HDL3-signalling in HepG2 cells involves glycosyl-phosphatidylinositol-anchored proteins.
In [3H]phosphatidylcholine (PC) prelabelled HepG2 cells, HDL3 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 microM D 609, RHC-80267 or U-73122 and less by 100 microM 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 microM propranolol. Cellular preincubation with 200 microg/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 microg/ml), digitonin (5 microg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL3-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 HDL3, with a DAG peak at 2-3 min. In the presence of protease-inhibitors, HDL3 releases in the culture medium several proteins with a residual IPG that binds Ab(IPG) after SDS-PAGE analysis and immunoblotting. HDL3-signalling pathways comprise tyrosine kinases, as preincubation with 100 microg/ml genistein or tyrphostin inhibits the HDL3-signal. HDL3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.
Nazih-Sanderson
F
F
Unité INSERM 325, Institut Pasteur, Lille, France.
Pinchon
G
G
Nion
S
S
Fruchart
J C
JC
Delbart
C
C
D000906:Antibodies / Q000276:immunology; D003470:Culture Media / Q000032:analysis; D004075:Diglycerides / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
C011439:1,2-diacylglycerol; D000906:Antibodies; D003470:Culture Media; D004075:Diglycerides; D017261:Glycosylphosphatidylinositols; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D011480:Protease Inhibitors; D010738:Type C Phospholipases