Transforming Growth Factor-β Regulates Collagen Gel Contraction by Increasing α2β1 Integrin Expression in Osteogenic Cells

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 of for in essential When cytokines and growth factors were cells were with in medium and they were also used during the were on with a and and by obtained by for were in 200 of the same 100 on with material was by for in cell was and an amount of was used in immunoprecipitation assays. did not changes in the of cellular as determined by and bovine serum were to the which were by with 50 of were with antibodies for were by to and the with and bovine serum and with and The were by on gels by were from by the FN were from cell of medium and 100 were with and with antiserum against human fibronectin et al., 1986) for were and as and were by and from cellular was by the method et al., were in to and with human α2 integrin cDNA and gels were by collagen of were with two of a of medium and and on Cells were and In were Collagen was by the for supplemented with 10% and cytokines was on gels the of the gels from the of the process was the cell were and the surface areas of the gels were from are as In one of or 10 human fibronectin Boehringer or fibronectin were the cells. In monoclonal antibodies were of a was by to of from of the fibronectin Boehringer type I collagen Boehringer or type collagen for serum was used to the on all were with bovine serum in for cell cultures were by and was by the cells with of Cells were in cells were each and for cells were by the with cells were with with in and with The were to and the to cells was 10% and with of human α2 integrin cDNA in the the provided by Dr. and 1989) was expression provided by Dr. et al., which the were by to the The was to the medium in the of cell clones were for and for their expression of α2 integrin as as α2 Three of clones (HOS and of clones expression for α2 cells used were transfected with same cDNA was to also in antisense Cells were transfected and as the first cell clones two expression of α2 integrin and the of antisense α2 integrin of in cell adhesion and in collagen gel contraction was with of when were the and the of with method Institute, was used when one treated with two were with test for Collagen by MG-63 and HOS-MNNG Cells by HOS gel contraction cell-type I collagen It is suggested to matrix reorganization during tissue healing. Type I collagen is the of bone its for osteogenic cells. TGF-β is a growth in the of fracture healing process. In this study we to the effects of TGF-β on collagen gel contraction by osteogenic cells and to the mechanism by which it other cells more of the gels We have used in two different osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed of HOS cells Cells were embedded in type I collagen and growth medium supplemented with 10% of was on gels was serum no contraction could be seen TGF-β was used in the of 200 pM and it enhanced collagen gel contraction by MG-63 of and HOS-MNNG cells of In HOS cells, TGF-β had no effect In MG-63 cells also could increase the but it to be TGF-β in TGF-β-induced Collagen of cellular FN in the process of collagen gel contraction by fibroblasts has been (Asaga et al., 1991). the fact that in many cell lines TGF-β can increase the production of this is a putative mechanism for TGF-β-induced contraction. We the gels both and FN but this did not have effect on the contraction We also FN synthesis by cells with and FN from the growth In HOS cells TGF-β increased the production of FN The fact that the contraction by HOS cells did not increase their production of FN increased when treated with TGF-β is also to the of effects of 10 fibronectin and cellular fibronectin on collagen gel contraction by MG-63 cells. Cells were with or collagen and were plated Collagen was by the for the was supplemented with 10% was on and the of the gels were from the of the MG-63 cells treated with 200 pM TGF-β were used as a contraction the areas of the gels were of is of α2β1 with Collagen studies have suggested the role of integrins (Gullberg et al., α2β1 et al., 1991; Klein et but also α1β1 in collagen gel contraction by Here, the first of the of from the that anti-β1 integrin can block the TGF-β-induced contraction by MG-63 cells one of Previous studies have shown that integrin antibodies are alone of the contraction et al., 1991). the effect of integrin was that of anti-β1 integrin was not had no effect and increased contraction one of We have the integrin pattern in MG-63 cells and shown that these cells amounts of α2 integrin whereas α3 integrin is the of β1 subunit (Heino and Massagué, TGF-β increases the expression of α2 subunit to (Heino and Massagué, 1989; also shown in and