Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol

  1. Vi T Tang
  2. Joseph McCormick
  3. Bolin Xu
  4. Yawei Wang
  5. Huan Fang
  6. Xiao Wang
  7. David Siemieniak
  8. Rami Khoriaty
  9. Brian T Emmer
  10. Xiao-Wei Chen
  11. David Ginsburg  Is a corresponding author
  1. Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, United States
  2. Life Sciences Institute, University of Michigan-Ann Arbor, United States
  3. College of Future Technology, Peking University, China
  4. Center for Life Sciences, Peking University, China
  5. State Key Laboratory of Membrane Biology, Peking University, China
  6. Howard Hughes Medical Institute, University of Michigan, United States
  7. Department of Internal Medicine, University of Michigan-Ann Arbor, United States
  8. Department of Cell and Developmental Biology, University of Michigan, United States
  9. Department of Human Genetics, University of Michigan, United States
  10. Department of Pediatrics and Communicable Diseases, University of Michigan, United States
6 figures, 2 tables and 5 additional files

Figures

Figure 1 with 1 supplement
Generation of hepatocyte-specific Surf4 deficient mice.

(A) Schematic presentation of the Surf4 conditional allele. Blue rectangles represent exons and black line segments represent introns. Red triangles denote loxP sites. Expression of a Cre

Figure 1—figure supplement 1
Read counts mapping to exon-exon junctions along the Surf4 transcript based on RNA-sequencing data.

n=3 per genotype. Crossbars represent the mean read count in each sample.

Figure 2 with 2 supplements
Deletion of hepatic Surf4 results in decreased serum PCSK9 level and profound hypocholesterolemia in mice.

(A) Serum PCSK9 levels measured by ELISA in Surf4fl/fl Alb-Cre+ mice and Surf4fl/fl Alb-Cre- littermate controls (n=6 per genotype). (B) Immunoblot for PCSK9 and HSP90 (loading control) in liver …

Figure 2—figure supplement 1
Normalized (Norm) Pcsk9 mRNA levels in Surf4fl/fl Alb-Cre-.and Surf4fl/fl Alb-Cre+ mice.

n=4 per genotype. Crossbars represent the mean, p-values were obtained from a two-sided Student’s t-test.

Figure 2—figure supplement 2
Hepatic Surf4 inactiavtion does not affect body mass while hypocholestrolemia is sustained through at least 1 year of age.

(A) Body mass of control (Surf4fl/fl Alb-Cre-), heterozygous (Surf4fl/+ Alb-Cre+), and homozygous (Surf4fl/fl Alb-Cre+) mice at 2 months of age (n=6 per genotype). p-value was obtained by a one-way …

Hepatic lipoprotein and APOB secretion defect in Surf4fl/fl Alb-Cre+ mice.

(A) Steady-state serum APOB levels in control and Surf4fl/fl Alb-Cre+ mice at 2 months of age (n=4 per genotype). (B) Representative immunoblot for APOB and HSP90 in liver lysates with and without …

Inactivation of hepatic Surf4 does not impact dietary lipid absorption, incorporation, and oxidation.

Mice were administered 3H-labelled triolein by oral gavage. Blood samples were collected over 4 hr and assayed for (A) glucose, (B) triglycerides, (C) non-esterified fatty acids (NEFA), and (D) 3H …

Figure 5 with 1 supplement
Hepatic Surf4 deletion results in mild hepatomegaly and an increase in liver lipid content, without apparent liver dysfunction or steatohepatitis.

(A) Relative liver mass in control and Surf4fl/fl Alb-Cre+ mice presented as percentage of total body mass (n=11 per genotype). (B) Relative fat and lean mass in the livers of control and Surf4fl/fl

Figure 5—figure supplement 1
Serum (A) albumin and (B) bilirubin levels in Surf4fl/fl Alb-Cre- and Surf4fl/fl Alb-Cre+ mice.

Crossbars represent the mean. p-values were obtained from two-sided Student’s t-test.

Figure 6 with 1 supplement
Depletion of hepatic Surf4 by siRNA recapitulates the hypolipidemia seen in Surf4fl/fl Alb-Cre+ mice.

(A) Normalized (Norm.) liver Surf4 mRNA levels in mice treated with scrambled siRNA (control) or varying concentrations of Surf4 targeting siRNA (n=5 per group). (B–D) Plasma PCSK9, cholesterol, and …

Figure 6—figure supplement 1
Immunoblots of plasma and liver lysates collected from mice treated with control or increasing doses of Surf4 targeting siRNA (Figure 6—figure supplement 1—source data 1).

Tables

Table 1
Genotype distribution of offspring of Surf4fl/fl Alb-Cre+and Surf4fl/+ Alb-Cre- intercrosses.
Genotype(Expected)Surf4fl/+ Alb-Cre-(25%)Surf4fl/+ Alb-Cre+(25%)Surf4fl/fl Alb-Cre-(25%)Surf4fl/fl Alb-Cre+(25%)p2)
MatingSurf4fl/fl Alb-Cre- X ♀ Surf4fl/+ Alb-Cre+
61
(25.8%)
71
(30.1%)
60
(25.4%)
44
(18.6%)
0.096
MatingSurf4fl/+ Alb Cre - X ♀ Surf4fl/fl Alb-Cre+
8
(18.6%)
9
(20.9%)
12
(27.9%)
14
(32.6%)
0.549
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (M. musculus)Surf4fl mice (C57BL/6 J)Wang et al., 2021b
Strain, strain background (M. musculus)Albumin-Cre mice (C57BL/6 J)Adams et al., 2014JAX 003574
Strain, strain background (M. musculus)Surf4fl/fl Alb-Cre+ mice (C57BL/6 J)This paper
Strain, strain background (M. musculus)SpCas9 micePlatt et al., 2014JAX 026556
Strain, strain background (M. musculus)C57BL/6 J miceJackson labJAX 0006640
Genetic reagent (include species here)AAV-Cre-sgRNAThis paper
AntibodyAnti-APOB - rabbit polyclonalFitzgerald Industries Internationa70 R-15771WB(1:1000)
AntibodyAnti-PCSK9 – rabbit polyclonalAbcamab31762WB (1:1000)
AntibodyAnti-LDLR – rabbit monoclonalAbcamab52818WB (1:1000)
AntibodyAnti-HSP90 – rabbit monoclonalCell Signaling Technology4877WB (1:1000)
AntibodyAnti-SURF4 – rabbit polyclonalWang et al., 2021bWB (1:1000)
AntibodyAnti-APOA1 – rabbit polyclonalFitzgerald Industries Internationa70 R-15769WB (1:1000)
AntibodyAnti-Albumin – mouse monoclonalProteintech66051WB (1:1000)
AntibodyAnti-Tubulin – rabbit polyclonalProteintech10094–1-APWB (1:1000)
AntibodyAnti-GAPDH – rabbit monoclonalAbcamab181602WB (1:1000)
Sequence-based reagentPrimersIDTSequences are listed in Supplement File 1
Sequence-based reagentCRISPR-gRNAIDTSequences are listed in Supplement File 2
Sequence-based reagentSURF4-targeting siRNAThis paperSequences are listed in Supplement File 3
Commercial assay or kitCholesterol assay kitSB-1010–225Fisher Scientific
Commercial assay or kitPCSK9 ELSIAMPC900R&D Systems
Commercial assay or kitAPOB ELISAab230932Abcam
Commercial assay or kitTriglycerides assay10010303Cayman Chemical
Commercial assay or kitRNeasy Plus Mini Kit74134Qiagen
Commercial assay or kitPower SYBR Green PCR Master Mix4367659Invitrogen

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/82269/elife-82269-mdarchecklist1-v2.pdf
Supplementary file 1

Primer sequences used in this study.

https://cdn.elifesciences.org/articles/82269/elife-82269-supp1-v2.docx
Supplementary file 2

Guide RNA (gRNA) sequence used for AAV-CRISPR mediated hepatic Surf4 inactivation.

https://cdn.elifesciences.org/articles/82269/elife-82269-supp2-v2.docx
Supplementary file 3

siRNA sequences used to target hepatic Surf4 in this study.

https://cdn.elifesciences.org/articles/82269/elife-82269-supp3-v2.docx
Supplementary file 4

DESeq2 output for differential gene expression analysis of RNA-seq data.

https://cdn.elifesciences.org/articles/82269/elife-82269-supp4-v2.csv

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