Collagen I regulates matrix metalloproteinase-2 activation in osteosarcoma cells independent of S100A4 Renate Elenjord1, Jasmine B Allen2, Harald T Johansen3, Hanne Kildalsen1, Gunbjørg Svineng2, Gunhild M Mælandsmo1,4, Thrina Loennechen1 and Jan-Olof Winberg2 Department of Pharmacy, University of Tromsø, Norway Department of Medical Biochemistry, University of Tromsø, Norway School of Pharmacy, University of Oslo, Norway Department of Tumor Biology, The Norwegian Radium Hospital, Oslo, Norway Keywords collagen I; extracellular matrix; inhibitors of matrix metalloproteinases; matrix metalloproteinases; S100A4 Correspondence J.-O Winberg, Department of Medical Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway Fax: +47 776 45350 Tel: +47 776 45488 E-mail: janow@fagmed.uit.no (Received December 2008, revised July 2009, accepted 20 July 2009) doi:10.1111/j.1742-4658.2009.07223.x This work investigates the effect of cell–collagen I interactions on the synthesis and activation of MMP-2, as well as synthesis of MT1-MMP and TIMP-1, by using an in vitro model with 3D fibrillar and 2D monomeric collagen In order to reveal whether the metastasis-associated protein S100A4 can influence the cell’s response to the two forms of collagen, osteosarcoma cell lines with high and low endogenous levels of S100A4 were used Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric collagen resulted in opposite effects on MMP-2 activation Attachment to 3D fibrillar collagen decreased activation of proMMP-2, with a corresponding reduction in MT1-MMP By contrast, attachment to monomeric collagen increased the amount of fully active MMP-2 This was caused by a reduction in TIMP-1 levels when cells were attached to monomeric 2D collagen The effect of collagen on proMMP-2 activation was independent of endogenous S100A4 levels, whereas synthesis of TIMP-1 was dependent on S100A4 When cells were attached to monomeric collagen, cells with a high level of S100A4 showed a greater reduction in the synthesis of TIMP1 than did those with a low level of S100A4 Taken together, this study shows that synthesis and activation of MMP-2 is affected by interactions between osteosarcoma cells and collagen I in both fibrillar and monomeric form Introduction The extracellular matrix (ECM) is an intricate network of macromolecules composed of a wide variety of locally secreted proteins and polysaccharides which are closely associated with the surface of the cell that produced them The ECM can be found in different forms, from the hard compositions of bone to the soft structures of connective tissue Collagens are the main components of ECM, and type I collagen is the most abundant form in bone and connective tissue [1] Controlled turnover of ECM is critical for a wide variety of normal physiological processes, such as wound healing and embryogenesis The matrix metalloproteinases (MMPs) are considered to be the major enzymes involved in ECM remodelling, and dysregulated MMPs have been implicated in several diseases such as arthritis, cancer and cardiovascular disease [2] The family of MMPs consists of over 20 secreted and membrane-bound enzymes which are involved in degrada- Abbreviations APMA, p-aminophenylmercuric acid; ECM, extracellular matrix; MMPs, matrix metalloproteinases; MT-MMPs, membrane type matrix metalloproteinases; TIMPs, tissue inhibitors of MMPs FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5275 Collagen I-modulated MMP expression R Elenjord et al tion and limited proteolysis of extracellular matrix Most MMPs are secreted as inactive proenzymes and latency is maintained by an interaction between a cystein residue in the prodomain and Zn2+ in the active site of the catalytic domain Two major types of endogenous inhibitors regulate the activity of MMPs; a2-macroglobulin and four tissue inhibitors of metalloproteinases, TIMP-1 to TIMP-4 [2] In order to identify potential drug targets, it is important to know the role of individual MMPs, their expression pattern and activation mechanisms The best-described activation mechanism for MMP-2 is the two-step cell-surface activation in which proMMP-2 is activated in a trimolecular complex including membrane type MMP (MT1-MMP) and the inhibitor TIMP-2, giving an MMP-2 intermediate that is autocatalysed to fully active MMP-2 [3] Other activators of MMP-2 can directly activate proMMP-2 to fully active MMP-2, for example, the lysosomal cysteine proteinase legumain, which is known to activate proMMP-2 by cleaving the Asn80–Tyr81 (i.e the autocatalytic site) and Asn82– Phe83 bonds [4], and MT6-MMP, which can cleave proMMP-2 at the Asn80–Tyr81 bond [5] Some activators are suggested primarily to take part in the second activation step, in which the MMP-2 intermediate is converted to the fully active MMP-2 Among these are TIMP-2 [6] and integrins such as aVb3 [7] However, there is some controversy regarding the role of the aVb3 integrin, because it is also reported to suppress collagen I-induced activation of proMMP-2 [8] Another MMP inhibitor, reversion-inducing cysteinerich protein with kazal motifs (RECK) is a cell membrane associated inhibitor of MMP-2 that is able to inhibit the second activation step of MMP-2 [9] The small calcium-binding protein S100A4 has been shown to regulate expression of MMPs and their inhibitors in several cell lines [10] The protein itself has no known enzymatic activity, but binds to distinct intracellular target proteins and regulates specific functions involved in tumour progression such as cell motility, proliferation and apoptosis [11] Although S100A4 is strongly associated with the stimulation of invasion and metastasis, the actual mechanism for the metastasis-promoting function of S100A4 is not completely understood The protein seems to have several functions, both intracellularly and extracellularly By reducing the S100A4 level in a human osteosarcoma cell line, and implementing these in mice, the capacity to metastasize has been shown to decrease [12] Cultivation of the same cell lines on plastic also showed decreased expression and activation of MMP-2 [13,14] Previously, we have shown that a reduced endogenous level of S100A4 in human osteosarcoma cell lines 5276 resulted in a reduced in vitro and in vivo invasive and metastatic capacity [12,13] Furthermore, we also showed that the reduction in the endogenous level of S100A4 in these cell lines resulted in altered levels of MMP-2, MT1-MMP, TIMP-1 and TIMP-2, as well as active MMP-2 [13,14] Therefore, these cell lines were used in this study to investigate the extent to which synthesis and activation of proMMP-2, as well as synthesis of MT1-MMP, TIMP-1 and TIMP-2, are affected by the interaction of the cell with various biological forms of collagen I A fibrillar 3D lattice and a 2D layer of monomeric collagen I will, to a certain extent, mimic the natural environment of osteosarcoma cells and were used in this study as an in vitro model to study effects of cell–collagen I interactions Results Cell morphology and actin cytoskeleton structure As observed by light microscopy (data not shown), pHb-1 and II-11b cells attached to plastic or monomeric 2D collagen were spread in a confluent cell layer and hence showed maximum cell–cell contact For cells seeded on a fibrillar 3D collagen matrix, the cells were rounded up and seemed to have a more spherical shape; hence they were separated from most adjacent cells, but were still attached to the surface Confocal microscopy revealed no differences in actin cytoskeleton organization for cells attached to the different surfaces In addition, whether the fibrillar 3D collagen gel was attached to or released from plastic, or whether the cells were attached on the top of or inside the fibrillar 3D matrix did not influence the organization of the actin cytoskeleton (data not shown) Cell viability As shown in Fig 1, during 48 h incubation in serumfree medium only minor changes in the number of viable cells were observed for both cell lines attached to plastic However, when the cells were attached to monomeric 2D collagen, the number of viable cells increased, whereas for cells attached to fibrillar collagen a small reduction in viable cells was observed S100A4 expression is not affected by the cells binding to collagen I In order to confirm the difference in S100A4 levels between the two cell lines, western blot analyses were performed on cell lysates from cells attached to plastic To ensure equal loading, the total amount of cellular FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS R Elenjord et al Collagen I-modulated MMP expression Relative cell viability (%) pHβ–1 200 A II-11b * 3h * 48 h pHβ-1 Mr (kDa) P 2D II-11b 3D P 2D 3D 72 64 100 62 3D 2D p 3D Act/Tot MMP-2 (rel) 2D p Fig Cell viability Relative cell viability (mean ± SEM) for pHb-1 and II-11b cells attached to plastic (P), monomeric 2D collagen I (2D) and fibrillar 3D collagen I (3D) *P < 0.05 for 48 h compared