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• W2016094576 abstract "The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-β (TGF-β), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-β-induced fibronectin synthesis is not sufficient for the process. Instead, anti-β1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely α1β1, α2β1, and α3β1. In MG-63 cells TGF-β increased the expression of α2β1 integrin and decreased the expression of α3β1 integrin, whereas α1β1 integrin is not expressed. HOS cells had no α2β1 integrin, neither did TGF-β induce its expression. However, HOS-MNNG cells expressed more α2β1 integrin when treated with TGF-β. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-β is the increased expression of α2β1 integrin heterodimer. To further test this hypothesis, we expressed a full-length α2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed α2β1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with α2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented α2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased α2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of α2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels. The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-β (TGF-β), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-β-induced fibronectin synthesis is not sufficient for the process. Instead, anti-β1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely α1β1, α2β1, and α3β1. In MG-63 cells TGF-β increased the expression of α2β1 integrin and decreased the expression of α3β1 integrin, whereas α1β1 integrin is not expressed. HOS cells had no α2β1 integrin, neither did TGF-β induce its expression. However, HOS-MNNG cells expressed more α2β1 integrin when treated with TGF-β. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-β is the increased expression of α2β1 integrin heterodimer. To further test this hypothesis, we expressed a full-length α2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed α2β1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with α2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented α2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased α2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of α2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels. INTRODUCTIONHydrated collagen lattices were first described as a method to mimic soft-tissue matrices (Elsdale and Bard, 1972). Watery milieu within fibrous collagen net has since been used as a substrate for growth and differentation for many types of cells. When fibroblasts are embedded in collagen they are able to reduce the areas of the collagen gels and form a tissue-like structure. The ultrastructure of fibroblasts during contraction differ from the same cells plated on a plastic surface. Differences in the prominence of cell coat, changes of the prevalence of cell processes and the state of aggregation of their associated microfilamentous material are seen (Bellows et al., 1981). Collagen gels can be used to study the contraction of either floating matrix or anchored matrix (Grinnell, 1994). In the two variations of in vitro collagen matrix reorganization model, the morphology and the behavior of fibroblasts differ. The first one is suggested to mimic dermis or scars and the latter granulation tissue (Grinnell, 1994). The exact molecular mechanism of collagen gel contraction is unknown. Previous studies have suggested an essential role for cell surface collagen receptors (Gullberg et al., 1990; Shiro et al., 1991; Klein et al., 1991). Also cellular fibronectin (FN) 1The abbreviations used are: FNfibronectinHOShuman osteogenic sarcoma cellsFCSfetal calf serumILinterleukinTGFtransforming growth factorMNNGN-methyl-N′nitro-N-nitrosoguanidineDMEMDulbecco's modified Eagle's mediumkbkilobase(s).has been suggested to be required in the collagen matrix contraction by fibroblasts (Asaga et al., 1991).Transforming growth factor-β (TGF-β) has been discovered to enhance collagen gel contraction by skin fibroblasts (Montesano and Orci, 1988). Later, a similar effect has been described with other growth factors and cytokines (Clark et al., 1989; Gullberg et al., 1990). Some others, like interferon-γ, can inhibit the process (Dans and Isseroff, 1994). TGF-β is a multifunctional regulator of cells which acts in the process of wound healing (for review, see Massagué, 1990). Many cell types can in vitro and in vivo produce TGF-β, and it has been found abundant in platelets and in bone. It stimulates the accumulation of extracellular matrix (Ignotz and Massagué, 1986) and regulates cell adhesion apparatus (Ignotz and Massagué, 1987). TGF-β is supposed to have a central role in tissue healing. The fact that it is present in large amounts in bone and that it increases bone formation in vivo in test animals (Noda and Camilliere, 1989; Joyce et al., 1990) indicates the possible role of TGF-β in the healing of bone fractures. Here, we have shown that TGF-β can increase collagen gel contraction by osteogenic cells. Since