CLCA1 Regulates Airway Mucus Production and Ion Secretion Through TMEM16A

Abstract

TMEM16A, a Ca²⁺-activated chloride channel (CaCC), and its regulator, CLCA1, are associated with inflammatory airway disease and goblet cell metaplasia. CLCA1 is a secreted protein with protease activity that was demonstrated to enhance membrane expression of TMEM16A. Expression of CLCA1 is particularly enhanced in goblet cell metaplasia and is associated with various lung diseases. However, mice lacking expression of CLCA1 showed the same degree of mucous cell metaplasia and airway hyperreactivity as asthmatic wild-type mice. To gain more insight into the role of CLCA1, we applied secreted N-CLCA1, produced in vitro, to mice in vivo using intratracheal instillation. We observed no obvious upregulation of TMEM16A membrane expression by CLCA1 and no differences in ATP-induced short circuit currents (Iscs). However, intraluminal mucus accumulation was observed by treatment with N-CLCA1 that was not seen in control animals. The effects of N-CLCA1 were augmented in ovalbumin-sensitized mice. Mucus production induced by N-CLCA1 in polarized BCi-NS1 human airway epithelial cells was dependent on TMEM16A expression. IL-13 upregulated expression of CLCA1 and enhanced mucus production, however, without enhancing purinergic activation of Isc. In contrast to polarized airway epithelial cells and mouse airways, which express very low levels of TMEM16A, nonpolarized airway cells express large amounts of TMEM16A protein and show strong CaCC. The present data show an only limited contribution of TMEM16A to airway ion secretion but suggest a significant role of both CLCA1 and TMEM16A for airway mucus secretion.

Keywords: CLCA1; KCNN4; MUC5AC; TMEM16A; airway epithelium; anoctamin 1; mucus.

Figure 1

N-CLCA1 increases mucus production in vivo. (A) Semiquantitative RT-PCR analysis of the expression of MUC5AC, TMEM16A, CFTR, SLC26A9 and KCNN4 in isolated tracheal epithelial cells from control mice and mice treated for 24 h with N-CLCA1. Mean ± SEM (number of animals/number of reactions). ^#—significant difference when compared to control or mock (p < 0.05; unpaired t-test). (B) Alcian blue staining indicating enhanced mucus secretion in airways exposed to N-CLCA1. Bar = 100 µm.

Figure 2

N-CLCA1 enhances mucus secretion in asthmatic mouse lungs in vivo. (A,B) Semiquantitative RT-PCR analysis of the expression of Tmem16a, Cftr, Slc26a9 and Kcnn4, and Muc5ac in lungs from control mice and OVA-treated mice. Expression of Tmem16a, Cftr, Kcnn4, and Muc5ac is enhanced in asthmatic (OVA) mice. Mean ± SEM (number of animals/number of experiments). ^#—significant difference when compared to control (p < 0.05; unpaired t-test). (C) Alcian blue staining of airways from OVA-sensitized mice treated with N-CLCA1 (upper panel) and OVA-sensitized mice without additional treatment with N-CLCA1 (lower panel). N-CLCA1 induced additional secretion of mucus with enhanced accumulation of the mucus in central and peripheral airways. Bar = 100 µm.

Figure 3

N-CLCA1 does not induce additional Ca²⁺-activated Cl⁻ secretion in mouse tracheas. (A,B) Original Ussing chamber recording of the transepithelial voltages measured in mouse trachea under open circuit conditions and summary of the calculated equivalent short circuit current (Isc’). No significant changes in amiloride-sensitive (10 µM) Na⁺ absorption was detected by 24 h exposure to N-CLCA1. (C,D) No significant changes in ATP (luminal; 100 µM)—induced negative voltage deflection and ATP-activated Isc’ was detected by N-CLCA1. (E,F) N-CLCA1 did not induce an effect of Ani9 (10 µM) or TRAM-34 (100 nM) on basal currents. (G,H) Effects of ATP in the presence of luminal Ani9 or basolateral TRAM-34 (T34) were independent of N-CLCA1. Mean ± SEM (number of animals/number of experiments). *—significant effect of amiloride and ATP (p < 0.05; paired t-test).

Figure 4

CLCA1 does not enhance expression of TMEM16A but stabilizes TMEM16A in the plasma membrane of Calu3 cells. (number of cover slips/number of cells)y. (A) Semiquantitative RT-PCR shows no increase in TMEM16A expression by exposure to N-CLCA1. (B) Western blot of TMEM16A indicates increase in expression by IL-13 but not by N-CLCA1. (C) Staining of TMEM16A in the plasma membrane indicates low expression in Calu3 cells. Exposure of Calu3 cells to IL-13 (20 ng/mL) increases expression of TMEM16A. N-CLCA1 does not increase the number of Calu3 cells expressing TMEM16A, but rather enhances membrane expression of TMEM16A in some cells. Bars = 50 µm. (D) Quantification of TMEM16A plasma membrane staining in the absence or presence of IL-13. (number of cover slips/number of cells). (E) Quantification of TMEM16A plasma membrane staining in the absence or presence of CLCA1. (number of cover slips/number of cells). Mean ± SEM ^#—significant difference when compared to control or control (p < 0.05; unpaired t-test).

Figure 5

TMEM16A supports upregulation of the master switch for goblet cell metaplasia, SPDEF. (A,B) RT-PCR analysis suggests inhibition of SPDEF expression by siRNA-knockdown of TMEM16A. (number of experiments). (C,D) Western blots indicating inhibition of SPDEF expression by inhibitors of TMEM16A. (number of experiments). Mean ± SEM. ^#—significant difference when compared to scrambled, control, and L-13, respectively (p < 0.05; unpaired t-test).

Figure 6

CLCA1-induced mucus production in polarized BCi-NS1 cells is TMEM16A-dependent. A,B) Western blot indicating upregulation of CLCA1 expression in BCi-NS1 cells exposed to IL-13 (20 ng/mL). Expression of TMEM16A was found to be attenuated in polarized grown cells (filter-grown ALI-cultures), when compared to nonpolarized (plastic-grown) cells. In contrast, CLCA1 expression was upregulated in ALI cultures. (number of experiments). (C,D) IL-13 does not enhance ATP-induced (100 µM) negative voltage deflections and activation of short circuit currents (Isc). Activation of Isc was fully transient, i.e., the current returned to the baseline. (E) The effect of the TMEM16A inhibitor Ani9 (10 µM) is not enhanced in IL-13 treated cells. (F,G) Individual examples and quantification of alcian blue staining in polarized BCi-NS1 cells under control conditions, after exposure to N-CLCA1 or in cells treated with N-CLCA1 and siTMEM16A. Mucus expression is enhanced in N-CLCA1-treated cells, but the effect of N-CLCA1 is blocked after knockdown of TMEM16A. (number of experiments). Scale bar = 50 µm. Mean ± SEM. ^# significant difference when compared to control (p < 0.05; unpaired t-test). * significant activation by ATP (p < 0.05; paired t-test).