with h (n = 7) Binding of cells to fibrillar 3D collagen I results in decreased proMMP-2 activation and reduced MT1-MMP expression A decrease in the total amount of MMP-2 (72, 64 and 62 kDa bands), as well as in the active MMP-2 forms Time (min) A * * B ProMMP-2 (rel) protein in the two cell lines was determined as described in Materials and methods, and 81 lg of protein was added to each well The amount of S100A4 in the II-11b cells was 1.2% of that in the pHb-1 cells after exposure of the film and 12% after exposure (Fig 2A) The level of S100A4 protein expression did not change when cells were attached to monomeric 2D or fibrillar 3D collagen surfaces pHb-1 cells maintained high S100A4 expression, whereas the II-11b cells maintained low S100A4 expression (Fig 2B) As shown in Fig 2B, equal amounts of protein were loaded based on equal amounts of actin pHβ-1 1.5 1.0 II-11b * 0.5 * * P 2D 3D P 2D 3D Fig Expression of MMP-2 in serum-free media from pHb-1 and II-11b cells (A) Gelatin zymography of harvested media from pHb-1 and II-11b cells attached to plastic (P), monomeric 2D collagen I (2D) or fibrillar 3D collagen I (3D) Typical zymograms showing proMMP-2 (72 kDa), intermediate MMP-2 (64 kDa) and fully activated MMP-2 (62 kDa) Box-plots illustrate the ratio of activated to total MMP-2 Open boxes denote pHb-1 cells while filled boxes denote II-11b cells Lines inside the boxes indicate median values, and dotted lines illustrate mean values (n = 12) (B) Harvested media from pHb-1 and II-11b cells attached to plastic (P), monomeric 2D collagen I (2D), or fibrillar 3D collagen I (3D) were analysed for proMMP-2 expression by ELISA Relative values (± SD) are adjusted for cell viability Open bars denote pHb-1 cells and filled bars denote II-11b cells (n = 3) *P < 0.05 compared with cells attached to plastic S100A4 pHβ B II-11b pHβ pHβ II-11b Mr (kDa) 20 II-11b S100A4 Actin 40 P 2D 3D P 2D 3D St Fig Expression of S100A4 in pHb-1 and II-11b cells (A) Determination of S100A4 by western blotting of cell lysates from pHb-1 and II-11b cells attached to plastic, using a total protein concentration of 81 lgỈmL)1, and the blot was exposed to the film for or (B) Western blot of cell lysates from pHb-1 and II-11b cells attached to plastic (P), monomeric 2D collagen I (2D) and fibrillar 3D collagen I (3D) Actin was used as loading control St: molecular mass standard (64 and 62 kDa bands), was observed for both cell lines attached to fibrillar 3D collagen compared with cells attached to plastic (Fig 3A) Because gelatin zymography is a semiquantitative method, the amount of proMMP-2 was also determined by ELISA As shown in Fig 3B, a large decrease in proMMP-2 was observed for cells attached to fibrillar collagen Reduction in proMMP-2 activation occurred independent of whether the fibrillar 3D collagen was attached to or released from plastic, or whether the cells were attached on top of or inside the fibrillar 3D matrix (data not shown) Some of the synthesized MMP-2 was adsorbed to the fibrillar collagen, and the ratio of active to total MMP-2 was the same as that detected in the conditioned medium (data not shown) Thus, adsorption may in part explain the reduction in the total amount of MMP-2 in the medium, but it does FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5277 Collagen I-modulated MMP expression A pHβ-1 R Elenjord et al Mr (kDa) II-11b 60 Actin pHβ-1 P 2D 64 62 Mr (kDa) II-11b TIMP-2 Fig The effect of monomeric 2D collagen I on MMP-2 activation Gelatin zymography of harvested media from pHb-1 and II-11b cells attached to plastic (P) and monomeric 2D collagen I (2D) Typical zymograms showing proMMP-2 (72 kDa), intermediate MMP-2 (64 kDa) and fully activated MMP-2 (62 kDa) 20 Actin 40 P 2D P 2D St Fig The expression of MT1-MMP and TIMP-2 in pHb-1 and II11b cells Determination of MT1-MMP (A) and TIMP-2 (B) by western blot of cell lysates from pHb-1 and II-11b cells (A) Cells were attached to plastic (P), monomeric 2D collagen I (2D) and fibrillar 3D collagen I (3D) Quantification of two blots gave mean values for pHb-1 cells: P = 100%, 2D = 103%, 3D = 34% and for II-11B cells: P = 100%, 2D = 90%, 3D = 33% (B) Cells were attached to plastic (P) and monomeric 2D collagen I (2D) Actin was used as loading control St: molecular mass standard not explain the reduction in active forms Because both cell lines showed reduction in active forms, S100A4 did not influence this alteration in activation Western blots of cell