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. 2004 Jan 6;101(1):266-71.
doi: 10.1073/pnas.2536800100. Epub 2003 Dec 26.

Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

Frank Kuhnert  1 , Corrine R Davis, Hsiao-Ting Wang, Pauline Chu, Mark Lee, Jenny Yuan, Roel Nusse, Calvin J Kuo
Affiliations

Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

Frank Kuhnert et al. Proc Natl Acad Sci U S A. .

Abstract

Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.

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Figures

Fig. 1.
Fig. 1.
Analysis of Ads expressing murine Dkk1. (A) Construction of Ad Dkk1. Murine Dkk1 cDNA bearing N-terminal IgK signal peptide and C-terminal FLAG and His6 tags was inserted into E1-E3- Ad strain 5 by homologous recombination (7, 8). (B) Inhibition of Wnt3a-stimulated TOPFLASH luciferase reporter activity by transfected Dkk1. Wnt3a and/or Dkk1 expression vectors were cotransfected into 293T cells with pTOPFLASH followed by luciferase measurement. (C). β-Catenin stabilization assay. Purified recombinant Dkk1 (125 ng/ml) was added to L cells 2 h before recombinant Wnt3A (1:8,000; Nusse Laboratory, Stanford, CA), followed after 3 h by harvest and Western blot analysis for β-catenin (BD Transduction Laboratories, San Diego). (D) Time course of Dkk1 expression in the circulation. Adult C57BL/6 mice received single i.v. tail vein injection of Ad Dkk1 (109 pfu) followed by phlebotomy at the indicated times. Dkk1 was detected by Western blotting using anti-His probe, Ab (Santa Cruz Biotechnology), and migrated as a doublet of 38–34 kDa. (E) Survival analysis of C57BL/6 mice after i.v. administration of 109 pfu Ad Dkk1 (IgK signal/3′ FLAG/His), Ad Dkk1-HA (IgK signal/5′HA/3′ FLAG/His), or Ad Fc treatment. Ad Fc or Ad Dkk1 doses =< 3 × 108 pfu have not exhibited any mortality over a 120-day time course.
Fig. 2.
Fig. 2.
Time course of histological changes in the gastrointestinal tract of Ad Dkk1-treated animals. Adult C57BL/6J mice (12–16 weeks old) received single i.v. tail vein injection of 109 pfu of either Ad Dkk1 or the negative control virus Ad Fc (109 pfu) followed by analysis of organs by hematoxylin/eosin staining at the indicated times. In duodenum and jejunum, Dkk1 induced crypt loss, villus blunting and fusion, and loss of mucosal integrity, followed by mucosal regeneration by day 10. The stomach was relatively unaffected. In cecum and colon, Dkk1 induced crypt loss with profound mucosal degeneration and ulceration by day 7 and regeneration by day 10 evidenced by irregular basophilic crypts at day 10. Stomach (st), duodenum (du), proximal jejunum (je), ileum (il), cecum (ce), and colon (co) are shown.
Fig. 3.
Fig. 3.
Spectrum of colonic lesions in Ad Fc- or Ad Dkk1-treated C57BL/6J (Upper) and SCID (Lower) mice. Colons were harvested for hematoxylin/eosin staining at day 7 after administration of 109 pfu of the appropriate Ads to 12- to 16-week-old mice. Moderate thinning of the ascending colon in C57BL/6J versus frequent ulceration in SCID is depicted. A spectrum of lesions was observed in descending colons of both strains, ranging from focal ulceration to frank effacement of architecture and replacement with inflammatory infiltrates, the latter being more severe in SCID animals.
Fig. 4.
Fig. 4.
Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (109 pfu). (Left) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. *, residual CD44 staining in nonepithelial lamina propria. (Right) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.
Fig. 5.
Fig. 5.
Analysis of proliferative state in gastrointestinal epithelium in Ad Dkk1-treated C57BL/6J mice by Ki67 immunohistochemistry. Arrowheads indicate the absence of Ki67 immunoreactivity in proliferative compartments 2 days after i.v. injection of Ad Dkk1 or Ad Fc (109 pfu). Note the strong repression of Ki67 immunoreactivity in duodenum, jejunum, cecum, and descending colon and moderate reduction in ascending colon. Ileum and stomach were not significantly affected.
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References

    1. Marshman, E., Booth, C. & Potten, C. S. (2002) BioEssays 24, 91-98. - PubMed
    1. Booth, C. & Potten, C. S. (2000) J. Clin. Invest. 105, 1493-1499. - PMC - PubMed
    1. Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P. J. & Clevers, H. (1998) Nat. Genet. 19, 379-383. - PubMed
    1. Wong, M. H., Rubinfeld, B. & Gordon, J. I. (1998) J. Cell Biol. 141, 765-777. - PMC - PubMed
    1. van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P., et al. (2002) Cell 111, 241-250. - PubMed

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