Canonical to noncanonical Wnt signaling switch in airway epithelial health and disease - PROJECT SUMMARY/ABSTRACT Cilia damage and ciliated cell loss brought on by epithelial remodeling contributes to defective mucociliary clearance in chronic inflammatory airway diseases such as cystic fibrosis (CF), chronic rhinosinusitis (CRS), asthma and chronic obstructive pulmonary disease (COPD). Proper ciliated cell formation requires coordination of ciliated cell fate acquisition and ciliogenesis programs, but our mechanistic understanding is incomplete. Current treatments do not directly target or fully reverse ciliary dysfunction, highlighting the need for novel molecular targets for specific therapies. We showed that ciliated cell formation and function depend on sequentially deployed Wnt signaling pathways. First, canonical, or β-catenin-dependent Wnt signaling (Wnt/β- cat) is required for ciliated cell fate acquisition, which then must be turned off. Next, noncanonical Wnt/planar cell polarity signaling (Wnt/PCP) is turned on and stays on to control ciliogenesis and polarized ciliary motility. In CF and CRS airways and in primary cultures remodeled by proinflammatory cytokine treatment Wnt/β-cat stays active, and Wnt/PCP is blocked. Wnt/β-cat inhibition alleviates ciliary dysfunction in vitro. Thus, we propose that instead of two independent Wnt pathways, a canonical to noncanonical Wnt signaling switch controls healthy ciliated cell formation, and “switch failure” is a mediator and therapeutic target in ciliary dysfunction. Our preliminary data show that DKK3, a canonical Wnt/β-cat inhibitor and WNT4, a noncanonical ligand, co-secreted by airway epithelial stem cells are required together to suppress Wnt/β-cat and to activate Wnt/PCP within ciliated cells. Downstream, we show that β-catenin, the central effector of Wnt/β-cat is inactivated and sequestered at the basal bodies of cilia. We demonstrate that DKK3 and WNT4 expression is disrupted in diseased, and cytokine treated epithelia. Thus, we hypothesize that DKK3/WNT4-dependent signaling and β-catenin basal body sequestration are required for healthy ciliated cell formation and that disruption of this Wnt switch mechanism mediates in ciliary dysfunction in inflamed airway epithelia. Here, we use transgenic mouse models, human primary cells, and tissues to test this hypothesis in the following aims: In Aim 1, we test the necessity and sufficiency of epithelial DKK3/WNT4 to regulate Wnt signaling and ciliated cell formation through the switch using Dkk3/Wnt4 knockout mice, CRISPR deletion in cells and ectopic expression. In Aim 2, we test if the switch is mediated by the DKK3/WNT4-dependent sequestration β-catenin at basal bodies where it then acts to control ciliogenesis. In Aim 3, we investigate the IL-1β cytokine as a driver of Wnt switch failure and test the ectopic DKK3/WNT4-mediated mitigation of ciliary dysfunction. The new paradigm of the canonical to noncanonical Wnt signaling switch explored in this proposal will establish molecular mechanistic insight into the role of airway epithelial Wnt signaling, elucidate switch failure as a cause of ciliated cell dysfunction, and provide the rationale and potential targets for Wnt modulation in a wide range of chronic lung diseases.