lysates showed reduced expression of MT1-MMP for both cell lines when attached to fibrillar 3D collagen compared with cells attached to plastic (Fig 4A) The level was reduced to 34% and 33% in pHb-1 and II-11b cells, respectively Cell binding to monomeric 2D collagen I increased the amount of fully activated MMP-2 For cells attached to both plastic and monomeric 2D collagen, pHb-1 cells produced more of the activated forms of MMP-2 than did II-11b cells (Fig 3A) For both cell lines, attachment to 2D monomeric collagen increased the amount of fully activated MMP-2 (62 kDa) and decreased the amount of the intermediate activated form (64 kDa) compared with cells attached to plastic (Figs 3A and 5) Although the amount of activated forms varied between experiments, the 64 kDa intermediate form was always weaker when cells were attached to monomeric 2D collagen than when cells were attached to plastic (Fig 5) However, the ratio of activated forms to total MMP-2 was approximately the same for cells attached to monomeric collagen and to plastic (Fig 3A) As shown by ELISA in Fig 3B, a decrease in proMMP-2 was observed when cells were attached to momomeric collagen 5278 2D 72 St 3D II-11b P 62 Western blots of cell lysates showed only small changes in MT1-MMP expression for both cell lines (+3% for pHb-1 and )10% for II-11b) when attached to monomeric 2D collagen compared with cells attached to plastic (Fig 4A) Neither of the cell lines showed any difference in TIMP-2 expression when comparing cells attached to plastic and to monomeric collagen (Fig 4B) This indicates that TIMP-2 was not involved in the observed difference in activation Cell binding to monomeric 2D collagen I results in an S100A4-dependent decrease in TIMP-1 expression Approximately twice as much TIMP-1 was secreted into the medium from pHb-1 cells compared with II-11b cells when attached to plastic (5.0 versus 3.1 lgỈmL)1Ỉ106 cells)1) (Fig 6) The difference was sustained when cells were attached to fibrillar 3D collagen However, when cells were attached to monomeric 2D collagen, pHb-1 cells produced significantly less TIMP-1 (2.8 lgỈmL)1Ỉ106 cells)1), whereas the level TIMP-1 (µg·mL–1·106 cells–1) B 3D 2D 2D 64 40 P P 72 50 50 MT1 pHβ-1 Mr (kDa) pHβ-1 II-11b * * P 2D 3D P 2D 3D Fig The effect of collagen I on TIMP-1 synthesis from pHb-1 and II-11b cells The cells were either attached to plastic (P), monomeric 2D collagen I (2D) or on the top of fibrillar 3D collagen gel I (3D) Harvested media were analysed for TIMP-1 expression by ELISA Open bars denote pHb-1 cells and filled denote for II-11b cells Mean values ± SEM are adjusted for cell viability (n = 6) *P < 0.05 compared with cells attached to plastic FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS R Elenjord et al Collagen I-modulated MMP expression was reduced to 2.3 lgỈmL)1Ỉ106 cells)1 for the II-11b cell line (Fig 6) A P TIMP-1 (ng·well–1) 2D 0 75 72 64 TIMP-1 prevents the second step in the MT1-MMP-induced activation of proMMP-2 62 Previously, it was shown that TIMP-1 prevents the p-aminophenylmercuric acid (APMA)-induced autoactivation of proMMP-2 and other MMPs [15–17] In order to test whether TIMP-1 inhibits the second step in the MT1-MMP-induced activation of proMMP-2, commercial recombinant proMMP-2 was activated for 24 h at 37 °C, either with membranes isolated from colchicines-stimulated pHb-1 cells containing a high level of MT1-MMP [18] or with a commercial recombinant soluble form of MT1-MMP containing only the catalytic domain As shown in Fig 7, both the membrane fraction and the recombinant soluble MT1MMP activated proMMP-2 As expected, TIMP-1 did not inhibit the first step where MT1-MMP cleaves the 72 kDa proMMP-2 form into the 64 kDa intermediate However, TIMP-1 inhibited the second step that is an autoactivation of the intermediate 64 kDa form to the fully active 62 kDa enzyme Further, autoactivation of the active 62 kDa form to a C-terminally truncated 45 kDa form was also inhibited by TIMP-1 Investigation of mechanisms that may explain the increased activation of MMP-2 when cells are attached to monomeric collagen I To investigate whether TIMP-1 affected MMP-2 activation, recombinant TIMP-1 was added to cells attached to monomeric 2D collagen As shown in 24 h 37 °C 0 rMT1-MMP 0 0 [MMP-2] Membr [TIMP-1] 72 64 62 Fig Activation of proMMP-2 by isolated cell membranes and MT1-MMP in the presence of TIMP-1 Human recombinant proMMP-2 (3 lgỈmL)1; 42 nM) was incubated for 24 h at 37 °C with membranes isolated from colchicine treated pHb-1 cells or recombinant human MT1-MMP catalytic domain in the presence of increasing concentrations of TIMP-1 as described in Materials and methods and analysed by gelatin zymography As controls, the proMMP-2 alone was either dirctly added to loading buffer without incubation or after 24 h incubation at 37 °C 2D B C E64 E64d Cys 72 64 62 Fig The effect of added inhibitors on MMP-2 activation Typical gelatin zymograms showing (A) the effect of TIMP-1 on the activation of MMP-2 using pHb-1 cells attached to monomeric 2D collagen (2D) compared with pHb-1 cells attached to plastic (P), (B) the effect of cystein proteinase inhibitors E-64, E-64d and cystatin (Cys) on the activation of MMP-2 using pHb-1 cells attached to monomeric 2D collagen (2D) C, control without added inhibitors Fig 8A, the intermediate 64 kDa band is stronger in the presence of TIMP-1 than in the absence of the inhibitor In order to determine whether other previously described mechanisms are also involved, several experiments were performed First, we studied whether an interaction between either the pro (72 kDa) or intermediate (64 kDa) MMP-2 and the underlying collagen layer caused the formation of a fully activated 62 kDa form of the enzyme Harvested media from cells attached to plastic were incubated for 24 h at 37 °C in culture wells with or without monomeric collagen Neither of these two conditions altered the relative amount of the activated forms, indicating that binding of the pro (72 kDa) or intermediate (64 kDa) forms of MMP-2 to collagen did not result in enhanced autoactivation (data not shown) Second, we wanted to investigate whether the cysteine proteinase legumain is involved in the activation The presence of this proteinase was shown in both cell lines, but incubation on monomeric 2D collagen did not change its level (data not shown) Third, treatment of cells attached to plastic or monomeric 2D collagen with the cysteine proteinase inhibitors egg white cystatin, E-64 or E-64d showed no effect on the synthesis and activation of proMMP-2 (Fig 8B) Fourth, in order to determine whether the observed activation took place intracellularly or extracellularly, surface proteins of cells attached to plastic and 2D collagen were labelled with biotin and removed as described in Materials and methods The unlabelled intracellular fraction of proteins were analysed by gelatin zymography No active MMP-2 was detected, demonstrating that the activation occurred outside the cell (data not shown) Fifth, FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5279 Collagen I-modulated MMP expression R Elenjord et al we investigated whether new activators on cell membranes from cells attached to monomeric 2D collagen could be responsible for the increased activation of proMMP-2 Isolated cell membranes from cells attached to plastic and monomeric 2D collagen did not show any difference in their capacity to activate proMMP-2 at pH 8.0 or at pH 5.8, indicating no new activators at the cell membrane of cells attached to the monomeric collagen Discussion The two osteosarcoma cell lines used in this study have characteristics suggesting they are of osteoblastic origin [19,20] In bone, active osteoblasts are embedded in a 3D ECM, whereas fully mature osteoblasts flatten out and line quiescent bone surfaces Collagen I is the main component of ECM, and interactions between the osteosarcoma cell lines and the 3D fibrillar collagen I network or the 2D layer of monomeric collagen I will, to a certain extent, mimic the in vivo situation for these cells Hence, this model can reveal to what extent cell–collagen I interactions will affect synthesis and activation of MMPs and their inhibitors Further, this model will discover whether the endogenous level of the metastasis-associated protein S100A4 is of importance for how the cells respond to interactions with these two forms of collagen I Included among cells that have been shown to increase the activation of proMMP-2 when they interact with fibrillar 3D collagen I are various human tumour cell lines [21,22], human skin fibroblasts [21,23–27], human umbilical vein and neonatal foreskin endothelial cells [28], human fetal lung fibroblasts [29], human hepatic stellate cells [30], rat capillary endothelial cells [31] and rat cardiac fibroblasts [32] In most cases, the increased activation of proMMP-2 is shown to be associated with an increase in MT1-MMP