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Integrin α2β1 Is a Positive Regulator of Collagenase (MMP-1) and Collagen α1(I) Gene Expression ∗

  • Terhi Riikonen
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland
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  • Jukka Westermarck
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland
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  • Leeni Koivisto
    Footnotes
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland
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  • Arsi Broberg
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland
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  • Veli-Matti Kähäri
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland

    Department of Dermatology, University of Turku, FIN-20520 Turku, Finland
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  • Jyrki Heino
    Correspondence
    To whom correspondence should be addressed: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. Tel.: 358-21-633-7005; Fax: 358-21-633-7000;
    Affiliations
    MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland

    Department of Medical Biochemistry, University of Turku, FIN-20520 Turku, Finland
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  • Author Footnotes
    ∗ This work was supported in part by grants from the Academy of Finland, the Sigrid Jusélius Foundation, the Paulo Foundation, the Turku University Foundation, the Finnish Cancer Association, and the SWF Cancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Has a scholarship from the Finnish Cancer Union.
Open AccessPublished:June 02, 1995DOI:https://doi.org/10.1074/jbc.270.22.13548
      A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins α1β1 and α2β1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only α1β1 integrin, MG-63 cells, which express only α2β1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, α1(I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with α2 integrin cDNA produced cell clones overexpressing α2β1 integrin. Transfection of MG-63 cells with α2 integrin cDNA in an antisense orientation reduced the expression level of α2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking α2β1 integrin were forced to express it, and this prevented the down-regulation in the levels of α1(I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor α2β1 integrin. The down-regulation of collagen α1(I) is mediated by another receptor. Integrin α2β1 may compete with it and thus be a positive regulator of collagen synthesis.

      INTRODUCTION

      The integrins are a large family of transmembrane proteins, which form heterodimers and mediate cell-matrix and cell-cell interactions (). Three integrins, α1β1, α2β1, and α3β1, have been proposed to be responsible for anchoring cells to collagenous matrices. The role of α1β1 and α2β1 integrins as the major cellular collagen receptors has been well documented, whereas in many cell lines α3β1 integrin is not a collagen receptor. Integrin subunits α1 and α2 contain an I-domain of about 190 amino acids, which is thought to be essential for binding to collagen (
      • Kern A.
      • Briesewitz R.
      • Bank I.
      • Marcantonio E.E.
      ;
      • Katama T.
      • Puzon W.
      • Takada Y.
      ). Both integrins also require the presence of Mg2+ for ligand binding (
      • Staatz W.D.
      • Rajpara S.R.
      • Wayner E.A.
      • Carter W.D.
      • Santoto S.A.
      ). Binding sites for α1β1 and α2β1 integrins in type I and type IV collagen are in the triple helical area (
      • Gullberg D.
      • Gehlsen K.R.
      • Turner D.C.
      • én K.
      • Zijenah L.S.
      • Barnes M.J.
      • Rubin K.
      ;
      • Eble J.A.
      • Golbik R.
      • Mann K.
      • Kühn K.
      ). Denatured collagen can be recognized by cells via RGD-binding integrins, whereas collagen binding by α1β1 and α2β1 integrins require a native conformation (
      • Gullberg D.
      • Gehlsen K.R.
      • Turner D.C.
      • én K.
      • Zijenah L.S.
      • Barnes M.J.
      • Rubin K.
      ).
      Given the fact that the role of integrins as signaling receptors has been established, α1β1 and α2β1 integrin heterodimers are the major candidates to mediate the well known cellular responses to extracellular collagen, i.e. decreased collagen gene expression and induction of matrix metalloproteinase-1 (MMP-1)1(
      The abbreviations used are: MMP
      matrix metalloproteinase
      HOS
      human osteogenic sarcoma cells
      TIMP
      tissue inhibitor of metalloproteinases
      PBS
      phosphate-buffered saline.
      ) production (
      • Grinnell F.
      ). Cells surrounded by matrix are also able to reorganize collagen fibrils, which is seen as the contraction of matrix (
      • Grinnell F.
      and references therein). Fibroblasts cultured inside floating or anchored three-dimensional collagen matrices are supposed to mimic dermis or scars and granulation tissue, respectively (
      • Grinnell F.
      ). The regulation of MMPs by matrix receptors may also have great importance for cancer cell invasiveness. The role of α2β1 integrin in the reorganization of collagenous matrix is well documented (Shiro et al., 1991;
      • Klein C.E.
      • Dressel D.
      • Steinmayer T.
      • Mauch C.
      • Eckes B.
      • Krieg T.
      • Bankert R.B.
      • Weber L.
      ;
      • Riikonen T.
      • Koivisto L.
      • Vihinen P.
      • Heino J.
      ), whereas the cellular receptors responsible for changes in collagen and MMP-1 gene expression are not known. Here, we show that the number of α2β1 integrin heterodimers at the cell surface can be critical for the expression level of α1(I) collagen and MMP-1.