the expression of α3 subunit (Heino and Massagué, HOS cells both α1 and α3 subunits, but no α2 subunit (Santala et al., 1994). Here, we show that in of the of α2β1 integrin HOS cells can contract the collagen gels can block this that other β1 in addition to α2β1, to mediate collagen gel contraction. TGF-β could not on the expression of α2 integrin in this cell as it could not increase their ability to contract collagen gels of TGF-β-induced collagen gel contraction by MG-63 cells with antibodies against different collagen-binding integrin were collagen with the cells. for the of the gels were from the of the and supplemented with 10% and 200 pM TGF-β was on The areas of the gels were of contraction. against α1, α2, and α3 subunits were of is of TGF-β on α2 integrin subunit cultures of MG-63 and HOS-MNNG cells were in medium with TGF-β for Cells were to medium 50 and 200 pM TGF-β. amount of from the cell was with subunit The were by by expression in and collagen gel contraction by HOS cells. immunoprecipitation of collagen-binding integrins from HOS cells. Cells were labeled with in medium for amount of from the cell was with antibodies against α2, and α3 integrin were by by of the collagen gel contraction of HOS cells by against β1 integrin subunit. cells were with collagen were to in the gels from the of the and supplemented with 10% on The areas of the gels were of The and anti-β1 of HOS cells on the expression of α2 integrin subunit (Santala et al., 1994). Here, TGF-β could increase the expression of α2 integrin in HOS-MNNG cells TGF-β also enhanced the contraction by these cells The data suggest that the increased expression of α2β1 integrin with the enhanced collagen gel contraction by of α2 in HOS Cells Collagen further the of α2 integrin to the we the full-length α2 integrin cDNA and 1989) to expression et al., a with the HOS cell clones were and by rabbit integrin two of and from the transfected HOS cells, the the α2 integrin subunit and the the β1 subunit the type cells and cells transfected with the no the expressed α2 subunit was of with β1 which is essential for the integrin to be expressed on the cell surface (Heino et al., Furthermore, the fact that these cell clones increased cell adhesion to type I collagen that the collagen was α2 integrin subunit were by HOS type cells no for α2 subunit (Santala et al., and MG-63 cells were used as a α2 integrin was seen in type MG-63 cells and a in cells transfected with the α2 integrin cDNA that α2 integrin seen in these cells is from and is not to of the type HOS cells to α2β1 integrin were more of collagen gels type cells or cells transfected with the the expression in transfected HOS cells. immunoprecipitation of α2 integrin in HOS cells transfected with full-length α2 integrin Human full-length α2 integrin cDNA was expression which the HOS cells were transfected by and cell clones were by clones were for their expression of α2 integrin in immunoprecipitation the is cells as were transfected with expression type HOS cells are as Cells were metabolically labeled with and an amount of from each cell was with α2 integrin were by gel and of from α2 HOS cells and and MG-63 type cells cDNA to α2 integrin was to different and collagen gel contraction by transfected HOS cells. adhesion of HOS cells α2 integrin and to type I collagen I type collagen and cellular fibronectin were with different matrix and bovine serum was used to the were with bovine serum cells were to for which the cells were with with and The cell was in and collagen gel contraction by HOS cells α2 integrin HOS cells transfected with and type HOS cells were used as a for a cell to be cells were in type I collagen and Collagen gel was by the for supplemented with 10% was on and the of the gels were from the of the of the areas of the gels were are of MG-63 Cells with α2 cDNA Collagen of MG-63 cells with α2 integrin cDNA increased their expression of α2β1 integrin and their ability to contract collagen gels of MG-63 cells with the same expression α2 integrin cDNA cell clones with decreased α2β1 expression cell clones were further by that α2 integrin had decreased to and of that in cells Furthermore, both cell clones expressed an which was supposed to the antisense The of integrin were one and Thus, we that is a of α2 integrin not by antisense a amount of antisense is However, it is not that all this is the decrease in α2 integrin synthesis to be in the cell clones decreased ability to contract collagen gels 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 of the expression of and antisense α2 integrin in of MG-63 cells transfected with or antisense clones α2 integrin MG-63 cells were transfected with expression the α2 integrin were by and cell clones were for their expression of α2 integrin by were by by of collagen gels by MG-63 cells transfected with or antisense clones and α2 integrin MG-63 cells transfected with were used as cells for and type MG-63 cells were used as for cell cells were and Collagen was by the was supplemented with 10% was on gels. the areas of the collagen gels were of is are a family of growth and other are supposed to be essential for wound healing and tissue processes 1990). In they increase the accumulation of tissue and 1990). are in the formation of of and also is bone In the healing of bone discovered of TGF-β bone are suggested to be in the of the healing process TGF-β is more in the other of the healing process, in the synthesis of matrix The role of extracellular matrix and growth factors for bone cells has also been in the by et have 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. cell lines could contract collagen and anti-β1 integrin antibodies could inhibit this contraction. We and et al., Heino and Massagué, 1989; and 1990; et al., 1994) have described the β1 integrin pattern in both cell There are three putative collagen namely α1β1, α2β1, and α3β1. In MG-63 cells α2β1 and α3β1 are whereas no α1β1 is HOS cells integrin α1β1 and α3β1 are but α2β1 is MG-63 cells and HOS cells fibronectin whereas is expressed in HOS cells (Santala et al., 1994). Previous have suggested the of β1 integrins in the collagen gel by skin fibroblasts et al., 1991; Shiro et al., 1991). Here, we show that osteogenic cells can induce the α1β1 or α2β1 heterodimers are Thus, the data suggest that these heterodimers can each other or that they both can be by a β1 integrin heterodimer. TGF-β could increase collagen gel contraction by MG-63 cells but not by HOS cells. In MG-63 cells TGF-β the synthesis of α2 integrin and the expression of α3 whereas it on the expression of α1 subunit (Heino and Massagué, that the effect of TGF-β is to increased expression of α2 Here, the increased contraction could be by anti-β1 integrin and by integrin whereas integrin had no Previous studies have shown that integrin antibodies can not alone inhibit collagen gel contraction by but enhance the effect of anti-β1 et al., 1991). in three of three contraction. The mechanism of this unknown. However, we have shown that in α3β1 is to a the expression of et al., The role of α2 subunit was also suggested by the fact that in HOS cells TGF-β induce its expression (Santala et al., 1994). We have shown that of HOS cells with the cells to 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 addition to integrin TGF-β other changes in cell that could have the increased collagen gel contraction. Here, we have the of TGF-β-induced increase in fibronectin we have shown that increased expression of α2 integrin subunit alone is sufficient to increase contraction by both MG-63 and HOS cells. was by α2 integrin cDNA in these cells. We have shown that both cell lines a large of β1 integrin subunit (Heino and Massagué, 1989; et al., 1994). it is possible to integrin heterodimers by expression of an α subunit. Furthermore, of MG-63 cells with α2 integrin cDNA cell clones with ability to contract behavior can be by both growth factors and extracellular The fact that a growth TGF-β, can regulate the synthesis of matrix and their cellular receptors the two mechanisms of the effects of TGF-β on cell growth and have been by the of extracellular matrix in cell cultures (Heino and Massagué, 1990; and The data show that TGF-β regulates the cell behavior also by their integrin We and have that in addition to TGF-β also other and interferon-γ, can regulate integrin expression in cell types (Santala and 1991; et al., 1991). In in vivo wound healing et al., and et al., cytokines are present in large it is possible to changes in integrin expression. The role of integrins in the healing of bone is unknown. studies have that cells in bone and in integrin However, α2β1 is expressed in all et al., and 1992; et al., 1992; et al., 1994). Our data suggest that TGF-β be in the reorganization of collagenous matrix also by osteogenic in the that these cells α2β1 We have suggested the of an other in HOS cells, which the expression of α2 integrin (Santala et al., 1994). of cells with both and sarcoma can induce the expression of α2 integrin in HOS cells (Santala et al., but we have not found a we have shown that TGF-β collagen gel contraction by osteogenic cells. The process is to increased expression of α2β1 collagen In cells which not α2 TGF-β can not on its expression or induce collagen gel contraction. The data show that TGF-β can regulate cellular by integrin Furthermore, we that TGF-β be one of the factors 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.