Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels

Abstract

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

Keywords: lung; repair; transforming growth factor-β.

Fig. 1.

Characterization of murine lung fibroblasts. A: vimentin and pan-cytokeratin expression by murine lung fibroblasts from matrix metalloproteinase (MMP)-9-deficient mice. After isolation from murine lung, cells were stained for vimentin and pan-cytokeratin (see materials and methods). Human bronchial epithelial cells (HBEC) were used as positive control for pan-cytokeratin staining. Data presented are one representative of three separate experiments. WT, wild type; KO, knockout. B: gelatin zymography. Fibroblasts were cultured in 3-dimensional (3D) collagen gels for 5 days in the presence or absence of cytokines [10 ng/ml tumor necrosis factor (TNF)-α and 5 ng/ml IL-1β]. Media in which the collagen gels were suspended were harvested after 5 days, and gelatin zymography was performed. Media from HT1080 cells, which secrete copious amounts of human MMP-9, which has an apparent molecular size of 90 kDa, and MMP-2, are included as a positive control. Data presented are one representative of three experiments.

Fig. 2.

Effect of murine gelatinase B (mGelB) transfection in MMP-9 KO murine fibroblasts. A: effect on collagen gel contraction. Fibroblast-populated collagen gels were released into 60-mm tissue culture dishes. Gel size was measured daily with an image analyzer. Vertical axis, gel size expressed as % initial size; horizontal axis, time (days of culture); WT, murine fibroblasts from wild-type mice; KO, fibroblasts from MMP-9-deficient mice; KO + pRcCMV, MMP-9-deficient murine fibroblasts transfected with vector only; KO + pRcCMV/mGelB, MMP-9-deficient murine fibroblasts transfected with vector containing mGelB (MMP-9) sequence. B: gelatin zymography. After transfection of expression vector for murine MMP-9 to the murine MMP-9-deficient cells (KO), media were harvested at 24 h, and gelatin zymography was performed. The arrow indicates an apparent molecular size of 105 kDa, consistent with latent murine MMP-9. Media from HT1080 cells, which secrete copious amounts of human MMP-9, which has an apparent molecular size of 90 kDa, and MMP-2 are included as a positive control. *P < 0.05 compared with KO cells. Data are shown as means ± SE. Data presented are from one representative experiment performed in triplicate on three separate occasions.

Fig. 3.

Effect of MMP inhibitor on fibroblast-mediated collagen gel contraction. Human lung fibroblast-populated collagen gels were released in 60-mm tissue culture dishes with or without the MMP inhibitor (GM-6001, 1 μM). Gel size was measured daily with an image analyzer. Vertical axis, gel size expressed as % initial size; horizontal axis, time (days of culture). *P < 0.05 compared with control cells. Data are shown as means ± SE.

Fig. 4.

Suppression of MMP-9 by small-interfering RNA (siRNA) and its effect on collagen gel contraction by fetal lung fibroblasts (HFL-1) or by human bronchial fibroblasts (HBF). A: gelatin zymography. After transfection of HFL-1 cells with MMP-9 siRNA or control siRNA, cells were cast into 3D collagen gel maintained in the presence or absence of cytokines (10 ng/ml TNF-α and 5 ng/ml IL-1β). Media in which the gels were suspended were harvested, and gelatin zymography was performed. Media from HT1080 cells, which secrete copious amounts of human MMP-9, which has an apparent molecular size of 90 kDa, and MMP-2 are included as a positive control. Data presented are from one experiment repeated with similar results on three separate occasions. B and C: effect of MMP-9 depletion on HFL-1 (B) or HBF (C) cell-mediated collagen gel contraction. After treatment with MMP-9 siRNA or control siRNA, HFL-1 or HBF cell-populated collagen gels were released in 60-mm tissue culture dishes with or without TGF-β1 (100 pM). Gel size was measured daily with an image analyzer. Vertical axis, gel size on day 2 expressed as %initial size; horizontal axis, culture medium with or without TGF-β1 (100 pM); open bars, control siRNA-transfected cells; hatched bars, MMP-9 siRNA-transfected cells. Data are shown as means ± SE. Data presented are from one representative experiment performed in triplicate on three separate occasions. *P < 0.05.

Fig. 5.

Effect of MMP-9 KO or suppression on transforming growth factor (TGF)-β1 production and activation in the gel culture. A: murine lung fibroblasts. WT and MMP-9 KO murine lung fibroblasts were cast into collagen gels. Gels were released and allowed to contract. After 2 days, culture medium was harvested for TGF-β1 quantification by ELISA. B: HFLs. After transfection of control or MMP-9 specific for siRNA into HFL-1 cells, fibroblast-populated collagen gels were prepared, and the media were harvested on day 2 after the gels were released and cultured in 60-mm dishes. TGF-β1 was measured by ELISA. Vertical axes, TGF-β1 concentration (pg/ml); horizontal axes, active form or total TGF-β1; open bars, WT murine lung fibroblasts (A) or control siRNA-transfected HFL-1 cells (B); hatched bars, MMP-9 KO murine lung fibroblasts (A) or MMP-9 siRNA-transfected HFL-1 cells (B). Data are shown as means ± SE. *P < 0.05; ND, not detectable.

Fig. 6.

Subcellular localization of Smad3 in HFL-1 cells. After transfection of MMP-9 siRNA or control siRNA, human lung fibroblasts were stimulated with TGF-β1 for 30 min followed by immunostaining for Smad3. Nuclear counterstaining was performed with DAPI. One representative datum of three experiments is shown.

Fig. 7.

Effect of MMP-9 suppression on transcriptional activity of Smad3. A: effect of MMP-9 depletion on Smad3 reporter activity. HLF-1 cells transfected with MMP-9 siRNA or control siRNA were cotransfected with CAGA12-Luc reporter plasmid together with pRL-TK. The cells were then treated with or without TGF-β1 for 30 min. Vertical axis, Smad3 reporter activity; horizontal axis, cells were treated with or without TGF-β1 (100 pM); open bars, cells transfected with control siRNA and reporter plasmid; hatched bars, cells transfected with MMP-9 siRNA and reporter plasmid. Data are shown as means ± SE. *P < 0.05. B: effect of MMP-9 depletion on fibronection production by HFL-1 cells. After collagen gel contraction assay, the media were harvested on day 2 and used for fibronection quantification by ELISA. Vertical axis, fibronection concentration (ng/ml); horizontal axis, culture medium with or without TGF-β1 (100 pM); open bars, control siRNA-transfected cells; hatched bars, MMP-9 siRNA-transfected cells. *P < 0.05.