BASIC RESEARCH: Inducing intestinal growth

The small-intestinal epithelium is composed of proliferative-crypt and differentiating-villus compartments.

Pluripotent stem cells (believed to reside near crypt bases) give rise to progenitors that proliferate and migrate either toward the villus while differentiating into enterocyte, goblet, and enteroendocrine cells or toward the crypt bases while differentiating into Paneth cells.

The signaling pathway mediated by the Wnt–{beta}-catenin cascade has a central role in determining the balance of these different types of cells — in other words, it has an important influence on the cell's tendency to differentiate or proliferate.

The discovery by Kim and colleagues1 that R-spondin1, a newly identified human protein, activates {beta}-catenin signaling and dramatically increases the proliferation and growth of the small and large intestines in mice is therefore of interest to those seeking new approaches to the treatment of intestinal and epithelium-related disorders.

Kim et al.1 discovered the effects of R-spondin1 during in vivo screening of secreted proteins. They engineered transgenic "knock-in" mice by inducing their B lymphocytes to express a specific human gene predicted to encode a secreted protein. They then screened the mice, each of which expressed a different protein, for particular phenotypes.

The R-spondin1 knock-in mice had dramatically enlarged small intestines that were more than twice as heavy as the intestines of wild-type mice and caused noticeable distention of the abdomen. Both the diameter and the length of the small intestine were affected.

Histochemical analysis revealed an expansion of the proliferative zones of the small and large intestines, but the numbers of differentiated goblet and Paneth cells were similar to those in wild-type mice.

Cells in the proliferative zone showed intense staining of cytoplasmic {beta}-catenin protein, and the small-intestinal expression of {beta}-catenin target genes was increased.

These findings are consistent with the previous observation that R-spondin2 activates the {beta}-catenin signaling cascade in Xenopus laevis.2

Normally, cytoplasmic {beta}-catenin forms complexes with a protein called APC (mutant APC causes familial adenomatous polyposis) and axin, and its levels are kept low as a result of continuous proteasome-mediated degradation (Figure 1A).3

On binding to a receptor complex on the cell surface, Wnt protein initiates signaling pathways that disrupt the degradation of {beta}-catenin.

This, in turn, results in an increase in {beta}-catenin levels in the cytoplasm and then the nucleus, where {beta}-catenin controls the transcription of genes involved in proliferation and differentiation.3

Activated R-spondin1 may exert some of its effects independently of Wnt signaling, because Kim et al. found that treatment of a human-embryonic-kidney cell line with a potent Wnt inhibitor only partially inhibited R-spondin1–mediated activation of {beta}-catenin.


Figure 1. Protective Effect of R-spondin1 on Chemotherapy-Induced Adverse Effects.

Mice were injected with carcinoma cells five days before intravenous treatment was initiated with fluorouracil (days 1 through 5) and either saline (Panel A, days 1 through 8), or R-spondin1 (Panel B, 50 µg per day intravenously on days 1 through 8) to assess the effects on chemotherapy-induced adverse effects and tumor size.1 In fluorouracil-treated mice, those that received R-spondin1 (Panel B) had a reduction in fluorouracil-mediated adverse effects (enteritis, diarrhea, and weight loss), whereas no such effects were observed in mice given saline (Panel A). In Panel A, pluripotent stem cells believed to reside near crypt bases give rise to progenitors that proliferate by means of Wnt signaling and the activation of {beta}-catenin and migrate toward the villus (enterocytes, goblet, and enteroendocrine cells) or crypt bases (Paneth cells). Normally, Wnt signaling and proliferation involve a decreasing gradient from stem cells to upper crypt regions. With decreased Wnt signaling (e.g., upper crypt regions), {beta}-catenin forms a complex with APC and axin (destruction complex), leading to the degradation of {beta}-catenin and low {beta}-catenin levels. Treatment with R-spondin1 (Panel B) led to increased {beta}-catenin levels, resulting in increased transcription of target genes, expansion of the proliferative zone, and enhanced epithelial repair. In the absence of fluorouracil (Panel C), mice treated with vehicle and R-spondin1 had similar tumor sizes. The sections of small intestine from treated and untreated mice, stained with hematoxylin and eosin, in Panels A and B are reprinted from Kim et al.,1 with the permission of the publisher.

 
Distinctions between the Wnt and R-spondin1 pathways may prove crucial to understanding the distinct pathways regulating the stabilization of {beta}-catenin.

Mutations in the APC gene can result in excessive activation of {beta}-catenin, increased proliferation of epithelial cells, and sporadic colon cancer and familial adenomatous polyposis, highlighting the deleterious effects of dysregulation of this pathway.

On the other hand, activation of {beta}-catenin is essential in intestinal development and physiology, and the induction of epithelial proliferation helps heal epithelial injuries.

Kim et al. observed that treatment with R-spondin1 mitigated the adverse gastrointestinal effects (enteritis, diarrhea, weight loss, and impaired epithelial integrity) associated with the chemotherapeutic agent fluorouracil without increasing tumor growth, as compared with fluorouracil treatment alone (Figure 1). 1

A single dose of R-spondin1 resulted in rapid proliferation of crypt progenitors, with normalization of the levels by 48 hours — demonstrating the potential short-term beneficial effects of this protein on chemotherapy-induced injuries.

Other intestinal diseases (such as inflammatory bowel disease, radiation-induced enteritis, and intestinal ischemia) involving acute and chronic epithelial injury and impaired epithelial healing may prove to be responsive to R-spondin1.

The effects of various members of the family of growth factors, such as fibroblast growth factors and glucagon-like peptide 2 (GLP-2), on epithelial growth and healing are being evaluated for their therapeutic benefit.

Kim et al.1 observed that R-spondin1 induced a more dramatic expansion of the proliferative zone than did fibroblast growth factor 7 or GLP-2, perhaps reflecting direct activation of {beta}-catenin by R-spondin1.

Epithelial proliferative effects may be mediated directly or indirectly — for example, through vascular endothelial cells and enteric neurons.

The sustained effects on intestinal growth observed in R-spondin1–chimeric mice also suggest that this protein may have therapeutic benefits in the short-gut syndrome.

However, how R-spondin1 affects the balance between intestinal epithelial healing and excessive growth and neoplasia, as well as its effects on other organs, will need to be evaluated carefully as it is considered for various therapeutic purposes.

Questions remain regarding the role and regulation of R-spondin1. What regulates its expression? With which receptors or receptor complexes does it interact? Does it initiate signaling pathways other than the {beta}-catenin pathway? What are the outcomes of these interactions? R-spondin1 is expressed in intestinal, pancreatic, and adrenal enteroendocrine cells, as well as in epithelial cells of the prostate and kidneys1; the effects of the protein on these other organs have not yet been reported.

Studies of knockout models would help define the degree to which this newly identified protein is essential for the maintenance of epithelial integrity. The elucidation of the full spectrum of the biologic effects of R-spondin1 will enable us to understand its physiological role as well as guide potential new therapeutic approaches.


NEJM.-Volume 353:2297-2299   November 24, 2005   Number 21

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Inducing Intestinal Growth
Clara Abraham, M.D., and Judy H. Cho, M.D.   Source Information

From the Department of Medicine, University of Chicago, Chicago.

References

  1. Kim KA, Kakitani M, Zhao J, et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 2005;309:1256-1259. [Abstract/Full Text]
  2. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, Wu W. R-spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 2004;7:525-534. [CrossRef][ISI][Medline]
  3. Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev 2005;19:877-890. [Abstract/Full Text]






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