Abstract

Background Reverse cholesterol transport by high density lipoproteins (HDL) is considered as an anti-atherogenic metabolic pathway. Hepatocytes determine the efficacy of this pathway by the production of apolipoprotein A-I (apoA-I) and its lipidation by ATP binding cassette transporter A1 (ABCA1), selective uptake of cholesterol via scavenger receptor BI (SR-BI) and uptake of entire HDL particles. The molecular determinants of the latter step are not well understood.

Methods We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent HDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by targeted in vitro experiments and the analysis of associations between their variants and HDL cholesterol levels in the databases of the Global Lipids Genetics Consortium and UK Biobank as well as inborn errors of metabolism and their respective mouse models.

Results The knockdown of 128 genes significantly inhibited HDL uptake. Six of them encode for components of the COPI coatomer, namely COPA, COPB1, COPB2, COPG1, ARCN1, and COPZ1. Knocking down any of them decreased the uptake of both fluorescently labeled proteins and lipids of HDL, the cell surface abundance of SR-BI as well as APOA1 expression and apoA-I secretion but increased the cell surface abundance of ABCA1 as well as cholesterol efflux. Single nucleotide polymorphisms of COPB1, ARCN1, and COPZ2 were associated with significantly higher HDL-cholesterol (HDL-C) levels in the population while rare COPA and COPG1 variants causing immunopathies in humans and mice were associated with low levels of HDL cholesterol.

Conclusions In hepatocytes, the COPI coatomer regulates HDL holoparticle uptake, selective lipid uptake, apoA-I secretion, and cholesterol efflux, and thereby, it influences plasma levels of HDL-C.

Highlights

By genome-wide RNA interference screening and replication experiments we found six components of the COPI coatomer, namely COPA, COPB1, COPB2, COPG1, ARCN1, and COPZ1 to limit the uptake of HDL holoparticles into Huh-7 hepatocarcinoma cells, possibly by a mechanism that involves ATP binding cassette transporter ABCA1

Loss of any of expression of any of these six COPI genes decreases HDL lipid uptake by interfering with the glycosylation and cell surface abundance of scavenger receptor SR-BI

Loss of any of expression of any of these six COPI genes decreases HDL secretion from Huh7 cells, possibly by decreasing gene expression of APOA1 and ABCA1, but despite increasing cell surface abundance of ABCA1 and ABCA1-mediated cholesterol efflux

Single nucleotide polymorphisms of ARCN1 are associated with significantly higher HDL-cholesterol (HDL-C) levels in the population while rare COPA and COPG1 variants causing immunopathies in humans and mice were associated with rather low levels of HDL cholesterol.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

Swiss National Science Foundation (31003A-160126 and 310030-185109) 7th Framework Program (FP7) granted by the European Commission (TransCard 603091) Swiss Systems X program (2014/267 (MRD) HDL-X) University of Zurich (Forschungskredit FK-20-037) Swiss Atherosclerosis Society (AGLA) DACH-Gesellschaft Praevention von Herz-Kreislauf-Erkrankungen grants RG3008 and PG008/08 from the British Heart Foundation

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Institutional Review Boards (IRB) for the protection of human subjects of the University of California in San Francisco (IRB protocol 10-02467) and Boston Childrens Hospital (IRB protocol 04-09-113R).

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Data Availability

All data produced in the present study are available upon reasonable request to the authors

Nonstandard Abbreviations and AcronymsRNAiRNA interferenceGWASGenome wide association studyWESWhole exome sequencingFPLCFast protein liquid chromatographyTLCThin layer chromatographyGC-MSGas chromatography mass spectrometryLDL-CLow-density lipoprotein cholesterolHDL-CHigh-density lipoprotein cholesterolTGTriglyceridesFCFree cholesterolCECholesterol esterAPOA1, apoA-IApolipoprotein A1COPICoat protein ICOPA, α-COPCOPI coat complex subunit alphaCOPB1, β-COPCOPI coat complex subunit beta 1COPB2, β’-COPCOPI coat complex subunit beta 2ARCN1, δ-COPCOPI coat complex subunit deltaCOPE, ε-COPCOPI coat complex subunit epsilonCOPG1, γ-COPCOPI coat complex subunit gamma 1COPG2, γ’-COPCOPI coat complex subunit gamma 2COPZ1, ζ-COPCOPI coat complex subunit zeta 1COPZ2, ζ’-COPCOPI coat complex subunit zeta 2SCARB1, SR-BIScavenger receptor class B type IABCA1ATP binding cassette subfamily A member 1