MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis
Jun-ichi Abe, … , Scott E. Evans, Nhat-Tu Le
Jun-ichi Abe, … , Scott E. Evans, Nhat-Tu Le
Published April 4, 2019
Citation Information: JCI Insight. 2019;4(7):e125570. https://doi.org/10.1172/jci.insight.125570.
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Research Article Cardiology Cell biology

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis

Abstract

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1–/+ mice inhibited d-flow–induced atherogenesis. In sum, EC activation and ER stress–mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

Authors

Jun-ichi Abe, Kyung Ae Ko, Sivareddy Kotla, Yin Wang, Jesus Paez-Mayorga, Ik Jae Shin, Masaki Imanishi, Hang Thi Vu, Yunting Tao, Miguel M. Leiva-Juarez, Tamlyn N. Thomas, Jan L. Medina, Jong Hak Won, Yuka Fujii, Carolyn J. Giancursio, Elena McBeath, Ji-Hyun Shin, Liliana Guzman, Rei J. Abe, Jack Taunton, Naoki Mochizuki, William Faubion, John P. Cooke, Keigi Fujiwara, Scott E. Evans, Nhat-Tu Le

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Figure 2

MAGI1-p90RSK association promotes EC activation.

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MAGI1-p90RSK association promotes EC activation. (A and B) p90RSK associ...
(A and B) p90RSK associates with MAGI1. HUVECs were exposed to d-flow or treated with Thb (10 U/ml (A and B), and their MAGI1-p90RSK interaction was assessed via co-IP with an anti-MAGI1 antibody followed by IB with an anti-p90RSK antibody (top row) and then with an anti-MAGI1 antibody (second row) to confirm pull-down of equal amounts of MAGI1 protein. The rows below the dotted line show the p90RSK and actin levels in the cell lysates. Simple Western blotting with Wes system (Proteinsimple) was used. A rabbit IgG was used as a negative control. Quantification of the ratio of coimmunoprecipitated p90RSK and MAGI1 after treatment with d-flow and Thb (B) and the fold increase in ECs from the mean value at 0 minutes. Data represent mean ± SEM (n = 3). (C) MAGI1-p90RSK interaction and pulled down MAGI1 (above the dotted line) and expression of p90RSK, IFWP (amino acid 362–543 fragment), and tubulin (below the dotted line) in HeLa cells expressing Flag-tagged p90RSK and MAGI1-WT in the presence or absence of Myc-tagged IFWP as in A were assessed. The asterisk indicates nonspecific bands. Representative blots from 3 independent experiments are shown. (D and E) MAGI1-p90RSK binding detected using a CheckMate Mammalian Two-Hybrid Assay was dose-dependently inhibited in cells expressing IFWP. The dose-dependent expression of IFWP was confirmed via Western blotting using anti-Myc (IFWP) and anti-tubulin antibodies (D). The Data represent mean ± SEM (n = 6–12). (F and G) p90RSK-MAGI1 binding increases NF-κB activity and adhesion molecule expression. (F) Bovine aortic ECs (left) and HUVECs (middle and right) expressing IFWP were subjected to an NF-κB activity assay under Thb (10 U/ml left) or TNF-α (10 ng/ml right) stimulation for 12 hours or d-flow stimulation (middle) for 24 hours. The relative NF-κB activity in the cells was measured. Data represent mean ± SEM (n = 5–9). (G) HUVECs transduced with Ad-LacZ or -IFWP (multiplicity of infection [MOI], 20) were treated with Thb (10 U/ml, 24 h), and expression of ICAM-1 and E selectin RNA was quantified. Data represent mean ± SEM (n = 5–6). **P < 0.01. Statistical differences were assessed using the 1-way ANOVA followed by Bonferroni’s post hoc testing for multiple groups.