      MATERIALS AND METHODS

      Cell Culture

      The human osteosarcoma cell lines MG-63, HOS, and KHOS-240 (HOS cells transformed with Kirsten murine sarcoma virus) were obtained from American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.). For collagen gel experiments, eight volumes of Vitrogen-100 (Celtrix) was neutralized with two volumes of 1:1 mixture of 10-fold concentrated medium and 0.1 N NaOH before adding detached cells.

      Plasmid Constructs and Transfections

      α2 integrin cDNA corresponding to nucleotides 1-4559 in the published sequence (
      • Takada Y.
      • Hemler M.E.
      ) was kindly provided by Dr. Hemler. It was ligated in both orientations into the pAWneo2 expression vector (a kind gift from Dr. Arthur Weiss) (
      • Ohashi P.S.
      • Mak T.W.
      • Van den Elsen P.
      • Yanagi Y.
      • Yoshikai Y.
      • Calman A.F.
      • Terhorst C.
      • Stobo J.D.
      • Weiss A.
      ), which carries the neomycin resistance gene.
      Transfections were carried out using Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. 400 μg/ml G418 (Life Technologies, Inc.) was added to the culture media of transfected and control cells. After 2-3 weeks of selection, the control cells were dead, and G418 resistant clones were isolated and analyzed for their expression of α2 integrin mRNA and protein (
      • Riikonen T.
      • Koivisto L.
      • Vihinen P.
      • Heino J.
      ).

      Northern Blot Hybridizations

      Total cellular RNA was isolated by the guanidium thiocyanate-CsCl method (
      • Chirgwin J.M.
      • Przybyla A.E.
      • MacDonald R.J.
      • Rutter W.J.
      ). Before isolating total RNA from the cells inside collagen gels, the gels were briefly treated with 0.5 mg/ml collagenase (type II, Sigma) in phosphate-buffered saline (PBS, pH 7.4) with 1 mM CaCl2. 20 μg of total cellular RNA was separated in formaldehyde-containing 1% agarose gels, transferred to nylon membranes (ZETA-probe, Bio-Rad), and hybridized with 32P-labeled (Amersham) cDNAs for human α1(I) collagen (
      • Mäkelä J.K.
      • Raassina M.
      • Virta A
      • Vuorio E.
      ), human MMP-1 (
      • Goldberg G.I.
      • Wilhelm S.M.
      • Kronberger A.
      • Bauer E.A.
      • Grant G.A.
      • Eisen A.Z.
      ), human α2 integrin (
      • Takada Y.
      • Hemler M.E.
      ), human tissue inhibitor of metalloproteinases-1 (TIMP-1) (
      • Carmichael D.F.
      • Sommer A.
      • Thompson R.G.
      • Anderson D.C.
      • Smith C.G.
      • Welgus H.G.
      • Stricklin G.P.
      ), and rat glyceraldehyde-3-phosphate dehydrogenase (a “housekeeping” enzyme used as a control) (
      • Fort P.L.
      • Marty L.
      • Piechaczyk M.
      • El Sabrouty S.
      • Dani C.
      • Jeanteur P.
      • Blanchard J.M.
      ) probes. Autoradiograms were quantified with Microcomputer Imaging Device version M4 (Imaging Research Inc.), and the resulting measurements were corrected for glyceraldehyde-3-phosphate dehydrogenase mRNA levels.

      Flow Cytometry

      Cells were grown to early confluence and detached with trypsin-EDTA, and trypsin activity was inhibited by medium supplemented with serum. Cells were washed with PBS (pH 7.4) and then incubated with PBS containing 10 mg/ml bovine serum albumin, 1 mg/ml glycine, and 0.02% NaN3 at 4°C for 20 min. Cells were collected by centrifugation, exposed to a saturating concentration of monoclonal antibody against α2 integrin (12F1) (
      • Pischel K.D.
      • Hemler M.E.
      • Huang C.
      • Bluetein H.G.
      • Woods V.L.
      ) in PBS/bovine serum albumin (1 mg/ml) containing NaN3 at +4°C for 30 min, and stained with rabbit anti-mouse IgG coupled to fluorescein (1:20 dilution; Dacopatts, Denmark) at 4°C for 30 min. Cells were washed twice with PBS containing NaN3 and suspended in the same buffer. To measure the amount of α2 integrin on the cell surfaces, the fluorescent excitation spectra were analyzed by using a FACScan apparatus (Becton Dickinson). Control samples were prepared by treating the cells without primary antibodies.

