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

Ellen Tufvesson* and Gunilla Westergren-Thorsson

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

* Author for correspondence (e-mail: Ellen.Tufvesson{at}medkem.lu.se)

Accepted 28 July 2003


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biglycan and decorin are small chondroitin/dermatan sulphate proteoglycans in the extracellular matrix of connective tissue that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins.

We show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in fibroblasts, resulting in an increase in migration. Biglycan changed the cell shape of fibroblasts with formation of long protruding filamentous processes. This was also seen for decorin but to a lesser extent. Using fluorescence staining of F-actin fibres it was possible to show that these long filamentous processes were supported by long thick bundles of actin, together with an induced formation of stress fibres after stimulation with biglycan and decorin. Moreover, a reorganisation of {alpha}-smooth muscle actin was clearly seen in these cultures. Decorin also stimulated {alpha}-smooth muscle actin expression in the cells. Using cDNA Atlas Arrays we were also able to show that the mRNA level of a number of the intracellular regulators and effectors involved in cell migration were increased. For example, the focal adhesion proteins paxillin and zyxin, and some of the small Rho GTPases such as RhoA, Rac1 and Cdc42 were upregulated. After treatment with biglycan or decorin, additional results showed an increased activation of RhoA (1.8- and 1.5-fold, respectively) and Rac1 (1.8- and 1.5-fold, respectively) after 15 minutes. These factors are known to be involved in fibroblast migration, and as expected a 1.3- to 1.6-fold increase in migration could be observed after stimulation with biglycan or decorin. This induced migration was caused by the core protein, as treatment with glycosaminoglycan chains alone did not have any effect.

In summary, these data indicate that biglycan- and decorin-induced fibroblast cytoskeletal and signalling changes result in an increased cell migration, and demonstrate their potential role in the remodelling process.

Key words: Biglycan, Decorin, Cytoskeleton, Fibroblast, Migration, Rho GTPases


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell migration is an important biological function in many physiological processes, such as wound healing, as well as during pathological conditions. Movement is controlled by a variety of growth factors and extracellular matrix proteins. When the cells migrate they change their shape by continuously remodelling their cytoskeleton.

The specialised sites of cell contact that are formed between the plasma membrane and the substratum are called focal complexes, or focal adhesions, and are composed of multiple structural and signalling proteins (Petit and Thiery, 2002Go). Integrins are transmembrane proteins that interact with extracellular matrix molecules such as fibronectin, laminin and collagen outside the cell. This binding leads to clustering of the integrins and subsequently their activation. Furthermore, the cytoplasmic domain of the integrins interacts with a complex of intracellular signalling molecules, consisting of, for example paxillin, vinculin, talin and zyxin (Geiger and Bershadsky, 2001Go) thus activating a signalling cascade that includes protein phosphorylation (Turner, 2000Go). These signalling events result in reorganisation of the actin cytoskeleton. Bundles of actin fibres anchor in focal adhesion complexes. They are termed stress fibres, and consist of mainly filamentous actin and myosin-II filaments, but also many of the proteins found in smooth-muscle filaments (Ridley, 1999Go). The main function of the stress fibres is to contract when the cells are released from the substratum, and they are therefore responsible for cell movement.

Members of the Rho family are major elements regulating cytoskeletal changes. The Rho family consists of small GTPases that cycle between an inactive GDP-bound and an active GTP-bound state. The GTP-bound form interacts with downstream targets and effector molecules to produce a biological response (Kjoller and Hall, 1999Go). Rho, Rac and Cdc42 are three members of the Rho family that are known to be involved in cell migration (Nobes and Hall, 1999Go). Rho induces stress fibre formation and is required for the formation and maintenance of focal adhesions (Hotchin and Hall, 1995Go; Ridley and Hall, 1992Go). Rac and Cdc42 regulate lamellipodia and filopodia formation, respectively, as well as formation of smaller focal complexes formed at their fronts (Kozma et al., 1995Go; Nobes and Hall, 1995Go; Ridley et al., 1992Go). These three small GTPases affect each other. Cdc42 rapidly induces activation of Rac leading to an assembled induction of filopodia and lamellipodia. Subsequently, Rac activates Rho, resulting in the formation of new sites of focal adhesion and filament assembly in the advancing lamellipodia. Together with stress fibre formation, this contracts the cell causing forward cell movement.

The cytoskeletal changes and activation of small GTPases are important in directional migration. These effects are influenced by the substratum to which the cells adhere, such as in the case of fibronectin (Barry et al., 1996Go; Clark et al., 1998Go; Price et al., 1998Go; Ren et al., 1999Go). In cooperation with the integrins, it has been shown that syndecan-2 participates selectively in the induction of stress fibre formation through specific binding to the fibronectin substrate, promoting substratum attachment (Kusano et al., 2000Go).

This study has focused on the role of the small proteoglycans, biglycan and decorin, in the migratory process of fibroblasts, of which little is known. These molecules belong to the family of small leucine-rich proteins (SLRPs) (Iozzo, 1997Go) and are in most cases substituted with two and one chondroitin/dermatan sulphate (CS/DS) chains, respectively. Both biglycan and decorin are known to bind to different types of collagens (Kresse et al., 1994Go). In addition, biglycan is known to bind to the membrane-bound proteoglycan dystroglycan (Bowe et al., 2000Go), while decorin is known to bind to other extracellular matrix components, such as fibronectin (Winnemöller et al., 1991Go). It has been reported that the induction of migration in wounded endothelial cell monolayers is accompanied by an increase in the synthesis of biglycan (Kinsella and Wight, 1988Go). In basic fibroblast growth factor-treated and wounded cultures of these cells, biglycan has been localized to the edges of lamellipodia on migrating cells (Kinsella et al., 1997Go). In osteosarcoma cells, decorin has been shown to impede the migration-promoting effect of matrix molecules, such as fibronectin and collagen type I, by a mechanism involving the DS chains (Merle et al., 1999Go). Similarly, biglycan and decorin inhibit the attachment of CHO cells and embryo fibroblasts to a cell-binding fibronectin fragment (Bidanset et al., 1992Go). It has also been shown that hyaluronan can induce lamellipodia formation, through activation of Rac1 via CD44 (Oliferenko et al., 2000Go).

Here, we show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in lung fibroblasts and activate the signalling pathways of RhoA and Rac1, known to be responsible for cell movement. All these changes result in increased migration.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Purification of tissue-derived biglycan, decorin, DS and CS
Biglycan and decorin were purified from bovine sclera as described previously (Cöster and Fransson, 1981Go). DS (iduronic acid content of 95%) was purified from porcine skin (known as DS-18) (Fransson and Malmström, 1971Go), and CS was purified from horse nasal septum (known as CS-4) (Rodén et al., 1972Go).

Morphological characterisation with Crystal Violet
Human lung fibroblasts (HFL-1, obtained from ATCC, Rockville, MD) were plated on four-well chamber slides (10,000 cells/well) and left to adhere overnight. Cells were stimulated with 10 µg/ml biglycan or decorin in DMEM with 0.4% donor calf serum (DCS). After 24 hours of stimulation the cells were fixed in 1% glutaraldehyde for 30 minutes and stained for 2 hours in 0.5% Crystal Violet. Cell shape was recorded as the ratio of the length versus the width of the cells.

Fluorescent staining
For stress fibre analysis, cells were seeded (5000 cells/well) and stimulated as described above. Thereafter, cells were fixed in 4% paraformaldehyde in PBS for 15 minutes. After permeabilisation in 0.5% Triton X-100 in PBS for 5 minutes, and blocking in 1% BSA in PBS for 30 minutes, the cells were incubated for 30 minutes with the Alexa FluorTM 488 phalloidin probe (Molecular Probes, Netherlands) diluted in blocking buffer. Cells were rinsed carefully between each step. A confocal laser scanning microscope (Multiprobe 2001 TM CLSM; Molecular Dynamics) was used to examine the cells. Monoclonal mouse antibody against human {alpha}-smooth muscle actin ({alpha}-SMA) (M0851, Dako, Dakopatts AB, Älvsjö, Sweden) or paxillin (Sigma-Aldrich, Saint Louis, MO, USA) was used, followed by Alexa Fluor® 584 goat anti-mouse IgG (Molecular Probes).

Immunostaining of {alpha}-SMA
Cells were seeded (5000 cells/well) and stimulated with 10 ng/ml transforming growth factor ß (TGFß), or with biglycan or decorin as described above. Cells were fixed in 3% glutaraldehyde and {alpha}-SMA was detected using an antibody against human {alpha}-SMA, as described above. The bound antibodies were stained with avidin-biotin vectorstatin AK-500 and thereafter with vector red (SK 5100). The monoclonal antibody against {alpha}-SMA was also used in western blotting for analysing the amounts of {alpha}-SMA in the cells after treatment with biglycan or decorin.

cDNA Atlas Array
Confluent cell cultures were incubated for 3 hours in 1% DCS and thereafter stimulated with 10 µg/ml biglycan or decorin in 0.4% DCS in DMEM. After 24 hours of stimulation the medium was decanted and RNA was isolated from the cells using the AtlasTM Pure Total RNA Labeling System (Clontech, Palo Alto, CA, USA). RNA was reverse transcribed to cDNA, radioactively labelled with 32P and hybridised to the Atlas Human cDNA Expression Array and Atlas Human Cell Interaction Array (7740-1 and 7746-1 from Clontech, Palo Alto, CA, USA).

GTPase activity assays with GST pull-down assay and western blotting for RhoA and Rac1
Cloning of the GST-C21 fusion protein has been described previously (Reid et al., 1996Go; Reid et al., 1999Go). GST-C21 contains the Rho-binding domain from the Rho effector protein Rhotekin. GST-PAKCD contains the Rac and Cdc42-binding region from human PAK1B (Sander et al., 1998Go). E. coli M15 bacteria transformed with either construct were grown at 37°C and recombinant fusion proteins were expressed and purified as described previously (Sander et al., 1998Go; Sander et al., 1999Go).

Fibroblasts were incubated for 24 hours in the absence of DCS and thereafter stimulated with 10 µg/ml biglycan or decorin for 15, 30 or 60 minutes. Cells were incubated for 5 minutes on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1% NP-40, 10% glycerol, 10 mM MgCl2) with inhibitors: 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 1 µg/ml PMSF, whereafter they were scraped off. The cell suspensions were centrifuged for 10 minutes at 10,000 g at 4°C. Aliquots were taken from the cell supernatant to compare protein amounts.

Cell supernatants were incubated with the bacterially produced GST-C21 or GST-PAK-CD fusion proteins bound to glutathionine-coupled Sepharose beads at 4°C for 45 minutes (Sander et al., 1998Go). The beads with GTP-loaded proteins bound to the fusion proteins were washed three times in an excess of lysis buffer and then analysed for RhoA and Rac1 molecules by western blotting using a monoclonal mouse antibody against human RhoA (Santa Cruz Biotechnology, Inc.) and Rac1 (Transduction Laboratories, Lexington, KY). The intensity of the bands was analysed using the Gel-ProTM Analyzer (version 2.0).

Migration assay
Cell migration assays were performed using Falcon cell culture inserts (diameter 10.5 mm, pore size 8 µm). 50,000 cells were added to the inserts, which were placed into the lower chamber containing DMEM supplemented with 10% DCS with or without 10 ng/ml platelet-derived growth factor-BB (PDGF-BB). These cultures were additionally stimulated with 10 µg/ml biglycan or decorin, or 100 µg/ml DS or CS. Cells were allowed to migrate to the underside of the insert for 8-24 hours. Non-migrated cells on the upper side of the chamber were removed with a cotton swab, and migrated cells attached to the underside were fixed for 30 minutes in 1% glutaraldehyde, stained for 2 hours in 0.5% Crystal Violet, washed and counted.

Statistical methods
Mean values ± standard errors of the mean (s.e.m.) were calculated. Student's t-test was used to evaluate the statistical significance. P<0.05 is considered significant.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biglycan and decorin induce morphological changes in lung fibroblasts
After stimulation of human lung fibroblasts with biglycan and decorin, a change in morphology was observed. Cells stimulated with biglycan (Fig. 1B) developed a more stretched cell shape with long, protruding filamentous processes when compared to control cells (Fig. 1A). Decorin (Fig. 1C) also changed fibroblast morphology in a similar manner, but to a lesser extent. This ratio of length to width was 13 for cells treated with biglycan compared with 7 for untreated cells (Fig. 1D). This demonstrates that biglycan induced a more stretched cell shape. A similar change, but not to the same extent, was seen after treatment with decorin, which gave a ratio of 9.



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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.

 

Stress fibre formation induced by biglycan and decorin
To examine how the morphological changes described above were supported by the cytoskeleton, cells were stained to visualise the stress fibres (indicated by arrows in Fig. 2). After stimulation with biglycan (Fig. 2B), the same long, extended filamentous processes could be seen (indicated by arrowheads), and were verified to contain thick, long stretched actin bundles. There was also an increase in stress fibre formation compared to controls (Fig. 2A). Similar results were obtained after stimulation with decorin (Fig. 2C).



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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.

 

Biglycan and decorin affect {alpha}-SMA in fibroblast cultures
Control cells were compared to cells stimulated with TGFß, a known stimulator of {alpha}-SMA production, and a marker for the differentiation of fibroblasts into myofibroblasts. As expected, after induction by TGFß, {alpha}-SMA expression was high (Fig. 3B). The whole of the cell was stained (Fig. 3A), where as in control cells only a light red staining was observed, indicative of a background level of {alpha}-SMA. After stimulation with biglycan (Fig. 3C) there was a specific increase in staining of the protruding edge (indicated by arrows in Fig. 3C). Furthermore, the morphological data described above, of a more stretched cell shape (Fig. 1B), was confirmed. Decorin (Fig. 3D) also increased staining of the protruding edge (indicated by arrows in Fig. 3D). Fluorescent staining for {alpha}-SMA in comparison to paxillin was done for a more detailed visualization of the {alpha}-SMA localization (Fig. 3E). Staining for {alpha}-SMA could clearly be seen in the protruding edge of the cell, such as the lamellipodia/filopodia, differing from the staining of the focal adhesion marker paxillin. Additionally, decorin induced a stimulation of {alpha}-SMA expression over the whole cell, which was not the case for biglycan. This was also confirmed using western blot analysis (Fig. 3F) in which decorin was found to significantly increased the level of total {alpha}-SMA (1.2 fold) in the cells (Fig. 3G). Biglycan did not significantly increase the level of total {alpha}-SMA, which may indicate a possible rearrangement of available cellular {alpha}-SMA.



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Fig. 3. Biglycan and decorin affect {alpha}-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. {alpha}-SMA was detected using a monoclonal mouse antibody against human {alpha}-SMA. Arrows indicate the protruding edge. (E) The protruding cell area stained for {alpha}-SMA and paxillin. Cells were stained with monoclonal mouse antibody against human {alpha}-SMA or paxillin, followed by Alexa Fluor® 584 goat anti-mouse IgG. (F,G) Western blot shows the total level of {alpha}-SMA in the cells after treatment. n=3.

 

mRNA changes detected on cDNA Atlas Arrays after stimulation with biglycan and decorin
To further evaluate fibroblast activities and intracellular pathways affected by biglycan and decorin, mRNA of stimulated cells was analysed on cDNA Atlas Arrays to screen for changed expression of a large number of genes (Fig. 4). The threshold for upregulation was set to twofold, and the proteins were divided into four groups (Table 1). In the first group there was an increase in many of the genes involved in cell-matrix interactions (Table 1), both matrix molecules themselves, such as collagens and fibronectin, and cell surface molecules involved in matrix attachment, such as integrins and CD44. There were also effects on molecules involved in remodelling of connective tissue, such as MMP-14, MMP-19 and TIMP-1. In the second group there was an increase in a number of genes involved in cell-cell interaction and cell recruitment (Table 1), such as ICAM-1. In the third group, genes involved in the stress response were affected, such as HSP27 and glutaredoxin (Table 1). Finally, and pertinent to this investigation, some of the cytoplasmic regulators and effectors known to be involved in focal adhesion and migration were affected (Table 1) (Fig. 4). Examples here were paxillin and zyxin, which are located at the sites of focal adhesion. Other important molecules that were upregulated were the small GTPases that belong to the Rho family, such as RhoA, Rac1 and Cdc42, and that are known to be involved in cell migration (Table 1; Fig. 4). Further down in the intracellular signalling cascade are the ERKs, which were also shown to be upregulated. The ERKs are known to be effectors in the migratory process.



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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.

 

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Table 1. Effect of biglycan and decorin on mRNA expression in lung fibroblasts analysed by cDNA Atlas Arrays

 

RhoA and Rac1 activity assays
To relate to the increased mRNA levels of the Rho family and to evaluate whether the Rho family GTPases were activated, the amount of the GTP-bound forms of RhoA and Rac1 was investigated. A GST pull-down assay, with fusion proteins for RhoA-GTP and Rac-GTP binding proteins was used. Both biglycan and decorin significantly stimulated the formation of the GTP-bound form of RhoA and Rac1, with maximum stimulation at 15 minutes (Fig. 5A-C). Levels of RhoA were stimulated 1.8-fold with biglycan and 1.5-fold with decorin (Fig. 5C). Similarly, both biglycan and decorin increased the amount of GTP-bound Rac1: biglycan a 1.8-fold increase and decorin a 1.5-fold increase (Fig. 5C). Activation of RhoA and Rac1 declined to the control level after 60 minutes treatment with both biglycan and decorin.



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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.

 

Stimulation with biglycan and decorin increases cell migration
It is well known that migration depends on focal adhesion formation and cellular outgrowths, so therefore we hypothesized that biglycan and decorin would affect fibroblast migration. As expected, both biglycan (Fig. 6B) and decorin (Fig. 6C) increased fibroblast migration compared to that of the control (Fig. 6A). By counting the cells (Fig. 6E) we showed that both biglycan and decorin significantly increased fibroblast migration by 1.3-fold. PDGF-BB is known to induce fibroblast migration, confirmed by a 4.3-fold increase of migration (Fig. 6D). The effects of biglycan and decorin were therefore evaluated together with PDGF-BB. The increase in migration induced by biglycan and decorin was further enhanced (1.6-fold increase) in the presence of PDGF-BB (Fig. 6F). To evaluate whether the effect of biglycan and decorin was due to the presence of glycosaminoglycan chains, purified CS and DS were added to the migrating cells. However, neither CS nor DS had any effect on fibroblast migration, either with or without stimulation by PDGF-BB (Fig. 6E,F). From this we can conclude that the increased migration induced by biglycan and decorin was most likely not due to the effect of the glycosaminoglycan chain, but rather due to the core protein or the intact protein.



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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.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study we show for the first time that the small proteoglycans biglycan and decorin cause lung fibroblasts to change shape. In addition, cytoskeletal changes were observed, such as an increase in stress fibres, together with an activation of the small GTPases RhoA and Rac1, which together orchestrate an increase in cell migration.

During cell movement, it is important that cell-substrate linkages at the front of the cell provide a stable traction, while the linkages at the rear detach to allow the cell to move forward. After stimulation with biglycan, and to some extent decorin, long protruding filamentous processes were seen. The function of these processes is not known, but they may represent long protrusions at the very front of the cell that extend in the direction of cell migration. Alternatively, they may indicate that the rear end of the cell is left behind, remaining attached to the substratum as a result of the forward movement.

The formation of stress fibres is also of importance for cell movement. They contract when the rear of the cell is released from the substratum and pull the cell body forward. They are anchored in the focal adhesion complexes, which are responsible for the attachment of the cell to the substratum. After treatment with biglycan and decorin we see an increase in stress fibre formation, indicating an increased potential for cell contraction. Correspondingly, {alpha}-SMA is preferentially assembled in the protruding edge, particularly after treatment with biglycan, but also in the presence of decorin. Decorin additionally induces an increase of {alpha}-SMA over the whole cell, suggesting a more contractile phenotype. Fibroblast migration was also increased after induction with both biglycan and decorin. Taken together we can conclude that these small proteoglycans affect cell movement through a reorganisation of the protruding edge, together with a contraction of the cell, allowing forward movement.

It is generally accepted that fibroblasts can differentiate into myofibroblasts. Myofibroblasts can be distinguished from fibroblasts by an increased level of {alpha}-SMA together with an increased assembly of stress fibres. Recently, protomyofibroblasts have been described as an intermediate form in the differentiation process (Tomasek et al., 2002Go). This phenotype forms stress fibres, but does not express increased levels of {alpha}-SMA. During wound healing, the protomyofibroblasts are initially formed in response to known factors such as mechanical tension and PDGF. In this form it rapidly migrates into the wounded area, and then develops into a differentiated myofibroblast, primarily in response to TGFß. The fibroblast phenotype induced by biglycan and decorin is similar to the proto-myofibroblast phenotype with an induction of stress fibre formation. Additionally, the amount of {alpha}-SMA is increased in the protruding edge, but the whole cell is not covered, as is the case with TGFß stimulation. With reference to the inflammatory role of biglycan and decorin, biglycan is upregulated early in the inflammatory process followed by a subsequent upregulation of decorin. This has been demonstrated in vivo in several pathological processes such as asthma (Westergren-Thorsson et al., 2002Go) and systemic sclerosis (Hesselstrand et al., 2002Go; Westergren-Thorsson et al., 1996Go). Consequently, in early events of the inflammatory process, such as wound healing, biglycan induces the fibroblasts to differentiate into proto-myofibroblasts that migrate into the wounded area. Subsequently, decorin is upregulated and induces an increased level of {alpha}-SMA that covers the cells, indicative of a more contractile phenotype. The amount of {alpha}-SMA is not as elevated as after treatment with TGFß, but may indicate a preliminary stage of a myofibroblast that ultimately generates the contractile force for closing the wound.

Little is known about the effects of biglycan and decorin on lung fibroblasts. However, we have shown here that they are both able to affect the morphology and migration of fibroblasts. Changes in mRNA levels of genes involved in cell-cell interactions were also affected by biglycan and decorin stimulation, e.g. ICAM-1 was shown to be involved in cell recruitment. This may be of interest because the recruitment of inflammatory cells to connective tissue occurs in the early stage of inflammation, simultaneously with an increase in biglycan. Similarly, expression of genes involved in stress responses was upregulated, e.g. HSP27 and glutaredoxin. This may also be of interest in the inflammatory process where these molecules are known to be affected. Several genes involved in cell-matrix interactions were also affected, e.g. matrix molecules, such as collagens and fibronectin, as well as molecules involved in connective tissue remodelling, e.g. MMPs and TIMPs. This shows that the small proteoglycans biglycan and decorin alone can induce changes in extracellular matrix composition, by inducing changes in the substratum for cell attachment. Furthermore, there was an increased expression of cell surface molecules, e.g. integrins and CD44. Several integrins were upregulated, enhancing cell attachment and initiation of focal adhesion formation. Moreover, it has been shown that CD44 activates Rac1, resulting in induction of lamellipodia formation (Oliferenko et al., 2000Go). In comparison to the resultant changes in morphology and cytoskeleton, changes in the expression of genes for some of the cytoplasmic regulators and effectors known to be involved in focal adhesion and migration were observed. Among these were paxillin, zyxin and integrins; all molecules located in the focal adhesion complex. This completes our results showing the induction of focal adhesion formation after treatment with biglycan and decorin, and the resultant increase in anchorage to the substratum. At the same time, there was an increase in the expression of the gene for the small GTPase RhoA, known to induce and sustain focal adhesions, as well as induce stress fibre formation. Cdc42 and Rac1 were also affected, reinforcing our results on increased migration. Increased mRNA levels of members in the Rho family were reflected in the GTP load, and thereby activation of RhoA and Rac1. The biglycan- and decorin-induced activation of RhoA and Rac1 could be the underlying mechanism of the forward fibroblast movement, as well as being responsible for the induction of additional intracellular pathways.

In summary, we conclude that biglycan and decorin affect fibroblast morphology and intracellular pathways with a subsequent increase in cell migration. These novel findings increase our understanding of the role of these proteoglycans both in physiological and pathological processes where the extracellular matrix is continuously being remodelled.


    Acknowledgments
 
We thank Prof. John Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for kindly providing us with the constructs of GST-C21 and GST-PAK-CD fusion proteins, and Prof. Peter Ekström (Cell and Organism Biology, Lund, Sweden) for valuable help with the confocal laser scanning microscope. This work was supported by grants from the Swedish Medical Research Council (11550), the Swedish Society for Medical Research, the Greta and Johan Kock Foundation, the Alfred Österlund Foundation, the Anna-Greta Crafoord Foundation, the Vårdal Foundation, the Swedish Rheumatism Association, Gustaf V's 80 years Fund, the Heart-Lung Foundation, and the Medical Faculty, Lund University.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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