Vascular calcification is a hallmark of advanced atherosclerosis, but the underlying mechanisms remain unknown. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells (vSMCs) of Low Density Lipoprotein receptor (LDLr) deficient mice fed an atherogenic high-cholesterol diet results in accelerated vascular calcification with chondrogenic metaplasia within the lesions. We demonstrate that vascular calcification in the absence of PPARγ requires the transmembrane receptor Low Density Lipoprotein receptor-related protein-1 (LRP1). LRP1 promotes a previously unknown Wnt5a dependent prochondrogenic pathway that activates the chondrogenic program. PPARγ protects against vascular calcification by activating sFRP2, which we show functions as a Wnt5a antagonist. Thus, targeting this signaling pathway has important clinical implications, impacting on common complications of atherosclerosis including coronary artery calcification and valvular sclerosis.

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (␣) chain of LRP1 mediates TGF␤-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (␤) chain of LRP1 suffices to limit cholesterol accumulation in LRP1؊/؊ cells. Cholesterol is a major component of mammalian cell membranes that accumulates in the vascular wall during atherosclerosis, the leading cause of death in industrialized societies (1, 2). The low density lipoprotein receptor-related protein 1 (LRP1), 3 a cell surface receptor that belongs to the LDL receptor family, endocytoses multiple ligands (3). It consists of an 85-kDa membrane-bound carboxyl fragment (␤ chain) and a non-covalently attached 515-kDa (␣ chain) amino-terminal fragment (4). We previously demonstrated that LRP1 limits cholesterol accumulation in the arterial wall. Mice deficient for LRP1 in vascular smooth muscle cells (vSMCs) (smLRP1 mice) develop vSMCs proliferation, cholesterol accumulation (5), and massive foam cell formation when fed a cholesterol-rich diet (6 -10). Whereas LRP1 integrates the platelet-derived growth factor (PDGF-BB) (8, 9) and transforming growth factor-␤ (TGF-␤) at the plasma membrane, two pathways known to regulate vSMCs proliferation (7), the physiological importance and function of LRP1 in regulating intracellular cholesterol homeostasis is still poorly understood. Several mechanisms have been proposed. LRP1 has been shown to promote cholesterol export in vSMCs through induction of ATP binding cassette transporter A1 (ABCA1) levels (5) and to induce a Wnt5a/␤-catenin pathway to limit cholesterol overload in mouse embryonic fibroblasts (11). Moreover, smLRP1 mice express very low levels of Wnt5a in vSMCs (12). TGF-␤ also stimulates a non-canonical Wnt5a pathway in airway smooth muscle cells (13). These data strongly suggest that a TGF-␤/LRP1/Wnt5a pathway limits intracellular cholesterol accumulation.How Wnt5a interferes with cholesterol homeostasis is unknown. It might increase cholesterol export and/or block cholesterol synthesis. ABCA1 and the ATP binding cassette transporter G1 (ABCG1) are two proteins that promote cholesterol efflux. Cholesterol synthesis is tightly regulated by a feedback system that senses the level of cholesterol and modulates the transcription of genes encoding enzymes of cholesterol biosynthesis and uptake (14, 15). For instance, when cholesterol levels rise in cells, the membrane-embedded protein of the endoplasmic reticulum (ER), Scap, senses the increase and binds to Insigs, proteins located to the ER. Insigs then limit cleavage and nuclear translocation of sterol regulatory element-binding proteins (SREBPs), in particular SREBP-2, an activator of cholesterol synthesis in liver and a...

Ras signaling originates from transient nanoscale compartmentalized regions of the plasma membrane composed of specific proteins and lipids. The highly specific lipid composition of these nanodomains, termed nanoclusters, facilitates effector recruitment and therefore influences signal transduction. This suggests that Ras nanocluster proteolipid composition could represent a novel target for future chemoprevention interventions. There is evidence that consumption of fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) may reduce colon cancer risk in humans, yet the mechanism underlying this effect is unknown. Here, we demonstrate that dietary n-3 PUFA reduce the lateral segregation of cholesterol-dependent and -independent nanoclusters, suppressing phosphatidic acid-dependent oncogenic KRas effector interactions, via their physical incorporation into plasma membrane phospholipids. This results in attenuation of oncogenic Ras-driven colonic hyperproliferation in both and murine models. These findings demonstrate the unique properties of dietary n-3 PUFA in the shaping of Ras nanoscale proteolipid complexes and support the emerging role of plasma membrane-targeted therapies. The influence of dietary long chain n-3 polyunsaturated fatty acids on plasma membrane protein nanoscale organization and KRas signaling supports development of plasma membrane-targeted therapies in colon cancer. http://cancerres.aacrjournals.org/content/canres/78/14/3899/F1.large.jpg .