. 2022 Apr 7;14(4):e15344.

doi: 10.15252/emmm.202115344. Epub 2022 Feb 22.

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression

Zehra Yildirim^{1

2}, Sabyasachi Baboo³, Syed M Hamid¹, Asli E Dogan^{1

2}, Ozlem Tufanli⁴, Sabrina Robichaud⁵, Christina Emerton⁵, Jolene K Diedrich³, Hasan Vatandaslar⁶, Fotis Nikolos⁷, Yanghong Gu⁸, Takao Iwawaki⁹, Elizabeth Tarling¹⁰, Mireille Ouimet⁵, David L Nelson⁸, John R Yates 3rd³, Peter Walter¹⁰, Ebru Erbay^{1

11}

Affiliations

¹ Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
² Department of Molecular Biology and Genetics, National Nanotechnology Center, Bilkent University, Ankara, Turkey.
³ Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.
⁴ Lagone Medical Center, New York University, New York, NY, USA.
⁵ Department of Biochemistry, Microbiology and Immunology, Heart Institute, University of Ottawa, Ottawa, ON, Canada.
⁶ Institute of Molecular Health Sciences, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
⁷ Samuel Oschin Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
⁸ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
⁹ Department of Life Science, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan.
¹⁰ Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA, USA.
¹¹ David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.

PMID: 35191199
PMCID: PMC8988208
DOI: 10.15252/emmm.202115344

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression

Zehra Yildirim et al. EMBO Mol Med. 2022.

. 2022 Apr 7;14(4):e15344.

doi: 10.15252/emmm.202115344. Epub 2022 Feb 22.

Authors

Affiliations

¹ Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
² Department of Molecular Biology and Genetics, National Nanotechnology Center, Bilkent University, Ankara, Turkey.
³ Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.
⁴ Lagone Medical Center, New York University, New York, NY, USA.
⁵ Department of Biochemistry, Microbiology and Immunology, Heart Institute, University of Ottawa, Ottawa, ON, Canada.
⁶ Institute of Molecular Health Sciences, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
⁷ Samuel Oschin Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
⁸ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
⁹ Department of Life Science, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan.
¹⁰ Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA, USA.
¹¹ David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.

PMID: 35191199
PMCID: PMC8988208
DOI: 10.15252/emmm.202115344

Abstract

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Keywords: ER stress; atherosclerosis; cholesterol homeostasis; efferocytosis; translational regulation.

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Figures

**Figure 1. FMRP is a novel IRE1 kinase substrate**
STING analysis of published IRE1 interactome proteins in relation to FMRP (Acosta‐Alvear *et al*, 2018).
HEK293T cells were co‐transfected with IRE1 and FMRP plasmids and stimulated with TG (600 nM) or TM (1 mg/ml) for 2 h. Protein lysates were immunoprecipitated (IP) with anti‐IRE1 or IgG (control) antibodies and analyzed by Western blotting using specific antibodies for FMRP and IRE1 (n = 3 biological replicates).
RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 h. Protein lysates were treated with λ Phosphatase (PPase) for 30 min and analyzed by Western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP fold induction is depicted above the blots (n = 6 biological replicates).
Apoe^−/− mice were fed with chow diet (CD) or western diet (WD) for 16 weeks followed by peritoneal macrophage (PM) isolation. Protein lysates were analyzed by Western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP‐fold induction is depicted above the blots (n = 5 mice per group).
Control‐ or IRE1‐siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 h. Protein lysates were analyzed by Western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP fold induction is depicted above the blots (n = 4 biological replicates).
Protein lysates of thioglycolate‐elicited PM from IRE1α^+/+ and IRE1α^−/− mice (after 16 weeks on WD) were analyzed by Western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP fold induction is depicted above the blots (n = 4 mice per group).
MEF cells were transfected with either empty vector, EGFP‐FMRP or 3xFLAG‐IRE1 plasmids then pre‐treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG‐18 (25 µM; 1 h) followed by TG (600 nM) stimulation for 4 h. Protein lysates were analyzed by Western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP fold induction is depicted above the blots (n = 4 biological replicates).
C57BL/6 were injected either with DMSO or AMG‐18 (30 mg/kg; 8 h), followed by TM injection (1 mg/kg; 8 h). Protein lysates of thioglycolate‐elicited PM were analyzed by Western blotting using antibodies for pFMRP, FMRP, pIRE1, IRE1, and β‐Actin. pFMRP/FMRP fold induction is depicted above the blots (n = 4 mice per group).
HEK293T cells were transfected with either empty vector (EV), IRE1‐WT, or IRE1–KD plasmids and stimulated by TG (600 nM; 1 h). Protein lysates from each transfection were separately immunoprecipitated (IP) with anti‐IRE1 antibody and subjected to a kinase reaction with purified hFMRP protein and ATP‐γ‐S (100 µM) in kinase buffer. The IP protein were analyzed by Western blotting using specific antibodies for thiophosphate esters (ThioP), IRE1, and FMRP (n = 3 biological replicates).
Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by Western blotting using specific antibodies for ThioP, IRE1, and FMRP (n = 3 biological replicates) and with LC‐MS/MS. Identified IRE1 kinase‐mediated FMRP phosphorylation sites (bottom).
Fmr1^−/− mouse embryonic fibroblasts (MEF) were transfected either with EV, WT‐FMRP, SA‐FMRP, or STSA‐FMRP plasmids followed by PA treatment (500 µM; 6 h). Protein lysates were analyzed by Western blotting using specific antibodies for FMRP, pFMRP, pIRE1, and β‐Actin (n = 3 biological replicates).
Data information: A representative blot is shown. In D, E, G, and H data are cumulative results of two independent experiments. Data are mean ± SEM. Unpaired t‐test with Welch’s correction or paired t‐test. Source data are available online for this figure.

Figure 2. FMRP deficiency enhances RCT while reducing foam cell formation *in vivo*
A
Fmr1^+/+ and Fmr1^−/− mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n = 7 mice per group; Scale bar = 50 µm).
B
Apoe^−/− mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG‐18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n = 5 mice per group; Scale bar = 50 µm).
C–E
Flow cytometry analysis of BMDMs after dil‐ac‐LDL (25 µg/ml) loading for 24 h; (C) control‐ or Fmr1‐siRNA transfected BMDM (n = 4 biological replicates), (D) Fmr1^+/+ and Fmr1^−/− BMDM (n = 4 biological replicates), (E) BMDM pre‐treated with either vehicle (DMSO) or AMG‐18 (5 µM; 1 h) (n = 6 biological replicates).
F, G
Macrophages were pre‐loaded with fluorescently labeled cholesterol (16 h) followed by incubation in efflux medium including APOA1 (25 µg/ml) or HDL (50 µg/ml) as acceptors for 6 h. % Efflux was calculated as cholesterol signal in medium/cholesterol signal in medium and cell: Cholesterol efflux in (F) Fmr1^+/+ and Fmr1^−/− BMDM (n = 4 biological replicates) and in (G) BMDM that were pre‐treated either with DMSO or AMG‐18 (5 µM; 1 h) (n = 4 biological replicates).
H–K
RCT experiment: (H) Schematic representation of C57BL6 mice were injected with [³H]‐cholesterol‐loaded foamy Fmr1^+/+ and Fmr1^−/− BMDM, (I) plasma cholesterol levels after 24 and 48 h, (J) liver cholesterol levels after 48 h, and (K) feces cholesterol levels after 48 h (n = 12 mice per group).
Data information: Data are mean ± SEM. Unpaired t‐test with Welch’s correction. Source data are available online for this figure.

Figure 3. FMRP deficiency increases efferocytosis *in vivo*
A–E
*In vitro* and *in vivo* efferocytosis experiments, where percentage of macrophages F4/80⁺ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)⁺ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1‐ or control‐siRNA and incubated CFSE‐labeled AC for the indicated hours (n = 4 biological replicates). (B) Fmr1^+/+ and Fmr1^−/− BMDMs were treated with PA (500 µM) for 6 h and then incubated with CFSE‐labeled ACs for 4 h (n = 4 biological replicates). (C) Fmr1^+/+ and Fmr1^−/− mice were fed WD (16 weeks) and injected intraperitoneally with CFSE‐labeled AC (1.5 h), followed by PM elicitation (n = 4–5 mice per group). (D) BMDM were pre‐treated either with vehicle (DMSO) or AMG‐18 (5 µM) for 1 h then incubated with CFSE‐labeled Acs for 4 h (n = 3 biological replicates). (E) C57BL/6 mice were injected with AMG‐18 (30 mg/kg) or vehicle (DMSO) for 8 h, followed by intraperitoneal injection with CFSE‐labeled ACs for 1.5 h and PM elicitation (n = 4 mice per group).
F, G
*In vitro* continuous *efferocytosis experiments*, where macrophages were stained for F4/80⁺ (red), AC were labeled with CFSE (AC‐1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80⁺, CFSE⁺, and Violet⁺ (triple positive) cells to total F4/80⁺ and CFSE⁺ (double positive) cells. (F) Fmr1^+/+ and Fmr1^−/− BMDM were incubated with AC‐1 for 2 h, and after 2 h interval, incubated with AC‐2 for 2 more hours (n = 4–3 biological replicates). (G) BMDM were pre‐treated either with vehicle (DMSO) or AMG‐18 (5 µM) for 1 h, incubated with CFSE‐labeled AC‐1 for 2 h, followed by incubation with Violet‐labeled AC‐2 for 2 h and PM collection (n = 4 biological replicates).
Data information: For all images scale bar = 50 µm; Red: Macrophages, Green: AC/AC‐1, Violet: AC‐2. Data are mean ± SEM. Unpaired t‐test with Welch’s correction. Source data are available online for this figure.

