HDL 3-signalling in HepG 2 cells involves glycosyl-phospatidylinositol-anchored proteins

Abstract

In [ 3H]phosphatidylcholine (PC) prelabelled HepG 2 cells, HDL 3 stimulates a biphasic increase in 1,2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 μM D 609, RHC-80267 or U-73122 and less by 100 μM propranolol. A phospholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 μM propranolol. Cellular preincubation with 200 μg/ml antibodies against the inositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab IPG), or depletion in GPI-anchored proteins by cellular pretreatment with 0.5 U/ml PI-PLC, 1 mM insulin and 2 HU/ml streptolysin-O, or depletion in membrane cholesterol content by filipin (5 μg/ml), digitonin (5 μg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL 3-signal, suggesting the involvement of a lipolytic cleavage of GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response time to HDL 3, with a DAG peak at 2–3 min. In the presence of protease-inhibitors, HDL 3 releases in the culture medium several proteins with a residual IPG that binds Ab IPG after SDS-PAGE analysis and immunoblotting. HDL 3-signalling pathways comprise tyrosine kinases, as preincubation with 100 μg/ml genistein or tyrphostin inhibits the HDL 3-signal. HDL 3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.