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Bcl-2 Suppresses Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPase Expression in Cystic Fibrosis Airways

Role in Oxidant-mediated Cell Death

¹ Department of Pediatrics, National Jewish Health, Denver; ² Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas; ³ Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, The University of North Carolina at Chapel Hill, North Carolina; and ⁴ Integrated Department of Immunology, National Jewish Health, Denver, Colorado

Correspondence and requests for reprints should be addressed to Shama Ahmad, Ph.D., Department of Pediatrics, National Jewish Medical and Research Center, A440, 1400 Jackson Street, Denver, CO 80206. E-mail: ahmads{at}njc.org

Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca²⁺ homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.

Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.

Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.

Measurements and Main Results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR_inh172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-.

Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.

Key Words: cystic fibrosis • SERCA2 • pulmonary epithelium • ER

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors are being tested to enhance epithelial Cl– transport; however, potential beneficial effects are unproven. The status of SERCA2 expression and activity in cystic fibrosis airway is unknown. What This Study Adds to the Field SERCA2 expression and activity are decreased in cystic fibrosis airway epithelium, resulting in enhanced susceptibility to oxidants.