R-spondin signaling as a pivotal regulator of tissue development and homeostasis

R-spondins (Rspos) are cysteine-rich secreted glycoproteins which control a variety of cellular functions and are essential for embryonic development and tissue homeostasis. R-spondins (Rspo1 to 4) have high structural similarity and share 60% sequence homology. It has been shown that their cysteine-rich furin-like (FU) domain and the thrombospondin (TSP) type I repeat domain are essential for initiating downstream signaling cascades and therefore for their biological functions. Although numerous studies have unveiled their pivotal role as critical developmental regulators, the most important finding is that Rspos synergize Wnt signaling. Recent studies have identified novel receptors for Rspos, the Lgr receptors, closely related orphans of the leucin-rich repeat containing G protein-coupled receptors, and proposed that Rspos potentiate canonical Wnt signaling via these receptors. Given that Wnt signaling is one of the most important developmental signaling pathways that controls cell fate decisions and tissue development, growth and homeostasis, Rspos may function as key players for these processes as well as potential therapeutic targets. Here, I recapitulate the Wnt signaling and then outline the biological role of Rspos in tissue development and homeostasis and explore the possibility that Rspos may be used as therapeutic targets.     

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.     

Axin2+ Peribiliary Glands in the Periampullary Region Generate Biliary Epithelial Stem Cells That Give Rise to Ampullary Carcinoma

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The structural basis of R-spondin recognition by LGR5 and RNF43

R-spondins (RSPOs) enhance Wnt signaling, affect stem cell behavior, bind to leucine-rich repeat-containing G-protein-coupled receptors 4–6, (LGR4–6) and the transmembrane E3 ubiquitin ligases RING finger 43/zinc and RING finger 3 (RNF43/ZNRF3). The structure of RSPO1 bound to both LGR5 and RNF43 ectodomains confirms their physical linkage. RSPO1 is sandwiched by LGR5 and RNF43, with its rod module of the cysteine-rich domain (CRD) contacting LGR5 and a hairpin inserted into RNF43. LGR5 does not contact RNF43 but increases the affinity of RSPO1 to RNF43, supporting LGR5 as an engagement receptor and RNF43 as an effector receptor. Disease mutations map to the RSPO1–RNF43 interface, which promises therapeutic targeting.     

人类新血小板反应素R-spondin 3的细胞定位及其对肿瘤细胞功能的影响

目的观察人类新血小板反应素R-spondin 3(Rspo 3)的细胞定位,并探讨其在肿瘤发生发展中的可能作用。方法利用荧光显微成像法观察EGFP-Rspo 3在HEK293细胞中的分布。将重组质粒pcDNA-Rspo 3转染结肠癌细胞HT-29、LoVo,采用流式细胞术观察Rspo 3基因过表达对肿瘤细胞周期与凋亡的影响;采用Matrigel、Transwell实验分别检测Rspo 3基因过表达对肿瘤细胞黏附及侵袭能力的影响。结果荧光显微成像法观察发现,EGFP-Rspo 3融合蛋白在细胞核表达,呈弥散点状分布。流式细胞术观察发现,Rspo 3基因过表达对肿瘤细胞生长周期没有明显影响;Rspo 3基因过表达不影响低恶性的HT-29细胞凋亡,但诱导高恶性的LoVo细胞凋亡(P<0.01)。Rspo 3基因过表达增强肿瘤细胞黏附性(P<0.01),降低肿瘤细胞的侵袭力(P<0.01),对肿瘤细胞移行有一定抑制作用。结论 Rspo 3有核定位功能,能诱导部分肿瘤细胞凋亡并抑制其转移扩散。     

R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice

BACKGROUND & AIMS: R-spondin 1 (Rspo1) is a novel epithelial mitogen that stimulates the growth of mucosa in both the small and large intestine. METHODS: We investigated the therapeutic potential of Rspo1 in ameliorating experimental colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) as well as nonsteroidal anti-inflammatory drug-induced colitis in interleukin (IL)-10-deficient mice. RESULTS: Therapeutic administration of recombinant Rspo1 protein reduced the loss of body weight, diarrhea, and rectal bleeding in a mouse model of acute or chronic DSS-induced colitis. Histologic evaluation revealed that Rspo1 improved mucosal integrity in both villus and/or crypt compartments in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration in DSS-treated mice. Moreover, Rspo1 significantly reduced DSS-induced myeloperoxidase activity and inhibited the overproduction of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, in mouse intestinal tissue, indicating that Rspo1 may reduce DSS-induced inflammation by preserving the mucosal barrier function. Likewise, Rspo1 therapy also alleviated TNBS-induced interstitial inflammation and mucosal erosion in the mouse colon. Furthermore, Rspo1 substantially decreased the histopathologic severity of chronic enterocolitis by repairing crypt epithelium and simultaneously suppressing inflammatory infiltration in piroxicam-exposed IL-10(-/-) mice. Endogenous Rspo1 protein was localized to villus epithelium and crypt Paneth cells in mouse small intestine. CONCLUSIONS: Our results show that Rspo1 may be clinically useful in the therapeutic treatment of inflammatory bowel disease by stimulating crypt cell growth, accelerating mucosal regeneration, and restoring intestinal architecture.     

LGR5 is associated with tumor aggressiveness in papillary thyroid cancer

PURPOSE

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker and a down-stream target in Wnt/β-catenin signaling. In human papillary thyroid cancer (PTC), over activation of Wnt/β-catenin has been associated with tumor aggressiveness.

PATIENTS AND METHODS

Using established human cell lines (TPC-1, KTC-1, Nthy-ori-3–1), we report LGR5 and R-spondin (RSPO1–3) overexpression in PTC and manipulate LGR5 and Wnt/β-catenin signaling via both pharmacologic and genetic interventions. We test the association of LGR5 tumor expression with markers of PTC aggressiveness using a Discovery Cohort (n = 26 patients) and a Validation Cohort (n = 157 patients). Lastly, we explore the association between LGR5 and the BRAFV600E mutation (n = 33 patients).

RESULTS

Our results reveal that LGR5 and its ligand, RSPO, are overexpressed in human PTC, whereby Wnt/β-catenin signaling regulates LGR5 expression and promotes cellular migration. In two separate cohorts of patients, LGR5 and RSPO2 were associated with markers of tumor aggressiveness including: lymph node metastases, vascular invasion, increased tumor size, aggressive histology, advanced AJCC TNM stage, microscopic extra thyroidal extension, capsular invasion, and macroscopic invasion. As a biomarker, LGR5 positivity predicts lymph node metastasis with 95.5% sensitivity (95% CI 88.8%-98.7%) and 61% specificity (95% CI: 48.4%–72.4%) and has a negative predictive value (NPV) of 91.3% (95% CI 79.2%–97.5%) for lymph node metastatic disease. In human PTC, LGR5 is also strongly associated with the BRAFV600E mutation (p = 0.005).

CONCLUSION

We conclude that overexpression of LGR5 is associated with markers of tumor aggressiveness in human PTC. LGR5 may serve as a future potential biomarker for patient risk stratification and loco regional metastases in PTC.     

Wnt Signaling Shapes the Histologic Variation in Diffuse Gastric Cancer

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