title: Lactosylceramide recruits PKCalpha/epsilon and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and adhesion to endothelial cells.
abstract: Despite the importance of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) in the adhesion and diapedesis of monocytes/lymphocytes, little is known about the mechanisms by which it is regulated. We explored the role of a glycosphingolipid, lactosylceramide (LacCer), in modulating PECAM-1 expression and cell adhesion in human monocytes. We observed that LacCer specifically exerted a time-dependent increase in PECAM-1 expression in U-937 cells. Maximal increase in PECAM-1 protein occurred after incubation with LacCer for 60 min. LacCer activated PKCalpha and -epsilon by translocating them from cytosol to membrane. This was accompanied by the activation of phospholipase A(2) (PLA(2)) and the increase of cell adhesion, which were abrogated by chelerythrine chloride, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (GO 6976) (PKC inhibitors). Similarly, bromoenol lactone (a Ca(2+)-independent PLA(2) inhibitor) and methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA(2) and Ca(2+)-independent PLA(2)) inhibited LacCer-induced PLA(2) activity. Bromophenacyl bromide (a PLA(2) inhibitor) abrogated LacCer-induced PECAM-1 expression, and this was bypassed by arachidonic acid. Furthermore, the arachidonate-induced up-regulation of PECAM-1 was abrogated by indomethacin [a cyclooxygenase (COX)-1 and -2 inhibitor] or N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (a COX-2 inhibitor) but not nordihydroguaiaretic acid (a lipoxygenase inhibitor). In sum, PKCalpha/epsilon are the primary targets for the activation of LacCer. Downstream activation of intracellular Ca(2+)-independent PLA(2) and/or cytosolic PLA(2) results in the production of arachidonic acid, which in turn serves as a precursor for prostaglandins that subsequently stimulate PECAM-1 expression and cell adhesion. These findings may be relevant in explaining the role of LacCer in the regulation of PECAM-1 and related pathophysiology.
authors.affiliation: Johns Hopkins Singapore-National Heart Centre Vascular Biology Program, 41 Singapore Science Park II, Singapore 117610.
authors.forename: NanLing
authors.initials: N
authors.lastname: Gong
authors.forename: Heming
authors.initials: H
authors.lastname: Wei
authors.forename: Sanaul Haq
authors.initials: SH
authors.lastname: Chowdhury
authors.forename: Subroto
authors.initials: S
authors.lastname: Chatterjee
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000494:pharmacology; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007790:Lactosylceramides / Q000494:pharmacology*; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D011453:Prostaglandins / Q000502:physiology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D014158:Transcription, Genetic / Q000187:drug effects; D020298:U937 Cells; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D007790:Lactosylceramides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011453:Prostaglandins; D016718:Arachidonic Acid; C009744:CDw17 antigen; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D010741:Phospholipases A; D054467:Phospholipases A2