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. 2005 Jan;3(1):163-9.
doi: 10.1111/j.1538-7836.2004.01054.x.

A novel mechanism of plasmin-induced mitogenesis in fibroblasts

S K Mandal  1 , L V M Rao, T T T Tran, U R Pendurthi
Affiliations

A novel mechanism of plasmin-induced mitogenesis in fibroblasts

S K Mandal et al. J Thromb Haemost. 2005 Jan.

Abstract

The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor-like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin-induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3H-thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose-dependent manner. Protease-activated receptor-1 (PAR-1)-specific antibodies, but not PAR-2-specific antibodies, reduced the plasmin-induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR-1-deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin-induced DNA synthesis. Plasmin-induced increase in the DNA synthesis was completely abrogated by anti-Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrix-associated Cyr61 and the conditioned media from plasmin-treated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR-1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.

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Figures

Fig. 1
Fig. 1
Effect of plasmin, thrombin and factor FVIIa on DNA synthesis of fibroblasts. (A) Quiescent monolayers of WI-38 cells were treated with plasmin (50 nm), thrombin (10 nm), FVIIa (100 nm) or fetal bovine serum (FBS) (10% v/v) for 48 h. 3H-methyl thymidine (1 μCi mL−1) was included in the medium. At the end of 48 h, 3H-thymidine incorporated into DNA was determined as described in Materials and methods (n = 6, mean ± SE). *P < 0.001; **P < 0.05, compared with the control value #(P = 0.004), compared with the values obtained with thrombin or FVIIa treatment. (B) Quiescent monolayers of WI-38 cells were treated with various concentrations of plasmin (1–100 nm) or FBS (10% v/v) and 3H-thymidine incorporated into DNA was determined as in (A) (mean ± SE, n = 6). *Values differ significantly (P < 0.01) from the control value. (C) Monolayers were treated with plasmin (50 nm), active site-inhibited plasmin (PPACK-plasmin, 50 nm) or plasmin (50 nm) pretreated with tissue factor pathway inhibitor (TFPI)-2 (250 nm) and 3H-thymidine incorporated into DNA was determined at the end of 48 h (mean ± SE from three experiments). *Values differ significantly (P < 0.001) from the value obtained with plasmin treatment.
Fig. 2
Fig. 2
Involvement of protease-activated receptor (PAR)-1 in plasmin-induced DNA synthesis. (A) Quiescent monolayers of wild-type mouse fibroblasts (filled bars) or PAR-1-deficient mouse fibroblasts (hatched bars) were treated with varying concentrations of plasmin or serum (10% v/v) in the presence of 3H-methyl thymidine (1 μCi mL−1). At the end of 48 h stimulation, 3H-thymidine incorporated into DNA was determined (mean ± SE, n = 3–6). *P < 0.05. (B) WI-38 cells were pretreated with PAR-1-specific monoclonal antibody, ATAP-2 (25 μg mL−1) or PAR-2-specific polyclonal antibodies (500 μg mL−1) for 1 h, followed by plasmin (50 nm) stimulation. At the end of 48 h stimulation, 3H-thymidine incorporated into DNA was determined (mean ± SE, n = 3–7). *P < 0.001; ns, no significant difference from the value obtained with plasmin treatment alone.
Fig. 3
Fig. 3
Involvement of p44/42 MAPK and p38 MAPK activation in plasmin-induced DNA synthesis. Quiescent monolayers of WI-38 cells were preincubated with or without specific inhibitors for 30 min and then stimulated with plasmin (50 nm) for 5 min (C) or 20 min (A,B). Cell lysates were subjected to Western blot analysis to detect phosphospecific and total p38 and p44/42 MAPK. (D) The effect of inhibitors on plasmin-induced DNA synthesis (mean ± SE, n = 4). Inhibitor concentrations as follows: PD98059, 50 μm; U0126, 10 μm; SB203580, 25 μm.Values obtained with the inhibitors significantly differ (P < 0.01) from the value obtained with plasmin alone.
Fig. 4
Fig. 4
Anti-Cyr61 antibodies abolish plasmin-induced DNA synthesis. Quiescent monolayers of WI-38 cells were pretreated with antihuman Cyr61 IgG (100 μg mL−1) or an irrelevant IgG as a control (100 μg mL−1) for 1 h. The cells were stimulated with plasmin (50 nm) for 48 h and 3H-thymidine incorporated into DNA was determined (n = 3).
Fig. 5
Fig. 5
Effect of plasmin and thrombin on Cyr61 protein expression in fibroblasts and recombinant Cyr61. Quiescent monolayers of WI-38 cells were treated with plasmin (50 nm) (A) or thrombin (10 nm) (B). At varying times, the overlying medium was removed, the cells were solubilized in cell lysis buffer and cell lysates were subjected to immunoblot analysis and probed with anti-Cyr61 antibodies. (C) Recombinant human Cyr61 (10 μg mL−1) was incubated with a control buffer (C), plasmin (10 nm) or thrombin (10 nm) for 1 h and the samples were subjected to immunoblot analysis with Cyr61 antibodies. Recombinant Cyr61 migrated at a higher Mr because it contained V5-His tag at the C-terminus.
Fig. 6
Fig. 6
Evidence for plasmin release of Cyr61 contributes to increased DNA synthesis in fibroblasts. Quiescent monolayers of WI-38 cells were exposed to control medium or medium containing plasmin (50 nm) or thrombin (10 nm). At the end of 2 h, the supernatant media were collected and the protease activity of plasmin in the conditioned media was inactivated with tissue factor pathway inhibitor (TFPI)-2 (250 nm, for 30 min). The conditioned media of plasmin-treated cells were incubated for 30 min with control IgG or anti-Cyr61 IgG (100 μg mL−1) before being added to fresh fibroblasts. 3H-thymidine incorporated into DNA was determined at the end of 48 h (mean ± SE, n = 4). *Paired values differ significantly from each other (P < 0.005). ns, Not significant.
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References

    1. Carmeliet P, Collen D. Development and disease in proteinase-deficient mice: role of the plasminogen, matrix metalloproteinase and coagulation system. Thromb Res. 1998;91:255–85. - PubMed
    1. Pepper MS. Role of the matrix metalloproteinases and plasminogen activator–plasmin system in angiogenesis. Arterioscler Thromb Vasc Biol. 2001;21:1104–17. - PubMed
    1. Lijnen HR. Plasmin and matrix metalloproteinases. Thromb Haemost. 2001;86:324–33. - PubMed
    1. Ploplis VA, Castellino FJ. Gene targeting of components of the fibrinolytic system. Thromb Haemost. 2002;87:22–31. - PubMed
    1. Lyons RM, Gentry LE, Purchio AF, Moses HL. Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin. J Cell Biol. 1990;110:1361–7. - PMC - PubMed

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