Abstract
Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.
Highlights
Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles
We used HT29-18N2 cells that differentiate into mucin producing cells after 6 days of incubation in protein-free medium as a means to unravel the requirements for mucin 2 (MUC2) and Mucin 5AC (MUC5AC) secretion [11]
Our results show that TRPM4, TRPM5, and Na؉/Ca2؉ exchanger 2 (NCX2) (SLC8A2 gene) are expressed in differentiated HT2918N2, whereas NCX1 and NCX3 (SLC8A1 and SLC8A3 genes, respectively) are not expressed in these cells (Fig. 1A)
Summary
We used HT29-18N2 cells that differentiate into mucin producing cells after 6 days of incubation in protein-free medium as a means to unravel the requirements for MUC2 and MUC5AC secretion [11]. To test the involvement of NCX in MUC5AC secretion, we applied a mixture containing 100 M ATP, 10 ng/ml of IL-13, and 300 nM PMA or a vehicle to CFT1-LC3 and NHBE cells (1 h at 37 °C) These exogenous stimuli promoted MUC5AC secretion from both NHBE– and CFT1-LC3– differentiated cells (Fig. 4, A and B, control bars, respectively) compared with cells treated with the vehicle alone. To test the requirement of extracellular calcium in MUC5AC secretion by differentiated CFT1-LC3 cells, the cells were treated with stimuli (100 M ATP, 10 ng/ml of IL-13, and 300 nM PMA) in the presence (1.2 mM CaCl2) or absence (0.5 mM EGTA) of extracellular Ca2ϩ (1 h at 37 °C). NCX inh., NCX inhibitor KB-R7943; TRPM4 inh., TRPM4 inhibitor 9-phenanthrol. *, p Ͻ 0.05; **, p Ͻ 0.01
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