Sphingosine kinase 1 regulates mucin production via ERK phosphorylation
Introduction
Mucus is a critical component of the innate host defense system in the respiratory tract. On the other hand, mucus hypersecretion is a major cause of airway obstruction in asthma and is sometimes associated with increased morbidity or mortality. Excessive mucus in asthmatic patients reflects over production of mucin due to goblet cell hyperplasia (GCH), that is a major pathological feature of asthma [1]. Airway GCH is especially marked in patients who die from asthmatic disease, with a 30-fold increase in the percentage of goblet cells compared with patients dying from non-asthmatic respiratory disease [2].
MUC5AC protein is a major component of airway mucus and is expressed in goblet cells of upper and lower respiratory tracts. Various stimuli such as lipopolysaccharide (LPS) [3], TNFα [4], and Th2 lymphocyte-derived cytokines, IL-4 [5] and IL-13 [6], [7] have been reported to induce MUC5AC both in vitro and in vivo. In mouse asthmatic models IL-13 is a central mediator of mucin production [8], [9], and mucin production induced by other Th2 cytokines is stimulated through IL-13 and the IL-13 receptor-mediated signals [10]. Moreover, it has been described that the downstream cascade of IL-13 involves signaling molecules such as signal transducer and activator of transcription 6 (STAT6), and mitogen-activated kinases (MAPKs) through epidermal growth factor receptor (EGFR) in airway epithelial cells [11], [12].
Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator of cellular functions such as proliferation, differentiation, apoptosis, tumor cell invasion, cell migration, and angiogenesis. Recently S1P has been shown to mediate asthmatic pathogenesis in vivo and in vitro. S1P levels are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after challenge with antigen and correlated with eosinophil numbers in the BAL fluid of asthmatic subjects [13]. S1P secreted by activated mast cells can promote allergic reactions by activating many types of immune cells including eosinophils, Th2 lymphocytes and neutrophils [14].
In a previous study, we demonstrated that inhibition of sphingosine kinase (SphK), a key enzyme that phosphorylates sphingosine to generate S1P, prevented airway mucin production and eosinophil inflammation [15]. We hypothesized that S1P produced by SphK may mediate airway mucin production in asthmatic pathogenesis. There is no information about the role of sphingosine metabolism in mucin production. In this study, we have clarified how SphK/S1P activation can affect the mucin production in human bronchial epithelial cells.
Section snippets
Materials
Recombinant human IL-13 was purchased from Strathmann Biotec (Hamburg, Germany). PD98059 and rabbit polyclonal antibodies against ERK1/2, phospho-ERK1/2, p38 MAPK, and phoshpo-p38 MAPK were from Cell Signaling Technology (Beverly, MA, USA). Anti-MUC5AC mouse monoclonal antibody was from Lab Vision (Fremont, CA, USA). N′,N′-dimethylsphingosine (DMS) and anti-β-actin mouse monoclonal antibody were from Sigma–Aldrich (St. Louis, MO, USA). Anti-STAT6 mouse monoclonal and anti-phospho-STAT6 rabbit
SphK1 expression in mouse asthmatic epithelium
We have previously demonstrated that SphK1 expression was increased in the bronchial epithelial walls of OVA-challenged asthmatic mice. Therefore, we hypothesized that SphK/S1P signaling may be involved in the molecular mechanisms underlying the etiology of airway mucin production in bronchial asthma.
To test this hypothesis, we first examined the localization of SphK and mucin proteins in mouse asthmatic models. After OVA inhalation for three consecutive days, C57BL/6 mice were sacrificed and
Discussion
Previous studies have demonstrated that SphK1 is expressed in airway epithelium, type II alveolar cells, serous glands, and endothelial cells of vessels in normal human lung tissue [15], [21]. We found that SphK1 was strongly stained in mouse airway epithelium treated with OVA and that SphK1 was co-localized with MUC5AC. On the other hand, SphK2 expression was not induced by IL-13 stimulation in bronchial epithelial cells. This observation is a novel finding to the best of our knowledge and
Conclusions
In summary we have demonstrated that SphK1 plays important roles in the regulation of IL-13 induced MUC5A production via ERK1/2 signaling pathway. These data also provide insights into novel mechanisms with potential therapeutic implications for mucin hypersecretion in asthmatic patients.
Acknowledgements
This work was supported by a grant-in-aid for Scientific Research (KAKENHI) from Japan Society for the Promotion of Science (JSPS18590848, Y. N., JSPS 21590965, Y. N.).
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