HDL₃Stimulates Multiple Signaling Pathways in Human Skin Fibroblasts

The influence of HDL3 on phospholipid breakdown was examined in human skin fibroblasts. HDL3 elicited phosphatidylcholine (PC) and phosphatidylinositol (PI) turnover and activated multiple phospholipases. In [14C]lyso-PC-labeled or [14C]choline (Cho)-labeled cells, a biphasic activation of PC-specific phospholipase D (PLD) with peak maxima 30 to 60 seconds and 5 to 7 minutes after stimulation with 20 micrograms/mL HDL3 was shown by (1) a 1.5- to 3-fold increase in Cho release, and (3) transphosphatidylation of PC to phosphatidylbutanol in the presence of 0.3% butanol. Activation of PC-specific PLD was paralleled by an activation of PC-specific phospholipase C (PLC). A significant increase in [14C]diacylglycerol (DG) was seen from 2 minutes after stimulation onward and remained for at least 2 hours. By means of butanol, the PA-phosphohydrolase (PPH) inhibitor propranolol, and the PC-PLC inhibitor D609, we demonstrated that the initial PC-derived DG formation occurred primarily by a coupled PLD/PPH pathway and that a major part of the sustained DG formation was derived directly from PC by PC-PLC. By down-regulating protein kinase C (PKC) we demonstrated that PKC activates PC-PLC and desensitizes PC-PLD at no longer incubation times. The sustained PC hydrolysis as well as HDL3-mediated PI turnover and PC resynthesis was observed on stimulation with 5 to 75 micrograms/mL HDL3, whereas the rapid activation of PC-PLD/PPH was detected only on stimulation with HDL3 at concentrations of between 10 and 75 micrograms/mL. Only the latter response could be mimicked by apolipoprotein A-I and apolipoprotein A-II proteoliposomes, and only this response was inducible by cholesterol loading. The HDL3-mediated second-messenger responses were inhibited by modification of HDL3 by tetranitromethane and could not be mimicked by protein-free liposomes. These data suggest that HDL3-induced cell signaling in human skin fibroblasts is mediated by specific protein-receptor interaction and that more than one agonist activity may be involved.