Abstract 408: A Novel PIP2-dependent Pathway Maintains Cellular Cholesterol Homeostasis

Introduction: ABCA1 acts as an anti-atherosclerotic protein by removing excess cholesterol from arterial foam cells via reverse cholesterol transport (RCT) pathway. ABCA1 mediated phosphatidylinositol 4, 5-bisphosphate (PIP2) flop (translocation of PIP2 from inner leaflet of plasma membrane to the cell-surface) promotes cholesterol efflux, indicating cross talk between cholesterol and PIP2 in maintaining lipid homeostasis. The mechanistic details of how cholesterol, PIP2, and ABCA1 are interlinked are not yet explored. Objectives: To determine the mechanism by which PIP2 maintains cellular cholesterol homeostasis. Methods and Results: To decipher link between PIP2, cholesterol, and ABCA1, we used macrophages (RAW264.7, BMDMs, and THP-1), HepG2, or PIP2 phosphatase (TMEM55b-/- KO) mice. For cholesterol loading, we used AcLDL (100 μg/ml for 24h) or cyclodextrin-cholesterol (50 μg/ml for 24 h). Cholesterol was depleted by cyclodextrin or simvastatin (2μM). PIP2 levels and localization were determined by Mass-ELISA and Nikon super-high resolution microscopy. Cholesterol loading increased PIP2 levels (~25-35% increase across various cell lines), while cholesterol depletion reduced PIP2 levels by ~30-40%. Cholesterol loading led to redistribution of PIP2 from plasma membrane to ER-PM junctions. Cholesterol loading reduced, while cholesterol depletion increased, TMEM55b levels. Expression of TMEM55b in BHK cells resulted in markedly reduced apoA1 binding, reduced ABCA1 levels, and resulted in ~ 70 % decrease in cholesterol efflux to apoA1 (N=4, P<0.0001). Importantly, expression of close homolog TMEM55a showed no effect on ABCA1 expression. Genetic or chemical ablation of PIP2 biosynthesis in THP-1 macrophages markedly reduced ABCA1 levels and cholesterol efflux. THP-1 macrophages supplemented with exogenous PIP2 showed increased ABCA1 expression. Conclusion: Cholesterol modulates PIP2 levels/localization, and PIP2 in-turn modulates ABCA1 expression to maintain cellular cholesterol homeostasis.