Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Nov 15;100(10):2457–2465. doi: 10.1172/JCI119788

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

R C Rubenstein 1, M E Egan 1, P L Zeitlin 1
PMCID: PMC508446  PMID: 9366560

Abstract

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation.

Full Text

The Full Text of this article is available as a PDF (4.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bear C. E., Duguay F., Naismith A. L., Kartner N., Hanrahan J. W., Riordan J. R. Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J Biol Chem. 1991 Oct 15;266(29):19142–19145. [PubMed] [Google Scholar]
  2. Brown C. R., Hong-Brown L. Q., Biwersi J., Verkman A. S., Welch W. J. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones. 1996 Jun;1(2):117–125. doi: 10.1379/1466-1268(1996)001<0117:ccctmp>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brusilow S. W. Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion. Pediatr Res. 1991 Feb;29(2):147–150. doi: 10.1203/00006450-199102000-00009. [DOI] [PubMed] [Google Scholar]
  4. Carducci M. A., Nelson J. B., Chan-Tack K. M., Ayyagari S. R., Sweatt W. H., Campbell P. A., Nelson W. G., Simons J. W. Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate. Clin Cancer Res. 1996 Feb;2(2):379–387. [PubMed] [Google Scholar]
  5. Chen W. Y., Bailey E. C., McCune S. L., Dong J. Y., Townes T. M. Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5798–5803. doi: 10.1073/pnas.94.11.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng S. H., Fang S. L., Zabner J., Marshall J., Piraino S., Schiavi S. C., Jefferson D. M., Welsh M. J., Smith A. E. Functional activation of the cystic fibrosis trafficking mutant delta F508-CFTR by overexpression. Am J Physiol. 1995 Apr;268(4 Pt 1):L615–L624. doi: 10.1152/ajplung.1995.268.4.L615. [DOI] [PubMed] [Google Scholar]
  7. Cheng S. H., Gregory R. J., Marshall J., Paul S., Souza D. W., White G. A., O'Riordan C. R., Smith A. E. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 1990 Nov 16;63(4):827–834. doi: 10.1016/0092-8674(90)90148-8. [DOI] [PubMed] [Google Scholar]
  8. Collins A. F., Pearson H. A., Giardina P., McDonagh K. T., Brusilow S. W., Dover G. J. Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial. Blood. 1995 Jan 1;85(1):43–49. [PubMed] [Google Scholar]
  9. Cunningham S. A., Worrell R. T., Benos D. J., Frizzell R. A. cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA. Am J Physiol. 1992 Mar;262(3 Pt 1):C783–C788. doi: 10.1152/ajpcell.1992.262.3.C783. [DOI] [PubMed] [Google Scholar]
  10. Dalemans W., Barbry P., Champigny G., Jallat S., Dott K., Dreyer D., Crystal R. G., Pavirani A., Lecocq J. P., Lazdunski M. Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation. Nature. 1991 Dec 19;354(6354):526–528. doi: 10.1038/354526a0. [DOI] [PubMed] [Google Scholar]
  11. Denning G. M., Anderson M. P., Amara J. F., Marshall J., Smith A. E., Welsh M. J. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992 Aug 27;358(6389):761–764. doi: 10.1038/358761a0. [DOI] [PubMed] [Google Scholar]
  12. Dietz H. C., Hamosh A. Nonstop treatment of cystic fibrosis. Nat Med. 1996 Jun;2(6):608–609. doi: 10.1038/nm0696-608b. [DOI] [PubMed] [Google Scholar]
  13. Dover G. J., Brusilow S., Samid D. Increased fetal hemoglobin in patients receiving sodium 4-phenylbutyrate. N Engl J Med. 1992 Aug 20;327(8):569–570. doi: 10.1056/NEJM199208203270818. [DOI] [PubMed] [Google Scholar]
  14. Egan M. E., Schwiebert E. M., Guggino W. B. Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells. Am J Physiol. 1995 Jan;268(1 Pt 1):C243–C251. doi: 10.1152/ajpcell.1995.268.1.C243. [DOI] [PubMed] [Google Scholar]
  15. Egan M., Flotte T., Afione S., Solow R., Zeitlin P. L., Carter B. J., Guggino W. B. Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. Nature. 1992 Aug 13;358(6387):581–584. doi: 10.1038/358581a0. [DOI] [PubMed] [Google Scholar]
  16. Hamosh A., Rosenstein B. J., Cutting G. R. CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Hum Mol Genet. 1992 Oct;1(7):542–544. doi: 10.1093/hmg/1.7.542. [DOI] [PubMed] [Google Scholar]
  17. Howard M., Frizzell R. A., Bedwell D. M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat Med. 1996 Apr;2(4):467–469. doi: 10.1038/nm0496-467. [DOI] [PubMed] [Google Scholar]