      RESULTS

      Down-regulation of Collagen α1(I) and Induction of MMP-1 Gene Expression Correlate with the Presence of α1β1 and α2β1 Integrins, Respectively

      We have taken advantage of the fact, that the pattern of collagen-binding integrins varies significantly in different osteogenic cell lines (
      • Takada Y.
      • Huang C.
      • Hemler M.
      ;
      • Heino J.
      • Massagué J.
      ;
      • Dedhar S.
      • Saulnier R.
      ;
      • Santala P.
      • Larjava H.
      • Nissinen L.
      • Riikonen T.
      • Määttä A.
      • Heino J.
      ). MG-63 cells express mainly α3β1 heterodimer and in smaller amounts α2β1 integrin. Integrin α1β1 is not usually detectable, although its expression can be induced, e.g. with cytokines (
      • Santala P.
      • Heino J.
      ). HOS cells express both α1β1 and α3β1 integrins, whereas α2β1 integrin expression is below the detection level (
      • Santala P.
      • Larjava H.
      • Nissinen L.
      • Riikonen T.
      • Määttä A.
      • Heino J.
      ). We have also used a third cell line KHOS-240, a Kirsten sarcoma virus transformed variant of HOS, which has all three putative integrin-type collagen receptors (
      • Santala P.
      • Larjava H.
      • Nissinen L.
      • Riikonen T.
      • Määttä A.
      • Heino J.
      ). When cultured inside collagenous matrices, only KHOS-240 cells showed fibroblast-like alterations in the expression of MMP-1 and collagen α1(I) (induction of MMP-1 and a 90% decrease in collagen α1(I) mRNA levels; Fig. 1). This observation is in accordance with the fact that fibroblasts and KHOS-240 cells have a similar pattern of integrin-type collagen receptors. Inside collagen gels, HOS cells showed markedly decreased mRNA levels (80%) for collagen α1(I), whereas no MMP-1 mRNA wes detected (Fig. 1). In MG-63 cells, there were no alterations in the mRNA levels of α1(I) collagen. MG-63 cells cultured in monolayer already expressed some MMP-1, and its expression was increased when the cells were grown in collagen gels (2-8-fold range in three measurements; Fig. 2 and 3). Thus, in these three cell lines, collagen α1(I) expression was down-regulated only when α1β1 integrin was present, and the induction of MMP-1 expression was seen only when α2β1 integrin was present.
      Figure thumbnail gr1
      Fig. 1Northern blot analysis of α1(I) collagen and interstitial collagenase (MMP-1) expression in HOS and KHOS-240 cells grown in monolayer or inside collagen. Cells were grown for 48 h, total cellular RNA was isolated, and the levels of mRNAs were analyzed by specific 32P-labeled probes and autoradiography. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control.
      Figure thumbnail gr2
      Fig. 2Northern blot analysis of interstitial collagenase (MMP-1) and TIMP-1 expression in MG-63 wild type (wt) and α2 integrin cDNA-transfected cells (sa2). MG-63 cells were transfected with sense-oriented α2 integrin cDNA in the pAWneo2 expression vector. A, the cells were grown in monolayer or inside collagen gels before isolating total RNA. 20 or 30 μg of cellular RNA was separated in 1% formaldehyde-containing agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for MMP-1, TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, autoradiograms were quantified with Microcomputer Imaging Device, and the values were corrected for glyceraldehyde-3-phosphate dehydrogenase mRNA levels.

      In MG-63 Cells, the Overexpression of α2β1 Integrin Enhances and Its Down-regulation Reduces MMP-1 mRNA Levels

      To get direct evidence about the role of α2β1 integrin in the regulation of MMP-1 expression, we specifically up- or down-regulated α2 integrin levels in MG-63 cells. We created a permanent MG-63 cell clone constantly overexpressing α2 integrin by transfecting cells with a cDNA construct containing the entire coding sequence of α2 integrin (
      • Riikonen T.
      • Koivisto L.
      • Vihinen P.
      • Heino J.
      ). In this cell clone, the cell surface level of α2 integrin was 5-fold higher than in wild type cells when measured by flow cytometry (not shown). Recent studies have shown that integrins might be sensitive to down-regulation by antisense strategies (
      • Lallier T.
      • Bonner-Fraser M.
      ). Instead of using oligonucleotides, we constructed a plasmid containing the α2 integrin cDNA in antisense orientation. This construct gave a continuous expression of an approximately 4.5-kilobase antisense mRNA in two MG-63 cell clones. In these clones, the expression level of α2 integrin protein was decreased (to 10% of control; not shown) as were the corresponding mRNA levels (about 50% of control; Fig. 3). Interestingly, in these two clones the ratio of antisense mRNA/α2 integrin mRNA were different (1 and 6), suggesting that they had different copy numbers of the antisense cDNA. The α2 integrin mRNA level, however, seemed to be the same even if the amount of antisense mRNA increased (Fig. 3). The levels of α2 integrin mRNA were strongly (6-fold) up-regulated when the cells were inside collagen gels (Fig. 3). Similar phenomena have been previously described with melanoma cells and fibroblasts (
      • Klein C.E.
      • Dressel D.
      • Steinmayer T.
      • Mauch C.
      • Eckes B.
      • Krieg T.
      • Bankert R.B.
      • Weber L.
      ). In antisense transfected cell clones grown in collagen gels, α2 integrin mRNA levels were also elevated, although they remained at a lower level than in control cells (35-50% of controls). Simultaneously with the increase in α2 integrin mRNA levels, the amount of antisense mRNA decreased (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Northern blot analysis of α1(I) collagen, interstitial collagenase (MMP-1), and α2 integrin mRNA expression in MG-63 wild type (wt) and two antisense α2 integrin-transfected cell clones (as#2, as#4). A, the antisense-oriented α2 integrin cDNA in the pAWneo2 expression vector was transfected into MG-63 cells. The two resulting clones, in which α2 integrin expression was reduced, were grown in monolayer or inside collagen gels; total cellular RNA was isolated, and specific mRNAs were analyzed. Autoradiograms were quantified, and the MMP-1 mRNA levels in cells grown in monolayer after correction for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are shown in B.
      In the MG-63 cell clone overexpressing α2 integrin MMP-1, mRNA levels were 9-fold higher than in wild type cells when the cells were cultured in monolayer (Fig. 2). Inside collagen gels, the difference was 6-fold (Fig. 2). In both antisense transfected cell clones, the basal level of MMP-1 was lower than in control cells (5-50% of control; Fig. 3). MMP-1 mRNA levels were elevated in antisense clone cells inside collagen gels, which is in accordance with the fact that the antisense construct could not entirely block the induction of α2 integrin by collagen. The data indicate that in MG-63 cells, elevated expression of α2 integrin enhances the expression of MMP-1, and diminished expression of α2 integrin leads to its down-regulation.
      In a second set of experiments, α2 integrin was overexpressed in HOS cells. The expression of α2 integrin on the cell surface was confirmed by flow cytometric measurements (not shown). Inside collagen gels, the level of the 4.5-kilobase plasmid-derived α2 mRNA was surprisingly elevated (Fig. 4), suggesting that the increased α2 integrin mRNA levels are partially due to increased stability of the mRNA. In HOS cells overexpressing α2 integrin, MMP-1 was detected in only two out of five experiments (not shown), suggesting that in HOS cells MMP-1 expression is also regulated by another factor more strongly than by α2 integrin.
      Figure thumbnail gr4
      Fig. 4The levels of α1(I) collagen and α2 integrin mRNAs in HOS cells transfected with sense-oriented α2 integrin cDNA into pAWneo2 expression vector (pα2AW#7, pα2AW#11). Total cellular RNAs from cells grown inside collagen gels or in monolayer were isolated, and 20 μg of total cellular RNA was separated in formaldehyde-containing 1% agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled α1(I) collagen, α2 integrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes.
      Many cell types produce, concomitantly with MMP-1, its specific inhibitor, TIMP-1 (see
      • Birkedahl-Hansen H.
      • Moore W.G.I.
      • Bodden M.K.
      • Windsor L.J.
      • Birkedahl-Hansen B.
      • DeCarlo A.
      • Engler J.A
      ). In α2 integrin overexpressing MG-63 cells, TIMP-1 mRNA levels were decreased (Fig. 2). This suggests that α2 integrin elicited up-regulation of MMP-1 expression may result in increased pericellular collagenase activity.

