Abstract
Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.
Highlights
Wnt signaling regulates immunomodulatory functions during infection and inflammation
When we examined the protein levels of various markers of Cancer stem cells (CSCs), both Dclk1 and CD44 levels were elevated in NCCIT and PA-1 cell lines, whereas increased levels of aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were recorded in both NCCIT and HCT116 cells, respectively (Fig. 1Biv)
In silico binding studies indicated the Employing cell lines with elevated levels of endogenous Wnt signaling and a murine model of enteric infection, we elucidate the mechanism of epigenetic regulation of Wnt/-catenin signaling initiated at the plasma membrane and provide evidence that selective targeting of individual CSCs is not sufficient to block spheroid growth in an environment fueled by high Wnt signaling
Summary
Wnt signaling regulates immunomodulatory functions during infection and inflammation. Aberrant activation of the Wnt pathway can be driven by inactivating mutations in zinc and RING finger 3 (ZNRF3) and RING finger protein 43 (RNF43), which encodes tumor suppressor E3 ubiquitin ligases [5] These mutations are frequently present in CRC and endometrial cancers, leading to stabilization and higher levels of FZD receptors [5, 6], which underscores a role for upstream Wnt signaling in these tumors. LRP6 variants have been identified in individuals with an early-onset CRC (Յ30 years of age) and contribute toward heterogeneous susceptibility to CRC [9] Despite these advances, a systematic study looking beyond mutational activation of Wnt signaling through signals generated at the plasma membrane in a nonneoplastic colonic epithelium is lacking.
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