Cystic Fibrosis F508del Patients Have Apically Localized CFTR in a Reduced Number of Airway Cells

Abstract

SUMMARY:Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n ϭ 12), in 42% of cells from F508del carriers (n ϭ 20), and in 56% of cells from healthy individuals (n ϭ 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p Ͻ 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation. (Lab Invest 2000, 80:857-868).

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…CFTR protein, as detected by immunolocalization, was first reported in a few unidentified columnar cells within the superficial epithelium (Engelhardt et al, 1992(Engelhardt et al, , 1994 and more recently in a restricted population of columnar cells (Penque et al, 2000;Carvalho-Oliveira et al, 2004). We identified the ciliated cell as the only cell type expressing CFTR in nasal, bronchial, and proximal bronchiolar surface epithelia by high resolution LCM and coimmunostaining with antibodies against cellular markers (e.g., tubulin).…”

“…(Engelhardt et al, 1992(Engelhardt et al, , 1194Puchelle et al, 1992;Kalin et al, 1999;Penque et al, 2000). With the availability of very sensitive anti-CFTR mAbs (Mall et al, 2004) and LCM technologies, we initiated a comprehensive study to explore two major issues controversial in CF research: 1) the cell type specific localization of CFTR in the normal lung; and 2) the localization of ⌬F508 CFTR in native airway epithelium.…”

“…There are several possible reasons why our data and conclusions differ from some previous reports. First, our mAbs exhibit very high affinity for CFTR and hence may be less cross-reactive with other apical PM proteins in the CF lung compared with CFTR antibodies (Claass et al, 2000) used in previous studies (Kalin et al, 1999;Penque et al, 2000;Carvalho-Oliveira et al, 2004). Second, we applied multiple, complementary techniques to the same specimens.…”

The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

“…In contrast to wt and F508del/F508del cells, in which the CFTR protein could be detected at the apical membrane (Figure 3a), apical CFTR staining was significantly reduced in nasal epithelial cells derived from F508del/3905insT compoundheterozygous patients (Figure 3c; Table 1 Effect of CFTR 3905insT at mRNA and protein level J Sanz et al number of cells from F508del homozygous patients have apically localized CFTR (Figure 3b). 21,36,37 Recent studies indicated that the CFTR C-terminus is not required for the biosynthesis and plasma membrane targeting of CFTR, but indispensable for maintaining the stability of the protein. 38 Benharouga et al 39 could also show that an ERAD-similar mechanism involving the proteasome-ubiquitin pathway may be responsible for the faster turnover and the short residence time at the apical membrane of truncated CFTR.…”

VIP as a Corrector of CFTR Trafficking and Membrane Stability

“…The most common mutation in CF is the deletion of a phenylalanine residue at position 508 (F508) that causes improper folding of the CFTR protein, resulting in its retention in the endoplasmic reticulum and proteosomal degradation of the majority of the newly synthesized CFTR proteins (Cheng et al, 1990;Kartner et al, 1992;Penque et al, 2000). However, part of the F508-CFTR protein can still mature and reach the cell membrane where it retains some chloride channel function (Bronsveld et al, 2000;Penque et al, 2000;Kopito, 1999).any efforts on CF research are devoted to attempt to rescueF508-CFTR defective trafficking to restore normal epithelial function.…”