      In HOS Cells, the Overexpression of α2β1 Integrin Prevents the Down-regulation of Collagen α1(I)

      In α2β1 integrin positive wild type MG-63 cells, collagen α1(I) mRNA levels were not down-regulated, indicating that α2β1 integrin is probably not the receptor mediating suppression of collagen synthesis by collagenous matrix. The data suggest that the unidentified receptor responsible for this phenomenon may not be expressed in MG-63 cells, making α1β1 integrin one of the candidate molecules. Therefore, it was not surprising that in MG-63 cells up-regulation or down-regulation of α2 integrin did not result in any dramatic changes in collagen mRNA levels (Fig. 2). In both antisense and sense transfected cell clones, there was some increase in the basal level of collagen synthesis that was considered to be nonspecific. In contrast to MG-63 cells, HOS cells responded to collagenous matrix by diminished collagen synthesis (Fig. 1). In HOS cells overexpressing α2 integrin, the levels of collagen α1(I) mRNA elevated in response to collagen matrix (Fig. 4). This elevation was seen in all experiments, but was not always as prominent as in Fig. 4.

      DISCUSSION

      Two integrin-type collagen receptors, α1β1 and α2β1 heterodimers, mediate the interaction of cells with collagenous matrices. To investigate the role of α2β1 integrin in the regulation of MMP-1 and collagen α1(I) expression, we used cDNA transfections to specifically up- and down-regulate its expression at the cell surface. The use of anti-integrin antibodies would have given indirect evidence about the role of integrins. That approach, however, would have been controversial because anti-integrin antibodies might either block integrin function or simulate ligand binding and activate signal transduction. Furthermore, anti-integrin α chain antibodies alone are usually ineffective and can only be used to potentiate the effect of anti-β1 antibodies (
      • Klein C.E.
      • Dressel D.
      • Steinmayer T.
      • Mauch C.
      • Eckes B.
      • Krieg T.
      • Bankert R.B.
      • Weber L.
      ;
      • Riikonen T.
      • Koivisto L.
      • Vihinen P.
      • Heino J.
      ).
      Previous studies with migrating keratinocytes (
      • Saarialho-Kere U.K.
      • Kovacs S.O.
      • Pentland A.P.
      • Olerud J.E.
      • Welgus H.G.
      • Parks W.C.
      ;
      • Sudbeck B.D.
      • Parks W.C.
      • Welgus H.G.
      • Pentland A.P.
      ) and with invasive melanoma cells (
      • Montgomery A.M.P.
      • Reisfeld R.A.
      • Cheresh D.A.
      ) have shown the regulation of MMP-1 after cell-collagen interactions. Here, we show that the amount of α2β1 integrin at the cell surface regulates the expression level of MMP-1 in response to collagen. Under different physiological conditions, the level of α2β1 integrin expression is not constant but can be up-regulated by growth factors and malignant transformation (
      • Heino J.
      • Massagué J.
      ;
      • Dedhar S.
      • Saulnier R.
      ;
      • Santala P.
      • Larjava H.
      • Nissinen L.
      • Riikonen T.
      • Määttä A.
      • Heino J.
      ). In melanoma cells, the α2 integrin subunit was originally described as the melanoma progression antigen (
      • Klein C.E.
      • Steinmayer T.
      • Kaufmann D.
      • Weber L.
      • Brocker E.B.
      ); also, the ability to contract collagen gels (an α2β1 integrin-related function) separates aggressive melanoma clones from less invasive clones (
      • Klein C.E.
      • Dressel D.
      • Steinmayer T.
      • Mauch C.
      • Eckes B.
      • Krieg T.
      • Bankert R.B.
      • Weber L.
      ). In osteogenic HOS cells, transformation with either a chemical mutagen, N-methyl-N′-nitro-N-nitrosoguanidine, or Kirsten murine sarcoma virus induces α2 integrin expression (
      • Santala P.
      • Larjava H.
      • Nissinen L.
      • Riikonen T.
      • Määttä A.
      • Heino J.
      ). In rhabdomyosarcoma cells, the forced expression of α2 integrin leads to an invasive cell phenotype (
      • Chan B.M.C.
      • Matsuura N.
      • Takada Y.
      • Zetter B.R.
      • Hemler M.
      ). The observation that α2β1 integrin-related signals regulate the expression of MMP-1 reveals a putative invasion mechanism and explains why α2β1 integrin seems to be important for cancer cells. Furthermore, this observation provides the basis for new specific strategies to prevent invasion by malignant tumors. Previous studies have shown that treatment of fibroblasts with RGD peptides, anti-α5β1 integrin antibodies, and fibronectin fragments induces the expression of MMPs (
