ABCMdb

PMID: 20398791

Kato T, Hayashi H, Sugiyama Y
Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11).
Biochim Biophys Acta. 2010 Sep;1801(9):1005-12. doi: 10.1016/j.bbalip.2010.04.002. Epub 2010 Apr 14., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. Login to comment 19 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We and other groups have indicated previously that the reduced cell surface expression of BSEP is responsible for the dysfunction of BSEP in PFIC2 patients with E297G or D482G mutation, both of which are observed frequently in European PFIC2 patients [12,14-17]. Login to comment 29 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We previously reported that 4-phenylbutyrate (4PBA), an approved drug for treating urea cycle disorders, is a promising agent for the treatment of intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of wild type (WT), and PFIC2-type (E297G and D482G) BSEP [18]. Login to comment 34 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Therefore, in this study, we investigated the potency of short-and medium-chain fatty acids against intrahepatic cholestasis by assessing the effects of these compounds on WT, E297G, and D482G BSEP expression and their transport functions using western blot analysis and transcellular transport, respectively. Login to comment 41 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Generation of recombinant adenovirus The BD Adeno-X Adenoviral Expression System (BD Biosciences) was used to establish the human WT, HA-WT, E297G, D482G BSEP and BCRP recombinant adenoviruses as described previously [3,14,22,23]. Login to comment 49 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly After 2 days of culture, confluent cells were infected by recombinant adenovirus containing cDNAs for WT, E297G, D482G BSEP or GFP at 20 MOI. Cells were cultured for 24 h after infection and subsequently treated with media containing 4PBA, formate, acetate, propionate, butyrate, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, or decanoic acid, each at 1 mM. Then, 24 h after treatment, transcellular transport assays were performed as described [18,24]. Login to comment 51 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly WT, E297G, and D482G BSEP-dependent PSapical (PSapical, WT BSEP-dependent, PSapical, E297G BSEP-dependent, and PSapical, D482G BSEP-dependent) were calculated by subtracting the PSapical value in MDCK II cells expressing GFP (PSapical, GFP) from that in MDCK II cells expressing WT, E297G, and D482G BSEP (PSapical, WT BSEP, PSapical, E297G BSEP, and PSapical, D482G BSEP), respectively. Login to comment 57 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Preparation of crude membrane fraction MDCKII cells were seeded in 12-well plates at a density of 2.5&#d7;105 cells per well. After 24 h of culture, confluent cells were infected by recombinant adenovirus containing cDNAs for E297G or D482G BSEP at 200 MOI. Login to comment 73 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Up-regulation of WT, E297G, and D482G BSEP-mediated transport by fatty acids We previously found that 4PBA treatment induces WT, E297G, and D482G BSEP expression at the cell surface by inhibiting the degradation of cell surface-resident WT, E297G, and D482G BSEP, and which facilitates the biliary excretion of bile acids. Login to comment 74 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly In this study, we first examined whether short-and medium-chain fatty acids, which have similar characteristics to 4PBA such as a carboxyl group and low-molecular-weight, can increase the cell surface expression of WT, E297G, and D482G BSEP and enhance WT, E297G, and D482G BSEP-mediated bile acid transport in a manner similar to that of 4PBA. Login to comment 80 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The effect of fatty acid treatment on transport function of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment 81 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly (A-C) MDCK II cells expressing WT (A), E297G (B), D482G BSEP (C), or GFP (A-C) were treated with formate, acetate, propionate, butyrate, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, or decanoic acid, each at 1 mM, for 24 h before the experiments. Login to comment 82 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Transcellular transport of 1 bc;M [3 H]TC across MDCK II monolayers expressing WT (A), E297G (B), D482G BSEP (C), or GFP (A-C) was examined. Login to comment 84 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The PSapical was expressed as the percentage of PSapical in MDCK II cells expressing WT (A), E297G (B), or D482G (C) BSEP under the untreated condition. Login to comment 85 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The closed and open bars represent WT (A), E297G (B), or D482G (C) BSEP- and GFP (A-C)-expressing MDCK II cells, respectively. Each bar represents the mean&#b1;S.E. of triplicate determinations. Asterisks represent statistically significant differences between the control and the compound-treated cells. Login to comment 89 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Butyrate and octanoic acid, the most potent short-and medium-chain fatty acids, respectively, also enhanced PSapical of [3 H]TC in MDCK II cells expressing E297G and D482G BSEP, both of which are the most frequently observed in European PFIC2 patients (Fig. 1B and C). Login to comment 90 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly PSapical, E297G BSEP-dependent and PSapical, D482G BSEP-dependent substantially increased with the treatment of butyrate and octanoic acid 8.4- and 3.1-fold in MDCK II cells expressing E297G BSEP and 3.6- and 1.7-fold in that expressing D482G BSEP, respectively. Login to comment 92 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Increased WT, E297G, and D482G BSEP expression after fattyacid treatment Results from a cell surface biotinylation study showed that WT BSEP expression at the cell surface of MDCK II cells increased 2.7-, and 6.1- Fig. 2. Login to comment 93 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The effect