Regulation of the endothelial cell urokinase-type plasminogen activator receptor. Evidence for cyclic AMP-dependent and protein kinase C-dependent pathways.
Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.
Langer
D J
DJ
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Kuo
A
A
Kariko
K
K
Ahuja
M
M
Klugherz
B D
BD
Ivanics
K M
KM
Hoxie
J A
JA
Williams
W V
WV
Liang
B T
BT
Cines
D B
DB
D015152:Blotting, Northern; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004792:Enzyme Precursors / Q000032:analysis* / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010960:Plasminogen Activators / Q000032:analysis* / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000187:drug effects; D055293:Receptors, Urokinase Plasminogen Activator; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000187:drug effects
D004792:Enzyme Precursors; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D005576:Colforsin; D012313:RNA; D003513:Cycloheximide; D000242:Cyclic AMP; D011493:Protein Kinase C; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator