Abstract
Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.
Highlights
Degradation of adhesive glycoproteins by plasmin is implicated in cell migration
We further show that gene transfer and expression of protease nexin-1 (PN-1) or plasminogen activator inhibitor-1 (PAI-1) inhibited plasminogen activation-induced anoikis of CHO-K1 cells
The activator, constitutively expressed by wild-type CHO-K1 cells, was identified as type plasminogen activator (t-PA) by (i) the inhibition in cell lysates by specific anti-t-PA polyclonal IgG antibodies (Fig. 2, inset B) of a fibrin zymography lysis band corresponding to the molecular mass of t-PA (Fig. 2, inset A) and (ii) the inhibition of plasminogen activation by 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone (IC50 ϭ 2 nM), a selective inhibitor for t-PA
Summary
The chromogenic substrate CBS0065 ((methylmalonyl)hydroxyprolylarginine-p-nitroanilide) was purchased from Stago (Asnieres, France),. Aprotinin (Trasylol®) from Bayer, and plastic cell culture dishes from TPP (Trasadingen, Switzerland). Goat anti-human t-PA polyclonal antibody was obtained from Biopool (Uppsala, Sweden). Rabbit anti-human fibronectin and anti-mouse laminin polyclonal antibodies were kindly provided by H. P. Erickson (Duke University Medical Center, Durham, NC). Recombinant rat PN-1 and anti-rat PN-1 monoclonal antibody 4B3 [33] were kind gifts of D. Other products were obtained as described previously [34]
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