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Activation of the Unfolded Protein Response by F508 CFTR

Rafal Bartoszewski¹, Andras Rab^1,2, Asta Jurkuvenaite^1,2, Marina Mazur², John Wakefield³, James F. Collawn^1,2 and Zsuzsa Bebk^1,2

¹ Department of Cell Biology, and ² Cystic Fibrosis Research Center, University of Alabama at Birmingham, and ³ Tranzyme Inc., Birmingham, Alabama

Correspondence and requests for reprints should be addressed to Zsuzsa Bebk, MD, Department of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd., MCLM 760, Birmingham, AL 35294-0005. E-mail: bebok{at}uab.edu

Environmental insults and misfolded proteins cause endoplasmic reticulum (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 F508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3F cells that express different levels of endogenous wild-type (WT) and recombinant F508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of 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 F508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate F508 CFTR mRNA levels. The results indicate that overexpression of 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 F508 CFTR expressed within the same cell.

Key Words: endoplasmic reticulum stress • unfolded protein response • F508 CFTR • quantitative PCR • Calu-3

CLINICAL RELEVANCE Chronic inflammation may cause endoplasmic reticulum (ER) stress and activate the unfolded protein response. Studies regarding ER stress responses are important to understand the pathomechanism of these disorders.