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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00808

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Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration

Section for Cell and Matrix Biology, Department of Cell and Molecular Biology, BMC C13, Lund University, 221 84 Lund, Sweden

View larger version (116K): [in a new window]   Fig. 1. Biglycan and decorin induce morphological changes. Human lung fibroblasts were plated on four well chamber slides (10,000 cells/well) and incubated without (A) or with 10 µg/ml biglycan (B) or decorin (C) for 24 hours. After fixation cells were stained with Crystal Violet. (D) The ratio of the length versus the width of the cells. Values are given as means±s.e.m.; n=150; *significant differences of treatment compared with control.

View larger version (33K): [in a new window]   Fig. 2. Stress fibre formation induced by biglycan and decorin. Human lung fibroblasts were seeded on four-well chamber slides (5000 cells/well) and incubated without (A) or with 10 µg/ml biglycan (B) or decorin (C) for 24 hours. Cells were stained with Alexa FluorTM 488 phalloidin, showing the F-actin, and analysed using a confocal laser scanning microscope. Arrows indicate stress fibres, and arrowheads indicate long thick actin bundles.

View larger version (72K): [in a new window]   Fig. 3. Biglycan and decorin affect -SMA. (A-D) Fibroblasts were plated (5000 cells/well) (A) without treatment or stimulated with (B) 10 ng/ml TGF-ß, (C) 10 µg/ml biglycan or (D) 10 µg/ml decorin for 24 hours. -SMA was detected using a monoclonal mouse antibody against human -SMA. Arrows indicate the protruding edge. (E) The protruding cell area stained for -SMA and paxillin. Cells were stained with monoclonal mouse antibody against human -SMA or paxillin, followed by Alexa Fluor® 584 goat anti-mouse IgG. (F,G) Western blot shows the total level of -SMA in the cells after treatment. n=3.

View larger version (82K): [in a new window]   Fig. 4. Changes of mRNA detected on a cDNA Atlas Array. Confluent cells were stimulated without (A) or with biglycan (B) for 24 hours. RNA was extracted, reverse transcribed to cDNA, radioactively labelled and hybridised using the Atlas Human Cell Interaction Array. The whole membranes are shown to the left and the small GTPases, RhoA, Rac and Cdc42, as well as paxillin and zyxin are indicated in the expanded figures.

View larger version (31K): [in a new window]   Fig. 5. Biglycan and decorin induce activation of RhoA and Rac1. (A) After stimulation of human lung fibroblasts with 10 µg/ml biglycan and decorin, the amount of active, GTP-loaded RhoA and Rac1 was determined by GST-pull down assays with GST-C21 and GST-PAK-CD, respectively. (B) To confirm similar amounts of protein, the cell lysate was also subjected to western blotting for measuring the total amounts of RhoA and Rac1. (C) The intensity of the western blot of the GTP-loaded RhoA and Rac1 in relation to the intensity of the total amount of RhoA and Rac1, respectively. A-C show representative experiments after stimulation with biglycan and decorin. Values are given as means±s.e.m. for n=3-5; *significant differences of treatment compared to control.

View larger version (73K): [in a new window]   Fig. 6. Stimulation with biglycan and decorin increases cell migration. Cell migration assays were performed in cell culture inserts with 50,000 cells added to the inserts in medium supplemented with or without 10 ng/ml PDGF-BB. (A-C) Cells were allowed to migrate for 8-24 hours as control (A), or stimulated with 10 µg/ml biglycan (B) or decorin (C) and thereafter fixed and finally stained with Crystal Violet. (D) Cell counting was performed after stimulation with or without PDGF-BB. (E,F) Cells were also counted after stimulation with biglycan, decorin, DS or CS in absence (E) or presence (F) of PDGF-BB. A-C show representative experiments of stimulation for 24 hours in presence of PDGF-BB. Values are given as means±s.e.m. for n=2-5; *significant differences of treatment compared to control.

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