Buserelin alleviates chloride transport defect in human cystic fibrosis nasal epithelial cells

Abstract

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl- channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl- efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/F508del) were treated with buserelin and we show here that the treatment alleviates Cl- channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.

Fig 1. Basal mRNA and protein expression of GnRH-receptor (GnRH-R) in normal and CF HNEC.

A. The left panel shows representative PCR bands for GnRH-R (319 bp) expression (2% agarose gel). A single band is observed in control HNEC, CF HNEC and human pituitary gland (positive control, spliced for the final figure). No signal is observed in the negative control. The bar graphs (right panel) represents the quantitative analysis of the mRNA expression (n = 4). No difference was observed between normal HNEC (expressing Wt-CFTR) and CF HNEC (expressing F508del-CFTR). B. The left panel shows a representative immunoblot analysis of the expression of GnRH-R (64 kDa) and G3PDH (37 kDa) proteins, in CF and control HNEC. Molecular weight was spliced for the final figure. G3PDH was detected to show that the loading was identical in each lane and to normalize the expression of GnRH-R. A quantitative analysis was performed (n = 4) and is represented as bar graphs (right panel). A significant increased expression of GnRH-R was observed in CF HNEC, when compared to normal HNEC.

Fig 2. Characterization of the cell types and detection of GnRH-R in ALI cultures of HNEC.

A. Cells were cultured at ALI for 40 days and were stained using antibodies directed against β-tubulin, ZO-1 and Muc-5Ac in order to label ciliated cells (a), tight junctions (b) and goblet cells (C), respectively. B. mRNA and protein expression of GnRHR were assessed in control and in CF HNEC in ALI cultures. The left panel shows representative PCR bands for GnRH-R (319 bp) expression in HNEC ALI cultures. A single band was observed in control HNEC, CF HNEC and human pituitary gland (positive control). No signal was observed in the negative control. The right panel shows representative immunoblots of GnRH-R (64 kDa) and G3PDH (37 kDa) protein expression, in ALI culture. The receptor was detected in CF HNEC, control HNEC and in CFBE41o-/F508del (positive control) cells. For each cell type, 40, 80 and 120 μg were loaded. G3PDH was detected as a loading control. Molecular weight was spliced for the final figure.

Fig 3. Patch-clamp analysis of the chloride channel function of CFTR in CF HNEC in the presence of buserelin.

A. Representative current traces were recorded in non-treated CF HNEC at the basal level (a), in the presence of CFTR activators (Fsk/Gst) (b), in the presence of CFTR inhibitor (CFTR_inh172) (c). Current traces were recorded in buserelin treated CF HNEC at the basal level (d), in the presence of CFTR activators (Fsk/Gst) (e), in the presence of CFTR inhibitor (CFTR_inh172) (f). B. I/V curves with normalized currents by cell capacitance (pA/pF) are presented for CFTR basal current in CF HNEC, with and without buserelin treatment (1, 2 or 4 hours). C. Mean CFTR-related normalized current amplitudes were recorded at +80 mV and are presented as bar graphs. The statistical analysis indicated that CFTR-related currents were highly increased in the presence of buserelin (2 and 4 hours). Data are presented as mean ± S.E.M. for n = 4. Student’s t test was used to evaluate significant differences (***: p<0.001).

Fig 4. Measurement of transepithelial currents in CF HNEC by Ussing Chamber.

A. Example of representative traces of short-circuit currents (Isc) in air-liquid culture in CF HNEC, recorded in the absence (left panel) and in the presence (right panel) of buserelin (2h, 10^-12M). Amiloride (20 μM), forskolin (Fsk, 10 μM), genistein (Gst, 50 μM) and the CFTRinh172 (10 μM) were added sequentially. B. Bar graph representation for transepithelial Cl^- conductance (ΔIsc μA/cm²) in CF HNEC at air-liquid culture in low Cl^- Krebs bath, after buserelin treatment (10^-12M; 1, 2, 4 and 8h) with Fsk/Gst (left panel) and CFTRinh172 (right panel). Whereas transepithelial Cl^- conductance was increased at the 2 and 4h time points, it was aboslished at 8h. Data are presented as mean ± S.E.M. (n = 4). Student’s t test was used to evaluate significant differences (*: p<0.05, **: p<0.01).

Fig 5. cAMP measurement in control and CF HNEC, with or without Buserelin treatment (10⁻¹² M, 2h).

cAMP was assessed in control (n = 2, in triplicate) and in CF HNEC (n = 2, in triplicate). Results are expressed in pmol cAMP/mg of total proteins. Beside an observed variability between patients, cAMP was increased in CF cells after buserelin treatment. Data are mean ± S.E.M. Student’s t test was used to evaluate the significance (*: p<0.05).

Fig 6. Identification of differentially expressed proteins in CF HNEC after buserelin treatment (10^-12M, 2h).

Two-dimensional gel analysis of differentially expressed proteins in untreated (upper gel) and in treated CF HNEC (lower gel) was performed. 14 spots with increased or decreased intensities (PDQuest analyzis) were submitted to MS. 7 Spots in blue and red circles were down-regulated and up-regulated, respectively, after treatment and were further analyzed regarding their possible involvement in CF (Table 1).

Fig 7. Localization and expression of GnRH-R in HBEC.

A. Representative confocal images of HBEC co-stained by cell markers CK13 (mucous cells, left panel, red labelling) and Muc5AC (ciliated cells, middle panel, red labelling) and GnRH-R (green labelling). Co-distribution of GnRH-R (green labelling) and CFTR (red labelling) was observed in ciliated cells, in apical cell membranes. B. Representative immunoblot performed to assess GnRH-R expression in 6 different HBEC cultures.