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Abstract 460: The Molecular Mechanism of Regulation of Macrophages Pinocytosis of Lipoproteins by Akt3

Akt, also known as protein kinase B (PKB), plays an important role in the regulation of numerous cellular processes, including cell growth, glucose metabolism, differentiation, proliferation, migration and apoptosis. We have recently demonstrated that deletion of Akt3 in macrophages promotes foam cell formation by increasing the uptake of native and modified LDL (Low-density lipoprotein) and elevating the level of ACAT-1, an enzyme esterifying excess cholesterol and promoting its storage in cytoplasmic lipid droplets. We ruled out increased scavenger receptor-dependent uptake and receptor-dependent endocytosis of lipoproteins as the mechanism of increased lipoprotein uptake, and demonstrated in vitro enhanced receptor-independent pinocytosis in Akt3-/- macrophages. The aim of this study was to investigate the mechanism of regulation of macrophage pinocytosis by Akt3. Actin assembly is the pivotal process in pinocytosis of lipoproteins. We observed increased actin assembly in Akt3-/- macrophages assessed by phaloidin staining, suggesting that actin assembly can be involved in the elevated pinocytosis in Akt3-/- macrophages. The increase in pinocytosis by Akt3-/- macrophages was reduced in the presence of actin assembly inhibitor, indicating that the enhanced pinocytosis in Akt3-/- macrophages is actin assembly dependent. Colocalization of actin filaments with Akt3 in wild-type murine macrophages using a confocal fluorescence microscopy as well as coimmunoprecipitation of β-actin with Akt3 in transfected HEK293 cells revealed a direct interaction between Akt3 and β-actin. We ruled out the possibility that Akt3 regulates macrophage pinocytosis via major downstream molecules of the Akt signaling pathway (mTOR, GSK3 and NFκB) by using specific pharmacological inhibitors. However, we observed that increased pinocytosis in Akt3-/- macrophages is Src kinases dependent. These results suggest that Akt3 may inhibit actin assembly and subsequent pinocytosis of LDL by direct interaction with β-actin. Taken together, our studies describe a novel mechanism of atheroprotective role of Akt3.