The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II (original) (raw)
Expression of Bsep-GFP in MDCK and HEK 293 cells. To examine the cellular localization of the rat Bsep PFIC II mutants, we first created a fusion construct, Bsep-GFP, in which GFP was fused to the N-terminus of Bsep, and Bsep-GFP was expressed in MDCK cells. We chose MDCK cells, a well-characterized polarized kidney cell line, for our expression studies because many apical and basolateral proteins (e.g., P-glycoprotein and NaK-ATPase) have the same membrane localization in these cells as in hepatocytes.
In transiently transfected MDCK cells, transfection efficiency was low (less than 5%). However, the green fluorescent signal colocalized well with the immunostaining signal detected using an antibody against rat Bsep (Figure 2a, top row). GFP also colocalized with gp-135, an apical marker of MDCK cells (Figure 2a, center row), but was distinct from the basolateral marker, NaK-ATPase (Figure 2a, bottom row), as shown by immunostaining. This demonstrates that Bsep-GFP was exclusively expressed on the apical membrane surface of MDCK cells. No specific apical fluorescent signal was detected in nontransfected MDCK cells, and the green fluorescent signal was observed throughout the cytoplasm when only GFP was transfected into MDCK cells (data not shown). Our data showing that Bsep-GFP was expressed at the apical membrane of MDCK cells is consistent with the normal canalicular/apical localization of Bsep in hepatocytes and suggests that the added GFP tag did not affect the membrane localization of Bsep as it trafficked through the secretory pathways in MDCK cells.
Expression of Bsep-GFP in MDCK cells and HEK 293 cells. (a) After transient transfection with Bsep-GFP, MDCK cells were processed for immunofluorescence using antibodies against rat Bsep, gp-135, and NaK-ATPase, and imaged to locate the GFP fusion protein and apical and basolateral membranes of MDCK cells. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image. Bsep-GFP colocalized with gp-135 but was distinct from NaK-ATPase. Bar, 5 μm. (b) Bsep-GFP was expressed in either a stable MDCK cell line or HEK 293 cells by transient expression. Both en face and Z-sectioning images of the GFP fusion protein are presented. Bar, 5 μm. Total membrane and cytosol fractions were prepared from (c) MDCK cell lines either stably expressing Bsep-GFP or expressing GFP alone, or (d) from transfected and mock-treated HEK 293 cells. These fractions (100 μg protein per lane) and a rat liver plasma membrane preparation (LPM) (100 μg protein per lane) were separated on a 6.5% Laemmli gel, and Bsep-GFP was detected by Western blotting using an antibody against rat Bsep. The position of Bsep-GFP in the total membrane fraction of MDCK cells is indicated by the arrow. Rat Bsep and Bsep-GFP have apparent molecular weights of approximately 160 kDa and 190 kDa, respectively.
Because the low transfection efficiency of transient transfection made detailed analyses of Bsep-GFP expression difficult, we next created an MDCK cell line that stably expressed Bsep-GFP. In the stable cell line, Bsep-GFP was exclusively expressed at the apical surface of MDCK cells (Figure 2b). When the total membranes from the stable cell lines were analyzed by SDS-PAGE followed by immunoblotting, the rat Bsep antibody recognized a band with an approximate molecular weight of 190 kDa, which is 30 kDa larger than the rat Bsep (∼160 kDa) detected from a rat liver plasma membrane fraction (Figure 2c). This difference in molecular weight is consistent with the size of the GFP tag. The Bsep-GFP band was not detected in MDCK cells stably expressing GFP alone, nor was it detected in the cytosolic fraction of MDCK cells.
We also compared the expression of Bsep-GFP in nonpolarized kidney epithelial cells (HEK 293 cells). After transient transfection, Bsep-GFP was expressed over the entire plasma membrane of HEK 293 cells and Bsep-GFP expression in plasma membrane was higher in HEK 293 cells than in MDCK cells, reflecting higher efficiency of transfection (Figure 2b). Bsep-GFP was detected by immunoblotting as a 190-kDa protein, consistent with the size of Bsep-GFP expressed in MDCK cells (Figure 2d). Taken together, expression studies in both MDCK and HEK 293 cells showed that Bsep-GFP was synthesized with the expected molecular weight and trafficked to the apical membrane surface only when expressed in the polarized kidney cells.
PFIC II mutations have a heterogeneous effect on Bsep-GFP localization in MDCK cells. G238V, E297G, C336S, D482G, G982R, R1153C, and R1268Q are published mutations in human BSEP that are associated with PFIC II. To analyze whether these PFIC II mutations affect the membrane localization of Bsep, each mutation was introduced into Bsep-GFP and the mutant proteins were expressed in MDCK cells by transient transfection. Contrary to the apical expression of wild-type Bsep-GFP (Figure 3a), five of these PFIC II mutations, G238V, E297G, G982R, R1153C, and R1268Q, caused the protein to be sequestered within the cell (Figure 3, b, c, and f–h). The intracellular localization of these mutants was confirmed by immunostaining with antibody against gp-135, which did not colocalize with the green fluorescent signals (data not shown). In the cases of E297G, G982R, R1153C, and R1268Q, the green fluorescent signals were widely distributed throughout the cytoplasm. The level of expression was much lower in G238V, although this mutant was also detected in the cytoplasm rather than on the apical membrane. Surprisingly, mutation C336S did not alter the apical expression of Bsep-GFP (Figure 3d). In the cells expressing D482G, a significant green fluorescent signal was also detected at the apical surface as well as in the cytoplasm (Figure 3e). Other studies in polarized hepatic C2rev7 cells also showed that mutant C336S was expressed at the canalicular membrane of the C2rev7 cells in a manner similar to the wild-type Bsep-GFP (our unpublished observation).
PFIC II mutations have a heterogeneous effect on the localization of Bsep-GFP in MDCK cells. MDCK cells were transiently transfected with wild-type (WT) Bsep-GFP (a) or Bsep PFIC II mutants (b–h) for 72 hours and imaged by confocal microscopy. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image. Bar, 10 μm.
Ubiquitin/proteasome-mediated degradation plays a role in the expression of some PFIC II mutants. Several studies with other ABC transporters, such as cystic fibrosis transmembrane conductance regulator (CFTR) and copper-transporting P-type adenosine triphosphatase (ATP7B), have shown that intracellularly sequestered membrane proteins that resulted from mutation-induced misfolding were subsequently degraded by the ubiquitin/proteasome system (13–15). This seemed a particularly interesting possibility for G238V because this mutant protein was expressed rather poorly in MDCK cells. Thus we examined the involvement of proteasomes in the expression of Bsep PFIC II mutants by adding MG-132, a specific proteasome inhibitor, for different lengths of time to the culture medium of the MDCK cells transfected with PFIC II mutants (Figure 4, top row). The effect of MG-132 on the expression of wild-type Bsep-GFP in MDCK cells was also examined (Figure 4, bottom row).
After treatment with MG-132 for 2 hours, we did not observe any increase in fluorescent signal intensity or any change in localization of wild-type Bsep-GFP. In contrast, a 2-hour treatment with MG-132 increased the fluorescent signal for G238V about 60% compared with untreated cells (after quantification by summation of the fluorescent signals from a series of Z-sectioning images); aggregate formation began to be seen in G238V-expressing cells after the 2-hour treatment. The presence of MG-132 significantly increased the fluorescence intensity of mutant G238V seen in the cells after an 8-hour incubation and persisted after 12 hours (Figure 4, top row). The G238V mutant accumulated at several perinuclear positions, reminiscent of the protein aggregates seen in a previous study of a mutant of ATP7B, which is associated with Wilson disease (15). These data together suggest that G238V in MDCK cells was very unstable and that active ubiquitin/proteasome-mediated degradation played a role in the expression of this mutant in MDCK cells. Mutants E297G, G982R, R1153C, and R1268Q (data not shown) also accumulated into aggregates after incubation with MG-132, suggesting the involvement of the ubiquitin/proteasome system in their expression, but the increase in fluorescence intensities was less significant with these mutants than with G238V. After a 12-hour treatment with proteasome inhibitor, some wild-type Bsep-GFP was detected in perinuclear aggregate structures, with some of the Bsep-GFP still localized on the apical surface (Figure 4, bottom row, far right). This is not surprising since several studies have shown that GFP chimeras containing wild-type proteins expressed in HEK 293 cells can form an aggresome or aggregates when the cells are treated with proteasome inhibitors for several hours (14, 16).
Taurocholate transport function of PFIC II mutants. As described above, two mutations (C336S and D482G) did not affect the ability of Bsep to reach the apical membrane. Yet it is not clear whether C336S and D482G disturb the folded structure of Bsep in such a way that the bile salt transport function of Bsep is affected. Therefore we used a baculovirus expression system to characterize the transport function of these mutants. In contrast to C336S and D482G, G238V, E297G, G982R, R1153C, and R1268Q prevented the apical trafficking of Bsep-GFP in MDCK cells. We also examined whether these mutants were able to transport bile salt despite their inability to reach the cell surface when expressed in MDCK cells.
Bsep cDNAs carrying the seven PFIC II missense mutations were expressed in insect Sf9 cells using recombinant baculovirus. We manipulated the inocula of the baculovirus used in the experiment in order to achieve an expression level in all mutants that was comparable to that of wild-type protein. When total membranes from infected Sf9 cells were analyzed by SDS-PAGE followed by Western blotting, six of the PFIC II mutants were expressed at a level comparable to the wild-type rat Bsep as detected by the Bsep antibody. In contrast, G238V mutant protein was below the level of detection (Figure 5a).
PFIC II mutations exhibit heterogeneous effects on Bsep taurocholate transport activity. (a) Sf9 cell vesicles (100 μg) expressing Bsep or Bsep PFIC II mutant were analyzed by Western blotting as indicated. (b) ATP-dependent [3H]taurocholate transport was measured using membrane vesicles isolated from Sf9 cells expressing Bsep or PFIC II mutants. Vesicle uptake of [3H]taurocholate (2.5 μM) was determined in the presence and absence of ATP (5 mM). Data represent the mean ± SD of three determinations. The difference between taurocholate uptake measured in the presence and absence of ATP was defined as ATP-dependent taurocholate uptake.
To examine whether these PFIC II missense mutations affected the bile salt transport function of Bsep, total membrane fractions were prepared from the infected Sf9 cells and analyzed for ATP-dependent taurocholate uptake. As can be seen in Figure 5b, significant [3H]taurocholate uptake (∼110 pmol/mg protein/10 min) was observed in membrane vesicles isolated from Sf9 cells infected with wild-type rat Bsep cDNA. Very little ATP-dependent taurocholate uptake was seen in vesicles from mock-treated Sf9 cells. The E297G, G982R, R1153C, and R1268Q mutations reduced taurocholate uptake of Bsep to the level of that in membrane vesicles from mock-treated Sf9 cells (Figure 5b). Because G238V decreased Bsep expression to an undetectable level (Figure 5a), we were unable to measure the taurocholate uptake by this mutant. The membrane vesicles expressing D482G showed a decrease in taurocholate uptake compared with wild-type Bsep, while the vesicles expressing C336S showed no significant difference from wild-type Bsep (Figure 5b).
Since glycochenodeoxycholate and glycocholate rather than taurochenodeoxycholate and taurocholate are the major bile salt species in human bile, we also investigated whether glycochenodeoxycholate and glycocholate inhibited [3H]taurocholate transport in Sf9 vesicles expressing C336S mutant and wild-type Bsep (Table 1). Glycocholate had a small inhibitory effect on both the wild-type Bsep and C336S (inhibited less than 10% of taurocholate transport) in this assay, while taurochenodeoxycholate inhibited more than 90% of the transport. Glycochenodeoxycholate and taurocholate inhibited about 40–50% of the [3H]taurocholate transport by both wild-type Bsep and C336S. Thus the relative inhibitory effects of these bile salts on the C336S mutant and the wild-type rat Bsep in this study is similar to the relative affinities of these bile salts for the rat and human bile salt transporters (3, 17, 18, 19). The similarity of the inhibitory effects of these bile salts on the C336S mutant and wild-type rat Bsep is consistent with the finding that C336S in rat Bsep did not affect the transport of taurocholate by this protein (Figure 5b).
Inhibition of ATP-dependent taurocholate uptake