Our work has recently defined an essential role for Wnt signaling in the maintenance of the extensive proliferation that allows complete regeneration of the intestinal epithelium every 5-7 days. Previously, the exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. This has been circumvented by adenoviral expression of Dickkopf-1 (Dkk1), a secreted Wnt antagonist which interacts with Wnt co-receptors of the LRP family. Using adenoviral expression of Dkk1, stringent, conditional and reversible Wnt inhibition in adult animals has been achieved, resulting in repression of expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, and marked inhibition of proliferation in small intestine and colon, accompanied by progressive architectural degeneration with loss of crypts, villi and glandular structure by 7 days. 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 implicate Wnt proteins as essential growth factors for crypt proliferation in small intestine and colon. Building upon these results, the current proposal will examine the role of Wnts in intestinal regeneration and repair, with the eventual goal of manipulating this pathway for therapy of mucosal disorders such as inflammatory bowel diseases. In the first aim, a systematic quantitative and spatial analysis of Wnt expression patterns in the resting and regenerating intestine will be performed to identify Wnts upregulated during regeneration after radiation or DSS treatment, Dkk1-mediated Wnt blockade, or in IL-10 knockout mice. The functional requirement of Wnt signaling during regeneration will be established by Ad Dkk1 treatment in these animal models. In the second aim, the therapeutic potential of Wnt agonists will be evaluated in the radiation, DSS and IL-10 injury models. Multiple strategies for Wnt stimulation will be utilized, including local/luminal and systemic administration of Wnt proteins, Wnt adenoviruses and small molecule agonists of the Wnt pathway embodied by GSK-3 inhibtors and lithium. In the third aim, the potential functional role of Dkk proteins as endogenous negative regulators of intestinal proliferation will be examined through adenoviral expression of soluble Kremen receptors to sequester and inactivate Dkk, and the examination of Dkk1 effects on Musashil1-expressing intestinal precursor cells.
