Cellular Pip2 Is Effluxed By Abca1 To Apoa1 And Pip2 Is Carried On Hdl That Can Be Delivered To Target Tissues Via Sr-B1

Introduction: apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport. Cell surface phosphatidylinositol 4,5- bisphosphate (PIP2) mediates this process, but whether PIP2 is effluxed via apoA1-ABCA1 pathway and is carried in plasma is unknown. Objective: To determine if PIP2 is effluxed during ABCA1-dependent nHDL biogenesis, whether PIP2 is found on plasma HDL, and if PIP2 can be delivered to target cells via SRB1. Methods and Results: We previously reported at this meeting that: 1) apoAI binds to PIP2 with a K d of ~100 nM; 2) ABCA1 expression translocates PIP2 from the inner to outer leaflet of the plasma membrane; 3) cell surface PIP2 is required for ABCA1-dependent cellular binding of apoA1 and cholesterol efflux. Here, we report that PIP2 incubation with lipid-free apoA1 promoted monomeric vs. dimeric apoA1. We found that ABCA1 expression increased efflux of PIP2 by ~20-fold in RAW264.7 cells labeled with [3H]myo-inositol, and chased with apoA1 (mean ± SD; ***, p<0.001, two-tailed t-test, n=3). Plasma from apoA1 knockout (A1 KO), wild type (WT), and human apoA1 transgenic (A1-Tg) mice contained apoA1-gene dosage dependent levels of both cholesterol and PIP2, with 64-fold higher PIP2 levels in the A1-Tg vs. A1 KO mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). PIP2 was also reverse transported in vivo from [3H]myo-inositol-labeled macrophages implanted s.c into mice. Labeled PIP2 was recovered in the plasma, with a 4-fold higher of the injected radioactivity found in the WT vs. A1 KO host mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). FPLC separation of human plasma determined that almost all of the plasma PIP2 was found in the HDL fraction; and, human HDL contained two PIP2 species with either 18:0, 20:4 fatty acids or 16:0, 20:4 fatty acids, as detected by liquid chromatography tandem mass spectrometry. SR-BI-inducible BHK cells exhibited 2-fold higher uptake of [3H]PIP2-labeled HDL after SR-BI induction, indicating that HDL can deliver PIP2 to target cells (mean ± SD; **, p<0.01, two-tailed t-test, n=3). Conclusions: PIP2 is effluxed from cells via the apoA1-ABCA1 pathway and is carried on HDL. HDL can deliver PIP2 to target tissues via SR-B1.