Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

The lipid kinase PI4KIIIß is highly expressed in breast tumors and activates Akt in cooperation with Rab11a.

Emerging evidence now implicates phosphatidylinositol 4-kinases (PI4K), enzymes that generate PI(4)P from phosphatidylinositol (PtdIns), in cancer. In this study, we investigate the role of PI4KIIIß, one of four mammalian PI4Ks, in breast cancer. Although PI4KIIIß protein levels are low in normal breast tissue, we find that approximately 20% of primary human breast tumors overexpress it. Expression of PI4KIIIß in breast carcinoma cells leads to increased Akt activation, dependent on increased PI(3,4,5)P3 production. However, a kinase-inactive version of PI4KIIIß also led to increased Akt activation, and no changes in PI(4)P or PI(4,5)P2 lipid abundance were detected in the PI4KIIIß-overexpressing cells. This implies that PI4KIIIß regulates PI(3,4,5)P3 and Akt independent of PI(4)P production. We find that the PI4KIIIß-binding protein, Rab11a, a small GTPase that regulates endosomal recycling, is involved in PI4KIIIß-mediated activation of Akt, as RNAi depletion of Rab11a impairs Akt activation. Furthermore, ectopic PI4KIIIß expression alters cellular Rab11a distribution and enhances recruitment of PI4KIIIß and Rab11a to recycling endosomes. This work suggests that PI4KIIIß affects PI3K/Akt signaling through Rab11a and endosomal trafficking, independent of its lipid kinase activity. Thus, PI4KIIIß likely plays a role in breast oncogenesis and that cooperation between Rab11a and PI4KIIIß represents a novel Akt activation pathway.