ABCMdb

PMID: 12359632

Zegarra-Moran O, Romio L, Folli C, Caci E, Becq F, Vierfond JM, Mettey Y, Cabrini G, Fanen P, Galietta LJ
Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07.
Br J Pharmacol. 2002 Oct;137(4):504-12., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07 *,1 Olga Zegarra-Moran, 1 Leila Romio, 1 Chiara Folli, 1 Emanuela Caci, 2 Frederic Becq, 3 Jean-Michel Vierfond, 3 Yvette Mettey, 4 Giulio Cabrini, 5 Pascale Fanen & 1 Luis J.V. Galietta 1 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, L.go G. Gaslini 5, Genova-16148, Italy; 2 Physiologie des Regulations Cellulaires, UniversiteÂ de Poitiers, 40 avenue du Recteur Pineau, 86022 Poitiers, France; 3 Laboratoire de Chimie Organique, FaculteÂ de MeÂ decine et de Pharmacie de Poitiers, 34 rue du Jardin des Plantes, 86005 Poitiers, France; 4 Laboratorio di Patologia Molecolare, Centro Regionale Fibrosi Cistica, Piazzale Stefani 1, Verona-37126, Italy and 5 Institut National de la Sante et la Recherche Medicale U.468, Service de Biochimie et Genetique, Hopital Henri Mondor, AH-HP, 94010 Creteil, France 1 This study compares the eect of three chemically unrelated cystic ®brosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. Login to comment 2 ABCC7 p.Gly551Asp The correction of G551D-CFTR defect was quanti®ed by comparison with maximal activity elicited in cells expressing wild type CFTR. Login to comment 3 ABCC7 p.Gly551Asp To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. Login to comment 5 ABCC7 p.Gly551Asp After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. Login to comment 6 ABCC7 p.Gly551Asp 4 Addition of genistein (10 ± 200 mM), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 ± 100 mM) or of the benzo[c]quinolizinium MPB-07 (10 ± 200 mM) to FRT-G551D epithelia pre-treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. Login to comment 8 ABCC7 p.Gly551Asp The dose-dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. Login to comment 9 ABCC7 p.Gly551Asp 6 On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. Login to comment 10 ABCC7 p.Gly551Asp The impressive eect of genistein suggests that it might correct the Cl7 transport defect on G551D patients. Login to comment 11 ABCC7 p.Gly551Asp British Journal of Pharmacology (2002) 137, 504 ± 512. doi:10.1038/sj.bjp.0704882 Keywords: CFTR; CF-G551D; monolayers; nasal epithelia; CFTR openers; genistein; CPX; MPB-07 Abbreviations: CF, cystic ®brosis; CFTR, cystic ®brosis transmembrane conductance regulator; CPT-cAMP, 8-(4-chlorophenylthio) adenosine-3'-5'-cyclic monophosphate; CPX, 8-cyclopentyl-1,3-dipropylxanthine; GFP, green ¯uorescent protein; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; DIDS, 4,4'-diisothiocyanato- stilbene-2,2'-disulfonic acid; FRT, Fisher rat thyroid; NBD, nucleotide binding domain. Login to comment 16 ABCC7 p.Gly551Asp Among the other mutations, the glycine-to-aspartic acid change at codon 551 (G551D) is the third commonest mutation with a worldwide frequency of 3.1% among CF chromosomes (Hamosh et al., 1992) although populations of Celtic descent display frequencies as high as 8% (Cashman et al., 1995). Login to comment 17 ABCC7 p.Gly551Asp The phenotype of patients carrying one F508del and one G551D allele is characterized by a clinical course as severe as that of homozygous for F508del but with a reduced risk of meconium ileus (Hamosh et al., 1992). Login to comment 18 ABCC7 p.Gly551Asp The G551D is a class III mutation that produces a protein that is well synthesized, processed and correctly inserted in the plasma membrane but with a strongly reduced channel activity (Welsh & Smith, 1993). Login to comment 19 ABCC7 p.Gly551Asp G551D is localized in the ®rst nucleotide binding British Journal of Pharmacology (2002) 137, 504 ± 512 ã 2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00 www.nature.com/bjp *Author for correspondence; E-mail: ozegarra@tin.it domain (NBD) of CFTR and interferes with ATP hydrolysis (Howell et al., 2000; Li et al., 1996; Logan et al., 1994). Login to comment 31 ABCC7 p.Gly551Asp Recent data indicate that the iso¯avone genistein is particularly eective on the G551D-CFTR Cl7 channel (Illek et al., 1999) and we showed that also the benzo[c]quinolizinium MPB-91 does activate this mutation (Derand et al., 2001). Login to comment 33 ABCC7 p.Gly551Asp This