SLC10A5 deficiency causes hypercholanemia

Abstract

Background and aims: Solute Carrier Family 10 Member 5 (SLC10A5) is a member of SLC10, comprising transporters of bile acids, steroidal hormones, and other substrates, but its function remains unclear. The aim of the current investigation was to clarify its function in the metabolism of bile acid and hypercholanemia.

Approach and results: Whole-exome sequencing and Sanger sequencing were used to identify and confirm the variant in the subjects of hypercholanemia. CRISPR/Cas9-mediated genome engineering was used to establish the knockout and point mutation mice. Primary mouse hepatocytes were isolated, and cell lines were cultured. SLC10A5 was silenced by siRNA and overexpressed by wild-type and mutant plasmids. The fluorescent bile acid derivative was used for the bile acid uptake assay. Bile acids were assessed with ultra-performance liquid chromatography tandem mass spectrometry. A heterozygous variant SLC10A5 : c.994_995del (p.D332X) was identified in subjects with elevated total bile acid or altered bile acid profiles. Bile acids were increased in the serum and liver of knockout and point mutation mice. The expressions of FXR and SHP, regulators involved in the negative feedback of bile acid synthesis, were downregulated, while the bile acid synthesis genes CYP7A1 and CYP8B1 were upregulated in both gene-edited mice. Both the wild and mutant SLC10A5 proteins were localized on the plasma membrane. Knockdown, knockout, or targeted mutation of SLC10A5 led to the inhibition of bile acid uptake by cell lines and primary mouse hepatocytes.

Conclusion: SLC10A5 is involved in the uptake of bile acid, and its deficiency causes hypercholanemia.

Graphical abstract

FIGURE 1

Identification of SLC10A5: c.994_995del in patients with hypercholanemia. (A) The pedigree. Squares represent males and circles represent females. The arrow points to the proband. (B) Sanger sequencing. The heterozygous c.994_995del mutation in SLC10A5 was confirmed by Sanger sequencing. (C) Chromosome localization of SLC10A5 and its mutation. (D) SLC10A5 is a predicted membrane protein containing 9 transmembrane segments, and the variant position is located in the extracellular loop between the sixth and seventh transmembrane domains. (E) Graphics depicting the structure of wild-type, truncation mutant type SLC10A5 protein. (F) Reduced SLC10A5 in PBMC. Compared with controls, SLC10A5 was significantly decreased in the PBMC of patients. Red points represented females and blue points represented males. **p < 0.01. Abbreviations: PMBC, peripheral blood mononuclear cells; SLC10A5, solute carrier family 10 member 5.

FIGURE 2

Slc10a5 ^−/− and Slc10a5 ^R328X/R328X induces hypercholanemia. (A) Slc10a5 ^−/− mice displayed elevated levels of serum and liver TBA in 10 weeks of age, while there were no significant differences in the ileum and fecal TBA compared to the age-matched WT mice. (B) The profiles of bile acid in the WT and Slc10a5 ^−/−- mice serum. (C) The profiles of bile acid in the WT and Slc10a5 ^−/−- mice liver. (D) Slc10a5 ^R328X/R328X mice displayed elevated levels of serum and liver TBA in 10 weeks of age, while there were no significant differences in the ileum and fecal TBA compared to the age-matched WT mice. (E) The profiles of bile acid in the WT and Slc10a5 ^R328X/R328X mice serum. (F) The profiles of bile acid in the WT and Slc10a5 ^R328X/R328X mice liver. *p < 0.05; **p < 0.01. Abbreviations: HCA, hyocholic acid; HDCA, hyodeoxycholic acid; MCA, muricholic acid; SLC10A5, solute carrier family 10 member 5; TBA, total bile acid; UCA, ursocholic acid; WT, wild-type.

FIGURE 3

Identification of primary mouse hepatocytes and bile acid uptake assay. (A) uptake of fluorescent bile acid in different kinds of primary mouse hepatocytes. Cyclosporine A (CsA, 10 uM) was used for a negative control. (B) PAS satin of primary mouse hepatocytes. (C) Cytokeratin 18 immunofluorescence of primary mouse hepatocytes. (D) Fluorescence intensity analysis of the fluorescent bile acid. The value for the control was set to 100. **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviation: PAS, periodic acid-schiff.

FIGURE 4

SLC10A5 was involved in the uptake of fluorescent bile acid in the HepG2 cell line. (A) Silencing of SLC10A5 significantly reduced the uptake of fluorescent bile acid. NC group. S1, S2, and S3: experimental groups with SLC10A5-specific small interfering RNAs. (B, E) Silencing of SLC10A5 or plasmids transfection was confirmed by western blotting. (D) SLC10A5^WT significantly increased the uptake of bile acid, while SLC10A5^D332X significantly decreased the uptake of bile acid. (C, F) Fluorescence intensity analysis of the fluorescent bile acid. The value for the control was set to 100. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Abbreviations: NC, normal control; SLC10A5, solute carrier family 10 member 5.

FIGURE 5

Knockout or knockdown of SLC10A5 inhibited the FXR signaling pathway. (A) The relative mRNA levels of CYP7A1, CYP8B1, FXR, and SHP between WT mice and Slc10a5 ^−/− mice. (B) Western blotting analysis of the relative protein expressions in WT mice and Slc10a5 ^−/− mice. (C) Grey value analysis of the western blotting results of B. (D) Western blotting analysis of FXR and SHP in HepG2 and HEK293T cell lines transfected with specific small interfering RNAs. (E) Grey value analysis of FXR and SHP in HepG2 and HEK293T cell lines transfected with specific small interfere RNAs. *p < 0.05, **p < 0.01. Abbreviations: CYP7A1, cholesterol 7α hydroxylase; CYP8B1, sterol 12α-hydroxylase; FXR, farnesoid X receptor; NC, normal control; SLC10A5, solute carrier family 10 member 5; SHP, Small heterodimer partner; WT, Wild-type.

FIGURE 6

The point mutation of SLC10A5 inhibited the FXR signaling pathway. (A) The relative mRNA levels of CYP7A1, CYP8B1, FXR, and SHP between WT mice and Slc10a5 ^R328X/R328X mice. (B) Western blotting analysis of the relative protein expressions in WT mice and Slc10a5 ^R328X/R328X mice. (C) The grey value analysis of the western blotting results of B. (D) Western blotting analysis of FXR and SHP in HepG2 and HEK293T cell lines transfected with plasmids. (E) The grey value analysis of FXR and SHP in HepG2 and HEK293T cell lines transfected with plasmids. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CYP7A1, cholesterol 7α hydroxylase; CYP8B1, sterol 12α-hydroxylase; FXR, farnesoid X receptor; SLC10A5, solute carrier family 10 member 5; SHP, small heterodimer partner; WT, wild-type.