Robert J. Lee

Background: Airway tuft cells, also called solitary chemosensory cells, are enriched in nasal polyps and are likely the main epithelial source of interleukin-25 (IL-25) in chronic rhinosinusitis with nasal polyps (CRSwNP). Tuft cells are activated through G protein-coupled receptors (GPCRs) that signal through Ca ²⁺ . These receptors may also lower cyclic AMP (cAMP), but whether the cAMP pathway can be used to suppress tuft cell responses is unknown. Methods: Ca ²⁺ and cAMP were measured by live cell imaging in isolated Trpm5-GFP mouse nasal tuft cells, and Ca ²⁺ responses were measured in primary human nasal epithelial air-liquid interface (ALI) cultures from patients with CRSwNP. Defensin and IL-25 release were measured by ELISA and ciliary beat frequency (CBF) by high-speed imaging. Phosphodiesterase (PDE) expression was assessed using publicly available single cell RNA sequencing datasets. Results: Agonists of bitter taste receptors (T2Rs), the succinate receptor SUCNR1, and the A1 adenosine receptor increased Ca ²⁺ in GFP-positive mouse nasal tuft cells. T2R and A1 receptor stimulation also lowered cAMP, and these decreases were blocked by pertussis toxin. Glucose, which activates taste family 1 receptors, increased cAMP. Tuft cells were enriched for Pde2a . PDE2A-selective inhibitors (PF-05180999 and BAY 60-7550) increased cAMP and reduced agonist-evoked Ca ²⁺ responses in mouse tuft cells. In human nasal ALIs, both compounds reduced denatonium-evoked Ca ²⁺ responses, defensin release, and IL-25 release without increasing CBF. Conclusions: GPCRs that activate nasal tuft cells also lower cAMP, and by raising cAMP through PDE2A inhibition, tuft cell Ca ²⁺ signaling and IL-25 release were reduced in vitro.

We previously found that skin tone correlates with intronic polymorphisms influencing the expression of the Joubert Syndrome (JS) ciliopathy gene TMEM138 , suggesting that primary cilia might regulate pigment production in skin melanocytes. Herein, we show that many ciliary proteins regulate basal pigmentation in wild-type mouse and human melanocytes by facilitating primary cilium-dependent Melanocortin-1 receptor signaling, and downstream activation of the Microphthalmia transcription factor and melanogenic gene expression. Additionally, we identify an extra-ciliary moonlighting role for TMEM138 and other JS proteins in endolysosomal organization required for the biogenesis of melanosomes, the pigment-producing lysosome-related organelle in melanocytes. Our results provide new mechanistic insights into the physiological regulation of pigmentation and suggest that JS-specific pathology might be mediated by ciliary-independent mechanisms.

Motile cilia demonstrate coordinated beating to propel fluids across epithelial tissues, and changes to their beating frequency are largely regulated by intracellular second messengers including Ca2+. In the airway epithelium, ciliary beating is essential to mucociliary clearance. Mucociliary clearance involves trapping inhaled pathogens and irritants in sticky mucus lining the airways for motile cilia to sweep away contaminated mucus, preventing infection and reducing general airway inflammation. Many chronic respiratory diseases, including chronic rhinosinusitis and asthma, are characterized by an acquired ciliary dysfunction. Despite the importance of Ca2+ signaling in cilia physiology, the identity and molecular mechanisms governing localized ciliary Ca2+ transport remain poorly understood. MS4A8B is an uncharacterized cilia-localized transmembrane protein. Other MS4A homologs have been indirectly linked to Ca2+ signaling via uncharacterized mechanisms. Using primary human nasal epithelial cells differentiated at air-liquid interface, we demonstrated that MS4A8B regulates motile cilia function. MS4A8B knockdown impairs ciliary beating and impacts cilia structure. Live-cell imaging combined with genetic analysis revealed that MS4A8B potentiates Orai1-mediated Ca2+ influx. Co-immunoprecipitation and FRET microscopy in ectopic expression systems demonstrated that MS4A8B interacts with Orai1 channels. Orai1 was further identified to reside in motile cilia of primary human nasal epithelial cells, allowing ciliary beat frequency to be stimulated by Orai1 agonists including arachidonic acid. MS4A8B functional coupling with Orai1 acts as an autonomous cilia signaling network. Targeting this compartmentalized signaling pathway offers a novel therapeutic approach to restore or enhance mucociliary clearance in airway diseases.

Cisplatin resistance remains a major barrier in head and neck squamous cell carcinoma (HNSCC) treatment. ATP-binding cassette (ABC) transporters contribute to chemoresistance by limiting intracellular drug accumulation. Bitter taste receptor 10 (T2R10) has been implicated in ABC transporter regulation, but its role in HNSCC remains undefined. HNSCC cell lines were treated with T2R10-agonist caffeine (100 or 200 μM), cisplatin, or a combination, and viability was assessed by crystal violet assay. T2R10 promoter activity and expression following caffeine exposure were evaluated using a promoter-driven mCherry reporter and RT-qPCR. ABC transporter expression was measured after caffeine treatment and T2R10 gene (TAS2R10) knockdown or overexpression. Associations between tumor TAS2R10 expression and survival were assessed using TCGA data through GEPIA2. Caffeine enhanced the cisplatin-associated reduction in viability in a cell line- and concentration-dependent manner, with the strongest effect seen in UM-SCC47. A significant effect was observed in FaDu at 200 μM of caffeine, and minimal response in RPMI 2650. RPMI 2650 cells and FaDu cells exhibited lower baseline TAS2R10 expression and RPMI 2650 cells did not demonstrate enhanced cisplatin sensitivity following caffeine treatment. Caffeine treatment increased TAS2R10 promoter activity and expression and was associated with decreased ABCG2 expression. TAS2R10 knockdown increased ABCG2 and ABCF1 expression, whereas TAS2R10 overexpression reduced ABCG2 and ABCC1 expression. High tumor TAS2R10 expression was associated with improved disease-free survival (log-rank p=0.0071; HR=0.61) but not overall survival. Caffeine enhances cisplatin sensitivity in selected HNSCC models. Caffeine exposure is associated with increased TAS2R10 expression and reduced expression of chemoresistance-associated transporters, particularly ABCG2.

Ca2+ responses with known T2R5-specific agonists.

Supplementary Figure 4 from T2R5 Agonist Phendione Decreases Cell Viability and Induces Apoptosis in Head and Neck Squamous Cell Carcinoma

Heterologous overexpression of TAS2R5 in FaDu cells show increased sensitivity to phendione.

Phendione acts on a pathway distinct from T2R14.

Dose dependent reduction in cell viability after 24-hour exposure to phenodione.