Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.
BACKGROUND
Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.


OBJECTIVE
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.


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(inh)172), 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-alpha.


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.
Ahmad
Shama
S
Department of Pediatrics, National Jewish Medical and Research Center, A440, 1400 Jackson Street, Denver, CO 80206, USA. ahmads@njc.org
Ahmad
Aftab
A
Dremina
Elena S
ES
Sharov
Victor S
VS
Guo
Xiaoling
X
Jones
Tara N
TN
Loader
Joan E
JE
Tatreau
Jason R
JR
Perraud
Anne-Laure
AL
Schöneich
Christian
C
Randell
Scott H
SH
White
Carl W
CW
D017209:Apoptosis; D016022:Case-Control Studies; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
C505032:CFTR protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases