Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.
Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4by extracellular ATP augments intracellular Ca2+signals and mucin secretion, whereas Ca2+signals and mucin secretion are dampened by inhibition of P2X4receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.
Ca2+-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described ‘fusion-activated’ Ca2+ entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca2+ signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca2+-binding to the C2 domains revealed that binding of Ca2+ to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca2+-dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase.
Exocytic post-fusion events play an important role determining the composition and quantity of cellular secretion. In particular, Ca2+-dependent regulation of fusion pore dilation/closure is a key regulator for fine-tuning vesicle content secretion. This requires a tight temporal and spatial integration of vesicle fusion with the PM, Ca2+ signals and translation of the Ca2+ signal into fusion pore dilation via auxiliary factors. Yet, it is still mostly elusive how this is achieved in slow and non-excitable secretory cells, where initial Ca2+ signals triggering fusions will abate before onset of the post-fusion phase. New results suggest, that the vesicles themselves provide the necessary itinerary to sense and link vesicle fusion to generation of local Ca2+ signals and fusion pore expansion.
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