of octanoic acid and butyrate treatment on expression level of WT, E297G, and D482G BSEP in MDCK II cells. Login to comment 94 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly (A-C) Determination of expression level of HA-WT (A), E297G (B), and D482G BSEP (C). Login to comment 95 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells expressing HA-WT (A), E297G (B), or D482G BSEP (C) were treated with 4PBA, butyrate, or octanoic acid, each at 1 mM, for 24 h before the experiments. Login to comment 97 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Quantification of band density indicating the 170 kDa forms of HA-WT (A), E297G (B), or D482G BSEP (C) was also shown (right). Login to comment 105 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The expression levels of 170 kDa form of E297G and D482G BSEP in crude membrane fractions, which are considered to be the mature form located on the cell surface [14,18], were also elevated 3.1and 2.7-fold for E297G BSEP and 2.6- and 2.2-fold for D482G BSEP with the treatment of butyrate and octanoic acid, respectively (Fig. 2B and C). Login to comment 106 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Since it is difficult to detect E297G and D482G in cell surface fractions due to low expression, crude membrane fractions were used instead to determine the alteration in those expression levels. Login to comment 107 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The increased expression rates of WT BSEP on the cell surface and of the 170 kDa forms of E297G and D482G BSEP in crude membrane fractions induced by these three compounds correlated with those of WT, E297G, and D482G BSEP-mediated [3 H]TC transport (Fig. 3A-C). Login to comment 108 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly This suggests that all three compounds increase WT, E297G, and D482G BSEP-mediated transport by inducing WT, E297G, and D482G BSEP expression at the cell surface, but not by the activating WT, E297G, and D482G BSEP transport function itself. Login to comment 111 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Neither compounds had any effect on these expression levels (Fig. 2D), suggesting that both compounds do indeed increase the expression of WT, E297G, and D482G BSEP with high specificity. Login to comment 119 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Correlation between expression level and transport capacity of WT, E297G, and D482G BSEP under octanoic acid- and butyrate-treated conditions. Login to comment 120 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The WT, E297G, and D482G BSEP-dependent clearance of [3 H]TC across the apical membrane of MDCK II monolayers (PSapical, WT BSEP-dependent (A), PSapical, E297G BSEP-dependent (B), PSapical, D482G BSEP-dependent (C)) treated with 4PBA, butyrate, or octanoic acid was determined as described in 'Materials and methods` using the results of Fig. 1. Login to comment 121 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The PSapical, WT BSEP-dependent (A), PSapical, E297G BSEP-dependent (B), and PSapical, D482G BSEP-dependent (C) are expressed as the percentages of these values under the untreated condition. Login to comment 122 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The expression level of the 170 kDa forms of HA-WT BSEP in cell surface fractions and E297G and D482G BSEP in crude membrane fractions are taken from those in Fig. 2A-C. Login to comment 136 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We previously demonstrated that 4PBA is a potential therapeutic agent to combat intrahepatic cholestasis, which induces the cell surface expression of WT and PFIC2-type (E297G and D482G) BSEP by slowing its degradation rate from cell surface [18]. Login to comment 143 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Both butyrate and octanoic acid were also effective on E297G and D482G BSEP (Figs. Login to comment 148 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Although there is no evidence for the molecular mechanism responsible for the effect elicited by octanoic acid, based on our previous study of 4PBA [22], it is likely that octanoic acid reduces the susceptibility of ubiquitination of WT, E297G, and D482G BSEP, an important regulatory signal for degradation of these proteins on the cell surface [22], by inactivating the enzymes involved in the ubiquitination reaction and consequently also increases their expressions at the cell surface. Login to comment 149 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Another possible explanation is that octanoic acid directly stabilizes the cell surface-resident WT, E297G, and D482G BSEP and interrupts their ubiquitinations at the cell surface. Login to comment 151 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Because the mRNA expression level of WT BSEP itself was not significantly increased by butyrate treatment under the experimental conditions we used in this study (Fig. 5), it is possible that butyrate induces a gene expression that promotes the translation and/or inhibits the ER-associated protein degradation pathway, and subsequently increases the amount of WT, E297G, and D482G BSEP in Fig. 5. Login to comment 172 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The rapid degradation of BSEP/Bsep from the cell surface partly contributes to the reduced BSEP/Bsep expression at the cell surface in PFIC2 patients with E297G or D482G mutations and in rat models of endotoxin- or drug-induced cholestasis [10-17], suggesting that octanoic acid may combat intrahepatic cholestasis. Login to comment 174 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Since octanoic acid induced the expression of E297G and D482G BSEP, accompanied with the enhanced [3 H]TC transport by them (Fig. 3B and C), particularly, PFIC2 patients with E297G or D482G mutations are expected to benefit from its effectiveness. Login to comment 182 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Considering the effectiveness on E297G and D482G BSEP, a particular benefit to PFIC2 patients with either an E297G or D482G mutation is expected. Login to comment