Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis.
In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.
Spisni
E
E
Department of Experimental Biology, University of Bologna, 40126 Bologna, Italy.
Griffoni
C
C
Santi
S
S
Riccio
M
M
Marulli
R
R
Bartolini
G
G
Toni
M
M
Ullrich
V
V
Tomasi
V
V
D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D011464:Epoprostenol / Q000096:biosynthesis*; D005372:Filipin / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D017612:Gold Compounds / Q000493:pharmacokinetics; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D017830:Octoxynol / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D000900:Anti-Bacterial Agents; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D017612:Gold Compounds; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D015121:6-Ketoprostaglandin F1 alpha; D005372:Filipin; D017830:Octoxynol; D003577:Cytochrome P-450 Enzyme System; D011464:Epoprostenol; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase