Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftr tm1Unc , cftr ؊/؊ ) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6 -11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr ؊/؊ mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr ؊/؊ mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A 2 . More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl Ϫ channels in the plasma membrane. ⌬F508-CFTR retains some ClϪ channel activity so increased expression of ⌬F508-CFTR in the plasma membrane can restore Cl Ϫ secretion deficiency. Recently, curcumin was shown to rescue ⌬F508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ⌬F508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ⌬F508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ⌬F508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ⌬F508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ⌬F508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ⌬F508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ⌬F508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments.With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Riordan et al., 1989). CF is characterized by abnormal chloride transport in many tissues, including lung, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive ducts. The most common mutation, ⌬F508, results in the production of an immature protein, which is retained in the endoplasmic reticulum (ER) for subsequent proteolytic degradation (Ward et al., 1995), preventing the correct localization of functional protein (Cheng et al., 1990;Ward and Kopito, 1994;Ward et al., 1995). However, it has been shown that, in certain tissues, in vivo, a small amount of ⌬F508-CFTR may reach the plasma membrane (Kalin et al., 1999) and function as a cAMP-activated Cl Ϫ channel but with a decreased chloride channel open probability compared with WT-CFTR (Dalemans et al., 1991;Benharouga et al., 2001).Among the different strategies that increase the delivery of ⌬F508-CFTR to the plasma membrane are low temperature, chemical chaperones, such as glycerol, and molecular chaperones, such as 4-phenylbutyrate (Buphenyl) or benzo[c-]quinolizinium components (MPB-07 and MPB-91) (for review see ...

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr ؊/؊ ). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr ؊/؊ mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr ؊/؊ mice. In an ex vivo assay, we found that the Ca 2؉ -dependent (carbacholstimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 M) was impaired in the distal colon of cftr ؊/؊ mice. These results support the conclusion that a ClCArelated function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.