TGF-β is known to regulate the integrin type collagen receptors in different cell lines (Heino and Massagué, 1989; Heino et al., 1989) and also to increase production of cellular FN (Ignotz and Massagué, 1986; Roberts et al., 1987), these are the candidate mechanisms to be responsible for the phenomenon. Integrins are a family of cell surface receptors consisting of two subunits, α and β (for review, see Hynes, 1992; Ruoslahti, 1991). Three different integrin heterodimers are known to mediate cell-type I collagen interaction, namely α1β1, α2β1, and α3β1. In addition to α1, α2, and α3 subunits, β1 subunit can form heterodimers with α4-α9 subunits and αv subunit. Several of these β1 integrins recognize FN as their ligand, namely α3β1, α4β1, α5β1, and αvβ1. Laminin is the ligand for α1β1, α2β1, α3β1, α6β1, and α7β1 integrin heterodimers.We have determined the effects of TGF-β on collagen gel contraction by osteogenic cell lines, in which the pattern of collagen-binding integrins differs from each other, and identified the molecular mechanism by which TGF-β enhances collagen gel contraction. Our data suggest that up-regulation of α2β1 integrin is required and alone sufficient to increase contraction.MATERIALS AND METHODSCell CulturesHuman osteosarcoma cells used were MG-63, HOS, and HOS-MNNG (HOS cells transformed with N-methyl-N′-nitro-N-nitrosoguanidine, tumorigenic) all from American Type Culture Collection. Cells were maintained in Dulbecco's modification of Eagle's medium (DMEM, Flow Laboratories, Irvine, United Kingdom) supplemented with 10% fetal calf serum (FCS, Flow Laboratories).Cytokines and Growth FactorsPurified bovine bone transforming growth factor-β1 (TGF-β1) was kindly provided by Dr. Joan Massagué (Sloan-Kettering Institute, New York). Human recombinant interleukin-1β (IL-1β) was purchased from Boehringer Mannheim. Concentrations used were 200 pM (TGF-β) and 10 units/ml (IL-1β).AntibodiesPolyclonal rabbit antiserum against human β1 (Heino et al., 1989), α2, and α3 integrin subunits (Santala et al., 1994) were used in immunoprecipitation assays. Functional monoclonal antibodies used to block the collagen gel contraction were against β1 (mab13; Akiyama et al., 1989), α2 (Gi9; Immunotech S.A, Marseille, France), α1 (SR-84; Rettig et. al., 1984), and α3 (B1B5; Wayner and Carter, 1987) subunits.ImmunoprecipitationsCell cultures were metabolically labeled with 50 or 100 μCi/ml of [35S]methionine (Tran 35S-label, ICN Biochemicals) for 24 h in methionine-free minimum essential medium. When cytokines and growth factors were used, cells were preincubated with them in serum-free medium overnight, and they were also used during the labeling. Cell monolayers were rinsed on ice with a solution containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 25 mM Tris-HCl (pH 7.4), and then detached by scraping. Cell pellets obtained by centrifugation at 500 × g for 5 min were solubilized in 200 μl of the same buffer containing 100 mMn-octyl-β-D-glucopyranoside (Sigma) on ice with occasional vortexing. Insoluble material was removed by centrifugation at 10,000 × g for 5 min at 4°C. Radioactivity in cell lysates was counted, and an equal amount of radioactivity was used in immunoprecipitation assays. Cytokines did not cause any systematic changes in the incorporation of [35S]methionine into cellular proteins as determined by trichloroacetic acid precipitation. Triton X-100 (0.5% v/v) and bovine serum albumin (0.5 mg/ml) were added to the supernatants, which were then precleaned by incubation with 50 μl of packed protein A-sepharose (Pharmacia LKB Biotechnology Inc.). Supernatants were immunoprecipitated with anti-integrin antibodies for 12 h at 4°C. Immunocomplexes were recovered by binding to protein A-sepharose and washing the beads four times with 25 mM Tris-buffered isotonic saline (pH 7.4) containing 0.5% Triton X-100 and 1 mg/ml bovine serum albumin and twice with 0.5 M NaCl and 25 mM Tris-HCl (pH 7.4). The immunoprecipitates were analyzed by electrophoresis on sodium dodecyl sulfate-containing 6% polyacrylamide gels under nonreducing conditions followed by fluorography. Integrin bands were quantified from fluorograms by the Microcomputer Imaging Device version M4 (Imaging Research Inc). FN immunoprecipitations were done from [35S]methionine-labeled methionine-free cell culture mediums. An equal volume of medium and phosphate-buffered isotonic saline containing 100 mMn-octyl-β-D-glucopyranoside were precleaned with packed protein A-sepharose and incubated with polyclonal antiserum against human plasma fibronectin (Chen et al., 1986) at 4°C for 12 h. Immunocomplexes were recovered and washed as mentioned previously, and reduced immunoprecipitants were analyzed by electrophoresis and quantified from fluorograms.Northern Blot HybridizationsTotal cellular RNA was isolated by using the guanidium thiocyanate-CsCl method (Chirgwin et al., 1979). RNAs were separated in 1% formaldehyde-containing agarose gels, transferred to nylon membranes (ZETA-probe, Bio-Rad), and