Cross-linking of F508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴Key words: cystic fibrosis, endoplasmic reticulum, oligomer, processing mutation, curcuminGiven the frequency of the ΔF508 processing mutation and the severity of its corresponding phenotype, much research has focused on identifying compounds that restore the trafficking and function of this mutant at the plasma membrane. Several synthetic ‘correctors’ of ΔF508 mis-processing and ‘potentiators’ of mutant channel activity have been identified.^5,6 Natural compounds such as curcumin also have generated interest. Curcumin is an organic phenolic compound abundant in turmeric, an Indian spice extracted from the rhizome of Curcuma longa.⁷ Earlier studies performed using ΔF508/ΔF508 mouse models and human airway epithelial cell lines suggested that curcumin may act as a ΔF508-CFTR trafficking corrector.⁸ Also, we and others showed that curcumin stimulates CFTR channel activity in excised membrane patches.^9,10 This stimulation occurs in the absence of ATP binding, which is normally required for channel opening.¹⁰ Binding sites of correctors and potentiators within the CFTR polypeptide as well as the molecular mechanisms underlying the rescue of CFTR trafficking and function remain to be elucidated. In our attempt to understand how curcumin could circumvent the normally critical step of ATP binding to promote CFTR channel activity we investigated the effect of curcumin on CFTR conformation by using biochemical assays. We showed that curcumin caused dimerization of several CFTR channel constructs (including ΔF508-CFTR) in a dose- and time-dependent manner both in microsomes and within intact cells. This effect of curcumin on CFTR oligomerization is attributable to its reactive β-diketone groups, which may undergo an oxidation reaction with CFTR nucleophilic amino acid residues.¹¹ Importantly, CFTR channel activation by curcumin is unrelated to its cross-linking effect. We identified cyclic derivatives of curcumin that lack this cross-linking activity but still promote CFTR channel function.¹¹Here we examined the possibility that the cross-linking of ΔF508-CFTR channels by curcumin promotes the delivery of this ER processing mutant to the cell surface. We were motivated to test this possibility for three reasons: (i) our previous evidence that curcumin-induced dimers of wild-type CFTR polypeptides were detected at the cell surface where they remained over an hour after the removal of curcumin;¹¹ (ii) the very efficient cross-linking of the immature (ER) forms of wild-type CFTR and the ΔF508-CFTR mutant that we observed earlier¹¹ and (iii) prior evidence from our group that the ER export and cell surface delivery of ΔF508-CFTR polypeptides could be promoted by the co-expression of this mutant with certain CFTR fragments (trans-complementation).¹² The latter result might be due to the existence of ER retention ‘signals’ that are exposed on the ΔF508-CFTR polypeptide but become buried by interacting (complementing) fragments.Figure 1 provides evidence that ΔF508-CFTR oligomers that form in response to curcumin treatment do indeed appear at the surfaces of cultured airway epithelial cells (CF bronchial epithelial (CFBE) cells stably transfected with this CFTR mutant). Surface biotinylation assays were performed to detect the appearance of ΔF508-CFTR polypeptides at the cell surface. MESNA, a cell impermeant reducing agent that cleaves the biotin label, was used to verify the surface accessibility of the labeled ΔF508-CFTR polypeptides. ΔF508-CFTR polypeptides were precipititated with streptavidinagarose (surface pool) or with a CFTR monoclonal antibody (total pool). In the absence of curcumin treatment the great majority of the ΔF508-CFTR protein existed as the ER form (monomeric band B), as previously observed by many investigators (Fig. 1, lane 5). No band B was detected in the surface pool before or after curcumin treatment (Fig. 1, lanes 1, 2). As we reported earlier, treatment of the cells with 50 µM curcumin for 15 mins at 37°C cross-linked nearly all of the ΔF508-CFTR polypeptides into higher order complexes (e.g., dimers, termed band D here; lanes 6–8 in Fig. 1). Interestingly, these higher order forms of ΔF508-CFTR were readily apparent in the surface pool (Fig. 1, lane 2).Open in a separate windowFigure 1ΔF508-CFTR oligomers detected at the surfaces of airway epithelial cells after curcumin treatment. ΔF508-CFTR expressing CFBE cells were treated with curcumin (50 µM) for 15 min at 37°C. Cell surface proteins were then biotinylated (Sulfo-NHS-SS-Biotin, 1 mg/ml) for 30 min at 4°C followed by cell lysis with 1% Triton X-100. Surface proteins were isolated by streptavidin pulldown and ΔF508-CFTR was isolated from the total cell protein pool by immunoprecipitation with an anti-CFTR C-terminus antibody (clone 24-1, R&D systems). After SDS-PAGE the ΔF508-CFTR signal was detected by immunoblotting using the 24-1 antibody described above. (SP: streptavidin pulldown; IP: immunoprecipitation). As an additional control curcumin-treated cells were treated with the cell impermeant MESNA after biotinylation to strip the biotin off the cell surface proteins with which it had reacted.CFTR oligomers also can be generated by standard chemical cross-linkers such as DSS, as previously reported by others and confirmed by us.