Elsevier

Matrix Biology

Volume 20, Issue 3, June 2001, Pages 193-203
Matrix Biology

Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form

https://doi.org/10.1016/S0945-053X(01)00135-4Get rights and content

Abstract

The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 μg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.

Introduction

Tissue remodelling is an important process in both physiological and pathological conditions. Among the potential enzymes involved in matrix degradation required for tissue remodelling are the matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases (Woessner, 1994). MMP-2 (gelatinase A) is an MMP family member with selective ability to degrade type IV collagen, a major structural component of the basement membrane, and gelatin, representing denatured collagen. It shares this capacity with MMP-9 (gelatinase B) by virtue of a common gelatin-binding domain. However, whilst MMP-9 is highly regulated at the transcriptional level, MMP-2 is not, and its rather general expression is regulated at the level of zymogen activation and/or inhibition by the family of tissue inhibitors of metalloproteinases (TIMPs). Increased levels of active MMP-2 have been associated with invasion and metastasis in various kinds of tumours (Koshiba et al., 1998, Nomura et al., 1995). Activation of the latent pro-MMP-2 zymogen is overwhelmingly effected by membrane type (MT) -MMPs, a novel subfamily of MMPs (Murphy et al., 1999, Seiki, 1999). MT-MMPs contain a furin-like enzyme cleavage motif, and are largely thought to be constitutively activated during cellular transport through the Golgi apparatus (Sato et al., 1999). However, endogenous expression of MT-MMP is insufficient to effect MMP-2 activation, and treatment with additional agents is usually required (reviewed in Murphy et al., 1999, Seiki, 1999). The plant lectin, concanavalin A (Con A), and a phorbol ester 12-O-tetradecanonylphorbol 13 acetate (TPA), potently stimulate activation in a wide variety of cells and thrombin stimulates activation in endothelial cells (Murphy et al., 1999, Seiki, 1999).

Induction of MMP-2 activation in response to three-dimensional collagen substratum has been shown by us and others in fibroblasts (Azzam and Thompson, 1992, Boyd and Balkwill, 1999, Gilles et al., 1997, Lee et al., 1997, Preaux et al., 1999, Seltzer et al., 1994, Tomasek et al., 1997), cancer cell lines (Azzam et al., 1993, Ellenrieder et al., 2000, Ellerbroek et al., 1999, Gilles et al., 1997, Kurschat et al., 1999) and endothelial cells (Haas et al., 1998, Haas et al., 1999). As seen with Con A (Yu et al., 1995), collagen-induced MMP-2 activation involves increased steady-state levels of MT1-MMP mRNA and protein (Gilles et al., 1997, Gilles et al., 1998), as well as a non-transcriptional component which can be seen in MCF-7 cells transfected with MT1-MMP driven by the heterologous CMV promoter (Gilles et al., 1998). Co-expression of collagen α1(I) and MT1-MMP mRNA transcripts has been detected in fibroblasts around the breast and pancreatic tumours (Gilles et al., 1997, Ellenrieder et al., 2000), suggesting that collagen-regulation of MT1-MMP may be a physiological counterpart to the artificial induction of MMP-2 activation by Con A. This appears to be an important mechanism for fibrillar matrix remodelling, given the over-abundance of collagen seen in mice lacking MT1-MMP (Holmbeck et al., 1999, Zhou et al., 2000).

A number of studies have indicated a specificity for collagen over other matrix components in this induction, and also a requirement for the three-dimensional structure of the collagen into fibrillar gels, since thin coatings of various collagen preparations, or denatured type I collagen (gelatin), failed to induce MMP-2 activation (Azzam et al., 1993, Gilles et al., 1997, Maquoi et al., 1998). In the current study, we have (i) extended our analysis to preparations enriched in various types of human collagen; (ii) characterised the activation seen in response to direct addition of soluble collagen to the culture medium; (iii) further evaluated the requirement for collagen fibril formation for induction of MMP-2 activation; and (iv) confirmed an essential role for MT1-MMP in this process. We employed normal human skin fibroblasts, which have shown a similar MMP-2 activational response profile to breast-derived fibroblasts (Azzam and Thompson, 1992), and also primary mouse fibroblast cultures derived from mice which lack MT1-MMP (Holmbeck et al., 1999).

