Myeloid cell–specific serine palmitoyltransferase subunit 2 haploinsufficiency reduces murine atherosclerosis
Mahua Chakraborty, Caixia Lou, Chongmin Huan, Ming-Shang Kuo, Tae-Sik Park, Guoqing Cao, Xian-Cheng Jiang
Mahua Chakraborty, Caixia Lou, Chongmin Huan, Ming-Shang Kuo, Tae-Sik Park, Guoqing Cao, Xian-Cheng Jiang
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Research Article Cardiology

Myeloid cell–specific serine palmitoyltransferase subunit 2 haploinsufficiency reduces murine atherosclerosis

Abstract

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2–haploinsufficient (Sptlc2+/–) macrophages. We found that Sptlc2+/– macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NF-κB and MAPK pathways, but also enhanced reverse cholesterol transport mediated by ABC transporters. LDL receptor–deficient (Ldlr–/–) mice transplanted with Sptlc2+/– bone marrow cells exhibited significantly fewer atherosclerotic lesions after high-fat and high-cholesterol diet feeding. Additionally, Ldlr–/– mice with myeloid cell–specific Sptlc2 haploinsufficiency exhibited significantly less atherosclerosis than controls. These findings suggest that SPT could be a novel therapeutic target in atherosclerosis.

Authors

Mahua Chakraborty, Caixia Lou, Chongmin Huan, Ming-Shang Kuo, Tae-Sik Park, Guoqing Cao, Xian-Cheng Jiang

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Figure 2

Sptlc2 haploinsufficiency in macrophages leads to significant reductions of SM, glucosylceramide, and GM3 in macrophage plasma membranes and lipid rafts, resulting in altered raft distribution.

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Sptlc2 haploinsufficiency in macrophages leads to significant reduction...
(A) Cell surface SM measurement by lysenin-mediated cell lysis assay, presented as percent cell mortality. (B) Plasma membrane isolation from primary macrophages. The purity of cytoplasm (Cyto), total subcellular organelle membranes (TM), and plasma membranes (PM) was determined by Western blot; Na+/K+ATPase was used as a plasma membrane marker, and cytochrome C as a mitochondrial marker. (C) SM and phosphatidylcholine (PC) measurements in macrophage plasma membranes and lipid rafts using LC/MS/MS. (D) Ceramide (Cer), glucosylceramide (GlcCer), and GM3 in macrophage plasma membranes and lipid rafts, as measured by LC/MS/MS. Plasma membrane measurements were done using equal numbers of macrophages (~50 × 106 cells per group), and lipid raft measurements were performed using equal quantities of proteins from macrophage lysates. (E) Lipid rafts were isolated from macrophages using sucrose density gradient centrifugation, and fractions were collected. Western blots were performed for CAV-1 (raft marker) and CD71 (non-raft marker) with respective antibodies. Data are representative of 3 independent experiments (n = 6 per group). *P < 0.05.