information is important for the development of improved CFTR activators eective on G551D and similar mutations. Login to comment 35 ABCC7 p.Gly551Asp Our results, obtained on a heterologous expression system, show that the three compounds are similarly eective on wild type CFTR but that only genistein is active on the G551D mutant. Login to comment 36 ABCC7 p.Gly551Asp This result is con®rmed on nasal monolayers obtained from a G551D CF patient, where the transport correction by genistein is as high as 20% that of wild type CFTR. Login to comment 62 ABCC7 p.Gly551Asp It has been well established that G551D mutation yields an amount of correctly processed protein similar to that of wild type CFTR (Logan et al., 1994; Welsh & Smith, 1993) therefore normalisation by CFTR mRNA relative levels (CFTR/ G3PDH ratio) was considered a good estimation of CFTR expression. Login to comment 65 ABCC7 p.Gly551Asp Two wild type (N8 and N10) and two G551D clones (6E and A1) were used in this study. Login to comment 69 ABCC7 p.Gly551Asp CF patient clinical feature and genotype, and nasal polyp cell cultures The CF patient is a 12-year-old female carrying the G551D mutation on one chromosome. Login to comment 70 ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Gly85Glu ABCC7 p.Gln552* The second mutation is presently unknown, but is not one of the 15 most frequent mutations found in the CF patients of Northeast Italy, namely F508del, I507del, R1162X, 2183AA4G, N1303K, 3849+10KbC4T, G542X, 1717-1G4A, R553X, Q552X, G85E, 711+5G4A, 3132delTG, 2789+5G4A, W1282X. Login to comment 89 ABCC7 p.Gly551Asp Results Northern blot and immunoprecipitation analysis of CFTR expression To assess the eect of dierent CFTR openers, we stably expressed wild type (wt) and G551D-CFTRs in Fisher rat thyroid (FRT) epithelial cells. Login to comment 92 ABCC7 p.Gly551Asp We considered two clones with wild type CFTR, labelled as N8 and N10, and two clones with G551D, A1 and 6E. Login to comment 96 ABCC7 p.Gly551Asp The G551D clones A1 and 6E showed a CFTR expression lower than N8, but considerably stronger than N10. Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The ratio of CFTR to G3PDH expression was 2.37, 0.12, 0.72 and 0.98 for wt-N8, wt-N10, G551D-6E and G551D-A1, respectively. Login to comment 101 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Response of wtand G551D-CFTR monolayers to cAMP elevating agents Application of the cAMP analogue CPT ± cAMP to intact FRT monolayers of cells transfected with wtor G551D-CFTR in symmetrical Cl7 was unable to stimulate a measurable Isc (Figure 2). Login to comment 108 ABCC7 p.Gly551Asp On monolayers expressing G551D-CFTR cells, CPT ± cAMP activated a Isc that was considerably lower. Login to comment 110 ABCC7 p.Gly551Asp Despite the dierence in the amplitude of Isc, the concentration that gave half of the maximal eect in G551D clones was 68.6+13.4 mM, not statistically dierent from that measured in wt-CFTR monolayers. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Response of wtand G551D-CFTR monolayers to genistein Apical application of genistein to wt-CFTR monolayers produced a sustained Isc increase (Figure 3). Login to comment 117 ABCC7 p.Gly551Asp (a) Northern analysis of RNA obtained from non transfected (NT) FRT cells and from cells stably transfected with G551D-CFTR (clones A1 and 6E) or with wt-CFTR (clones N8 and N10) CFTR. Login to comment 123 ABCC7 p.Gly551Asp (b) Immunoprecipitation of G551D- and wt-CFTR proteins was done using a monoclonal antibody that recognises the C-terminus of CFTR. Login to comment 124 ABCC7 p.Gly551Asp Fully glycosylated (band C, 175 kDa) and core glycosylated (band B, 150 kDa) CFTR can be observed in wild type and G551D clones, but not in non-transfected FRT cells (lane NT). Login to comment 128 ABCC7 p.Gly551Asp In contrast to wild type CFTR, the G551D mutant did not respond to genistein (10 ± 200 mM). Login to comment 135 ABCC7 p.Gly551Asp The currents activated by CPT ± cAMP and/or genistein in wild type and G551D-CFTR cells were totally blocked by 500 mM glibenclamide. Login to comment 140 ABCC7 p.Gly551Asp CPX alone was unable to activate G551D-CFTR. Login to comment 145 ABCC7 p.Gly551Asp Representative traces depicting the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) monolayers to 500 mM CPT ± cAMP and the inhibition caused by 500 mM glibenclamide. Login to comment 148 ABCC7 p.Gly551Asp (e) Dose-response relationships to CPT ± cAMP of wild type and G551D-CFTR monolayers. Login to comment 149 ABCC7 p.Gly551Asp Each point is the mean of 8 (wt) or 9 (G551D) experiments and vertical bars are s.e. Figure 3 Isc response to genistein. Login to comment 150 ABCC7 p.Gly551Asp Representative traces depicting the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) permeabilized monolayers to apical application of genistein. Login to comment 152 ABCC7 p.Gly551Asp (e) Dose-response relationship to genistein of wild type and G551D-CFTR epithelia after preactivation with 30 mM or 500 mM CPT ± cAMP, respectively. Login to comment 154 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Response of wtand G551D-CFTR to the benzo[c]quinolizinium MPB-07 Apical MPB-07 added to wt-CFTR monolayers after a low CPT ± cAMP concentration (30 mM) evoked a sustained Isc increase (126.3+17.4 mA cm72 , n=3, see Figure 5e). Login to comment 155 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Nevertheless, when MPB-07 was added without prestimulation it had no signi®cant eect on wtor on G551D-CFTR monolayers. Login to comment 157 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Relative efficacy of genistein, CPX and MPB-07 on G551D FRT cells To determine the relative eect of CFTR openers on the mutant CFTR, Isc activated on G551D cells in response to the dierent compounds in the presence of 500 mM CPT ± cAMP was compared with the response of wild type CFTR monolayers to CPT ± cAMP alone, which we assume as Figure 4 Isc response to CPX. Login to comment 158 ABCC7 p.Gly551Asp Traces depict the response of wt-CFTR (a and c, clone N8) and of G551D-CFTR (b and d, clone A1) permeabilized monolayers to apical addition of 100 mM CPX. Login to comment 162 ABCC7 p.Gly551Asp Traces depict the response of wt-CFTR (a and c, clone N10) and of G551D-CFTR (b and d, clone 6E) permeabilized monolayers to apical application of 200 mM MPB-07. Login to comment 166 ABCC7 p.Gly551Asp Mean of wt-CFTR epithelia were obtained pooling data from clones N8 and N10, and mean of G551D-CFTR epithelia were obtaining pooling data from clones 6E and A1. Login to comment 170 ABCC7 p.Gly551Asp Genistein (200 mM) increased the G551D-CFTR Isc from 0.8+0.3% to 32.2+4.4% of the wild type value (from 4.2+0.5 to 168.1+23.1 mA cm72 , see Figure 5e). Login to comment 174 ABCC7 p.Gly551Asp Response to CFTR openers of nasal G551D and non-CF primary cultures To strengthen the results obtained with transfected FRT monolayers, we decided to test the openers on human airway epithelial cells. Login to comment 175 ABCC7 p.Gly551Asp Primary cultures of nasal polyps were obtained from four non-CF subjects and from one G551D CF patient. Login to comment 176 ABCC7 p.Gly551Asp Application of 500 mM CPT ± cAMP to amiloride treated G551D epithelia elicited a small Isc increase of 0.20+0.05 mA cm72 (n=13). Login to comment 179 ABCC7 p.Gly551Asp In nasal G551D epithelia, genistein eect was also dose-dependent and the dose-response relationship was bell-shaped. Login to comment 182 ABCC7 p.Gly551Asp The response of G551D nasal epithelia to genistein required the presence of the cAMP analogue (see Figure 6d). Login to comment 183 ABCC7 p.Gly551Asp Neither CPX nor MPB07 elicited a signi®cant stimulatory eect on G551D epithelia despite a good eect on non-CF nasal monolayers (not shown). Login to comment 185 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Figure 6f depicts mean responses of non-CF and G551D-CF epithelia to cAMP and the correction of G551D monolayers obtained with 200 mM genistein. Login to comment 186 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Chronic stimulation with genistein To determine if chronic stimulation with genistein alters the electrophysiological characteristics and viability of epithelia, wtand G551D-FRT monolayers and CF and non-CF nasal epithelia were incubated for 48 h with genistein (50 or 200 mM). Login to comment 188 ABCC7 p.Gly551Asp For example, resistance and PD were 4.388+0.192 and 4.200+0.236 kO cm2 and 76.4+0.5 and 75+0.7 mV for control and genistein treated G551D-CFTR FRT monolayers. Login to comment 189 ABCC7 p.Gly551Asp For control and genistein treated G551D nasal epithelia, resistance and PD were 1.910+0.103 and 2.092+0.149 kO cm2 and 758+1 and 757+4 mV, respectively. Login to comment 197 ABCC7 p.Gly551Asp In the present study we compare the pharmacological properties of three major CFTR openers, namely genistein, CPX, and MPB- Figure 6 G551D and non-CF nasal polyps. Login to comment 198 ABCC7 p.Gly551Asp Representative traces showing the response of G551D nasal epithelia to CPT ± cAMP and genistein (a and c, respectively). Login to comment 207 ABCC7 p.Gly551Asp G551D is a relatively common CF mutation with the characteristic of being correctly processed and inserted in apical membrane, therefore a good target for a comparative pharmacological study. Login to comment 213 ABCC7 p.Gly551Asp We found that the maximal cAMP-dependent Isc in permeabilised G551D-CFTR was less than 1% of the response of wt monolayers. Login to comment 215 ABCC7 p.Gly551Asp Indeed, while CPX only doubled the conductance of G551D monolayers, genistein potentiated it to values that correspond to 7, 15 and 30% of wild type monolayers (with 50, 100 and 200 mM genistein, respectively). Login to comment 217 ABCC7 p.Gly551Asp Therefore, the results obtained in our FRT cell model indicate that genistein, but not CPX or MPB-07, might correct the G551D CF phenotype. Login to comment 218 ABCC7 p.Gly551Asp We recently found that MPB-91, an improved derivative of MPB-07, is able to increase the conductance of G551D transfected FRT monolayers. Login to comment 222 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Given that only one allele carries the G551D mutation in the nasal epithelia that we analysed, the recovery of 20% of CFTR function by 200 mM genistein is in good agreement with the 30% correction found in FRT cells which carry exclusively the G551D mutant. Login to comment 224 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Nevertheless, the pattern of response to genistein, including the shift of the dose-response relationship to higher concentrations, and to CPX and MPB-07, is similar in G551D nasal polyp and in G551D-transfected FRT cells. This suggests that the response to openers in the nasal polyp is mainly due to stimulation of G551D-CFTR. Login to comment 226 ABCC7 p.Gly551Asp The meaningful correction of G551D monolayers conductance with genistein supports recent data by Illek et al. (1999). Login to comment 227 ABCC7 p.Gly551Asp These authors observed with genistein an increase of isoproterenol-dependent nasal PD of G551D CF patients and calculated that the hyperpolarization obtained corresponded to 17% of the response of normal subjects. Login to comment 228 ABCC7 p.Gly551Asp The maximal eective genistein concentration on G551D-CFTR was far higher than that observed on wild type CFTR. Login to comment 229 ABCC7 p.Gly551Asp Indeed, it was 200 ± 300 mM in our experiments on G551D FRT and nasal epithelia while 30 ± 50 mM genistein is the maximal eective concentration observed on wild type CFTR in our experiments and in single cell measurements by other authors (Illek et al., 1995; Wang et al., 1998). Login to comment 232 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The most appealing explanation for the dierence between wild type and G551D-CFTR data, is that G551D mutant is displaying genistein-dependent activation, but the typical inhibition is shifted to much higher opener concentrations due to a dicult interaction with mutated NBD1. Login to comment 234 ABCC7 p.Gly551Asp If genistein is inhibiting K+ channels on our airway epithelia, this action seems much less important than the stimulatory eect on G551D-CFTR since the net result is a substantial increase on Cl7 secretion. Login to comment 238 ABCC7 p.Gly551Asp These measurements also con®rm that genistein alone does not activate G551D-CFTR in the absence of an intracellular cAMP increase (see Figure 3b) since CFTR activation would have reduced resistance of the monolayers. Login to comment 239 ABCC7 p.Gly551Asp In conclusion, our study shows that genistein is a particularly potent CFTR agonist able to correct the G551D functional defect to therapeutic levels in polarized preparations. Login to comment 243 ABCC7 p.Gly551Asp Our study suggests that ¯avonoid- based combinatorial libraries will be a promising source of potent and more selective derivatives that could show a signi®cant eect on G551D and similar CFTR mutants. Login to comment