Aquaporin 5 increases keratinocyte-derived chemokine expression and NF-κB activity through ERK activation
Introduction
Aquaporins (AQPs) are a family of transmembrane channels that allow rapid movement of water and, in some cases, small molecules across the plasma membrane [1], [2]. Currently, 13 mammalian AQPs (AQP0–12) have been identified [3]. Among them, AQP5 is selectively expressed in the apical plasma membrane of various secretory glands, such as the airway submucosal glands, and alveolar type I epithelial cells in the lungs [4], [5]. Targeted deletion of AQP5 in mice results in considerable reduction of fluid secretion from submucosal glands [6] and a 10-fold decrease of osmotic water permeability of the alveolar-capillary barrier in distal lungs [7]. These previous findings suggest that AQP5 plays a critical role in maintenance of normal lung water homeostasis, and changes in the amount of cell surface expression of AQP and/or its function may contribute to abnormal water metabolism in various lung diseases such as lung edema.
A recent study has indicated that AQPs regulate not only water metabolism, but also various cellular functions such as cell migration, proliferation, and invasion. For example, AQP3 expression in keratinocytes has been shown to facilitate migration in vitro, and targeted deletion of AQP3 in mice results in considerably delayed wound healing [8], [9]. Reduced migratory activity has also been reported in vascular endothelial cells from AQP1 null mice. In addition, AQP1 and/or AQP5 have been shown to increase tumor cell growth [10], [11], [12].
AQP5 exists on apical cellular membranes at immunologically active tissues such as submucosal glands and the alveolar epithelium in the lungs. Therefore, in the present study, we examined whether AQP5 affects inflammatory responses in terms of chemokine expression. We propose a new function of AQP5, which enhances chemokine expression through activation of ERK.
Section snippets
Reagents
TNF-α and MG132 were obtained from Sigma (St. Louis, MO). A rabbit anti-AQP5 antibody was from Millipore (Temecula, CA), and rabbit anti-phospho-p44/p42 mitogen-activated protein kinase (MAPK) (ERK1/2) (Thr202/Thr204), anti-p44/p42 MAPK (ERK1/2) and anti-IκB-α antibodies were from Cell Signaling Technology (Danvers, MA). PD98059, SB203580, U0126, actinomycin D, and tetraethylammonium chloride were from Wako (Saitama, Japan).
Cell culture and treatments
MLE-12 (immortalized murine lung epithelial) cells and NIH-3T3
Results
MLE-12 cells show high expression of AQP5 protein. To investigate whether AQP5 affects inflammatory responses, the cells were transfected with specific siRNA against AQP5, and then KC secretion induced by TNF-α was measured by ELISA. As a result, endogenous AQP5 expression was decreased in a dose-dependent manner (Fig. 1A). KC levels in TNF-α (20 ng/ml)-containing medium were significantly higher than those in control medium (Fig. 1B). Interestingly, AQP5 siRNA transfection resulted in
Discussion
KC is one of the most important mediators secreted from airway epithelial cells, which activates neutrophil influx into lung tissue [18], [19]. In the present study, we examined the effect of AQP5 on KC secretion and mRNA expression induced by TNF-α. All data presented here indicate that AQP5 increases KC expression in response to TNF-α. In MLE-12 cells, which highly express AQP5, knockdown of endogenous AQP5 resulted in a considerable decrease of both KC mRNA and protein expression induced by
Acknowledgments
This work was supported in part by a grant-in-aid for scientific research (23590109) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and grant-in-aid for the Cooperative Research Project from Institute of Natural Medicine, University of Toyama in 2012.
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