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Published ahead of print on May 5, 2008, doi:10.1165/rcmb.2008-0065OC

Am. J. Respir. Cell Mol. Biol., Volume 39, Number 4, October 2008, 448-457

A more recent version of this article appeared on October 1, 2008
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Submitted on February 8, 2008
Revised on May 1, 2008

Activation of the Unfolded Protein Response by {Delta}F508 CFTR

Rafal Bartoszewski1, Andras Rab2, Asta Jurkuvenaite2, Marina Mazur3, John Wakefield4, James F Collawn2, and Zsuzsa Bebok2*

1 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA, 2 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA; University of Alabama at Birmingham, Cystic Fibrosis Research Center, Birmingham, Alabama, USA, 3 University of Alabama at Birmingham, Cystic Fibrosis Research Center, Birmingham, Alabama, USA, 4 Tranzyme Inc, Birmingham, Alabama, USA

* To whom correspondence should be addressed. E-mail: bebok{at}uab.edu.

Environmental insults and misfolded proteins cause ER stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient {Delta}F508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3{Delta}F cells that express different levels of endogenous WT and recombinant {Delta}F508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of {Delta}F508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and {Delta}F508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate {Delta}F508 CFTR mRNA levels. The results indicate that over-expression of {Delta}F508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and {Delta}F508 CFTR expressed within the same cell.




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