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The LRP6 functional mutation rs2302685 contributes to individual susceptibility to alcoholic liver injury related to the Wnt/β-catenin-TCF1-CYP2E1 signaling pathway

  • Ying Xu
  • Dan Chen
  • Xiu-Xian Lin
  • Qing Zhao
  • Jing Guo
  • Li-jie Chen
  • Wei Zhang
  • Jian Xiao
  • Guang-Hui Lian
  • Shi-Fang Peng
  • Dong Guo
  • Hong Yang
  • Obinna Obianom
  • Yan Shu
  • Yao ChenEmail author
Organ Toxicity and Mechanisms
  • 68 Downloads

Abstract

Low-density lipoprotein receptor-related protein 6 (LRP6) is an important coreceptor in the Wnt/β-catenin upstream signaling pathway. Rs2302685 is a common functional mutation of LRP6 that has been previously associated with reduced alcoholic liver injury among alcoholic liver disease (ALD) patients, and the present research was designed to study the underlying mechanisms of that finding. A total of 107 ALD patients and 138 non-ALD patients were recruited from hospitalized alcoholics in China. Their venous blood samples were collected for DNA extraction and genotyped using Sequenom MassARRAY. We found that the rs2302685 mutation, which impaired the function of LRP6, was present in higher frequency among alcoholics with ALD than those without ALD. We also conducted a mouse model experiment in which LRP6(+/−) knockdown mice and LRP6(+/+) wild-type mice received daily intragastric doses of ethanol (2.4 g/kg) as well as a larger dose of ethanol (4 g/kg) every 7 days for 28 days. The mouse blood and liver specimens were subsequently collected for laboratory analysis, and cell experiments were performed to compare the inhibition, activation, over-expression, and siRNA of LRP6 in the treatment versus the control HL7702 cells. Expression of the targeted molecules was detected by real-time PCR or western blot analysis. Stably transfected cells with pRL3-CYP2E1 vector were used to further study the underlying mechanisms. The total bile acid (TBA), direct bilirubin, total bilirubin (TBIL), aspartate aminotransferase (AST), mitochondrial aspartate aminotransferase, and AST/ALT values were significantly lower in carriers of the rs2302685 mutation than in the wild-type patients, by 63.4, 60.6, 82.1, 44.8, 45.7, and 21.4%, respectively. Compared to the LRP6(+/+) wild-type mice, the LRP6(+/−) knockdown mice had lower ALT, TBIL, TBA, and ALB/GLO values, as well reduced liver tissue damage, in accordance with their reduced expressions of LRP6, β-catenin, and CYP2E1. In HL7702 cells exposed to ethanol, AST, ALT, lipid accumulation, and ROS generation decreased in cells that were treated with LRP6 inhibitors or siRNA but increased in cells treated with LRP6 activators or over-expressed LRP6. TCF1 was the transcriptional factor most likely to connect the LRP6-Wnt/β-catenin signaling pathway to the regulation of CYP2E1. We concluded that the LRP6 functional mutation rs2302685 contributes to individual differences in susceptibility to alcoholic liver injury related to the Wnt/β-catenin-TCF1-CYP2E1 signaling pathway.

Keywords

LRP6 Alcoholic liver injury ALD Wnt/β-catenin CYP2E1 

Abbreviations

LRP6

Low-density lipoprotein receptor-related protein 6

ALD

Alcoholic liver disease

LDLR

Low-density lipoprotein receptor

TCF

T cell factor

LEF

Lymphoid enhancer factor

ROS

Reactive oxygen species

PBS

Phosphate-buffered saline

IHC

Immunohistochemical

CHB

Han Chinese in Beijing

CHS

Han Chinese South

WB

Western blot

SPF

Specific pathogen free

SDS

Sodium dodecyl sulfate

DTT

Dithiothreitol

ADH

Alcohol dehydrogenase

ALDH

Aldehyde dehydrogenase

TBA

Total bile acid

BMI

Body mass index

TP

Total protein

ALB

Albumin

GLO

Globulin

GGT

Gamma-glutamyl transpeptidase

ALT

Alanine transaminase

AST

Aspartate aminotransferase

TG

Triglyceride

TC

Total cholesterol

LDL

Low-density lipoprotein

HDL

High-density lipoprotein

AFU

A-l-fucosidase

ALP

Alkaline phosphatase

GSP

Glycated serum protein

TBIL

Total bilirubin

AST

Aspartate aminotransferase

DBIL

Direct bilirubin

Notes

Acknowledgements

This work was supported by the National Scientific Foundation of China (Grant 81302850), the National Science and Technology Plan of China (Grant 2017ZX09304014), the Natural Science Foundation of Hunan Province (2018JJ2629), and the Changsha Science and Technology Plan (Kq1602014). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors have declared that no competing interests exist.

Supplementary material

204_2019_2447_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1154 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Ying Xu
    • 1
    • 2
    • 3
    • 4
  • Dan Chen
    • 1
    • 2
    • 3
    • 4
  • Xiu-Xian Lin
    • 1
    • 2
    • 3
    • 4
  • Qing Zhao
    • 1
    • 2
    • 3
    • 4
  • Jing Guo
    • 1
    • 2
    • 3
    • 4
  • Li-jie Chen
    • 1
    • 2
    • 3
    • 4
  • Wei Zhang
    • 1
    • 2
    • 3
    • 4
  • Jian Xiao
    • 5
    • 7
  • Guang-Hui Lian
    • 6
  • Shi-Fang Peng
    • 7
  • Dong Guo
    • 8
  • Hong Yang
    • 8
  • Obinna Obianom
    • 8
  • Yan Shu
    • 8
  • Yao Chen
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of Clinical PharmacologyXiangya Hospital, Central South UniversityChangshaChina
  2. 2.Institute of Clinical Pharmacology, Central South UniversityChangshaChina
  3. 3.Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of EducationChangshaPeople’s Republic of China
  4. 4.National Clinical Research Center for Geriatric DisordersChangshaPeople’s Republic of China
  5. 5.Department of Hepatology and Infectious DiseasesXiangya Hospital, Central South UniversityChangshaChina
  6. 6.Department of GastroenterologyXiangya Hospital, Central South UniversityChangshaChina
  7. 7.Department of PharmacyXiangya Hospital, Central South UniversityChangshaChina
  8. 8.Department of Pharmaceutical Sciences, School of PharmacyUniversity of MarylandBaltimoreUSA

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