Gastroenterology

Gastroenterology

Volume 126, Issue 1, January 2004, Pages 32-41
Gastroenterology

Clinical-alimentary tract
The ΔF508 mutation results in loss of CFTR function and mature protein in native human colon

https://doi.org/10.1053/j.gastro.2003.10.049Get rights and content

Abstract

Background & Aims: Deletion of the codon for phenylalanine at position 508 (ΔF508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In heterologous cells, defective processing of the ΔF508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent plasma membrane Cl conductance. However, data with respect to the processing block of ΔF508 protein in native epithelia are limited and conflicting. Methods: To characterize both the fate and function of ΔF508 protein in a native epithelium, we measured CFTR-mediated Cl secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and ΔF508 homozygous patients with cystic fibrosis (CF). Results: Ussing chamber studies showed that cAMP-dependent and cholinergic Cl secretion was absent from rectal tissues freshly excised from ΔF508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not ΔF508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in ΔF508 homozygous tissues. Conclusions: Collectively, these data show that there is insufficient maturation and transport of ΔF508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl secretion in the CF colon.

Section snippets

Patients

Rectal biopsy specimens were studied from 12 ΔF508 homozygous patients with CF (ΔF/ΔF; mean age, 10.4 ± 2.8 years; range, 1 month to 32 years; 7 male patients and 5 female patients), 15 age-matched normal individuals (wt/wt; mean age, 8.9 ± 1.4 years; range, 2–22 years; 9 male patients and 6 female patients), and 8 ΔF508 heterozygous subjects (ΔF/wt; mean age, 8.8 ± 1.8 years; range, 3–18 years; 5 male patients and 3 female patients). All ΔF508 homozygous patients with CF had chronic lung

cAMP-dependent and cholinergic Cl secretion in normal and ΔF508 homozygous CF rectal tissues

In freshly excised normal colonic epithelia, CFTR-mediated secretion is at approximately 50% maximal activity under basal conditions due to endogenous cellular cAMP accumulation.22, 24 To assess the absolute magnitude of CFTR-mediated secretion, endogenous cAMP formation was inhibited by pretreatment with indomethacin (10 μmol/L, basolateral, 60 minutes) and electrogenic Na+ absorption was eliminated by exposure to amiloride (10 μmol/L, luminal). As previously reported for patients with CF with

Discussion

The elucidation of the molecular and cellular fate of ΔF508 CFTR in native epithelial tissues is crucial for the development of therapeutic strategies for the treatment of patients with CF. In contrast to studies of ΔF508 maturation and function in heterologous cells,5, 6, 7, 8, 9 the data describing ΔF508 maturation and function in native epithelia are discordant.12, 13, 14, 15, 16, 17, 18, 19 In an attempt to reconcile these conflicting data, we studied a native epithelium affected by CF

Acknowledgements

The authors thank the patients with CF as well as the volunteers for their participation in this study, Dr. Peter Greiner for performing rectoscopy procedures, Dr. Joachim Kuehr and Dr. Tanja Gonska for clinical evaluation of patients with CF, Dr. Sherif Gabriel for the kind gift of the MTE18 and MTE18-CFTR cells, Tracy Eldred and Kim Burns for tissue processing, Transgene (France) for the kind gift of CFTR antibody MATG1061, and Lisa Brown for editing of the manuscript.

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  • Cited by (0)

    Supported by the Mukoviszidose e.V. and the Deutsche Forschungsgemeinschaft (DFG MA 2081/2-1) (to M.M.), the Cystic Fibrosis Foundation (KREDAO0110) and the Mary Lynn Richardson Fund (to S.M.K.), and National Institutes of Health grants DK51870 (to J.R.R.) and HL34322 and HL60280 (to R.C.B.).

    1

    M.M. and S.M.K. contributed equally to this work.

    2

    J.R.R. and R.C.B. contributed equally to this work as senior authors.

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