Date Published: December 24, 2009
Publisher: Public Library of Science
Author(s): Mathieu Kerbiriou, Ling Teng, Nathalie Benz, Pascal Trouvé, Claude Férec, Dominik Hartl. http://doi.org/10.1371/journal.pone.0008436
Abstract: In cystic fibrosis (CF), the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER). We previously showed that the unfolded protein response (UPR) may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt) and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.
Partial Text: Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene –. The most common mutation in CF is a missing phenylalanine at position 508 (F508del-CFTR) in the first nucleotide-binding domain of the CFTR protein. The misfolded F508del-CFTR protein does not traffic correctly to the plasma membrane and is degraded by proteasome –. Nevertheless, some F508del-CFTR is retained in the endoplasmic reticulum (ER) . Beside the accumulation of F508del-CFTR in the ER, inflammation and infection are the major features of CF . Eukaryotic cells respond to the accumulation of misfolded proteins in the ER, to inflammation and infection by activating the unfolded protein response (UPR) –. Some lines of evidence suggest that UPR is triggered in F508del-CFTR expressing cells due to the mutated protein itself or by exogenous factors –. UPR induces the transcription of genes encoding ER chaperones, protein-folding enzymes and components of the ER-associated degradation system, limiting new protein synthesis –. Whereas it is an adaptive process aimed to restore the ER homeostasis, it may lead to apoptosis due to an increased intracellular calcium ([Ca2+]i) content followed by the activation of the calpain (Cal-1 and -2), caspase (Csp) -12 and Csp-3 cascade –.
The first step of the studied cascade is a sustained increase in intracellular free [Ca2+] . Therefore, we compared the basal free [Ca2+] between 16HBE14o- (wt-CFTR expressing cells) and CFBE41o- cells (F508del-CFTR expressing cells), using Fura-2 AM as a probe. No difference between 16HBE14o- and CFBE41o- cells regarding the basal [Ca2+]i was observed (Fig. 1). 16HBE14o- and CFBE41o- cells were further submitted to Tg treatment for 24, 30, 36 and 48 hours to induce ER stress. At the 24 hours time point the [Ca2+]i level was increased in both cell types indicating that the Tg treatment was efficient. The observed increased [Ca2+]i remained high until 48 hours. Nevertheless, the increased [Ca2+]i lower in CFBE41o- cells.
Mutations in the gene encoding CFTR are responsible for CF. The most common mutation F508del-CFTR, whose pathology is primarily due to a decrease in Cl permeability through the CFTR. The associated pathology is maintained by repeated lung infections, which provokes inflammatory responses that lead to lung fibrosis and respiratory failure in which lung necrosis is involved . Furthermore, infected epithelial cells expressing mutant CFTR are less sensitive to apoptosis than cells expressing normal CFTR . Because the released debris by cells undergoing necrosis initiate inflammatory response which is harmful in CF, it is important to understand why cells expressing mutant CFTR are more resistant to apoptosis than normal cells in the absence of infection. Consistent with this view, are the typical large DNA fragments of necrotic cells which are released by CF epithelia, increasing the viscosity of the mucus.