Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Dec;35(6):705-13.
doi: 10.1165/rcmb.2006-0006OC. Epub 2006 Jul 13.

Plasminogen-mediated activation and release of hepatocyte growth factor from extracellular matrix

Hiroto Matsuoka  1 , Thomas H Sisson, Teruaki Nishiuma, Richard H Simon
Affiliations

Plasminogen-mediated activation and release of hepatocyte growth factor from extracellular matrix

Hiroto Matsuoka et al. Am J Respir Cell Mol Biol. 2006 Dec.

Abstract

Interventions that enhance plasminogen activation within the lung consistently limit the fibrosis that follows alveolar injury. However, this protective effect cannot be attributed solely to accelerated clearance of fibrin that forms as a provisional matrix after lung injury. To explore other mechanisms, we considered interactions between the plasminogen activation system and hepatocyte growth factor (HGF). HGF is known to have antifibrotic activity, but to do so, it must be both released from its sites of sequestration within extracellular matrix (ECM) and activated by proteolytic cleavage. A recent study using bleomycin-exposed mice showed that manipulations of the plasminogen activation system influenced the amount of free HGF within bronchoalveolar lavage fluid without affecting total lung HGF mRNA or protein. To elucidate the mechanisms, we studied the role of plasminogen activation in fibroblast-mediated HGF release and activation. We found that NIH3T3 and mouse lung fibroblasts release ECM-bound HGF in a plasminogen-dependent fashion. The plasminogen effect was lost when lung fibroblasts from urokinase-type plasminogen activator (uPA)-deficient mice were used, and was increased by fibroblasts from plasminogen activator inhibitor (PAI)-1-deficient mice. Plasminogen addition to NIH3T3 or mouse lung fibroblasts increased conversion of pro-HGF to its active form. The plasminogen effect on activation was lost when uPA-deficient fibroblasts were used and accentuated by PAI-1-deficient fibroblasts. In conjunction with the previous in vivo study, these results suggest that plasminogen activation can protect the lung against fibrosis by increasing the availability of active HGF.

PubMed Disclaimer

Figures

<b>Figure 1.</b>
Figure 1.
HGF binding to NIH3T3-derived ECM and release by plasmin. (A) Soluble heparin inhibits HGF binding to ECM. Fibroblast-derived ECMs were incubated with 10 ng/ml 125I-HGF together with various concentration of heparin for 2 h at 4°C. After extensive washing, ECMs were collected and their radioactivities in cpm were measured. Values are means ± SEM (n = 3). (B) Plasmin releases HGF from ECM in a dose- and time-dependent fashion. 125I-HGF–labeled ECMs were incubated without plasmin (open triangle), or with 10 mU/ml (open circle) or 100 mU/ml (closed circle) plasmin in PBS for the indicated times. The supernatants were collected and their radioactivities (cpm) were measured. Values are means ± SEM (n = 3). Statistically significant differences (P < 0.01) are present between all treatments at all time intervals.
<b>Figure 1.</b>
Figure 1.
HGF binding to NIH3T3-derived ECM and release by plasmin. (A) Soluble heparin inhibits HGF binding to ECM. Fibroblast-derived ECMs were incubated with 10 ng/ml 125I-HGF together with various concentration of heparin for 2 h at 4°C. After extensive washing, ECMs were collected and their radioactivities in cpm were measured. Values are means ± SEM (n = 3). (B) Plasmin releases HGF from ECM in a dose- and time-dependent fashion. 125I-HGF–labeled ECMs were incubated without plasmin (open triangle), or with 10 mU/ml (open circle) or 100 mU/ml (closed circle) plasmin in PBS for the indicated times. The supernatants were collected and their radioactivities (cpm) were measured. Values are means ± SEM (n = 3). Statistically significant differences (P < 0.01) are present between all treatments at all time intervals.
<b>Figure 2.</b>
Figure 2.
HGF release from ECM by fibroblasts and/or plasminogen and inhibition by ε-aminocaproic acid. ECMs were produced by NIH3T3 cells in 24-well plates, the fibroblasts were removed, and the ECMs were labeled with 10 ng/ml 125I-HGF. (A) HGF is released from ECM synergistically by fibroblasts and plasminogen. NIH3T3 cells (3T3) (2 × 105) and various concentrations of plasminogen (Plg) were added to wells, and the plates were incubated for 6 h. Media were collected, and the amounts of radioactivity released into the media were measured. Values are means ± SEM (n = 3). Connecting lines between designated groups indicate statistically significant differences (*P < 0.001). (B) The plasmin inhibitor, ε-aminocaproic acid, inhibits fibroblast/plasminogen-induced release of HGF from ECM. NIH3T3 cells (3T3)(2 × 105) and various concentrations of plasminogen (Plg) and/or ε-aminocaproic acid (εACA) were added to wells, and the plates were incubated for 6 h. Media were collected and their radioactivities measured. Values are means ± SEM. Connecting lines between designated groups indicate statistically significant differences (*P < 0.01; **P < 0.001).
<b>Figure 2.</b>
Figure 2.
HGF release from ECM by fibroblasts and/or plasminogen and inhibition by ε-aminocaproic acid. ECMs were produced by NIH3T3 cells in 24-well plates, the fibroblasts were removed, and the ECMs were labeled with 10 ng/ml 125I-HGF. (A) HGF is released from ECM synergistically by fibroblasts and plasminogen. NIH3T3 cells (3T3) (2 × 105) and various concentrations of plasminogen (Plg) were added to wells, and the plates were incubated for 6 h. Media were collected, and the amounts of radioactivity released into the media were measured. Values are means ± SEM (n = 3). Connecting lines between designated groups indicate statistically significant differences (*P < 0.001). (B) The plasmin inhibitor, ε-aminocaproic acid, inhibits fibroblast/plasminogen-induced release of HGF from ECM. NIH3T3 cells (3T3)(2 × 105) and various concentrations of plasminogen (Plg) and/or ε-aminocaproic acid (εACA) were added to wells, and the plates were incubated for 6 h. Media were collected and their radioactivities measured. Values are means ± SEM. Connecting lines between designated groups indicate statistically significant differences (*P < 0.01; **P < 0.001).
<b>Figure 3.</b>
Figure 3.
Molecular size and biological activity of HGF released from ECM. (A) Fibroblasts/plasminogen release intact HGF from ECM. ECMs were labeled with 50 ng/ml 125I-HGF and, after washing, were incubated in media alone (media) or with 10 μg/ml plasminogen (Plg) and/or 2 × 105 NIH3T3 cells (3T3) per well. After 5 h, media were collected and separated by SDS-PAGE and were analyzed by autoradiography. The left lane contains a sample of the stock 125I-HGF used to label the matrices. The autoradiogram is representative of the pattern of results seen in three separate studies. (B) HGF released from ECM by fibroblasts/plasminogen is biologically active. ECMs were labeled with 50 ng/ml HGF and incubated with media alone (media) or 10 μg/ml plasminogen (Plg) and/or 2 × 105 NIH3T3 cells (3T3) for 5 h. Conditioned media (100 μl) were