hybridized with [32P]-labeled (Amersham) human α2 integrin cDNA probe (Takada and Hemler, 1989).Collagen Gel ContractionsCollagen gels were prepared by using Vitrogen-100 collagen (Celtrix). Eight volumes of Vitrogen were mixed with two volumes of a 1:1 mixture of 10-fold concentrated medium and 0.1 N NaOH and kept on ice. Cells were detached, counted, and mixed gently into neutralized Vitrogen solution before transferred into 24-well plates (50,000 cells/well). In some experiments 96-well plates were used. Collagen polymerization was initiated by incubating the plates at 37°C for 45 min. DMEM supplemented with 10% FCS and appropriate cytokines was then added on gels before detaching the edges of the gels from the sides of the wells. Contraction process was observed daily. After 2-6 days the cell culture wells were photographed and the surface areas of the gels were measured from prints. Data are presented as relative values (experiment/control). In one set of experiments 1 or 10 μg/ml human plasma fibronectin (pFN, Boehringer Mannheim) or cell-derived fibronectin (cFN, Fibrogenex, Mortons Grove, IL) were added into neutralized Vitrogen solution before adding the cells. In antibody blocking experiments monoclonal antibodies were added similarly.Cell Adhesion AssaysThe coating of a 96-well immunoplate (Maxi Sorp, Nunc, Denmark) was done by exposure to 0.2 ml of phosphate-buffered saline (pH 7.4) containing 4-5 μg/cm2 laminin (purified from basement membranes of the Engelbreth-Holm-Swarm mouse tumor, Collaborative Research), fibronectin (human plasma fibronectin, Boehringer Mannheim), type I collagen (from lathyric rat skin, Boehringer Mannheim), or type IV collagen (Sigma) for 12 h at 4°C. Bovine serum albumin was used to measure the nonspecific binding. Residual protein absorption sites on all wells were blocked with 1% bovine serum albumin in phosphate-buffered saline for 1 h at 37°C. Confluent cell cultures were detached by using 0.01% trypsin and 0.02% EDTA. Trypsin activity was inhibited by washing the cells with 1 mg/ml of soybean trypsin inhibitor (Sigma). Cells were suspended in MEM (Life Technologies, Inc.). 10,000 cells were transferred into each well and incubated at 37°C for 45 min. Non-adherent cells were removed by rinsing the wells with medium. Adherent cells were fixed with 2% paraformaldehyde, stained with 0.5% crystal violet in 20% ethanol, and washed with distilled water. The immunoplates were allowed to air-dry, and the stain bound to cells was dissolved into 10% acetic acid and spectrophotometrically measured at 600 nm with Multiscan Plus (Lab-Systems).TransfectionsA 4.5-kb fragment of human α2 integrin cDNA (nucleotides 1-4559 in the published sequence), containing the full translated sequence (kindly provided by Dr. Martin Hemler; Takada and Hemler, 1989) was linked into pAWneo2 expression vector (kindly provided by Dr. Arthur Weiss; Ohashi et al., 1985) which carries the neomycin resistance gene. Transfections were done by using Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. The neomycin analog G418 (Life Technologies, Inc.) was added to the culture medium in the concentration of 400 μg/ml. G418-resistant cell clones were selected for 2-3 weeks, isolated, and analyzed for their expression of α2 integrin mRNA as well as α2 protein. Three out of 24 clones (HOS cells), and 3 out of 19 clones (MG-63 cells) showed high expression levels for α2 integrin. Control cells used were transfected with pAWneo2 plasmid only.The same 4.5-kb cDNA was linked to pAW vector also in antisense orientation. Cells were transfected and selected as above. Among the first 28 cell clones analyzed, two showed markedly reduced expression of α2 integrin and the presence of antisense α2 integrin mRNA.Statistical AnalysisThe statistical significance of differences in cell adhesion and in collagen gel contraction assays was assessed with general mixed model analysis of variance (BMDP/Dynamic, 7.0, BMDP Statistical Software Inc., Cork, Ireland), when multiple independent experiments were analyzed (random factor being the experiment and fixed factor being the treatment). One way analysis of variance with Dunnett's method (SAS statistical program package; SAS Institute, Cary, NC) was used when one experiment contained several differentially treated groups. Experiments with two groups were analyzed with Student's t test for independent samples.RESULTSTGF-β Increases Collagen Gel Contraction by MG-63 and HOS-MNNG Cells But Not by HOS CellsCollagen gel contraction assays cell-type I collagen interaction. It is suggested to simulate matrix reorganization during tissue healing. Type I collagen is the major component of bone matrix, proposing its importance for osteogenic cells. TGF-β is a growth factor involved in the regulation of fracture healing process. In this study we wanted to elucidate the effects of TGF-β on collagen gel contraction by osteogenic cells and to find out the mechanism by which it makes other cells more capable of contracting the gels than others. We have used in our experiments two different osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed variant of HOS cells (HOS-MNNG). Cells were embedded in neutralized type I collagen solution, and growth medium supplemented with 10% of FCS was added on gels after polymerization was complete. Without serum no contraction could be seen (not shown). TGF-β was used in the concentration of 200 pM and it constantly enhanced collagen gel contraction by MG-63 (p <0.001; mixed model analysis of variance) and HOS-MNNG cells (p <0.05; mixed model analysis of variance; Fig. 1). In HOS cells, however, TGF-β had no effect (Fig. 1). In MG-63 cells also IL-1 could increase the contraction, but it seemed to be less potent than TGF-β (Fig. 1).Cellular Fibronectin Is Not Involved in TGF-β-induced Collagen Gel ContractionThe importance of cellular FN in the process of collagen gel contraction by fibroblasts has been proposed (Asaga et al., 1991). Knowing the fact that in many cell lines TGF-β can increase the production of FN, this is a putative mechanism for TGF-β-induced contraction. We added into the gels both plasma- and cell-derived FN (1-10 μg/ml), but this did not have any effect on the contraction (Fig. 2). We also measured FN synthesis by labeling cells with [35S]methionine and immunoprecipitating FN from the growth media. In HOS cells TGF-β increased the production of FN 10-fold (not shown). The fact that the contraction by HOS cells did not increase although their production of FN increased when treated with TGF-β is also contradictory to the involvement of FN.Figure 2The effects of 10 μg/ml plasma fibronectin (pFN) and cellular fibronectin (cFN) on collagen gel contraction by MG-63 cells. Cells were added together with pFN or cFN into neutralized Vitrogen-100 collagen solution and were then plated into 24-well culture plates. Collagen polymerization was initiated by incubating the plates at 37°C for 45 min. After the polymerization was complete, DMEM supplemented with 10% FCS was added on gels, and the sides of the gels were gently removed from the sides of the wells. MG-63 cells treated with 200 pM TGF-β were used as a contraction control. After 4 days the areas of the gels were measured. Mean ± S.D. of four parallel measurements is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of α2β1 Integrin Correlates with Collagen Gel ContractionPrevious studies have suggested the role of collagen-receptor integrins (Gullberg et al., 1990), especially α2β1 (Shiro et al., 1991; Klein et al. 1991), but also α1β1 in collagen gel contraction by fibroblasts. Here, the first evidence of the involvement of β1-integrins came from the experiments showing that anti-β1 integrin antibody can almost completely block the TGF-β-induced contraction by MG-63 cells (p <0.05; one way analysis of variance; Fig. 3). Previous studies have shown that anti-α integrin antibodies are often alone incapable of inhibiting the contraction (Klein et al., 1991). Additionally, here the blocking effect of anti-α2 integrin antibody was much weaker than that of anti-β1 integrin antibody (inhibition was not statistically significant; Fig. 3). Anti-α1 antibody had no effect and anti-α3 antibody constantly increased contraction (p <0.05; one way analysis of variance; Fig. 3). We have previously analyzed the integrin pattern in MG-63 cells and shown that these cells express small amounts of α2 integrin subunit, whereas α3 integrin is the major partner of β1 subunit (Heino and Massagué, 1989). TGF-β increases the expression of α2 subunit to about 6-8-fold (Heino and Massagué, 1989; also shown here in Fig. 4) and concomitantly down-regulates the expression of α3 subunit about 60-80% (Heino and Massagué, 1989). HOS cells express both α1 and α3 subunits, but no α2 subunit (Santala et al., 1994). Here, we show that in spite of the lack of α2β1 integrin HOS cells can slightly contract the collagen gels (Fig. 5). Anti-β1 antibody can block this phenomenon (Fig. 5). This suggests that other β1 integrins, in addition to α2β1, can, at least to some extent, mediate collagen gel contraction. More importantly, TGF-β could not turn on the expression of α2 integrin in this cell line (not shown) as it could not increase their ability to contract collagen gels (Fig. 1).Figure 3The blocking of TGF-β-induced collagen gel contraction by MG-63 cells with functional antibodies against different collagen-binding integrin subunits. Antibodies were added into neutralized Vitrogen-100 collagen together with the cells. After incubation at 37°C for 45 min, the edges of the gels were detached from the sides of the wells, and DMEM supplemented with 10% FCS and 200 pM TGF-β was added on them. The areas of the gels were measured after 4 days of contraction. Antibodies against β1, α1, α2, and α3 subunits were used. Mean ± S.D. of four parallel measurements is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of TGF-β on α2 integrin subunit biosynthesis. Confluent cultures of MG-63 and HOS-MNNG cells were incubated in serum-free medium with TGF-β for 12 h. Cells were transferred to methionine-free medium containing 50 μCi/ml [35S]methionine and 200 pM TGF-β. An equal amount of radioactivity from the cell lysates was precipitated with anti-α2 subunit antibody. The immunoprecipitants were analyzed by electrophoresis followed by fluorography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Integrin expression in and collagen gel contraction by HOS cells. A, immunoprecipitation of collagen-binding integrins from HOS cells. Cells were labeled with [35S]methionine in methionine-free medium for 24 h. An equal amount of radioactivity from the cell lysates was incubated with antibodies against β1, α2, and α3 integrin subunits. Immunoprecipitants were analyzed by SDS-PAGE under nonreducing conditions followed by fluorography. B, blocking of the collagen gel contraction of HOS cells by functional antibody against β1 integrin subunit. 10,000 cells were added together with mAb13 into neutralized Vitrogen-100 collagen solution. Gels were let to polymerize in 96-well plates before detaching the gels from the sides of the wells and adding DMEM supplemented with 10% FCS on them. The areas of the gels were measured after 6 days of incubation. The figure shows four parallel control and anti-β1 antibody-containing wells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chemical transformation of HOS cells turns on the expression of α2 integrin subunit (Santala et al., 1994). Here, TGF-β could slightly (about 2.5-fold) increase the expression of α2 integrin in HOS-MNNG cells (Fig. 4). TGF-β also enhanced the contraction by these cells (Fig. 1). The data suggest that the increased expression of α2β1 integrin correlates with the enhanced collagen gel contraction by TGF-β.Forced Expression of α2 Integrin in HOS Cells Increases Collagen Gel ContractionTo further examine the involvement of α2 integrin to the phenomenon, we linked the full-length α2 integrin cDNA (Takada and Hemler, 1989) to pAWneo2 expression vector (Ohashi et al., 1985) under a constitutive Friend's spleen focus forming virus long terminal repeat promoter. After transfection with the plasmid, G418-resistant HOS cell clones were selected and analyzed by immunoprecipitation. Polyclonal rabbit anti-α2 integrin antibody immunoprecipitated two polypeptides of 140 and 130 kDa (non-reduced) from the transfected HOS cells, the 140-kDa polypeptide representing the α2 integrin subunit and the 130-kDa representing the β1 subunit while the wild type cells and cells transfected with only the vector gave no signal (Fig. 6). Importantly, the expressed α2 subunit was capable of forming dimers with β1 subunit, which is essential for the integrin heterodimer to be expressed on the cell surface (Heino et al., 1989). Furthermore, the fact that these cell clones showed increased cell adhesion to type I collagen (p <0.001; t test; Fig. 7) suggests that the collagen receptor was functional. mRNAs encoding α2 integrin subunit were analyzed by Northern blot hybridization. HOS wild type cells give no signal for α2 subunit mRNA (Santala et al., 1994), and MG-63 cells were used as a positive control. A 8-kb α2 integrin mRNA was seen in wild type MG-63 cells and a smaller about 4-kb transcript in cells transfected with the construct containing α2 integrin cDNA (Fig. 6). This suggests that α2 integrin seen in these cells is translated from cDNA-derived mRNA and is not due to artificial activation of the wild type gene. HOS cells forced to express α2β1 integrin heterodimer were more capable of contracting collagen gels than wild type cells or cells transfected with only the vector carrying the neomycin resistance gene (p <0.001; t test; Fig. 7).Figure 6Integrin expression in pAWα2 transfected HOS cells. A, immunoprecipitation of α2 integrin in HOS cells transfected with full-length α2 integrin cDNA. Human full-length α2 integrin cDNA was linked into pAWneo2 expression vector, which carries the neomycin resistance gene. HOS cells were transfected by using Lipofectin reagent, and stable cell clones were selected by using G418. G418-resistant clones were tested for their expression of α2 integrin protein in immunoprecipitation assays (in the figure clone 16 is shown). Control cells (marked as C) were transfected with pAWneo2 expression vector only. Wild type HOS cells are marked as Wt. Cells were metabolically labeled with [35S]methionine, and an equal amount of radioactivity from each cell lysate was precipitated with α2 integrin antibody. Immunoprecipitants were analyzed by gel electrophoresis and fluorography. B, Northern blot hybridization of total RNA from α2 integrin-transfected HOS cells (clones 11 and 16) and MG-63 wild type cells (MG). cDNA probe specific to α2 integrin was used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Adhesion to different substrates and collagen gel contraction by pAWα2 transfected HOS cells. A, adhesion of HOS cells expressing α2 integrin (clones 16 and 7) to type I collagen (T I COL), laminin (LM), type IV collagen (T IV COL), and cellular fibronectin (cFN). 96-well immunoplates were coated with different matrix molecules, and bovine serum albumin (BSA) was used to measure the nonspecific binding. Residual protein absorption sites were blocked with 1% bovine serum albumin. 10,000 cells were let to adhere for 45 min after which the adherent cells were fixed with paraformaldehyde, stained with crystal violet, and air-dried. The cell bound stain was dissolved in acetic acid and measured spectrophotometrically at 600 nm. B, collagen gel contraction by HOS cells expressing α2 integrin (clone 16). HOS cells transfected with pAWneo2 vector and wild type HOS cells were used as a control for a cell clone to be tested. 50,000 cells were added in neutralized type I collagen solution and immediately transferred into 24-well plates. Collagen gel polymerization was initiated by incubating the plates at 37°C for 45 min. DMEM supplemented with 10% FCS was added on gels, and the edges of the gels were gently removed from the sides of the wells. After 2 days of incubation at 37°C, the areas of the gels were measured. Two parallel experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Transfection of MG-63 Cells with Antisense α2 Integrin cDNA Decreases Collagen Gel ContractionThe transfection of MG-63 cells with α2 integrin cDNA increased their expression of α2β1 integrin and concomitantly their ability to contract collagen gels (p <0.001; t test; Fig. 8). Transfection of MG-63 cells with the same expression vector containing antisense-oriented α2 integrin cDNA produced cell clones with decreased α2β1 expression (Fig. 8). Two cell clones were further analyzed by Northern blot hybridization revealing that α2 integrin mRNA levels had decreased to 46 and 56% of that in control cells (not shown). Furthermore, both cell clones expressed an approximately 4-kb transcript which was supposed to represent the antisense mRNA. The ratios of antisense/α2 integrin mRNA were one and six (not shown). Thus, we suggest, that there is a pool of α2 integrin mRNA not reached by antisense mRNA, even if a larger amount of antisense mRNA is synthesized. However, it is not probable that all this mRNA is translated because the decrease in α2 integrin protein synthesis seems to be larger than in the corresponding mRNA levels. Both cell clones showed decreased ability to contract collagen gels (p <0.001; t test; Fig. 8). Thus, both in MG-63 and in HOS cells the increased α2 integrin expression alone is sufficient for the increased contraction of collagen gels. Furthermore, the amount of α2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.Figure 8Effects of the expression of sense and antisense α2 integrin mRNA in MG-63-cells. Immunoprecipitation of MG-63 cells transfected with sense (A, clone 4) or antisense (B, clones 1-4)-oriented α2 integrin cDNA. MG-63 cells were transfected with pAWneo2 expression vector containing the α2 integrin cDNA. Transfections were done by using Lipofectin reagent, and G418-resistant cell clones were tested for their expression of α2 integrin by immunoprecipitation. Immunoprecipitants were analyzed by SDS-PAGE followed by fluorography. Contraction of collagen gels by MG-63 cells transfected with sense (C, clone 4) or antisense (D, clones 1 and 4)-oriented α2 integrin cDNA. MG-63 cells transfected with pAWneo2 vector only (pAW1) were used as control cells for sense-transfection clones, and wild type MG-63 cells were used as controls for antisense-transfected cell clones. 50,000 cells were added into neutralized Vitrogen solution and poured into 24-well plates. Collagen polymerization was initiated by incubating the plates at 37°C. After polymerization was complete DMEM supplemented with 10% FCS was added on gels. After 4 days the areas of the collagen gels were measured. Mean ± S.D. of four parallel measurements is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONTGFs-β are a family of growth and differentiation factors. Among other biological functions, TGFs-β are supposed to be essential for wound healing and tissue repair processes (Massagué, 1990). In general, they increase the accumulation of connective tissue macromolecules and angiogenesis (Massagué, 1990). Both phenomena are important in the formation of scars. A major source of TGF-β1, and also TGF-β2, is bone matrix. In the healing of bone fractures, recently discovered members of TGF-β superfamily called bone morphogenetic proteins (BMP 2-7) are suggested to be involved in the initiation of the healing process (Reddi, 1992). TGF-β is more probably involved in the other stages of the healing process, e.g. in the synthesis of new matrix (Reddi, 1992). The important role of extracellular matrix and growth factors for bone forming cells has also been emphasized in the recent review by Robey et al.