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

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.

Figure 1. Functional rescue of murine F508del CFTR in mouse ileum after low temperature incubation.

Pieces of normal and F508del mutant murine ileal mucosa were incubated for different times at 26°C. The same piece of tissue was subjected to short- circuit current measurements (Isc) at the start of the experiment (t = 0) and following incubation in supplemented William's E medium (4 h–14 h). A. Representative tracing showing temperature rescue (14 h at 26°C) of mutant F508del CFTR activity compared to 0 h. B. Time course of temperature rescue of ▪: ileum from homozygous F508del-Cftr mice, compared to □: WT ileum. Delta Isc represents the change in short-circuit current from baseline after addition of forskolin plus genistein. C. Overnight incubation (t = 14 h) of F508del CFTR ileal mucosa at low temperature. Bioelectric characteristics of F508del ileum at t = 0, (□) and after incubation at reduced temperature (26°C, t = 14 h, ▪). Delta Isc represents the change in short-circuit current from baseline after addition of carbachol (100 µM; S), STa (0.1 µM; M), 8-pCPT-cGMP (50 µM; S), bradykinin (2 µM; S), or ATP (100 µM; M+S). N = 4–6 mice, 2 ileal pieces/mouse. Bars represent averages +/− SD (N = 6 mice, 2 samples per mouse. * indicates significantly different from 0 h, P<0.01 by ANOVA).

Figure 2. Maturation of F508del protein at low temperature.

Pieces of ileal mucosa (∼5 cm) isolated from WT or homozygous F508del CF mice were mounted in large aperture tissue holders and incubated at 26°C for 14 h under carbogen gassing. Subsequently, epithelial cells were harvested by high-frequency vibration and a crude membrane fraction was isolated and subjected to SDS-PAGE (20 µg protein/lane). Wild type (WT) and F508del Cftr was detected by Western blotting using the R3195 (1∶1000) antibody. A representative result of four different experiments is shown. Band intensities were estimated by digital scanning (see Methods), showing an increase of F508del CFTR band C to approximately 25% (+/−5 SEM, N = 4) of wild type levels.

Figure 3. F508del CFTR is detectable by immunostaining after low temperature incubation.

Immunohistochemical staining of CFTR in ileum of wild type (WT, A and B) and homozygous F508del (F508del, C and D) mice prior to (A,C) and following low temperature incubation (t = 14 h, B, D). F508del CFTR is only detectable after low temperature incubation. Ileal sections of homozygous Cftr^tm1Cam (FVB) knockout mice were used as negatieve controls for the immunostaining (CF- KO, E). Magnifications: A, C, E: 400× and B, D: 200×.

Figure 4. Functional rescue of F508del CFTR in native ileal mucosa after treatment with proteasome inhibitors.

A. Representative Isc tracing of ileum from homozygous mutant F508del mice in the Ussing-chamber before (t = 0 h) and after treatment with the proteasome inhibitor ALLN (t = 6 h). Residual forskolin and genistein mediated chloride secretion at the start of the experiment (t = 0 h) was observed in all experiments. The dotted line indicates the average short circuit current level following forskolin/genistein addition for WT ileum (N = 24). ALLN treatment resulted in complete correction of CFTR activity compared to wild type. B. Ileal mucosa from F508del/F508del mutant mice was mounted in Ussing- chamber holders and incubated under carbogen gassing in William's E medium for 4 h at 37°C in the presence of vehicle (DMSO, 0.1%), MG-132 (10 µM), epoxomicin (1 µM ), PS341 (30 nM) or ALNN (20 µM). Subsequently the Isc response to forskolin plus genistein was measured in Ussing chambers. C. At the end of each measurement the viability of each sample was tested by addition of 10 mM glucose to the mucosal bath. The average glucose response after treatment with the various proteasome inhibitors is not significantly different from the vehicle control. All experiments were performed with N = 4 mice, two samples per mouse. Averages plus or minus SD are shown, statistical analysis was by one way ANOVA with Tukey post hoc test for multiple averages, *: indicates significantly different P<0.05 from vehicle).

Figure 5. Improved CFTR maturation after treatment with proteasome inhibitors.

Pieces of ileal mucosa (∼5 cm) isolated from homozygous F508del CF mice were mounted in large aperture tissue holders and exposed for 4 h (panel A) or 2–6 h (panel B) at 37°C to various PIs (see legend Fig. 4). Subsequently, epithelial cells were harvested by high-frequency vibration and a crude membrane fraction was isolated and subjected to western blot analysis of CFTR as described in Methods. Autoradiographs of 3 independent experiments were scanned, representative figures are shown here. Slots were loaded with equal amounts of protein to facilitate comparison. Panel A, left: dilution series of crude membranes derived from wild type mice (WT; 20, 10 and 4 µg protein/lane) used to calibrate the intensity of the wt CFTR signal. Panel A, right: crude membranes (20 µg protein) derived from homozygous F508del mice treated for 4 h with epoxomicin (1 µM); vehicle (0.1%DMSO), or MG-132 (10 µM). Average band C intensity increased from 5% (vehicle) to 20% (epoxomicin) and 15% (MG-132) of WT. Band B intensity increased twofold after epoxomicin and MG-132, compared to vehicle. Panel B: Crude membranes from wild type mice (WT; 20 µg protein/lane); F508del: crude membranes derived from homozygous F508del mice, untreated (lane 1); following low temperature incubation (26°C, t = 14 h) (lane 2); after treatment at 37°C with ALLN (20 µM) for 2 h (lane 3); 4 h (lane 4); and 6 h (lane 5). Band C/Band B ratio increased from 0.08 (2 h) to 0,36 (4 h) and 4,8 (6 hr).

Figure 6. Effect of proteasome inhibitors on CFTR immunostaining.

Histochemical staining of CFTR in ileum from FVB wild type mice (A); ileum from untreated homozygous Cftr^tm1Eur F508del mice (B); ileum from homozygous Cftr^tm1Cam knockout mice (C); PI-treated ileum from homozygous Cftr^tm1Eur F508del mice (D–G); ileum was treated for 4 h at 37°C with 1 µM epoxomicin (D), 10 µM MG-132 (E), 20 µM ALLN (F), or 30 nM PS341 (G). Panel H: Vehicle-treated (0.1% DMSO) ileum from homozygous Cftr^tm1Eur (FVB) F508del mice. Magnifications: 200×.

Figure 7. ALLN rescue of F508del CFTR function persists in the presence of brefeldin.

Short-circuit current responses to forskolin plus genistein were measured in stripped ileum from homozygous mutant Cftr^tm1eur mice (t = 0 h), and again after 4 h of incubation with ALLN as described in the legend of Fig. 4 (4 h). The tissues were then incubated for an additional 2 h in the presence of ALLN alone (closed circles), ALLN plus BFA (closed squares) or BFA alone (closed triangles). At t = 6 h the tissues were again analysed in the Ussing-chamber. Delta Isc represents the change in short-circuit current from baseline after addition of forskolin and genistein (average ±SEM of 3 experiments). All three averages at 6 hrs are significantly different (P<0.05 by one way ANOVA, with Tukey post hoc test for multiple averages).