transferred to wells in a 96-well plate containing compact colonies of MDCK cells. Serum-free DMEM (Bkgd) or DMEM containing 50 ng/ml recombinant HGF (rHGF) were added to a set of wells as negative and positive control conditions, respectively. To verify that scatter activity depended on HGF, neutralizing anti-human HGF antibody (anti-HGF) or isotype-matched control antibody (iso) were used. After overnight incubation at 37°C, the percent of colonies that showed scattering were measured for each well. Values are means ± SEM (n = 3). The figure is representative of three independent experiments. Connecting lines between designated groups indicate statistically significant differences (*P < 0.01, **P < 0.001). Anti-HGF antibody blocked the scatter activity generated by fibroblasts plus plasminogen and by recombinant HGF (P < 0.001).
<b>Figure 3.</b>
Figure 3.
Molecular size and biological activity of HGF released from ECM. (A) Fibroblasts/plasminogen release intact HGF from ECM. ECMs were labeled with 50 ng/ml 125I-HGF and, after washing, were incubated in media alone (media) or with 10 μg/ml plasminogen (Plg) and/or 2 × 105 NIH3T3 cells (3T3) per well. After 5 h, media were collected and separated by SDS-PAGE and were analyzed by autoradiography. The left lane contains a sample of the stock 125I-HGF used to label the matrices. The autoradiogram is representative of the pattern of results seen in three separate studies. (B) HGF released from ECM by fibroblasts/plasminogen is biologically active. ECMs were labeled with 50 ng/ml HGF and incubated with media alone (media) or 10 μg/ml plasminogen (Plg) and/or 2 × 105 NIH3T3 cells (3T3) for 5 h. Conditioned media (100 μl) were transferred to wells in a 96-well plate containing compact colonies of MDCK cells. Serum-free DMEM (Bkgd) or DMEM containing 50 ng/ml recombinant HGF (rHGF) were added to a set of wells as negative and positive control conditions, respectively. To verify that scatter activity depended on HGF, neutralizing anti-human HGF antibody (anti-HGF) or isotype-matched control antibody (iso) were used. After overnight incubation at 37°C, the percent of colonies that showed scattering were measured for each well. Values are means ± SEM (n = 3). The figure is representative of three independent experiments. Connecting lines between designated groups indicate statistically significant differences (*P < 0.01, **P < 0.001). Anti-HGF antibody blocked the scatter activity generated by fibroblasts plus plasminogen and by recombinant HGF (P < 0.001).
<b>Figure 4.</b>
Figure 4.
HGF release from ECM by primary lung fibroblasts obtained from WT, uPA−/−, and PAI-1−/− mice. Lung fibroblasts (5 × 104 cells/well) from mice of each genotype were plated onto radiolabeled ECM (10 ng/ml 125I-HGF) in 24-well plates and cultured in serum-free DMEM with or without 10 μg/ml plasminogen. After 10-h incubation, media were collected and their radioactivities were measured. Values are means ± SEM (n = 3). The addition of plasminogen increased the release of radiolabel from ECM by WT and PAI-1−/− mice significantly (*P < 0.001). However the release of radioactivity by uPA−/− fibroblasts was unchanged with the addition of plasminogen (P > 0.05).
<b>Figure 5.</b>
Figure 5.
Activation of endogenously generated HGF by NIH3T3 cells. (A) Leupeptin inhibits the FBS-induced activation pro-HGF. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid, with or without 20 μg/ml leupeptin, for 7 d. Equal volumes of media were analyzed by SDS-PAGE run under reducing conditions, followed by immunoblotting for HGF. Results of a representative experiment (n > 10) are displayed for cells grown in the absence (Cont) or presence (Leup) of leupeptin. The locations of migration of pro-HGF and active, α-chain HGF are displayed. (B and C) NIH3T3 fibroblasts activate HGF in a plasminogen-dependent process that is inhibitable by α2-antiplasmin and aprotinin. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid with 20 μg/ml leupeptin. After 7 d in culture, cells were washed with PBS and media were changed to serum-free, leupeptin-free DMEM containing various concentrations of plasminogen (B) or plasmin (C) ± 20 μg/ml α2-antiplasmin or 10 μg/ml aprotinin. After 24 h, conditioned media were collected and subjected to SDS-PAGE, followed by immunoblotting for HGF. The data displayed are representative of results from three independent experiments.
<b>Figure 5.</b>
Figure 5.
Activation of endogenously generated HGF by NIH3T3 cells. (A) Leupeptin inhibits the FBS-induced activation pro-HGF. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid, with or without 20 μg/ml leupeptin, for 7 d. Equal volumes of media were analyzed by SDS-PAGE run under reducing conditions, followed by immunoblotting for HGF. Results of a representative experiment (n > 10) are displayed for cells grown in the absence (Cont) or presence (Leup) of leupeptin. The locations of migration of pro-HGF and active, α-chain HGF are displayed. (B and C) NIH3T3 fibroblasts activate HGF in a plasminogen-dependent process that is inhibitable by α2-antiplasmin and aprotinin. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid with 20 μg/ml leupeptin. After 7 d in culture, cells were washed with PBS and media were changed to serum-free, leupeptin-free DMEM containing various concentrations of plasminogen (B) or plasmin (C) ± 20 μg/ml α2-antiplasmin or 10 μg/ml aprotinin. After 24 h, conditioned media were collected and subjected to SDS-PAGE, followed by immunoblotting for HGF. The data displayed are representative of results from three independent experiments.
<b>Figure 5.</b>
Figure 5.
Activation of endogenously generated HGF by NIH3T3 cells. (A) Leupeptin inhibits the FBS-induced activation pro-HGF. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid, with or without 20 μg/ml leupeptin, for 7 d. Equal volumes of media were analyzed by SDS-PAGE run under reducing conditions, followed by immunoblotting for HGF. Results of a representative experiment (n > 10) are displayed for cells grown in the absence (Cont) or presence (Leup) of leupeptin. The locations of migration of pro-HGF and active, α-chain HGF are displayed. (B and C) NIH3T3 fibroblasts activate HGF in a plasminogen-dependent process that is inhibitable by α2-antiplasmin and aprotinin. NIH3T3 cells (1 × 105 cells/well) were plated into 24-well tissue culture plates and incubated in 10% FBS/DMEM containing 50 μg/ml of ascorbic acid with 20 μg/ml leupeptin. After 7 d in culture, cells were washed with PBS and media were changed to serum-free, leupeptin-free DMEM containing various concentrations of plasminogen (B) or plasmin (C) ± 20 μg/ml α2-antiplasmin or 10 μg/ml aprotinin. After 24 h, conditioned media were collected and subjected to SDS-PAGE, followed by immunoblotting for HGF. The data displayed are representative of results from three independent experiments.
<b>Figure 6.</b>
Figure 6.
Plasminogen-dependent pro-HGF activation by lung fibroblasts from WT, uPA−/−, and PAI-1−/− mice. (A) Primary lung fibroblasts (5 × 105 cells/well) obtained from WT, uPA−/−, and PAI-1−/− mice were placed into 6-well tissue culture plates and incubated for 7 d in 15% FBS/DMEM containing 50 μg/ml ascorbic acid with 20 μg/ml leupeptin. The monolayers were washed with PBS, and media were changed to serum-free DMEM (cont) containing 10 μg/ml plasminogen (Plg) or 370 mU/ml plasmin. After 24 h, equal volumes of conditioned media were collected and subjected to SDS-PAGE under reducing conditions, followed by immunoblotting for HGF. (B) Bar graph showing the density ratio of α-chain HGF to pro-HGF for the three genotypes, with and without plasminogen, as displayed in (A).
<b>Figure 6.</b>
Figure 6.
Plasminogen-dependent pro-HGF activation by lung fibroblasts from WT, uPA−/−, and PAI-1−/− mice. (A) Primary lung fibroblasts (5 × 105 cells/well) obtained from WT, uPA−/−, and PAI-1−/− mice were placed into 6-well tissue culture plates and incubated for 7 d in 15% FBS/DMEM containing 50 μg/ml ascorbic acid with 20 μg/ml leupeptin. The monolayers were washed with PBS, and media were changed to serum-free DMEM (cont) containing 10 μg/ml plasminogen (Plg) or 370 mU/ml plasmin. After 24 h, equal volumes of conditioned media were collected and subjected to SDS-PAGE under reducing conditions, followed by immunoblotting for HGF. (B) Bar graph showing the density ratio of α-chain HGF to pro-HGF for the three genotypes, with and without plasminogen, as displayed in (A).
<b>Figure 7.</b>
Figure 7.
Proposed mechanisms by which the plasminogen activation system modulates lung fibrosis. uPA produced by lung fibroblasts converts plasminogen to plasmin. The amount of plasmin generated depends upon the relative amounts of uPA and its inhibitor, PAI-1. The plasmin in turn, directly or indirectly, (1) releases HGF from its binding sites on heparan sulfate proteoglycans contained within the ECM, and (2) activates it. Although uPA can directly activate HGF, this process appears to be minor compared with the plasmin pathway. HGF once released and activated contributes to lung repair and prevention of fibrosis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151. - PubMed
    1. Sisson TH, Simon RH. Cell specific control of coagulation and fibrinolysis within the lung. Thromb Haemost 2004;92:435–437. - PubMed
    1. Chapman HA, Allen CL, Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 1986;133:437–443. - PubMed
    1. Idell S, James KK, Levin EG, Schwartz BS, Manchanda N, Maunder RJ, Martin TR, McLarty J, Fair DS. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989;84:695–705. - PMC - PubMed
    1. Bertozzi P, Astedt B, Zenzius L, Lynch K, LeMaire F, Zapol W, Chapman HA Jr. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990;322:890–897. - PubMed

Publication types

MeSH terms

Substances