  18. Jensen T. J., Loo M. A., Pind S., Williams D. B., Goldberg A. L., Riordan J. R. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell. 1995 Oct 6;83(1):129–135. doi: 10.1016/0092-8674(95)90241-4. [DOI] [PubMed] [Google Scholar]
  19. Li C., Ramjeesingh M., Reyes E., Jensen T., Chang X., Rommens J. M., Bear C. E. The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR. Nat Genet. 1993 Apr;3(4):311–316. doi: 10.1038/ng0493-311. [DOI] [PubMed] [Google Scholar]
  20. Maestri N. E., Brusilow S. W., Clissold D. B., Bassett S. S. Long-term treatment of girls with ornithine transcarbamylase deficiency. N Engl J Med. 1996 Sep 19;335(12):855–859. doi: 10.1056/NEJM199609193351204. [DOI] [PubMed] [Google Scholar]
  21. McGrath S. A., Basu A., Zeitlin P. L. Cystic fibrosis gene and protein expression during fetal lung development. Am J Respir Cell Mol Biol. 1993 Feb;8(2):201–208. doi: 10.1165/ajrcmb/8.2.201. [DOI] [PubMed] [Google Scholar]
  22. Morales M. M., Carroll T. P., Morita T., Schwiebert E. M., Devuyst O., Wilson P. D., Lopes A. G., Stanton B. A., Dietz H. C., Cutting G. R. Both the wild type and a functional isoform of CFTR are expressed in kidney. Am J Physiol. 1996 Jun;270(6 Pt 2):F1038–F1048. doi: 10.1152/ajprenal.1996.270.6.F1038. [DOI] [PubMed] [Google Scholar]
  23. Pasyk E. A., Foskett J. K. Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem. 1995 May 26;270(21):12347–12350. doi: 10.1074/jbc.270.21.12347. [DOI] [PubMed] [Google Scholar]
  24. Perrine S. P., Ginder G. D., Faller D. V., Dover G. H., Ikuta T., Witkowska H. E., Cai S. P., Vichinsky E. P., Olivieri N. F. A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders. N Engl J Med. 1993 Jan 14;328(2):81–86. doi: 10.1056/NEJM199301143280202. [DOI] [PubMed] [Google Scholar]
  25. Pind S., Riordan J. R., Williams D. B. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1994 Apr 29;269(17):12784–12788. [PubMed] [Google Scholar]
  26. Qu B. H., Thomas P. J. Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway. J Biol Chem. 1996 Mar 29;271(13):7261–7264. doi: 10.1074/jbc.271.13.7261. [DOI] [PubMed] [Google Scholar]
  27. Sato S., Ward C. L., Krouse M. E., Wine J. J., Kopito R. R. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem. 1996 Jan 12;271(2):635–638. doi: 10.1074/jbc.271.2.635. [DOI] [PubMed] [Google Scholar]
  28. Thomas P. J., Pedersen P. L. Effects of the delta F508 mutation on the structure, function, and folding of the first nucleotide-binding domain of CFTR. J Bioenerg Biomembr. 1993 Feb;25(1):11–19. doi: 10.1007/BF00768063. [DOI] [PubMed] [Google Scholar]
  29. Thomas P. J., Shenbagamurthi P., Sondek J., Hullihen J. M., Pedersen P. L. The cystic fibrosis transmembrane conductance regulator. Effects of the most common cystic fibrosis-causing mutation on the secondary structure and stability of a synthetic peptide. J Biol Chem. 1992 Mar 25;267(9):5727–5730. [PubMed] [Google Scholar]
  30. Trapnell B. C., Chu C. S., Paakko P. K., Banks T. C., Yoshimura K., Ferrans V. J., Chernick M. S., Crystal R. G. Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6565–6569. doi: 10.1073/pnas.88.15.6565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trapnell B. C., Zeitlin P. L., Chu C. S., Yoshimura K., Nakamura H., Guggino W. B., Bargon J., Banks T. C., Dalemans W., Pavirani A. Down-regulation of cystic fibrosis gene mRNA transcript levels and induction of the cystic fibrosis chloride secretory phenotype in epithelial cells by phorbol ester. J Biol Chem. 1991 Jun 5;266(16):10319–10323. [PubMed] [Google Scholar]
  32. Ward C. L., Omura S., Kopito R. R. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995 Oct 6;83(1):121–127. doi: 10.1016/0092-8674(95)90240-6. [DOI] [PubMed] [Google Scholar]
  33. Yang Y., Janich S., Cohn J. A., Wilson J. M. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9480–9484. doi: 10.1073/pnas.90.20.9480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zeitlin P. L., Lu L., Rhim J., Cutting G., Stetten G., Kieffer K. A., Craig R., Guggino W. B. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection. Am J Respir Cell Mol Biol. 1991 Apr;4(4):313–319. doi: 10.1165/ajrcmb/4.4.313. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES