ABCMdb

PMID: 17538928

Hayashi H, Sugiyama Y
4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps.
Hepatology. 2007 Jun;45(6):1506-16., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We previously reported that E297G and D482G BSEP, which are frequently found mutations in European patients, result in impaired membrane trafficking, whereas both mutants retain their transport function. Login to comment 3 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Because sodium 4-phenylbutyrate (4PBA) has been shown to restore the reduced cell surface expression of mutated plasma membrane proteins, in the current study, we investigated the effect of 4PBA treatment on E297G and D482G BSEP. Login to comment 4 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Transcellular transport and cell surface biotinylation studies using Madin-Darby canine kidney (MDCK) II cells demonstrated that 4PBA treatment increased functional cell surface expression of wild-type (WT), E297G, and D482G BSEP. Login to comment 7 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Conclusion: 4PBA treatment with a clinically achievable concentration enhances the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells, and also induces functional BSEP expression at the canalicular membrane and bile acid transport via canalicular membrane in vivo. Login to comment 8 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 4PBA is a potential pharmacological agent for treating not only PFIC2 patients with E297G and D482G mutations but also other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced. Login to comment 21 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 1506 ing junction mutations in the BSEP gene.1 Among them, E297G and D482G, two missense mutations in the second intracellular loop and in the first ATP-binding domain, respectively, are frequently observed in PFIC2 patients. Login to comment 23 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly However, both mutated BSEPs per se exhibit normal transport functions.10 Accordingly, restoration of the reduced cell surface expression of these mutated BSEPs is an important task for achieving the therapeutic goal for PFIC2 patients with E297G and D482G mutations. Login to comment 24 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Sodium 4-phenylbutyrate (4PBA) has been shown to be capable of restoring the reduced cell surface expression of cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) with the deletion of phenylalanine at 508 (CFTR ⌬F508).11 CFTR ⌬F508 is the most common mutation in cystic fibrosis patients12 and has similar features to E297G and D482G BSEP in that this mutated molecule accumulates in the endoplasmic reticulum (ER), followed by degradation in the proteasomes, but maintains its normal function as a chloride channel.13-15 4PBA is a nontoxic butyrate analog that was originally approved for clinical use as an ammonia scavenger in subjects with urea cycle disorders.16 Clinical trials of this agent in cystic fibrosis patients with ⌬F508 demonstrated that CFTR function in the nasal epithelia is induced by 4PBA therapy.17 Considering that a surgical procedure such as liver transplantation is the only therapy to cure PFIC2, this compound may offer a new medical therapy for PFIC2. Login to comment 26 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We evaluated the effectiveness and mechanism of action of 4PBA by biological and transport functional experiments with Madin-Darby canine kidney (MDCK) II cells expressing wild-type (WT), E297G, and D482G BSEP. Login to comment 36 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The BD Adeno-X Adenoviral Expression System (BD Biosciences, Palo Alto, CA) was used to establish the human WT, E297G, and D482G BSEP recombinant adenoviruses as previously described.10 The virus titer was checked with an Adeno-X Rapid Titer Kit (Clontech). Login to comment 39 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were seeded in six-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 200 multiplicity of infection (MOI). Login to comment 47 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly After 2 days` culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, the transcellular transport assays were performed as described.19 The apparent efflux clearance across the apical membrane (PSapical) was calculated by dividing the steady-state rate for the transcellular transport of [3H]TC determined over 2 hours by the cellular concentration of [3H]TC determined at the end of the experiments (2 hours). Login to comment 49 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 200 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, cell surface biotinylation was performed as described.10 When the degradation rate of the cell surface-resident protein was examined, biotinylated MDCK II cells were incubated for various periods at 37°C, with or without 1 mM 4PBA, before solubilization. Login to comment 52 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were seeded in six-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP, and GFP at 50 MOI. Cells were cultured for 24 hours after infection and subsequently treated with 1 mM 4PBA. Then, 24 hours after treatment, RNA was isolated using ISOGEN (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer`s instructions. Login to comment 57 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, and D482G BSEP at 200 MOI. Cells were cultured for 36 hours after infection and subsequently treated, with or without 5 ␮g/ml Act D (Sigma, St. Louis, MO), to inhibit mRNA synthesis and, with or without 20 ␮g/ml CHX (Sigma, St. Louis, MO), to inhibit protein synthesis. Login to comment 59 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Subsequently, 6 hours (E297G, D482G BSEP) or 8 hours (WT) after 4PBA treatment, the crude membrane fraction was prepared as described previously.19 The specimens were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment 61 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were seeded in 6-well plates at a density of 2.5 ϫ 105 cells per well. After a 24-hour culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, and D482G BSEP at 200 MOI. Cells were cultured for 12 hours after infection, and subsequently treated, with or without 5 ␮g/ml brefeldin A (BFA) (Sigma, St. Louis, MO), to inhibit the translocation of BSEP from ER to the Golgi complex. Login to comment 82 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G were treated with 4PBA for various periods and at various concentrations (Fig. 1A,B). Login to comment 84 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 4PBA treatment altered the expression level of the mature form of not only E297G and D482G BSEP but also WT BSEP in a concentration-dependent and time-dependent manner, the optimal condition being 1 mM for 24 hours. Login to comment 85 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The expression level of the mature form of WT, E297G, and D482G BSEP was increased 2.5-fold to 3-fold in response to 1 mM 4PBA treatment for 24 hours, which is a clinically achievable concentration.21-23 We then examined the increase in cell surface expression of BSEP by 4PBA treatment using transcellular transport assay and cell surface biotinylation methods. Login to comment 87 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Vectorial transport of [3H]TC in the apical direction was observed in MDCK II monolayers expressing WT, E297G, and D482G BSEP, and hardly detected in MDCK II monolayers expressing GFP. Login to comment 88 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Similar to hepatocyte, coexpression of Naϩ-taurocholate cotransporting polypeptide (NTCP), basolateral uptake transporter, and BSEP, apical efflux transporter, is needed to detect the vectorial transport of [3H]TC in the apical direction in LLC-PK1 and MDCK monolayers.19,24 The finding that the expression of BSEP alone is sufficient to induce the vectorial transport of [3H]TC in the apical direction suggests that MDCK II cells endogenously express uptake transporter for bile acids in the basolateral membrane as suggested.25 The basal-to-apical flux of [3H]TC across MDCK II monolayers expressing WT, E297G, and D482G BSEP was increased 1.5-fold, 2.5-fold, and 3-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours), whereas the increase in the basal-to-apical flux of [3H]TC was not detected in MDCK II cells expressing GFP. Login to comment 93 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G BSEP were treated for the indicated periods with 1 mM 4PBA before the preparation of crude membrane fractions. Prepared specimens (40 ␮g ) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment 95 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G BSEP were treated with the indicated 4PBA concentration for 24 hours before the preparation of crude membrane fractions. Prepared specimens (40 ␮g) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment 97 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly PSapical was increased in MDCK II cells expressing WT, E297G, and D482G BSEP, but not affected in MDCK II cells expressing GFP by 4PBA treatment. Login to comment 98 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly BSEP-dependent PSapical (PSapical, BSEP) in MDCK II cells expressing WT, E297G, and D482G BSEP, which was calculated by subtracting the PSapical value in MDCK II cells expressing GFP from that in MDCK II cells expressing WT, E297G, and D482G BSEP, was enhanced 1.7-, 3.0-, and 2.8-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours). Login to comment 100 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Cell surface expression of WT, E297G, and D482G BSEP was increased 1.8-fold, 3.1-fold, and 2.6-fold, respectively, by 4PBA treatment under optimal conditions (1 mM, 24 hours), whereas cell surface expression of exogenously expressing P-gp and endogenously expressing DPPIV was not affected (Fig. 2D). Login to comment 101 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The increase in cell surface expression of BSEP by 4PBA treatment was to an equivalent degree to that in PSapical, BSEP, suggesting that 4PBA treatment with a clinically achievable concentration can enhance the transport capacity of BSEP in MDCK II cells expressing WT, E297G, and D482G BSEP by increasing the cell surface expression of WT, E297G, and D482G BSEP. Login to comment 104 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly A possible mechanism is that 4PBA treatment promotes transcription or translation of WT, E297G, and D482G BSEP and consequently increases the cell surface expression of WT, E297G, and D482G BSEP by mass action. Login to comment 107 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly WT, E297G, and D482G BSEP mRNA expression levels were slightly increased by 4PBA treat- Fig. 2. Login to comment 109 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, D482G BSEP, and GFP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment 112 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Transcellular transport of 1 ␮M [3H]TC across MDCK II monolayers expressing WT, E297G, D482G BSEP, and GFP was examined as a function of time. Login to comment 118 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The clearance of the transport of [3H]TC across the apical membrane of MDCK II monolayers expressing WT, E297G, D482G BSEP, and GFP (PSapical) was determined as described in Materials and Methods. Login to comment 123 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ment, but the difference was not statistically significant [P ϭ 0.10 (WT), 0.20 (E297G, D482G)] (Fig. 3A). Login to comment 126 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The inhibition of transcription with Act D and translation with CHX did not affect the 4PBA-mediated increase in the mature form of BSEP in MDCK II cells expressing WT, E297G, and D482G BSEP. Login to comment 127 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These results indicate that a post-translational mechanism mainly contributes to the 4PBA-mediated increase in the cell surface expression of WT, E297G, and D482G BSEP. Login to comment 135 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment 140 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 6 hours (E297G, D482G BSEP) or 8 hours (WT BSEP), with or without 1 mM 4PBA, in the presence or absence of 5 ␮g/ml Act D (B), and 20 ␮g/ml CHX (C) before the preparation of crude membrane fractions. Prepared specimens (40 ␮g) were separated by 6% SDS-PAGE and subjected to western blot analysis. Login to comment 144 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing WT, E297G, and D482G BSEP were treated for 12 hours, with or without 1 mM 4PBA, in the presence or absence of 5 ␮g/ml BFA. Login to comment 151 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The expression level of the immature ER-resident form of WT, E297G, and D482G BSEP was almost identical in non- and 4PBA-treated MDCK II cells just after BFA washout (Fig. 4A; 0 hours), and that of the mature form was similar in non- and 4PBA-treated MDCK II cells until 3 hours after BFA washout, at which time the mature form of BSEP linearly increased. Login to comment 153 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly This result suggests that 4PBA treatment does not promote WT, E297G, and D482G BSEP maturation, but stabilizes the mature form of these proteins. Login to comment 156 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To examine whether 4PBA treatment can inhibit the cell surface-resident BSEP from entering the degradation pathway, we measured the degradation rate of the cell surface-resident BSEP using biotin-labeling methods in MDCK II cells expressing WT, E297G, and D482G BSEP. Login to comment 157 ABCB11 p.Glu297Gly The half-life of cell surface-resident WT and E297G BSEP was prolonged 1.8- and 2.5-fold, respectively, by 4PBA treatment, whereas those of exogenously expressing P-gp and endogenously expressing DPPIV was not affected (Fig. 5A,B). Login to comment 158 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The degradation rate of cell surface-resident D482G BSEP could not be determined under the same conditions as WT and E297G because of its low expression level. Login to comment 161 ABCB11 p.Glu297Gly (A) The degradation rate of cell surface-resident WT and E297G BSEP. Login to comment 162 ABCB11 p.Glu297Gly MDCK II cells expressing WT and E297G BSEP were treated for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment 165 ABCB11 p.Glu297Gly (B) Quantification of band density indicating WT and E297G BSEP in (A). Login to comment 166 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The intensity of the band indicating WT and E297G BSEP was quantified by Image Gauge software and expressed as a percentage of the BSEP present at 0 hours, respectively. Closed and open circles represent remaining cell surface WT BSEP or E297G BSEP in MDCK II cells treated, with and without 4PBA, respectively. Login to comment 168 ABCB11 p.Glu297Gly By the same methods as WT and E297G BSEP, the densitometric analysis was also performed for exogenously expressing P-gp and endogenously expressing DPPIV. Login to comment 169 ABCB11 p.Asp482Gly (C) Cell surface expression of D482G BSEP at a low temperature. Login to comment 170 ABCB11 p.Asp482Gly MDCK II cells expressing D482G BSEP were cultured for 24 hours at 27°C before the cell surface biotinylation. Login to comment 172 ABCB11 p.Asp482Gly (D) The degradation rate of cell surface-resident D482G BSEP. Login to comment 173 ABCB11 p.Asp482Gly MDCK II cells expressing D482G BSEP were cultured at 27°C for 24 hours, with or without 1 mM 4PBA, before the experiments. Login to comment 175 ABCB11 p.Asp482Gly (E) Quantification of band density indicating D482G BSEP in (D). Login to comment 176 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly The intensity of the band indicating D482G BSEP was quantified by Image Gauge software and expressed as a percentage of the BSEP present at 0 hours, respectively. Closed and open circles represent remaining cell surface D482G BSEP in MDCK II cells treated, with and without 4PBA, respectively. Login to comment 178 ABCB11 p.Asp482Gly D482G (Fig. 5C). Login to comment 179 ABCB11 p.Asp482Gly The cell surface expression of D482G increased 4.0-fold by 24-hour culture at a lower temperature (27°C). Login to comment 180 ABCB11 p.Asp482Gly We further examined the degradation rate of cell surface-resident D482G at 37°C after 24 hours` culture at a lower temperature (Fig. 5D,E). Login to comment 181 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The half-life of the cell surface-resident D482G BSEP was prolonged 3.3-fold by 4PBA treatment such as WT and E297G BSEP. Login to comment 182 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The prolonged half-life of the cell surface-resident WT, E297G, and D482G BSEP is consistent with the result of the BFA washout study, which suggests that 4PBA treatment stabilizes the mature form of BSEP (Fig. 4A,B). Login to comment 184 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 4PBA treatment can increase the functional cell surface expression of WT, E297G, and D482G BSEP in MDCK II cells (Fig. 2). Login to comment 205 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Discussion We previously reported that E297G and D482G BSEP, which are frequently found mutants in European patients, result in impaired membrane trafficking, whereas the transport function of both mutated BSEPs remains almost normal.10 Restoration of the reduced cell surface expression of these mutated BSEPs is a therapeutic goal for PFIC2 patients with E297G and D482G mutations, because both mutated BSEPs per se exhibit normal transport functions. Login to comment 206 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In the current study, we analyzed the potential therapeutic effect of 4PBA against PFIC2 by examining the effect of 4PBA treatment on the cell surface expression of E297G and D482G BSEP. Login to comment 208 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Initially, we examined the effect of 4PBA treatment on WT, E297G, and D482G BSEP using MDCK II cells. Login to comment 210 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Together with the finding that the increase in PSapical, BSEP by 4PBA treatment correlates with the cell surface expression of WT, E297G, and D482G BSEP (Fig. 2C), 4PBA treatment with a clinically achievable concentration can enhance the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment 214 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Furthermore, the expression levels of the immature ER-resident form of WT, E297G, and D482G BSEP were almost identical in non- and 4PBA-treated MDCK II cells just after BFA washout (Fig. 4A; 0 hours). Login to comment 215 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These results indicate that a post-translational mechanism mainly contributes to the 4PBA-mediated increase in the cell surface expression of WT, E297G, and D482G BSEP. Login to comment 216 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Although we investigated whether 4PBA treatment promotes the maturation of WT, E297G, and D482G BSEP as the possible post-translational mechanism, such an effect of 4PBA treatment was not observed in the BFA washout study (Fig. 4A,B). Login to comment 217 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly However, the results of this study showed that 4PBA treatment could stabilize the mature form of WT, E297G, and D482G BSEP (Fig. 4A,B). Login to comment 230 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly E297G, and D482G BSEP (Fig. 5A-E). Login to comment 231 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Because cell surface-resident BSEP constitutively cycles between the canalicular membrane and the intracellular compartment, and is finally removed from this cycle to the degradation pathway,30,31 4PBA treatment may interrupt the internalization process or promote the recycling process and, consequently, increase the cell surface expression of WT, E297G, and D482G BSEP. Login to comment 233 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Although the molecular mechanism of this effect remains unknown, taking into the consideration that the prolonged half-life with 4PBA treatment was only detected in cell surface-resident WT, E297G, and D482G BSEP, but not for other membrane proteins such as P-gp and DPPIV (Fig. 5B,E), 4PBA treatment possibly induces a specific interaction of WT, E297G, and D482G BSEP with adaptor proteins. Login to comment 238 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly If the prolonged half-life of cell surface-resident Bsep with 4PBA treatment is responsible for this result in vivo as well as in vitro, the protective effect of 4PBA treatment against hepatocellular damage by increasing biliary excretion of bile acids and lowering intrahepatic bile acids may be remarkably observed in PFIC2 patients with E297G and D482G mutations. Login to comment 249 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In conclusion, we demonstrated that 4PBA treatment with a clinically achievable concentration induces the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells and also enhances functional Bsep expression at the canalicuar membrane and bile acid transport via canalicular membrane in vivo. Login to comment 251 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These results suggest that 4PBA is a potential pharmacological agent not only for PFIC2 patients with E297G and D482G mutations but also for other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced. Login to comment