¹³ Figure 2 shows that oligomers of ΔF508-CFTR that are induced by DSS treatment also appear in the surface pool. These experiments were performed using transiently transfected HEK-293T cells with 30 µM curcumin as a positive control. Quantitative densitometry results are shown in Figure 3. By titrating the DSS concentration we observed a dose-dependent disappearance of the monomeric band B form, a corresponding increase in the band D (dimer) pool and the appearance of higher order oligomers (band E) which prevailed at higher DSS concentrations (see total cell pool data in right-hand). A small amount of the band D form was detected in the absence of DSS or curcumin treatment, which might represent some spontaneous cross-linking of ΔF508-CFTR polypeptides under these conditions. The DSS and curcumin-induced ΔF508-CFTR oligomers were readily detected in the surface pool. The densitometry analysis revealed that 20 ± 5% and 33 ± 19% of the total oligomer pool (combined bands D and E) was found in the surface pool after treatment with 0.1 mM DSS (n = 3) or 30 µM curcumin (n = 3), respectively, which corresponded to a 17 ± 7 and 26 ± 20 fold increase compared to the control condition (i.e., no DSS or no curcumin).Open in a separate windowFigure 2ΔF508-CFTR oligomers detected at the surfaces of HEK cells after DSS or curcumin treatment. ΔF508-CFTR expressing HEK cells were treated with the indicated concentrations of DSS or with 30 µM curcumin (*) for 15 min at 37°C. Cell surface proteins were then biotinylated and isolated by streptavidin pulldown as described above. ΔF508-CFTR was immunoprecipitated from the total cell protein pool with the 24-1 antibody and detected by immunoblotting as before (SP: streptavidin pulldown; IP: immunoprecipitation). Band B corresponds to ΔF508 monomer (ER form). Band D corresponds to ΔF508 dimer. Band E corresponds to a higher degree of ΔF508 oligomerization. Each panel corresponds to a different exposure of the same blot.Open in a separate windowFigure 3Dose-dependent expression of ΔF508-CFTR oligomers at the surfaces of HEK cells after DSS treatment. CFTR signals detected by the 24-1 antibody from three different experiments as the one described in Figure 2 were analyzed using the ImageJ software (from the National Institute of Health). (A) band B signal intensity is plotted as a function of the DSS concentrations. Signals analyzed correspond to ΔF508-CFTR band B immunoprecipitated by the 24-1 antibody. (B) band D plus band E signal intensities are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E immunoprecipitated by the 24-1 antibody. (C) band D plus band E signal intensities at the cell surface are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E isolated from the surfaces of ΔF508-CFTR expressing HEK cells by biotinylation and streptavidin pulldown. (D) the ratio between the amount of band E and D at the surfaces of ΔF508-CFTR expressing HEK cells is plotted as a function of the DSS concentration. Error bars are SEMs.Altogether these data indicate that the cross-linking of ΔF508-CFTR band B into oligomers by curcumin or DSS allows ΔF508-CFTR to traffic to the cell surface. This effect might be caused by the burial of ER retention motifs within the oligomer, which also could explain our previous trans-complementation results in which we observed that certain CFTR fragments promote the cell surface delivery of this processing mutant.¹² Although non-specific protein cross-linkers like DSS would not be therapeutically beneficial, more specific CFTR cross-linkers (perhaps curcumin?) may be worth considering for treating CF disease linked to ER processing mutations in CFTR. In this regard, we note that cross-linked CFTR polypeptides appear to retain chloride channel activity. Namely, in our prior excised patch clamp studies we observed stable CFTR channel activity when these patches were exposed to curcumin at doses and times that promote robust cross-linking of CFTR polypeptides.^10,11