**Figure 4. FMRP targets in macrophages**
A, B
RNA lysates from Fmr1^+/+ and Fmr1^−/− BMDM that were treated with PA (500 µM; 6 h) were fractionated using a 10–50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n = 3 biological replicates). (A) Representative profile for RNA distribution from genotypes based on UV absorbance readings after sucrose gradient fractionation. (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47, and Rac1 mRNA in polysome to NTR fraction (n = 3 biological replicates).
C
BMDM were isolated from Fmr1^+/+ and Fmr1^−/−, and protein lysates were analyzed by Western blotting using specific antibodies for ABCA1, ABCG1, MerTK, LRP1, FMRP, and β‐Actin antibodies and fold inductions relative to β‐Actin are depicted above the blots (n = 6 biological replicates).
D
Fmr1^−/− MEF cells were transfected with EV, WT‐FMRP, or STSA‐FMRP plasmids followed by PA treatment (500 µM; 6 h). Protein lysates were analyzed by Western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP, and β‐Actin and fold inductions relative to β‐Actin are depicted above the blots (n = 5 biological replicates).
Data information: A representative blot is shown. In C and D, data are cumulative results of 2 and 3 independent experiments, respectively. In Western blots, the protein expression fold change was calculated relative to β‐Actin and depicted above the blots and a representative blot was shown. Data are mean ± SEM. Unpaired t‐test with Welch’s correction. Source data are available online for this figure.

**Figure 5. FMRP‐deficiency alleviates atherosclerosis**
Atherosclerosis experiment design in Fmr1^+/+ and Fmr1^−/− mice that were injected with AAV_PCSK9 and fed WD (16 weeks).
Lesion area calculated from *en face* aorta, stained with ORO (n = 12–13 mice per group; Scale bar = 5 mm).
Total plaque area was calculated from hematoxylin & eosin (H&E)‐stained aortic root sections (n = 8 mice per group; Scale bar = 300 µm).
Foam cell area was calculated from ORO‐stained aortic root sections (n = 8 mice per group; Scale bar = 300 µm).
Necrotic area was calculated from H&E‐stained aortic root sections (n = 8 mice per group; Scale bar = 100 µm).
Atherosclerosis experiment design in myFmr1^+/+ and myFmr1^−/− mice that were injected with AAV_PCSK9 and fed WD (16 weeks).
Lesion area calculated from *en face* aorta, stained with ORO (n = 9 mice per group; Scale bar = 5 mm).
Total plaque area was calculated from H&E‐stained aortic root sections (n = 9–6 mice per group; Scale bar = 300 µm).
Foam cell area was calculated from ORO‐stained aortic root sections (n = 9–6 mice per group; Scale bar = 300 µm).
Necrotic area was calculated from H&E‐stained aortic root sections (n = 9–6 mice per group; Scale bar = 100 µm).
Data information: Data are mean ± SEM; Mann Whitney U test. Source data are available online for this figure.

**Figure 6. IRE1 Kinase inhibition alleviates atherosclerosis**
Atherosclerosis experiment design in Apoe^−/− mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG‐18 (30 mg/kg) once a day in the last 4 weeks of WD.
Lesion area calculated from *en face* aorta, stained with ORO (n = 5 mice per group; Scale bar = 5 mm).
Total plaque area was calculated from H&E‐stained aortic root sections (n = 5 mice per group; Scale bar = 300 µm).
Foam cell area was calculated from ORO‐stained aortic root sections (n = 5 mice per group; Scale bar = 300 µm).
Necrotic area was calculated from H&E‐stained aortic root sections (n = 5 mice per group; Scale bar = 100 µm).
Atherosclerosis experiment design in Apoe^−/− mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG‐18 (30 mg/kg) twice a day in the last 4 weeks of WD.
Lesion area calculated from *en face* aorta, stained with ORO (n = 6 mice per group; Scale bar = 5 mm).
Total plaque area was calculated from H&E‐stained aortic root sections (n = 5 mice per group; Scale bar = 300 µm).
Foam cell area was calculated from ORO‐stained aortic root sections (n = 5 mice per group; Scale bar = 300 µm).
Necrotic area was calculated from H&E‐stained aortic root sections (n = 5 mice per group; Scale bar = 100 µm).
Data information: Data are mean ± SEM; Mann Whitney U test. Source data are available online for this figure.

**Figure 7. IRE1‐FMRP signaling controls cholesterol efflux and efferocytosis pathways in macrophages**
IRE1‐mediated FMRP phosphorylation suppresses translation of mRNA for key cholesterol transporters and efferocytosis receptors in macrophages and promotes atherosclerosis.

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Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression

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Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression

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