Rescue of murine F508del CFTR activity in native intestine by low temperature and proteasome inhibitors

PLoS One. 2012;7(12):e52070. doi: 10.1371/journal.pone.0052070. Epub 2012 Dec 21.

Abstract

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • Chlorides / metabolism
  • Cold Temperature*
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis / metabolism
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Intestinal Mucosa / drug effects
  • Intestinal Mucosa / metabolism*
  • Intestines / drug effects*
  • Mice
  • Mice, Knockout
  • Mutation
  • Proteasome Inhibitors / pharmacology*

Substances

  • Chlorides
  • Proteasome Inhibitors
  • Cystic Fibrosis Transmembrane Conductance Regulator

Grant support

This work was supported by grants from the EU (grant QLG1-CT-2001-01005), the Cystic Fibrosis Foundation (CFF-USA; grant DEJONG04G0), The Dutch Stomach Liver –Intestine Society (MLD-Stichting: grant 6–11), and the Sophia- Foundation (grant number 506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.