Section snippets

Human collagen preparation

Fibril-forming collagens were prepared from foetal human skin, obtained with Royal Children Hospital Institutional Ethics approval, using established procedures (Chan et al., 1990). The sample was extracted sequentially with acetic acid and pepsin, and the latter subjected to differential salt precipitation with 0.7 M, 0.7–0.9 M, 0.9–1.2 M and 1.2–2.4 M NaCl to prepare different collagen fractions. The composition and purity of these preparations were analysed by 5% SDS-PAGE and protein

Effect of different collagen preparations on the activation of MMP-2 and MT1-MMP

When used as a three-dimensional gel, each of the fibril-forming collagen preparations containing either collagens types I, II, or III alone, or mixtures of types I and III collagens induced a similar degree of activation of the endogenous MMP-2 in human skin fibroblast cultures (Fig. 1a). Time course for induction of MMP-2 activation by the various collagen preparations also revealed no significant differences between each collagen preparation, with MMP-2 activation becoming evident by 24 h

Discussion

The physiological regulation of MT-MMP-mediated MMP-2 activation at the cell surface is not well understood. A growing number of factors, including ConA, phorbol ester and cytochalasin D (Murphy et al., 1999, Seiki, 1999) may mimic certain aspects of the regulation induced by extracellular matrices such as collagen. Collagen-induced activation is well documented in fibroblasts, endothelial cells and invasive cancer cells. Although thin layers of type IV collagen have been shown not to induce

Acknowledgements

We thank Dr John T. Price for many helpful discussion in this study and Susan Yamada for the primary culture of MT1-MMP knockout mouse fibroblasts. This work has been supported by Victorian Breast Cancer Research Consortium (N.R., E.W.T.), National Health and Medical Research Council, Australia (D.C., J.F.B.), and NIH RO1 Grant AR45879 to FibroGen, Inc (J.P., C.Y.). Neeracha Ruangpanit is supported by the Ministry of University Affairs, Thailand.

References (50)

  • S. Matsumoto et al.

    Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA

    Biochim. Biophys. Acta

    (1997)
  • T. Nakamura et al.

    Platelet adhesion to native type I collagen fibrils. Role of GPVI in divalent cation-dependent and -independent adhesion and thromboxane A2 generation

    J. Biol. Chem.

    (1998)
  • T. Nakamura et al.

    Activation of the GP Iib–IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP VI

    J. Biol. Chem.

    (1999)
  • E. Ohuchi et al.

    Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules

    J. Biol. Chem.

    (1997)
  • D.J. Prockop et al.

    Inhibition of the self-assembly of collagen I into fibrils with synthetic peptides. Demonstration that assembly is driven by specific binding sites on the monomers

    J. Biol. Chem.

    (1998)
  • S.A. Santoro et al.

    Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

    Biochem. Biophys. Res. Commun.

    (1988)
  • T. Sato et al.

    Furin-independent pathway of membrane type 1-matrix metalloproteinase activation in rabbit dermal fibroblasts

    J. Biol. Chem.

    (1999)
  • J.L. Seltzer et al.

    Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction

    Exp. Cell Res.

    (1994)
  • J.J. Tomasek et al.

    Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton

    J. Biol. Chem.

    (1997)
  • W. Vogel et al.

    The discoidin domain receptor tyrosine kinases are activated by collagen

    Mol. Cell

    (1997)
  • M. Yu et al.

    Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation

    FEBS Lett.

    (1997)
  • H.S. Azzam et al.

    Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

    J. Natl. Cancer Inst.

    (1993)
  • H.S. Azzam et al.

    Collagen-induced activation of the Mr 72,000 type IV collagenase in normal and malignant human fibroblastoid cells

    Cancer Res.

    (1992)
  • R.S. Boyd et al.

    MMP-2 release and activation in ovarian carcinoma: the role of fibroblasts

    Br. J. Cancer

    (1999)
  • D. Chan et al.

    Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro

    Biochem. J.

    (1990)
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    Current Address: Department of Biochemistry, University of Hong Kong, Hong Kong.

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