R‐spondin signaling in the stomach: isthmal Lgr4 rules

Abstract

R‐spondins are critical regulators of gastric epithelial cells, with Lgr5 receptor historically considered as their main signaling transducer. Recent work by Wizenty et al (2022) now revealed distinct roles for Lgr4 and Lgr5 in directing gland reconstitution following H. pylori infection, shedding new light on the complexities of Rspo signaling during gastric regeneration and raising questions about antral stem cell hierarchy.

Recent work shows a specific role for Lgr4 receptor in driving NF‐kB‐dependent gastric epithelial gland reconstitution upon H. pylori infection.

R‐spondin (Rspo) and Wnt signaling appear to be crucial for gastrointestinal (GI) stem cell maintenance. Leucine‐rich repeat‐containing G‐protein coupled receptor 5 (Lgr5) was originally discovered as a downstream target of Wnt signaling in intestinal crypts and was shown to mark both intestinal and gastric stem cells (Barker et al, 2007, 2010). Lgr5 was later identified as an Rspo receptor, as it mediates responses to Rspo agonism (de Lau et al, 2011). However, Lgr5 is part of a larger family of Lgr receptors that includes Lgr4 and Lgr6, which all participate in regulating Frizzled receptors turnover. However, functional differences between these Lgr receptors are yet to be fully elucidated.

As suggested for the small intestine, several groups reported the presence of an Lgr5‐negative isthmal stem cell population in the gastric antrum. Among them, Sigal et al (2017) found that Axin2, a Wnt‐regulated gene, is expressed broadly in the base and isthmus regions above the Lgr5⁺ cells, and that depletion of Lgr5⁺ cells resulted in their repopulation of antral glands by Axin2⁺/Lgr5⁻ cells. Thus, Axin2⁺/Lgr5⁻ cells may serve as gastric stem cells. The group further examined the behavior of these different antral progenitor populations in the setting of H. pylori infection, which increases proliferation and turnover in antral glands in part through release of Rspo3 from adjacent myofibroblasts. Interestingly, the investigators showed that Rspo3 induce proliferation in Axin2⁺/Lgr5⁻ cells but not in Axin2⁺/Lgr5⁺ cells, raising the questions as to what the non‐proliferative Lgr5⁺ cells represented and what Lgr5⁻ cellular lineage was mediating antral proliferative responses (Fig 1).

Figure 1. Rspo/Lgr axis during H. pylori colonization.

(1) Following H. pylori colonization myofibroblasts located at the bottom of gastric glands upregulate Rspo3. (2) Increased levels of Rspo3 modulate the activity of different epithelial cells population in a Lgr4‐dependent manner. In Lgr4⁺/Axin2⁺Lgr5^neg isthmal stem cells, Rspo3/Lgr4 signaling promotes proliferation as well as generation of Lgr5⁺ Secretory progenitors (via NFkB signaling). In Axin2⁺/Lgr5⁺ Secretory progenitors Rspo3 promotes differentiation toward Itln1⁺/Lgr5⁺ secretory cells. (3) Increased production of Itln1 in turn reduces H. pylori colonization.

In a subsequent study, the group addressed these questions regarding the nature of the non‐proliferative Lgr5⁺ cells and their response to Rspo3 signaling (Sigal et al, 2019) and showed that Rspo3 induces differentiation of basal Lgr5⁺ cells into secretory cells expressing antimicrobial factors such as intelectin‐1. Thus, depletion of Lgr5⁺ cells (or knockout of Rspo3) resulted in greater colonization of the antral gastric glands with H. pylori. This study makes clear that in addition to serving as a proliferative factor, Rspo3 functions as differentiation agent for secretory cells. Indeed, R‐spondins promote differentiation of secretory lineages at gastrointestinal sites. In the gastric corpus, Lgr5 marks chief cells residing at gland base that secrete digestive enzymes, while in the small intestine, Rspo1 stimulation has been shown to increase progenitor cell proliferation and differentiation of secretory Paneth cells (Stange et al, 2013; Yan et al, 2017).

In the current study, Wizenty et al (2022) investigate the mechanisms by which Rspo signaling promotes expansion of antral gastric glands in response to H. pylori infection by dissecting specific roles of Lgr4 and Lgr5 receptors. Using conditional knockout mice, they demonstrated that loss of Lgr4 abrogates the proliferative responses to Rspo signaling and renders antral glands more susceptible to injury, while deletion of Lgr5 does not impair homeostasis or injury repair; suggesting that the previously described R‐spondin mediated proliferation of Axin2⁺/Lgr5⁻ cells is dependent on Lgr4. Additionally, the authors observe that loss of Lgr4 diminishes Lgr5 expression and impairs production of antimicrobial Lgr5⁺ secretory cells following infection. Using whole transcriptomic analysis, they find that H. pylori infection induces activation of NF‐kB signaling, which promotes pro‐inflammatory signaling and gland base secretory differentiation in a Lgr4‐dependent manner. Systemic exogenous Rspo3 delivery in fact upregulates p65, a central mediator of NF‐kB signaling, at the gland base across the GI tract. Additionally, exposure of gastric organoids to ADP heptose, a bacterial sugar, triggers NF‐kB activation only when Rspo is present. Thus, Lgr4⁺ isthmal cells appear to be responsible for epithelial homeostasis and proliferation in response to R‐spondins, although Lgr5⁺ cells, which are increased by H. pylori infection, are needed to suppress (but not eliminate) H. pylori glandular colonization.

The work of Wizenty et al better clarifies the molecular mechanisms of Rspo‐driven gastric response to H. pylori infection, but the findings clearly upend many previous assumptions regarding the hierarchical organization of the gastric antrum. Indeed, these recent observations demonstrating a requirement for Lgr4 but not Lgr5 in antral regeneration, clearly places Lgr4 above Lgr5 in the hierarchy of gastric progenitor cells. While the original assumption was that most if not all Lgr5⁺ cells in the GI tract are stem cells, it does appear in the stomach that majority of Lgr5⁺ cells actually belong to the secretory lineage (including chief cells in the corpus). The two studies taken together (Sigal et al, 2019; Wizenty et al, 2022) raise new questions about molecular mechanisms that lead to such different roles in proliferation and differentiation by Lgr4 and Lgr5, respectively.

Furthermore, although not explicitly stated, these observations strongly imply that Axin2⁺/Lgr4⁺ cells in the isthmus comprise antral stem and/or progenitor cells. Little is known regarding the heterogeneity of this Axin2⁺/Lgr4⁺ isthmal population. Previous studies have identified other markers in this region that appear to be expressed in antral stem cells such as Cck2r (Chang et al, 2020; Hayakawa et al, 2021). Searching for possible overlap or combined expression of several markers may be a more productive approach to discriminate antral stem cells from progenitors. In addition, although the authors have suggested that all Axin2⁺ isthmus cells showing proliferative responses to Rspo are Lgr5⁻, Lgr5⁺ cells have been shown to lineage trace antral glands. Therefore, one might suspect that at least some of these cells are Lgr5^low and thus would account for the lineage tracing seen. Finally, the findings in this study likely have relevance to the hierarchical organization of other portions of the gastrointestinal tract, which should be explored in future work.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

The EMBO Journal (2022) 41: e111696.