Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Matthew C. Wolfgang,Nanette B. Fulcher,Carla M.P. Ribeiro,Wanda K. O'Neal,Mary E.B. Martino,John C. Olsen

doi:10.1074/jbc.m809180200

Matthew C. Wolfgang, Nanette B. Fulcher

Open Access

https://doi.org/10.1074/jbc.m809180200

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Abstract

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Highlights

Because total protein synthesis is increased in inflamed human bronchial epithelia (HBE) [2], and the increased synthesis of inflammatory mediators and defensive factors should result in an increased flux of newly synthesized, unfolded proteins into the endoplasmic reticulum (ER), we have hypothesized that airway epithelial inflammation alters ER homeostasis and triggers an ER stress response, the unfolded protein response (UPR) [2,3,4,5,6,7,8,9]
The relevance of X-box binding protein-1 (XBP-1) in airway inflammation was further investigated in native airway epithelia from ER stress-activated indicator (ERAI) mice exposed to the bacterial pathogen Pseudomonas aeruginosa and freshly isolated normal versus cystic fibrosis (CF) human bronchial epithelia
Airway Inflammation Activates the UPR in Vivo—We have reported that ER Ca2ϩ stores are expanded in native HBE harvested directly from infected/inflamed CF airways as compared with HBE harvested from uninfected/non-inflamed donors [1]

Summary

Freshly Isolated Human Bronchial Epithelia

Bronchial epithelial cells were isolated from main stem or lobar bronchi from 3 normal and 3 CF human lungs as previously described [1, 2] and immediate total RNA extraction was performed according to standard methods for determination of XBP-1 mRNA splicing

Pseudomonas aeruginosa Challenge of Mouse Airways

Calreticulin Staining in Murine Airway Epithelia

Assessment of Venus Fluorescence in ERAI Mice Airway Epithelia

Supernatant from Mucopurulent Material from CF Airways

RESULTS

DISCUSSION