It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54 JNK
Cholesterol efflux, an important mechanism by which high density lipoproteins (HDL) protect against atherosclerosis, is initiated by docking of apolipoprotein A-I (apoA-I), a major HDL protein, to specific binding sites followed by activation of ATP-binding cassette transporter A1 (ABCA1) and translocation of cholesterol from intracellular compartments to the exofacial monolayer of the plasma membrane where it is accessible to HDL. In this report, we investigated potential signal transduction pathways that may link apoA-I binding to cholesterol translocation to the plasma membrane and cholesterol efflux. By using pull-down assays we found that apoA-I substantially increased the amount of activated Cdc42, Rac1, and Rho in human fibroblasts. Moreover, apoA-I induced actin polymerization, which is known to be controlled by Rho family G proteins. Inhibition of Cdc42 and Rac1 with Clostridium difficile toxin B inhibited apoA-I-induced cholesterol efflux, whereas inhibition of Rho with Clostridium botulinum C3-exoenzyme exerted opposite effects. Adenoviral expression of a Cdc42(T17N) dominant negative mutant substantially reduced apoA-I-induced cholesterol efflux, whereas dominant negative Rac1(T17N) had no effect. We further found that two downstream effectors of Cdc42/Rac1 signaling, c-Jun N-terminal kinase (JNK) and p38 mitogenactivated protein kinase (p38 MAPK), are activated by apoA-I. Pharmacological inhibition of JNK but not p38 MAPK decreased apoA-I-induced cholesterol efflux, whereas anisomycin and hydrogen peroxide, two direct JNK activators, could partially substitute for apoA-I in its ability to induce cholesterol efflux. These results for the first time demonstrate activation of Rho family G proteins and stress kinases by apoA-I and implicate the involvement of Cdc42 and JNK in the apoA-I-induced cholesterol efflux.
Objective:
ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P
1
(S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport.
Approach and Results:
HDL were isolated from plasma of wild-type mice,
Apom
knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P
1
) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P
1
) endothelium-specific knockin of S1P
1
. The binding, association, and transport of HDL from
Apom
knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of
125
I-HDL by human aortic endothelial cells was increased by an S1P
1
agonist but decreased by an S1P
1
inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of
125
I-HDL transport by the S1P
1
agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P
1
, apoE-haploinsufficient mice with endothelium-specific knockin of S1P
1
showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium.
Conclusions:
ApoM and S1P
1
promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.
We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R mice that are mediated by favourable modulation of endothelial function.
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