using whole-exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high-fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1 ablation resists HFD-induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4-Wnt/𝜷-catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4-Wnt/𝜷-catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain-of-function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator, suppress fat thermogenesis and contribute to obesity in humans.

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Differential activities and mechanisms of the four R-spondins in potentiating Wnt/β-catenin signaling

Cited by 80 publications

References 43 publications

The most common RNF43 mutant G659Vfs41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

The most common RNF43 mutant G659Vfs41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

A Holistic Analysis of the Intestinal Stem Cell Niche Network

Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

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