Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.
Serum-free rat hepatocyte cultures can be stimulated to divide by the inactive, single-chain form of hepatocyte growth factor (scHGF), suggesting that hepatocytes contain a protein that can cleave scHGF to its biologically active, two-chain (tcHGF) form. We added radiolabeled scHGF to serum-free cultures and confirmed that tcHGF was being generated. Because scHGF can be cleaved to tcHGF by plasminogen activators (PAs), we next tested the cultures for active PA. Although little PA activity was initially present, the majority was of the urokinase type (u-PA) as determined by neutralization studies using either a polyclonal antibody against u-PA or, since u-PA functions in the context of its receptor (u-PAR), a monoclonal antibody against u-PAR. Considerable PA activity developed within 24 h, which was also neutralizable with antibody. To test whether the active, receptor-bound u-PA from the cell cultures was cleaving scHGF, iodinated scHGF was added to intact cells in the presence of the antibody against u-PAR. Comparison to control cultures determined that the antibody prevented scHGF cleavage. Analysis of cultures treated with HGF, epidermal growth factor, and transforming growth factor alpha (TGF-alpha) alpha showed these growth factors increased the hepatocyte PA activity in parallel with the mRNA for u-PA. TGF-beta had the opposite effect, and when TGF-beta was added to the culture system, conversion of scHGF to tcHGF was prevented in concert with the production of the type 1 PA inhibitor. When liver remnants from hepatectomized animals were assayed for active TGF-beta, elevated protein was found just prior to the appearance of PA inhibitor 1 message and protein. Collectively, our data show that in culture, active u-PA is present and cleaves scHGF to tcHGF in the context of its receptor. It also suggests that modulation of u-PA activity by various growth factors is relevant for regulating cleavage of scHGF to tcHGF both in vitro and in vivo.
Mars
W M
WM
Department of Pathology, University of Pittsburgh, Pennsylvania 15261, USA.
Kim
T H
TH
Stolz
D B
DB
Liu
M L
ML
Michalopoulos
G K
GK
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004815:Epidermal Growth Factor / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors; D055293:Receptors, Urokinase Plasminogen Activator; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; C524900:Plaur protein, rat; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D016211:Transforming Growth Factor alpha; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator