Abstract-Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1 Ϫ/Ϫ mice, and this phenotype persists in PDZK1Ϫ/Ϫ mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity. Key Words: PDZK1 Ⅲ high-density lipoprotein Ⅲ SR-BI Ⅲ endothelium T he risk of atherosclerosis is inversely related to circulating high-density lipoprotein (HDL) cholesterol levels, 1,2 and there is also evidence that a lower HDL level is associated with a greater likelihood of restenosis after a vascular intervention. 3,4 HDL classically functions in reverse cholesterol transport, removing cholesterol from peripheral tissues and delivering it to the liver and to steroidogenic organs by binding of the major HDL apolipoprotein, apolipoprotein (apo)A-I, to the high-affinity HDL receptor scavenger receptor B type I (SR-BI). 5,6 In mouse models of atherosclerosis, apoA-I and SR-BI both provide atheroprotection, 7,8 and in the context of experimental hypercholesterolemia, the provision of apoA-I or HDL attenuates neointima formation after artery injury. 9,10 The protective nature of HDL has been previously attributed to its role in reverse cholesterol transport. However, evidence is accumulating that HDL has a number of additional actions that also afford cardiovascular protection, and many of these entail direct modulation of endothelial cell phenotype. 11 The direct actions of HDL on the endothelium are multiple. In particular, HDL promotes the production of the atheroprotective signaling molecule NO by upregulating endothelial NO synthase (eNOS) expression, 12 by maintaining the lipid environment in caveolae, where eNOS is colocalized with partner signaling molecules, 13 and by stimulating eNOS enzymatic activity. 14,15 As importantly, HDL protects endothelial cells from...

Abstract-Scavenger receptor B type I (SR-BI) plays an important role in mediating cholesterol exchange between cells, high-density lipoprotein (HDL) cholesterol, and other lipoproteins. SR-BI in hepatocytes is essential for reverse cholesterol transport and biliary secretion of HDL cholesterol; thus, it is atheroprotective. More recently, it has been discovered that the HDL-SR-BI tandem serves other functions that also likely contribute to HDL-related cardiovascular protection. A number of the latter mechanisms, particularly in endothelial cells, involve unique direct signal initiation by SR-BI that leads to the activation of diverse kinase cascades. SR-BI signaling occurs in response to plasma membrane cholesterol flux. It requires the C-terminal PDZ-interacting domain of the receptor, which mediates direct interaction with the adaptor molecule PDZK1; and the C-terminal transmembrane domain, which directly binds membrane cholesterol. In endothelium, direct SR-BI signaling in response to HDL results in enhanced production of the antiatherogenic molecule nitric oxide; in a nitric oxide-independent manner, it serves to maintain endothelial monolayer integrity. The role of SR-BI signaling in the numerous other cellular targets of HDL, including hepatocytes, macrophages, and platelets, and the basis by which SR-BI senses plasma membrane cholesterol movement to modify cell behavior are unknown.

Rationale Signal initiation by the HDL receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain (CTTM). The CTTM uniquely interacts with plasma membrane (PM) cholesterol. Objective The molecular basis and functional significance of SR-BI interaction with plasma membrane cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a plasma membrane cholesterol sensor. Methods and Results In studies performed in COS-M6 cells, mutation of a highly-conserved CTTM glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin (CD) was also prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or CD in cultured enterocytes, and it is required for HDL activation of eNOS and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusion Through interaction with plasma membrane cholesterol, SR-BI serves a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.