Figure 1.
IL-17A-dependent induction of Pendrin mRNA.
A. Mature, well-differentiated HBE cells were stimulated with IL-17A (50 ng/ml, 48 h) prior to collection of total RNA, reverse-transcription, and analysis of Pendrin mRNA expression by quantitative PCR (n = 5 inserts, ** p<0.01). B. IL-17A increases Pendrin mRNA in a time-dependent fashion (Note logarithmic scale, n = 2 inserts per time point, one from each of 2 donors compared to its own control). C. HBE cells were stimulated with IL-17A (50 ng/ml, 48 h) or vehicle (PBS) after which conditioned media were collected and used to stimulate naïve cells. Conditioned media were treated with either an IL-17A neutralizing antibody (NA) or an isotype control IgG (IgG) (n = 2 inserts per condition, one from each of two donors compared to its own vehicle control). D. Quantitative PCR for SLC26A3 (DRA) and SLC26A6 (PAT1) in the presence and absence of IL-17A (n = 3 inserts per condition).
Figure 2.
IL-17A induces Pendrin protein expression at the apical plasma membrane of normal HBE cells.
A. Immunoblot of Pendrin from total cell lysates at 24 h and 48 h of IL-17A stimulation. B. Confocal immunofluorescence images of vehicle and IL-17A stimulated HBE cells. Note that Pendrin (red) is only detected in IL-17A stimulated cells and is primarily localized to non-ciliated cells, which lack of Type IV Tubulin (green). Images are z-stacks with 0.5 micron sections from the Transwell support to the tips of the cilia. For each set of images, the main image is a single slice from the z-stack. Below is an x–z projection and to the right is a y–z projection. The yellow lines indicate the location from which the projections are taken.
Figure 3.
IL-17A induces Cl−/HCO3− exchange at the apical membrane of normal HBE cells.
A. Calibration curve of pHi versus F640/580. An insert of HBE cells was loaded with SNARF-5/6-AM and then incubated in high-potassium buffer (pH 6.8, 7.2, or 7.6) containing 20 µM each valinomycin and nigericin for 40 minutes. Five serial measurements over 2 minutes were taken for each filter and the average used to calculate a single calibration point for each pH. Linear fit was performed in Microsoft Excel. B. Intracellular pH changes in response to chloride removal from the apical perfusate (open circles: vehicle, n = 6 inserts; shaded circles: IL-17A (50 ng/ml for 48 h, n = 5 inserts). C. Intracellular pH changes in response to chloride removal from the apical perfusate in the absence of soluble HCO3− and CO2 (only IL-17A treated cells are shown). Please see the results section in the text for a complete discussion of two-way ANOVA applied to 3b.
Figure 4.
IL-17A increases Pendrin and Cl−/HCO3− exchange in CF HBE cells.
A. Pendrin mRNA is increased in CF HBE cells treated with IL-17A (shaded bar) (n = 9 inserts from 2 donors, *** p<0.01 compared to vehicle controls (open bar)). B. Consistent with the increase in Pendrin expression, apical membrane Cl−/HCO3− is also increased in CF HBE cells treated with IL-17A (closed circles, n = 3 inserts).
Figure 5.
Inhibition of Pendrin expression by siRNA reduces IL-17A-induced Cl−/HCO3− exchange.
A. and B. Normal HBE cells from two different donors were transfected with either non-targeting (scrambled) siRNA (closed circles) or one of two Pendrin-targeting siRNA from Invitrogen: HSS 107794 (siRNA 1, closed squares) and HSS 107796 (siRNA 2, open triangles). After 14 days, cells were tested for Cl−/HCO3− exchange. Panels A and B represent data from the individual donor. C. To account for differences in baseline pHi, pH was normalized to the mean of the first minute (pHt0) and data were then combined. Normalized mean data encompassing both donors are shown in panel C. D. Quantitative PCR measuring Pendrin mRNA expression in targeting siRNA-treated cells compared with scrambled control siRNA-treated cells. E. Immunoblot comparing Pendrin protein expression in targeting siRNA-treated cells compared with scrambled control siRNA-treated cells.
Figure 6.
IL-17A-induced Pendrin expression is dependent on NF-κB.
Cells were incubated with NF-κB inhibitor II (20 µM) for 6 hours and then with NF-κB inhibitor II and IL-17A (10 ng/ml) for 24 h prior to analysis. Quantitative PCR (A) and immunoblotting (B) from normal HBE cells demonstrating that inhibition of NF-κB prevents IL-17A induced Pendrin expression.