TMEM16A channel function does not influence goblet cell numbers in the human airway epithelium
Henry Danahay1,2, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK.
A role for the calcium activated chloride channel, TMEM16A, in the regulation of airway goblet cell populations has been recently reported (Kondo et al., 2017; Lin et al., 2015; Qin et al., 2016). The published data to support a role for TMEM16A in driving the formation of goblet cells, are largely based on gene silencing and the use of non-specific TMEM16A blockers (e.g. niflumic acid). It is unclear whether this proposed function is therefore dependent on the ion channel activity of TMEM16A or through an alternative aspect of the proteins function. Using a potent TMEM16A channel blocker (Ani9) and a recently identified TMEM16A potentiator compound (Enterprise Therapeutics; proprietary), we have evaluated whether channel function can regulate goblet cell numbers in primary cultures of human bronchial epithelial (HBE) cells.
Primary HBE (3 donor codes; non-CF) were cultured for 2 weeks at air-liquid interface (ALI) on permeable supports and formed a well-differentiated mucociliary epithelium. On ALI day 15, cells were treated with either: 1) vehicle, 2) IL-13 (10 ng/mL) or ET003861 (1 µM) each group in either the absence or presence of the TMEM16A blocker, Ani9 (10 µM). ET003861 is a TMEM16A potentiator with an EC50 of 150-200 nM for the potentiation of chloride secretion in patch clamp (FRT-TMEM16A) and ion transport (HBE). Ani9 fully blocks TMEM16A function in both patch clamp and HBE ion transport studies. Cells were cultured under these conditions for 96h before fixation and staining with antibodies directed against MUC5AC (goblet cells) and acetylated α-tubulin (ciliated cells). Goblet and ciliated cell numbers were quantified using an automated image acquisition (Zeiss Axiovert) and analysis system (Image J).
IL-13 induced a significant increase in the density of goblet cells (7-25-fold depending on donor code) based on the increased staining for MUC5AC, that was unaffected by co-administration of Ani9. ET003861 also failed to influence goblet cell numbers and Ani9 was likewise without effect. Finally, the co-administration of ET003861 with IL-13 also failed to modify goblet cell numbers.
Together, these data do not support a role for the ion channel function of TMEM16A in the regulation of goblet cell numbers in primary HBE.
Kondo M et al. (2017) Clin Exp Allergy. 47(6):795-804.
Lin J et al. (2015) Exp Cell Res. 334(2):260-9.
Qin et al. (2016) Int Immunopharmacol. 40:106-114