Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells

Abstract

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

Keywords: acridine orange; autophagosome; autophagy flux inhibition; bitter taste receptor; calcium; cariogenic bacteria; serum starvation.

Figure 1

T2R14-dependent autophagy flux in gingival epithelial cells (GECs). (A) Effect of Baf A1 and Rapa on GEC viability. Flow cytometry cell-based assay suggested the effect of Baf A1(10 nM) and Rapa (1000 nM) on the cell growth in both WT-mock and T2R14KO cells over 72 h. The bar plots represent the SEM of three independent experiments performed in triplicates. (B–D) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Flow cytometry evaluation of the red, green, and red/green fluorescence ratio for detecting the autophagy flux in WT-mock and T2R14KO in GECs under serum-starved conditions and their respective normal growth condition. The bar graphs were generated using GraphPad PRISM 9.0. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post hoc test * p < 0.05, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant. The data represent the SEM of 3–4 independent experiments. (E) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Representative raw traces showing the flow cytometric detection of red and green fluorescence in Acridine Orange-stained GECs (WT-mock and T2R14 KO) that were serum-starved or treated with Baf A1 (10 nM) and Rapa (1000 nM) for 72 h for detecting the autophagy flux. (F–H) Transmission electron microscopy (TEM) evaluation of autophagic vacuoles in WT-mock and T2R14KO cells. The white-headed arrow indicates the phagophore (incomplete autophagosome) and autophagosome double-membrane structure (Insets) in respective GECs (F,G). Scale = 2 microns at magnification of 10,500×. (H) Quantification of autophagosome based on the morphology (double membrane) in WT-mock and T2R14 KO cells. The results represent the mean value of autophagic vesicles per cell.

Figure 1

T2R14-dependent autophagy flux in gingival epithelial cells (GECs). (A) Effect of Baf A1 and Rapa on GEC viability. Flow cytometry cell-based assay suggested the effect of Baf A1(10 nM) and Rapa (1000 nM) on the cell growth in both WT-mock and T2R14KO cells over 72 h. The bar plots represent the SEM of three independent experiments performed in triplicates. (B–D) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Flow cytometry evaluation of the red, green, and red/green fluorescence ratio for detecting the autophagy flux in WT-mock and T2R14KO in GECs under serum-starved conditions and their respective normal growth condition. The bar graphs were generated using GraphPad PRISM 9.0. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post hoc test * p < 0.05, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant. The data represent the SEM of 3–4 independent experiments. (E) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Representative raw traces showing the flow cytometric detection of red and green fluorescence in Acridine Orange-stained GECs (WT-mock and T2R14 KO) that were serum-starved or treated with Baf A1 (10 nM) and Rapa (1000 nM) for 72 h for detecting the autophagy flux. (F–H) Transmission electron microscopy (TEM) evaluation of autophagic vacuoles in WT-mock and T2R14KO cells. The white-headed arrow indicates the phagophore (incomplete autophagosome) and autophagosome double-membrane structure (Insets) in respective GECs (F,G). Scale = 2 microns at magnification of 10,500×. (H) Quantification of autophagosome based on the morphology (double membrane) in WT-mock and T2R14 KO cells. The results represent the mean value of autophagic vesicles per cell.

Figure 2

ATG7-independent autophagy flux in gingival epithelial cells (GEC). (A) Western blot analysis of ATG7 knockout in WT and T2R14KO GECs. ATG7 KD in WT and T2R14 KO GECs was performed using an shRNA lentiviral approach. WT denoted WT-sc shRNA, and T2R14KO denoted T2R14 KO sc shRNA. (B–D) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Flow cytometry evaluation of the red, green, and red/green fluorescence ratio for detecting the autophagy flux in ATG7KD and T2R14KO-ATG7KD in GECs under serum-starved conditions and their respective normal growth condition. The bar graphs were generated using GraphPad PRISM 9.0. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post hoc test * p < 0.05, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant. The data represent the SEM of 3–4 independent experiments. (E) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Representative raw traces showing the flow cytometric detection of red and green fluorescence in Acridine Orange-stained GECs (ATG7KD and T2R14-ATG7KD) that were serum-starved for 72 h for detecting the autophagy flux.

Figure 3

Intracellular calcium mobilization assay in GECs. (A) Pharmacological characterization of autophagy flux inhibitor, Baf A1 (10 nM) and autophagy inducer Rapa (1000 nM) on the calcium mobilization in GECs. WT, T2R14KO, ATG7 KD, and T2R14KO-ATG7KD cells (approx. 30,000/96 wells) were loaded with Fluo-4NW calcium dye and the intracellular calcium response was measured after Baf A1 and Rapa treatment. The data shown are the mean ± SEM of three independent experiments performed in triplicate. (B) Effect of S. aureus autoinducer peptide (AIP-1) and S. mutans competence-stimulating peptide (CSP-1) on calcium mobilization in GECs. Intracellular calcium release measurement after the application of autoinducer peptides (AIP from S. aureus and CSP-1 from S. mutans (50 µM each) in GECs. Results are from three independent experiments performed in triplicate. (C) Combined effect of Baf A1 and CSP-1 on calcium signaling in GECs. WT, T2R14KO, ATG7 KD, and T2R14KO-ATG7KD cells (approx. 30,000/96 wells) were loaded with Fluo-4NW calcium dye and the intracellular calcium response was measured after CSP-1 and CSP-1 plus Baf A1 treatment. The data shown are the mean ± SEM of three independent experiments performed in triplicate. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post -hoc test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant.

Figure 4

Immune cell (dHL-60) migration. (A,B) Conditioned medium (CM) obtained from with treatments in WT, T2R14KO, ATG7KD, and T2R14KO-ATG7KD cells with or without S. aureus and S. mutans on the migration of differentiated HL-60 cells (dHL-60). The changes in cellular impedance or cell index (CI) were measured every 5 min over 24 h using RTCA software. The bar graph shows change in the migration of dHL-60 cells over a period of 24 h. The bar plots represent the SEM of three independent experiments and were generated using GraphPad PRISM 9.0. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post hoc test * p < 0.05, and ns denotes non-significant. (C) Effect of conditioned medium (CM) obtained from (with or without BafA1 (10 nM) treatment) WT, T2R14KO, and ATG7KD and T2R14KO-ATG7KD cells on the migration of differentiated HL-60 cells (dHL-60). The changes in cellular impedance or cell index (CI) were measured every 5 min over 24 h using RTCA software. FMLP was included as a positive control for neutrophil chemoattractant. The bar plots represent the SEM of 2–3 independent experiments performed in triplicates. * p < 0.05 showed statistical significance.