Munc13‑4 mediates human neutrophil elastase‑induced airway mucin5AC hypersecretion by interacting with syntaxin2

Abstract

The overexpression and hypersecretion of mucus is a hallmark of chronic pulmonary inflammatory disease. Mucin5AC (MUC5AC) is a major component of airway gel‑forming mucin. Members of the Unc13 (Munc13) protein family act as important activators of granule exocytosis from various types of mammalian cells. The present study aimed to determine the role of Munc13 family proteins in MUC5AC secretion via an in vitro study with BEAS‑2B and Calu‑3 cell lines. Reverse transcription‑quantitative polymerase chain reaction and western blotting indicated that stimulation of the cells with 100 nM human neutrophil elastase (hNE) for 1 h did not affect the expression of either unc13 homolog B (Munc13‑2) or unc13 homolog D (Munc13‑4), but immunofluorescence analysis demonstrated that hNE treatment was associated with the recruitment of Munc13‑4 to the plasma membrane. Co‑immunoprecipitation analysis indicated increased binding between Munc13‑4 and syntaxin2 followingh NE stimulation; however, Munc13‑2 formed a stable interaction with syntaxin2 with or without hNE stimulation. Subsequently, Munc13‑2 and Munc13‑4 expression levels were downregulated in BEAS‑2B and Calu‑3 cells using small interfering RNA (siRNA). ELISAs and immunofluorescence analysis were performed to assess MUC5AC secretion and intracellular retention, respectively. Munc13‑2 siRNA transfection did not alter the expression levels of intracellular or secreted MUC5AC following hNE stimulation in either cell line; however, it increased the baseline intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC. Conversely, Munc13‑4 siRNA transfection increased the intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC following hNE stimulation, but did not affect their baseline quantities. The results of the present study indicate that Munc13‑2 may be an essential regulator of basal MUC5AC exocytosis, while Munc13‑4 appears to be a Munc13 protein subtype that may to be sensitive to hNE stimulation during airway MUC5AC hypersecretion.

Lentiviral Gene Therapy for Familial Hemophagocytic Lymphohistiocytosis Type 3, Caused by UNC13D Genetic Defects

“…31,32 Munc13-4 is part of the UNC13 family of proteins, all of which are involved in processes relating to vesicle exocytosis. Munc13-4 has been implicated in the exocytosis of lytic granules from cytotoxic cells, 8,33 tertiary, and mucincontaining granules from neutrophils, 34,35 dense granules from platelets, 36 and secretory granules from mast cells during regulated exocytosis. 37 Although much of HLH research has focused on pathology arising from impairment of CD8 T cell and natural killer cell cytotoxic function, 8 it is worth noting that FHL3 patients also suffer from susceptibility to fungal infection, 38 neurological symptoms, 39 exacerbated response to lung infection, 26,39 and macrophage activation syndrome, 26 which could be related to impaired degranulation from other cell types.…”