Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Soat1 mediates the mouse strain effects on cholesterol loading-induced endoplasmic reticulum stress and CHOP expression in macrophages
Introduction
Atherosclerosis is an important contributor to cardiovascular diseases. The hallmark of atherosclerosis is the formation and accumulation of lipid-laden macrophages in the subendothelial intimal region. These lipid-loaded macrophages promote inflammatory responses in the arterial wall and multiple pathological consequences may ensue [1]. ApoE-deficient mice bred onto different inbred mouse strains have varying aortic root atherosclerotic lesion areas, demonstrating genetic effects that modify the atherosclerosis phenotype [2]. We previously found that the lesion areas in 16-week old chow diet-fed apoE-deficient mice on the DBA/2 genetic background were ~10-fold larger than apoE-deficient mice on the AKR background [3]. As macrophages play a key role in early atherosclerosis lesion development, we performed transcriptomics on bone marrow derived macrophages (BMDM) from both strains in the presence or absence of cholesterol loading using the non-physiological scavenger receptor ligand acetylated LDL (AcLDL) [4]. We found that CHOP expression, a downstream modulator of endoplasmic reticulum (ER) stress encoded by the Ddit3 (DNA-damage inducible transcript 3) gene, was differentially induced in the two strains, with robust CHOP mRNA induction in the AKR BMDM, but a small reduction in the DBA/2 BMDM, leading to a highly significant mouse strain-cholesterol loading interaction effect [4]. Tabas and colleagues, in an elegant series of papers, have shown that free cholesterol loading of C57BL/6 mouse macrophages, by incubation with AcLDL and an inhibitor of acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), induces ER stress via activation of the unfolded protein response (UPR), leading to CHOP induction, and if severe to apoptotic cell death [[5], [6], [7]].
We previously demonstrated that after AcLDL loading, AKR cells accumulate more free cholesterol (FC), while DBA/2 cells accumulate more cholesteryl esters (CE) [8]. Using mouse genetics and Crispr-Cas9 gene editing, we determined that the major genetic effect leading to this FC/CE phenotype was due to an N-terminal truncation of the Soat1 gene on chromosome 1, encoding ACAT1, the enzyme that converts FC to CE [9]. Here we performed studies to probe the mechanism that accounts for the differential induction of CHOP by cholesterol loading in AKR and DBA/2 BMDM.
ER stress signaling is triggered by three upstream proteins: inositol requiring 1 (IRE1), activating transcription factor 6 (ATF6) and, RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) [10]. We also interrogated which of these pathways is responsible for the cholesterol loading induction of CHOP expression in embryonic stem cell derived macrophages.
Section snippets
Bone marrow-derived macrophages
AKR (stock #648) and DBA/2 (stock #671) mouse strains were obtained from JAX. Mouse studies were performed under a protocol approved by the Cleveland Clinic Institutional Animal Care and Use Committee and were in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Bone marrow cells were flushed from femur bones of apoE-deficient mice bred onto the AKR and DBA/2 genetic background [8], resuspended, and plated in macrophage growth medium (DMEM, 10%
CHOP induced after cholesterol loading in AKR but not DBA/2 BMDM
BMDM derived from AKR and DBA/2 mice were incubated overnight with AcLDL in order to load them with cholesterol. CHOP protein induction was determined as an indicator of ER stress, which was observed by western blot only in the AKR macrophages after AcLDL loading (Fig. 1). To determine if the DBA/2 macrophages were competent to induce CHOP using an alternative ER stress inducer, the BMDMs were incubated with tunicamycin, which specifically leads to the accumulation of unfolded proteins in the
Discussion
In the study, we investigated the mechanism by which two mouse strains respond differently to cholesterol loading in the induction of ER stress, as monitored by the expression of CHOP. The Ddit3 gene encodes CHOP, a transcription factor whose expression is upregulated by ER stress response [10]. We previously noted that the Ddit3 mRNA was highly upregulated by cholesterol loading with AcLDL in AKR BMDM, but not DBA/2 BMDM [4]. Here, we confirmed this result on the mRNA level, and extended it to
Conclusion
The genetic alteration in the Soat1 gene in AKR macrophages is sufficient to induce the ER stress response after cholesterol loading with AcLDL, confirming the role of FC in the ER stress induction of CHOP expression in macrophages. This explains our prior differential response to AcLDL in mediating gene expression comparing macrophages derived from the AKR and DBA/2 mouse strains.
Funding
This research was supported by National Institutes of Health [grant P01HL029582] to J.D.S., by American Heart Association Scientist Development Grant [#15SDG25310009] to P.R., and by China Scholarship Council to M.L.
Data statement
All original data and full western blot scans are available upon request to the corresponding author.
CRediT authorship contribution statement
Mengdie Luo: Investigation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Emmanuel Opoku: Investigation, Visualization, Writing - original draft. C. Alicia Traughber: Investigation, Formal analysis, Visualization, Writing - original draft. Qimin Hai: Investigation, Writing - review & editing. Peggy Robinet: Conceptualization, Supervision, Funding acquisition, Writing - review & editing. Stela Berisha: Conceptualization, Supervision, Writing - review &
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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- 1
These authors contributed equally.
- 2
Current address: ProEd communications, Inc., Beachwood, OH, USA.
- 3
Current address: Center for Human Genetics, University Hospitals Cleveland Medical Center, Cleveland, OH, USA.