Abstract 198: The Role of PI(4,5)P 2 in LDL Receptor Lysosomal Decay

Abstract

Upregulation of low density lipoprotein receptor (LDLR) activity reduces LDL levels and coronary disease risk. LDLR releases LDL in endosomes after internalization and then is either recycled to the cell surface or transported to lysosomes for decay. The transmembrane protein 55B (TMEM55B) is a phosphatase that hydrolyzes phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ) and has been found to affect lysosome function. We reported TMEM55B regulates cellular cholesterol metabolism by modulating LDLR protein decay. To evaluate whether Tmem55b affects cholesterol metabolism in vivo , we treated western diet fed C57BL/6J mice with antisense oligonucleotides against either Tmem55b or a non-targeting control for 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ~70%, resulting in increased plasma total (1.5-fold, p<0.0001) and non-HDL-cholesterol (1.8-fold, p<0.0001). FPLC and ion mobility analyses revealed increased levels of small LDL particles that were enriched in apoE as determined by immunoblot. Notably, Tmem55b knockdown had no effect on plasma cholesterol levels in Ldlr -/- mice. Increased LDLc (1.3-fold, p<0.05) was also observed in a murine model that lacks Ocrl, another phosphatase which hydrolyzes PI(4,5)P 2 . Given the role of the lysosome in LDLR decay, we tested whether TMEM55B regulates LDLR via lysosomes. Using confocal microscopy, TMEM55B knockdown in HepG2 cells significantly increased PI(4,5)P 2 , decreased LDLR, increased lysosome staining, and reduced LDLR-lysosome colocalization. Impairment of lysosome function by incubation with NH 4 Cl or knockdown of the lysosomal proteins LAMP1 or RAB7 abolished the effect of TMEM55B knockdown on LDLR. Although there was no change in RAB11 levels, a marker of recycling endosomes, LDLR-RAB11 colocalization was reduced by 50% upon TMEM55B knockdown. Finally, incubation of HepG2 cells with PI(4,5)P 2 increased LDL uptake and reversed the inhibitory effect of TMEM55B overexpression . Together, these findings suggest that TMEM55B increases plasma cholesterol through stimulating LDLR lysosomal degradation, reducing LDLR recycling to the plasma membrane through PI(4,5)P 2 , and thereby inhibiting plasma clearance of apoE-containing LDL particles.