Expression of SLC26A9 in Airways and Its Potential Role in Asthma

Abstract

SLC26A9 is an epithelial anion transporter with a poorly defined function in airways. It is assumed to contribute to airway chloride secretion and airway surface hydration. However, immunohistochemistry showing precise localization of SLC26A9 in airways is missing. Some studies report localization near tight junctions, which is difficult to reconcile with a chloride secretory function of SLC26A9. We therefore performed immunocytochemistry of SLC26A9 in sections of human and porcine lungs. Obvious apical localization of SLC26A9 was detected in human and porcine superficial airway epithelia, whereas submucosal glands did not express SLC26A9. The anion transporter was located exclusively in ciliated epithelial cells. Highly differentiated BCi-NS1 human airway epithelial cells grown on permeable supports also expressed SLC26A9 in the apical membrane of ciliated epithelial cells. BCi-NS1 cells expressed the major Cl− transporting proteins CFTR, TMEM16A and SLC26A9 in about equal proportions and produced short-circuit currents activated by increases in intracellular cAMP or Ca2+. Both CFTR and SLC26A9 contribute to basal chloride currents in non-stimulated BCi-NS1 airway epithelia, with CFTR being the dominating Cl- conductance. In wtCFTR-expressing CFBE human airway epithelial cells, SLC26A9 was partially located in the plasma membrane, whereas CFBE cells expressing F508del-CFTR showed exclusive cytosolic localization of SLC26A9. Membrane localization of SLC26A9 and basal chloride currents were augmented by interleukin 13 in wild-type CFTR-expressing cells, but not in cells expressing the most common disease-causing mutant F508del-CFTR. The data suggest an upregulation of SLC26A9-dependent chloride secretion in asthma, but not in the presence of F508del-CFTR.

SLC26A9 in airways and intestine: secretion or absorption?

“…These data have been obtained under thin film conditions in highly differentiated human airway epithelial cells. As mentioned above, we found equal expression of SLC26A9 in airways from CFTR+/+ and CFTR-/- knockout piglets [ 56 ], indicating expression of SLC26A9 also in the absence of CFTR. Interestingly, short circuit currents (Isc) were activated in CFTR+/+ but not CFTR-/- primary piglet airway epithelial cells by S9-A13 ( Figure 4 ).…”

“…Apart from Pendrin, also SLC26A9 is expressed in airway epithelial cells. Using immunohistochemistry, we demonstrated location of SLC26A9 in the apical membrane of ciliated epithelial cells of human, mouse, and piglet airways, as well as ciliated Bci-NS1 and CFBE airway epithelial cells [ 21 , 56 ]. In contrast, SLC26A9 was not detected in airways of CF-patients homozygous for F508del-CFTR.…”

“…As pointed out above, airways of piglets lacking expression of CFTR (CFTR-/-) showed normal apical localization of SLC26A9, comparable to wild type animals. This suggests normal trafficking of SLC26A9 to the apical membrane in the absence of CFTR, while F508del-CFTR inhibits expression of SLC26A9 in the apical membrane [ 56 ]. In contrast to superficial airways, SLC26A9 was not expressed in submucosal glands, which makes sense since submucosal glands are the true place of secretion.…”

The SLC26A9 inhibitor S9‐A13 provides no evidence for a role of SLC26A9 in airway chloride secretion but suggests a contribution to regulation of ASL pH and gastric proton secretion

“…Sections or cells were incubated with rabbit anti‐SCL26A9 antibody (1:100, raised against mouse SCL26A9 aa 11–29, DRAAYSLSLFDDEFEKKDR, Davids Biotechnologie, Regensburg, Germany) overnight at 4°C. Nonspecific binding of the antibody to the airway epithelium was excluded in previous publications showing lack of binding in airways from patients carrying the F508del‐CFTR/F508del‐CFTR mutation, which causes a lack of apical expression of SLC26A9 24 . Antigen retrieval was performed in preheated Tris‐EDTA buffer (pH 9.0) for 15 min, using a microwave before blocking.…”

“…Nonspecific binding of the antibody to the airway epithelium was excluded in previous publications showing lack of binding in airways from patients carrying the F508del-CFTR/F508del-CFTR mutation, which causes a lack of apical expression of SLC26A9. 24 Antigen retrieval was performed in preheated Tris-EDTA buffer (pH 9.0) for 15 min, using a microwave before blocking. Cells were incubated with secondary anti-rabbit antibody conjugated with Alexa Fluor 488 or Alexa Fluor 546 (1:400) for 1 h at room temperature.…”