Domain-dependent action of urokinase on smooth muscle cell responses.
BACKGROUND
Single-chain urokinase-type plasminogen activator (sc-uPA) is one of the key serine proteases involved in modulating cellular and extracellular matrix responses during tissue remodeling. Sc-uPA is composed of three domains: aminoterminal fragment (ATF), kringle domain, and carboxyterminal fragment (CTF). sc-uPA is readily cleaved into these three domain fragments in vitro, each of which is biologically active; however, their roles in the microenvironment of the vessel wall are poorly understood.


OBJECTIVE
The purpose of this study was to determine the role of each domain of sc-uPA on vascular smooth muscle cell (SMC) proliferation and migration.


METHODS
SMCs were cultured in vitro. Assays of DNA synthesis, cell proliferation, and migration were performed in response to sc-uPA, ATF, kringle, and CTF in the presence and absence of the plasmin inhibitors epsilon-aminocaproic acid (EACA) and aprotinin, the Galphai inhibitor pertussis toxin, and the mitogen-activated protein kinase 1 (the upstream regulator of the extracellular-signal regulated kinase [ERK]) inhibitor PD98059.


RESULTS
sc-uPA produced dose-dependent increases in DNA synthesis and cell proliferation. These responses were dependent on the CTF domain and were sensitive to plasmin inhibitors, pertussis toxin, and PD98059. Sc-uPA also induced SMC migration, which could be elicited by both ATF and kringle. Migration to sc-uPA, ATF, and kringle was both pertussis toxin and PD98059 sensitive, but importantly was plasmin-independent.


CONCLUSIONS
sc-uPA induces SMC proliferation and migration, which are domain-dependent and mediated in part by Galphai-linked, ERK-dependent processes, while only the mitogenic response is protease dependent. These findings suggest that migration is linked to a G-protein coupled nonprotease receptor, while proliferation is associated with a G-protein coupled protease receptor.
Tanski
William J
WJ
Department of Surgery, University of Rochester, Strong Memorial Hospital, Box SURG, 601 Elmwood Avenue, Rochester, NY 14642, USA. wtanski@rochester.rr.com
Fegley
Allison J
AJ
Roztocil
Elisa
E
Davies
Mark G
MG
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D018082:Kringles; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology; D010960:Plasminogen Activators / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000494:pharmacology*
D010446:Peptide Fragments; D004247:DNA; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator