Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor.
We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.
Anichini
E
E
Institute of General Pathology, Florence University, Italy.
Fibbi
G
G
Pucci
M
M
Caldini
R
R
Chevanne
M
M
Del Rosso
M
M
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D002999:Clone Cells; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015290:Second Messenger Systems; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014161:Transduction, Genetic; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D008934:Mitogens; C524898:PLAUR protein, human; D010446:Peptide Fragments; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011493:Protein Kinase C; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate