Organic Anion Transporting Polypeptide (OATP) 1B3 is a Significant Transporter for Hepatic Uptake of Conjugated Bile Acids in Humans

Abstract

Background & aims: OATP1B3/SLCO1B3 is a human liver-specific transporter for the clearance of endogenous compounds (eg, bile acid [BA]) and xenobiotics. The functional role of OATP1B3 in humans has not been characterized, as SLCO1B3 is poorly conserved among species without mouse orthologs.

Methods: Slc10a1-knockout (Slc10a1^-/-), Slc10a1^hSLCO1B3 (endogenous mouse Slc10a1 promoter-driven human-SLCO1B3 expression in Slc10a1^-/- mice), and human SLCO1B3 liver-specific transgenic (hSLCO1B3-LTG) mice were generated and challenged with 0.1% ursodeoxycholic-acid (UDCA), 1% cholic-acid (CA) diet, or bile duct ligation (BDL) for functional studies. Primary hepatocytes and hepatoma-PLC/RPF/5 cells were used for mechanistic studies.

Results: Serum BA levels in Slc10a1^-/- mice were substantially increased with or without 0.1% UDCA feeding compared with wild-type (WT) mice. This increase was attenuated in Slc10a1^hSLCO1B3-mice, indicating that OATP1B3 functions as a significant hepatic BA uptake transporter. In vitro assay using primary hepatocytes from WT, Slc10a1^-/-, and Slc10a1^hSLCO1B3-mice indicated that OATP1B3 has a similar capacity in taking up taurocholate/TCA as Ntcp. Furthermore, TCA-induced bile flow was significantly impaired in Slc10a1^-/- mice but partially recovered in Slc10a1^hSLC01B3-mice, indicating that OATP1B3 can partially compensate the NTCP function in vivo. Liver-specific overexpression of OATP1B3 markedly increased the level of hepatic conjugated BA and cholestatic liver injury in 1% CA-fed and BDL mice. Mechanistic studies revealed that conjugated BAs stimulated Ccl2 and Cxcl2 in hepatocytes to increase hepatic neutrophil infiltration and proinflammatory cytokine production (eg, IL-6), which activated STAT3 to repress OATP1B3 expression by binding to its promoter.

Conclusions: Human OATP1B3 is a significant BA uptake transporter and can partially compensate Ntcp for conjugated BA uptake in mice. Its downregulation in cholestasis is an adaptive protective response.

Keywords: Bile Acid Transporter; Cholestasis; OATP1B3; Proinflammatory Cytokine.

Graphical abstract

Figure 1

Generation and characterization of Slc10a1^-/-and Slc10a1^hSLCO1B3mice. (A) A schematic diagram of the strategy for generation of Slc10a1^-/- mice. (B) Genotyping of Slc10a1^-/- mice. Genomic DNA was extracted from mouse tails and analyzed by PCR, followed by agarose gel electrophoresis. (C) A schematic diagram of the strategy for generation of Slc10a1^hSLCO1B3 mice. (D) Genotyping of Slc10a1^hSLCO1B3 mice. (E) Western blot analysis of the expression of OATP1B3 and Ntcp in different strains of mice. (F) The levels of intracellular [³H]-TCA in primary hepatocytes from WT, Slc10a1^-/-, and Slc10a1^hSLCO1B3 mice were analyzed by [³H]-TCA uptake after these cells were treated with 1 pM [³H]-TCA for 30 min (n = 12 per group). (G) Bile flow was measured in WT, Slc10a1^-/-, and Slc10a1^hSLC01B3 mice with the tail vein injection of TCA. Bile samples were collected every 10 minutes and continued for up to 120 minutes following TCA (30μmol/L) infusion (n = 3 per each group). ∗P < .05 vs the WT group at the same timepoint. Slc10a1^-/-, Slc10a1 knockout mice; Slc10a1^hSLCO1B3, Slc10a1^{hSLCO1B3/hSLCO1B3}Slc10a1^-/- homozygous mice with both endogenous Slc10a1 promoter-driven human SLCO1B3 expression and Slc10a1 knockout; WT, wild-type C57BL/6 mice.

Figure 2

Generation and characterization of hSLCO1B3 LTG. (A) A schematic diagram of the hSLCO1B3 LTG construct. (B) Genotyping of hSLCO1B3 LTG mice. Genomic DNA was extracted from mouse tails and analyzed by PCR, followed by agarose electrophoresis. (C) Western blot analysis of the distribution of OATP1B3 protein expression in hSLCO1B3 LTG mice. (D) HPLC/MS analysis of conjugated BA and (E) unconjugated BA levels in liver tissues from WT and hSLCO1B3 LTG mice after feeding them with 1% CA for 14 days (n = 7 for each group). hSLCO1B3 LTG, human SLCO1B3 liver-specific transgenic mice.

Figure 3

Human OATP1B3 liver-specific overexpression aggravates cholestatic liver injury in BDL mice. (A) Representative gross appearance of the liver. (B) Hematoxylin and eosin staining of liver morphology. (C) Liver histologic assessments, including scores for inflammation, necrosis, fibrosis, and bile-duct proliferation. The histologic assessments were blinded and assessed by expert pathologists. BDL, Mice receiving bile duct-ligation; hSLCO1B3 LTG, human SLCO1B3 liver-specific transgenic mice; Sham, mice receiving a sham operation; WT, wild-type mice. ∗P < .05 vs the Sham-WT group; #P < .05 vs the Sham-hSLCO1B3 LTG group; $P < .05 vs the BDL-WT group (n = 5–9 per group).

Figure 4

Human OATP1B3 liver-specific overexpression disturbs hepatic expression of genes for the adaptive response to cholestasis in BDL mice or 1% CA-fed mice for 14 days. The livers in BDL or 1% CA-fed mice were sampled and the relative levels of gene mRNA transcript in individual liver samples were quantitatively analyzed by RT-qPCR. The relative levels of liver mRNA transcripts of (A, G) canalicular membrane transporters of Bsep, Mrp2, Mdr1a, Mdr1b, and Mdr2; (B, H) basolateral membrane transporters of Mrp3, Mrp4, Ostα/β, Ntcp, and Oatp1a1; (C, I) BA synthesis and detoxification enzymes of Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, Cyp2b10, and Cyp3a11; (D, J) nuclear receptors of Fxr, Shp, Rxrα, and Rarα; (E, K) cytokines of Tnfα, Il1β, Il6, Icam1, and Mcp1; and (F, L) fibrotic and bile duct cell proliferation genes of α-Sma, Cola1, Col1a2, Mmp2, Mmp9, Ck19, Tgfβ1, Tgfβ2, and Tgfβ3. (The value in the WT sham group was designated as 1). TG-BDL, human SLCO1B3 liver-specific transgenic mice with bile duct-ligation; TG-1% CA, human SLCO1B3 liver-specific transgenic mice fed with 1% CA diet group; TG-CTR, human SLCO1B3 liver-specific transgenic mice with control diet group; TG-Sham, human SLCO1B3 liver-specific transgenic mice receiving sham procedure; WT-BDL, wild-type bile duct-ligation group; WT-1% CA, wild-type mice fed with 1% CA diet group; WT-CTR, Wild-type control diet group; WT-Sham, wild-type sham group. ∗P < .05 vs the WT sham or control diet group; #P < .05 vs the hSLCO1B3 LTG sham group; $P < .05 vs the WT-BDL or 1% CA diet group. (n = 3–8 per group).

Figure 5

Hepatic OATP1B3 downregulation is associated with increased levels of serum proinflammatory cytokine IL-6 in patients with OC. (A) RT-qPCR analysis of the relative levels of SLCO1B3 mRNA transcripts in control (n = 13) and patients with OC (n = 20). (B) Representative Western blots of hepatic OATP1B3 protein expression. C: controls; OC: obstructive cholestasis. ∗P < .05 vs the controls. (C) The levels of serum IL-6, IL-1β, TNFα, and IL-8 in patients with OC (n = 39) and controls (n = 13). (D) The levels of SLCO1B3 mRNA transcripts in PLC/RPF/5 cells after treatment with cytokines (100 ng/mL) or BAs (100 μM). ∗P < .05 vs CTR (control). (E) IL-6 reduced SLCO1B3 mRNA expression in a dose-dependent manner in PLC/RPF/5 cells. ∗P < .05 vs control (0 ng/mL). (F) Treatment with IL-6 also reduced OATP1B3 protein expression in PLC/RPF/5 cells in a dose-dependent manner. n = 3; ∗P < .05 vs control (0 ng/mL).

Figure 6

The activation of IL-6/STAT3 signaling transcriptionally downregulates OATP1B1 expression in hepatocytes. (A) Western blot analysis of the relative levels of hepatic STAT3 expression and phosphorylation in patients with OC (n = 20) and control patients (n = 13). ∗P < .05 vs controls. (B) IL-6 induced STAT3 phosphorylation and (C) nuclear STAT3 protein expression in PLC/RPF/5 cells in a dose-dependent manner. n = 3; ∗P < .05 vs control (0 ng/mL). (D) Dual luciferase reporter assays revealed that treatment with IL-6 and STAT3 o/e attenuated the SLCO1B3 promoter activity (pGL3-SLCO1B3-1967 or pGL3-SLCO1B3-261) in PLC/RPF/5 cells in vitro. PLC/PRF/5 cells were transiently transfected with the plasmid for Renilla luciferase expression and control pGL3-basic or plasmid for the pGL3-SLCO1B3-1967 or pGL3-SLCO1B3-261, together with control plasmid (CTR o/e) or the plasmid for STAT3 overexpression (STAT3 o/e). One day later, the cells were treated with, or without, 100 ng/mL IL-6 for 12 hours. The luciferase activity of each group of cells was quantified by dual-luciferase reporter assays. ∗P < .05 or #P < .05 vs CTR o/e. (E) ChIP assays (RT-qPCR method) indicated that IL-6-activated STAT3 bound to the SLCO1B3 promoter (ChIP 2 site: −215 to −205) in PLC/PRF/5 cells in a dose-dependent manner. ∗P < .05 vs the 0 ng/mL IL-6 group; #P < .05 vs. the 1 ng/mL IL-6 group; $P < .05 vs the 10 ng/mL IL-6 group. (F) Treatment with IL-6 and/or STAT3 o/e inhibited the pGL3-SLCO1B3-261 promoter activity, but not its mutant promoter activity (pGL3-SLCO1B3-261MUT) in PLC/RPF/5 cells. ∗P < .05 vs the CTR o/e. (G) ChIP assays (RT-qPCR method) unveiled that treatment with APTSTAT3-9R, a specific STAT3-binding peptide, abrogated the increased activity of IL-6-activated STAT3 binding to the SLCO1B3 promoter (ChIP 2 site: −215 to −205) in PLC/RPF/5 cells. ∗P < .05 vs the 0 ng/mL group; #P < .05 vs the 100 ng/mL group. (H) ChIP assays (RT-qPCR method) revealed that there were more activated STAT3 bound to the SLCO1B3 promoter in the liver of patients with OC than control patients.

Figure 7

Analysis of the SLCO1B3 promoter. (A) Putative STAT3 cis-elements were identified in the proximal promoter region of the human SLCO1B3 gene (http://jaspar.genereg.net). (B) A diagram for illustration of ChIP and luciferase reporter assays of the human SLCO1B3 promoter. (C) The key motif mutation of STAT3 potential response elements in the pGL3- SLCO1B3-261 construct.

Figure 8

Conjugated BAs stimulate Ccl2 and Cxcl2 production in WT and Slc10a1^hSLCO1B3hepatocytes, but not in Slc10a1^-/-hepatocytes to attract neutrophil migration, producing IL-6 in a co-culture system. (A) A diagram for illustration of the co-culture system in transwell plates. (B) and (C) Examinations of migration ability of co-cultured WT neutrophils across the transwell membrane after co-cultured with the primary hepatocytes from WT, Slc10a1^-/- or Slc10a1^hSLCO1B3mice in the presence or absence of 100 μM GCA. (D) RT-qPCR analysis of the relative levels of Cxcl2 and Cxl2 mRNA transcripts in co-cultured WT, Slc10a1^-/- or Slc10a1^hSLCO1B3 primary hepatocytes. ∗P < .05 vs WT primary hepatocyte group; #P < .05 vs WT primary hepatocyte-GCA group; $P < .05 vs Slc10a1^-/- primary hepatocyte-GCA group. (E) Representative Western blots of hepatic EGR1 protein expression. C: controls. ∗P < .05 vs controls. (F) A schematic diagram for illustration of a novel negative feedback mechanism against cholestasis.