Bmal1 is involved in the regulation of macrophage cholesterol homeostasis
Xiaoyue Pan, John O’Hare, Cyrus Mowdawalla, Samantha Mota, Nan Wang, M. Mahmood Hussain
Xiaoyue Pan, John O’Hare, Cyrus Mowdawalla, Samantha Mota, Nan Wang, M. Mahmood Hussain
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Research Article Metabolism Vascular biology

Bmal1 is involved in the regulation of macrophage cholesterol homeostasis

Abstract

Atherosclerotic cardiovascular disease is a major contributor to the global disease burden. Atherosclerosis initiation depends on cholesterol accumulation in subendothelial macrophages (Mφs). To clarify the role of Bmal1 in Mφ function and atherosclerosis, we used several global and myeloid-specific Bmal1-deficient mouse models. Myeloid-specific Bmal1-deficient mice had higher Mφ cholesterol and displayed greater atherosclerosis compared with controls. Bmal1-deficient Mφs exhibited: (a) elevated expression of Cd36 and uptake of oxLDL; (b) diminished expression of Abca1 and Abcg1, and decreased cholesterol efflux and reverse cholesterol transport; and (c) reduced Npc1 and Npc2 expression and diminished cholesterol egress from lysosomes. Molecular studies revealed that Bmal1 directly regulates basal and cyclic expression of Npc1 and Npc2 by binding the E-box motif (CANNTG) sequence recognized by Bmal1 in their promoters and indirectly regulates the basal and temporal regulation of Cd36 and Abca1/Abcg1 involving Rev-erbα and Znf202 repressors, respectively. In conclusion, Mφ Bmal1 is a key regulator of the uptake of modified lipoproteins, cholesterol efflux, lysosomal cholesterol egress, and atherosclerosis and, therefore, may be a master regulator of cholesterol metabolism in Mφs. Restoration of Mφ Bmal1 expression or blocking of factors that decrease its activity may be effective in preventing atherosclerosis.

Authors

Xiaoyue Pan, John O’Hare, Cyrus Mowdawalla, Samantha Mota, Nan Wang, M. Mahmood Hussain

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Figure 7

Bmal1 deficiency decreases Npc1 and Npc2 expression in J774A.1 Mφs.

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Bmal1 deficiency decreases Npc1 and Npc2 expression in J774A.1 Mφs. (A) ...
(A) Mouse J774A.1 Mφs were transfected with siBmal1 or siControl for 48 hours and subjected to 2 hours serum shock (9:30–11:30 am). Cells were collected at the indicated times to measure mRNA levels (n = 3). Values at time 0 (11:30 am) were normalized to 1. (B) J774A.1 Mφs were treated with various siRNAs, and mRNA and protein levels were measured after 48 hours (n = 4). (C) Cells were transfected with siControl or siBmal1. After 48 hours, they were labeled for 4 hours, and cholesterol accretion in lysosomes was quantified. For egress studies, cells were labeled with [3H]-cholesterol for 4 hours. A zero-time point was collected. Other cells were then incubated in normal medium for 2 hours. Cholesterol counts in lysosomes at 0 hours and 2 hours were used to calculate egress (n = 4). (D and E) J774A.1 Mφs were transduced with Adv-Control or Adv-Bmal1. After 48 hours, Mφs were used to measure mRNA (n = 3) and protein levels (D, n = 3), as well as lysosomal cholesterol accretion and egress (E, n = 4–6). (F and G) BMDMs from 7-month-old male M-Bmal1−/− Apoe−/− and Bmal1fl/fl Apoe−/− mice were cultured for 7 days and used to study the binding of various circadian transcription factors to the Npc1 and Npc2 promoters with ChIP (n = 3). All values are mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with Control group, 2-tailed, unpaired t tests (C, E, and F) or multiple t tests followed by Holm-Šídák method (B and D) or Cosinor analysis (A).