ATP Stimulates Caveolar Endocytosis of P2X 7R in MC3T3-E1 Osteoblasts

Abstract

Purinergic signaling plays a critical role in mechanotransduction in bone. The nucleotides released by the bone cells in response to mechanical stimulation initiate a cascade of signaling events in osteoblasts. Purinergic signaling plays a critical role in mechanotransduction in bone The nucleotides released by the bone cells in response to mechanical stimulation initiate a cascade of signaling events in osteoblasts. P2X7 activation is required for PGE2 release, COX2 synthesis and bone formation, however current research lacks to identify the detailed signaling events and mechanism leading to this activation. We determined the localization of P2X7 receptors on the plasma membrane and it's co-loclaization using the family of Image Correlation Spectroscopy combined with molecular biological methods. Additionally, we used detergent free isolation of lipid rafts to prove that P2X7 receptors are present in caveolae and that release of ATP leads to caveolae endocytosis. To visualize the caveolar endocytosis in response to ATP treatment, MC3T3-E1 cells were transfected with caveolin-1 GFP and visualized under Zeiss 5 Live confocal microscope. We used AF-555 albumin as a selective marker for caveolar endocytosis. The cells treated with 250μM ATP for 40 minutes showed increased AF-555 albumin uptake and its co-localization with caveolin-1 GFP demonstrates endocytosis via caveolae. These data suggest ATP stimulation translocates caveolin-1 from the membrane lipid rafts into the cytosol. Presence of P2X7 receptors in the same fraction as caveolin-1 demonstrates its existence in caveolae of MC3T3-E1 osteoblasts and ATP stimulation induces caveolar endocytosis of P2X7 receptor. The caveolae is a major signaling hub in mammalian cells, the presence P2X7 receptor and its endocytosis in response to ATP stimulation could be a vital process in mechanotransduction in osteoblasts.