(1993).We have tested two osteogenic cell lines, MG-63 and HOS, for their ability to contract collagen gels and studied the effect of TGF-β on the process. Both cell lines could contract collagen gels, and anti-β1 integrin antibodies could inhibit this contraction. We and others (Takada et al., 1987; Heino and Massagué, 1989; Dedhar and Saulnier, 1990; Santala et al., 1994) have previously described the β1 integrin pattern in both cell lines. There are three putative integrin-type collagen receptors, namely α1β1, α2β1, and α3β1. In MG-63 cells α2β1 and α3β1 are expressed, whereas no α1β1 is present. HOS cells express distinct integrin pattern: α1β1 and α3β1 are present, but α2β1 is missing. Both MG-63 cells and HOS cells express α5β1 fibronectin receptor, whereas α6β1 laminin receptor is expressed only in HOS cells (Santala et al., 1994). Previous reports have suggested the involvement of β1 integrins in the collagen gel contraction, especially by skin fibroblasts (Klein et al., 1991; Shiro et al., 1991). Here, we show that osteogenic cells can still induce contraction, even if the α1β1 or α2β1 heterodimers are missing. Thus, the data suggest that these heterodimers can replace each other or that they both can be replaced by a third β1 integrin containing heterodimer. TGF-β could increase collagen gel contraction by MG-63 cells but not by HOS cells. In MG-63 cells TGF-β strongly induces the synthesis of α2 integrin and concomitantly decreases the expression of α3 subunit, whereas it cannot turn on the expression of α1 subunit (Heino and Massagué, 1989). This suggests that the effect of TGF-β is due to increased expression of α2 integrin. Here, the increased contraction could be inhibited totally by anti-β1 integrin antibody and partially by anti-α2 integrin antibody, whereas anti-α1 integrin antibody had no effect. Previous studies have shown that anti-α integrin antibodies can not alone inhibit collagen gel contraction by fibroblasts, but only enhance the effect of anti-β1 antibody (Klein et al., 1991). Interestingly, anti-α3 antibody constantly, in three out of three experiments, stimulated contraction. The mechanism of this phenomenon stays unknown. However, we have recently shown that in keratinocytes α3β1 heterodimer is connected to a signal transduction pathway regulating the expression of gelatinases (Larjava et al., 1993a). The role of α2 subunit was also suggested by the fact that in HOS cells TGF-β cannot induce its expression (Santala et al., 1994). We have previously shown that transformation of HOS cells with MNNG induces the cells to express α2 integrin subunit (Santala et al., 1994). Here, we show that TGF-β increases both α2 integrin expression and collagen gel contraction by HOS-MNNG cells.In addition to altered integrin expression, TGF-β induces several other changes in cell metabolism that could have explained the increased collagen gel contraction. Here, we have excluded the involvement of TGF-β-induced increase in fibronectin synthesis. More importantly, we have shown that increased expression of α2 integrin subunit alone is sufficient to increase contraction by both MG-63 and HOS cells. This was done by expressing α2 integrin cDNA in these cells. We have previously shown that both cell lines express a large intracellular pool of excess precursor β1 integrin subunit (Heino and Massagué, 1989; Santala et al., 1994). Therefore, it is possible to get functional integrin heterodimers by forced expression of an α subunit. Furthermore, transfection of MG-63 cells with antisense-oriented α2 integrin cDNA generated cell clones with reduced ability to contract gels.Cell behavior can be regulated by both growth factors and extracellular matrix. The fact that a growth factor, TGF-β, can regulate the synthesis of matrix molecules and their cellular receptors connects the two mechanisms together. Previously, some of the effects of TGF-β on cell growth and differentiation have been partially explained by the altered structure of extracellular matrix in TGF-β-treated cell cultures (Heino and Massagué, 1990; Nugent and Newman, 1989). The data presented here show that TGF-β regulates the cell behavior also by altering their integrin pattern. We and others have shown, that in addition to TGF-β also other cytokines, including interleukin-1, tumor necrosis factor-α, and interferon-γ, can regulate integrin expression in numerous tissue-cultured cell types (Santala and Heino, 1991; Defilippi et al., 1991). In in vivo conditions, including wound healing (Larjava et al., 1993b) and chronic inflammation (Nikkari et al., 1993), where cytokines are present in large amounts, it is possible to detect dramatic changes in integrin expression. The role of osteoblast integrins in the healing of bone fractures is unknown. Recent studies have shown, that osteoblastic cells in bone and in culture express several integrin heterodimers. However, α2β1 is expressed only in small quantities, if at all (Hughes et al., 1993; Brighton and Albelda, 1992; Clover et al., 1992; Saito et al., 1994). Our data suggest that TGF-β might be involved in the reorganization of collagenous matrix also by osteogenic cells; however, only in the case that these cells already express α2β1 integrin. We have previously suggested the presence of an inhibitory element, other than DNA methylation, in HOS cells, which prevents the expression of α2 integrin gene (Santala et al., 1994). Transformation of cells with both MNNG and Kirsten murine sarcoma virus can induce the expression of α2 integrin in HOS cells (Santala et al., 1994), but we have not yet found a physiological inducer.To conclude, we have shown that TGF-β induces collagen gel contraction by osteogenic cells. The process is due to increased expression of α2β1 integrin-type collagen receptor. In cells which do not express α2 subunit, TGF-β can not turn on its gene expression or induce collagen gel contraction. The data show that TGF-β can regulate cellular functions by altering integrin pattern. Furthermore, we propose that TGF-β might be one of the factors involved in reorganization of bone matrix during the healing of fractures. INTRODUCTIONHydrated collagen lattices were first described as a method to mimic soft-tissue matrices (Elsdale and Bard, 1972). Watery milieu within fibrous collagen net has since been used as a substrate for growth and differentation for many types of cells. When fibroblasts are embedded in collagen they are able to reduce the areas of the collagen gels and form a tissue-like structure. The ultrastructure of fibroblasts during contraction differ from the same cells plated on a plastic surface. Differences in the prominence of cell coat, changes of the prevalence of cell processes and the state of aggregation of their associated microfilamentous material are seen (Bellows et al., 1981). Collagen gels can be used to study the contraction of either floating matrix or anchored matrix (Grinnell, 1994). In the two variations of in vitro collagen matrix reorganization model, the morphology and the behavior of fibroblasts differ. The first one is suggested to mimic dermis or scars and the latter granulation tissue (Grinnell, 1994). The exact molecular mechanism of collagen gel contraction is unknown. Previous studies have suggested an essential role for cell surface collagen receptors (Gullberg et al., 1990; Shiro et al., 1991; Klein et al., 1991). Also cellular fibronectin (FN) 1The abbreviations used are: FNfibronectinHOShuman osteogenic sarcoma cellsFCSfetal calf serumILinterleukinTGFtransforming growth factorMNNGN-methyl-N′nitro-N-nitrosoguanidineDMEMDulbecco's modified Eagle's mediumkbkilobase(s).has been suggested to be required in the collagen matrix contraction by fibroblasts (Asaga et al., 1991).Transforming growth factor-β (TGF-β) has been discovered to enhance collagen gel contraction by skin fibroblasts (Montesano and Orci, 1988). Later, a similar effect has been described with other growth factors and cytokines (Clark et al., 1989; Gullberg et al., 1990). Some others, like interferon-γ, can inhibit the process (Dans and Isseroff, 1994). TGF-β is a multifunctional regulator of cells which acts in the process of wound healing (for review, see Massagué, 1990). Many cell types can in vitro and in vivo produce TGF-β, and it has been found abundant in platelets and in bone. It stimulates the accumulation of extracellular matrix (Ignotz and Massagué, 1986) and regulates cell adhesion apparatus (Ignotz and Massagué, 1987). TGF-β is supposed to have a central role in tissue healing. The fact that it is present in large amounts in bone and that it increases bone formation in vivo in test animals (Noda and Camilliere, 1989; Joyce et al., 1990) indicates the possible role of TGF-β in the healing of bone fractures. Here, we have shown that TGF-β can increase collagen gel contraction by osteogenic cells. Since TGF-β is known to regulate the integrin type collagen receptors in different cell lines (Heino and Massagué, 1989; Heino et al., 1989) and also to increase production of cellular FN (Ignotz and Massagué, 1986; Roberts et al., 1987), these are the candidate mechanisms to be responsible for the phenomenon. Integrins are a family of cell surface receptors consisting of two subunits, α and β (for review, see Hynes, 1992; Ruoslahti, 1991). Three different integrin heterodimers are known to mediate cell-type I collagen interaction, namely α1β1, α2β1, and α3β1. In addition to α1, α2, and α3 subunits, β1 subunit can form heterodimers with α4-α9 subunits and αv subunit. Several of these β1 integrins recognize FN as their ligand, namely α3β1, α4β1, α5β1, and αvβ1. Laminin is the ligand for α1β1, α2β1, α3β1, α6β1, and α7β1 integrin heterodimers.We have determined the effects of TGF-β on collagen gel contraction by osteogenic cell lines, in which the pattern of collagen-binding integrins differs from each other, and identified the molecular mechanism by which TGF-β enhances collagen gel contraction. Our data suggest that up-regulation of α2β1 integrin is required and alone sufficient to increase contraction." @default.
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• W2016094576 title "Transforming Growth Factor-β Regulates Collagen Gel Contraction by Increasing α2β1 Integrin Expression in Osteogenic Cells" @default.
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