      • Werb Z.
      • Tremble P.M.
      • Behrendtsen O.
      • Howley E.
      • Damsky C.H.
      ). Transformation can, however, down-regulate the expression of α5β1 integrin () or inhibit its activity (
      • Akiyama S.K.
      • Larjava H.
      • Yamada K.M.
      ), and therefore, α5β1 integrin is probably not involved in the regulation of MMP-1 expression in transformed cells. Moreover, the forced overexpression of α5β1 integrin prevents the malignant behavior of Chinese hamster ovary cells, suggesting that α5β1 integrin might function as a tumor suppressor (
      • Giancotti F.G.
      • Ruoslahti E.
      ).
      In addition to MMP-1, other genes are regulated by signals generated by integrin-type collagen receptors. Our data suggest that α2 integrin itself is positively regulated by collagenous matrix, whereas collagen α1(I) is down-regulated in cells surrounded by collagen. The suppression in collagen gene expression was seen in α1β1 positive-α2β1 negative HOS cells, suggesting that α1β1 integrin rather than α2β1 integrin mediates the signals required. In HOS cells the overexpression of α2β1 heterodimer not only prevented the down-regulation of collagen α1(I) mRNA levels but even elevated them. The molecular mechanism of this phenomenon is not clear. Previous studies have suggested that the regulation of collagen synthesis in cells inside collagenous matrices is a complex phenomenon involving both transcriptional and posttranscriptional mechanisms (
      • Eckes B.
      • Mauch C.
      • Huppe G.
      • Krieg T.
      ). Due to the fact that in α1β1 negative-α2β1 positive MG-63 cells, the altered expression of α2β1 integrin did not have any significant effect on collagen gene expression, we suggest that α2β1 integrin is a positive regulator of collagen synthesis by competing with a “negative regulator” collagen receptor, possibly α1β1 integrin. Inhibition of the down-regulation of collagen synthesis by overexpression of α2 integrin suggests that the accumulation of collagen in tissues can also be regulated by the expression pattern of different collagen receptors. In a progressive fibrotic skin disorder (scleroderma), there are alterations in the expression of integrin-type collagen receptors (
      • Ivarsson M.
      • McWhirter A.
      • Black C.M.
      • Rubin K.
      ). The fact that in scleroderma cells the ratio of α1β1 integrin/α2β1 integrin is decreased and that these cells show reduced response to collagenous matrix (
      • Ivarsson M.
      • McWhirter A.
      • Black C.M.
      • Rubin K.
      ) is in full accordance with our observations.
      Signal transduction via integrins has been associated with protein tyrosine phosphorylation (
      • Schaller M.D.
      • Parsons J.T.
      ). A specific focal adhesion kinase (pp125FAK) is claimed to be directly linked to integrins, and it is a strong candidate to be one of the first components in the cascade (
      • Schaller M.D.
      • Parsons J.T.
      ). Furthermore, the regulation of MMP-1 by cell-collagen interactions can be prevented by inhibitors of protein kinase C and protein tyrosine kinases (
      • Sudbeck B.D.
      • Parks W.C.
      • Welgus H.G.
      • Pentland A.P.
      ). Our observations suggest that different collagen receptors might generate distinct signals, having opposite effects on cell behavior (Fig. 5). It is an important challenge for future research to elucidate the molecular mechanisms giving rise to the specificity of signals generated by different matrix receptors.
      Figure thumbnail gr5
      Fig. 5Model for the distinct roles of the integrin-type collagen receptors in the regulation of (1) collagenase (MMP-1) and (2) collagen α1(I) expression (left). Rightpanel summarizes the main results of this paper supporting the model. It is not clear whether α2β1 integrin up-regulates α1(I) collagen mRNA levels directly or whether overexpression of α2β1 integrin competes with another collagen receptor. The α2 integrin subunit itself is up-regulated in cells exposed to collagen. This phenomenon might be mediated by α2β1 integrin-related signals.

      Acknowledgments

      We thank Drs. M. Hemler, A. Weiss, E. Vuorio, P. Fort, E. Bauer, V. Woods, and D. Carmichael for cDNAs, antibodies, and vectors, Dr. S. Edwards for critical comments, and M. Potila for technical assistance.

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