Furthermore, cells have a changed morphology when attached to fibrillar 3D collagen compared with the same cells attached to a 2D surface such as monomeric collagen or plastic [21–23,29–31,33] This was also shown for the osteosarcoma cells in our study where the cells appeared more rounded in shape In human skin fibroblasts there is an increase in proMMP-2 activation that is independent of the lattice contraction [23] It has also been shown that cells grown on fibrillar 3D collagen I attached to a plastic surface contain actin stress fibres, whereas stress fibres are missing when collagen is released from the surface Only under conditions where the cells lack stress fibres, fibroblasts produce increased amounts of activated MMP-2 [29] A lack of stress fibres is also necessary for the 5280 increase in the activation of proMMP-2 in smooth muscle endothelial cells [34] In several of the studies referred to above, it has also been shown that treatment of cells attached to a planar substrate (such as monomeric collagen or plastic) with compounds that dissolve actin stress fibres (cytochalasin D, vascular endothelial growth factor), results in increased activation of proMMP-2 However, treating cells with compounds that dissolve the tubulin network (colchicine, nocodazole) did not induce proMMP-2 activation In contrast to this, we have previously shown that colchicine-induced rearrangements of the microtubule network in osteosarcoma cell lines increase activation of proMMP-2 along with an increased level of MT1-MMP [18] This study shows that the interaction between osteosarcoma cells and fibrillar 3D collagen reduces the activation of proMMP-2 because of a decrease in MT1-MMP The reduction was independent of whether the 3D collagen lattice was attached to plastic or not In contrast to the cell lines discussed above, the actin cytoskeleton in the osteosarcoma cells was not affected by the surface the cells were attached to Hence, the reduction in MT1-MMP and active forms of MMP-2 could not be attributed to changes in the actin cytoskeleton This adds to previous investigations showing that these osteosarcoma cell lines respond differently to various stimuli compared with other cells We also show that the osteosarcoma cells produce an increased amount of fully active MMP-2 when bound to 2D monomeric collagen (Figs 3A and 5) which is another example of a different characteristic trait of these cells compared with fibroblasts and endothelial cells No drastic change in the amount of MT1-MMP was observed in osteosarcoma cells attached to monomeric collagen compared with plastic Although MT1-MMP here may participate in the activation of proMMP-2, it cannot account for the increased amount of fully activated enzyme MT1-MMP induces the conversion of proMMP-2 to the intermediate 64 kDa form by cleaving the Asn37–Leu38 bond [35,36], whereas the 64 kDa intermediate is further processed to the fully activated 62 kDa species in an autoactivation step In this study, various experiments were performed to determine whether one of the following mechanisms was responsible for the increase in fully activated MMP-2 when cells were attached to monomeric collagen: (a) increased expression of an activator enzyme that cleaves proMMP-2 in or near the autocatalytic site (Asn80–Tyr81), (b) increased expression of a factor that stimulates the second step of the MT1-MMP induced activation, or (c) reduced expression of an inhibitor of the second step of the activation FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS R Elenjord et al The two osteosarcoma cell lines used in this study produce legumain, a potential intracellular activator of proMMP-2 However, because we were not able to detect any intracellular fully activated MMP-2, nor could we inhibit MMP-2 activation with E-64, we can rule out legumain or other lysosomal cysteine proteinases as activators when the cells are attached to monomeric 2D collagen Furthermore, there was no difference in activation of exogenously added recombinant proMMP-2 between isolated cell membranes from cells attached to plastic and monomeric 2D collagen, irrespective of the pH used This result excludes the existence of some enzyme in membranes from cells attached to monomeric collagen that cleaves proMMP2 either in or close to the autoactivation site Nor could the activation be explained by autoactivation caused by a direct binding of either the 72 kDa proform or the 64 kDa intermediate to the cell membrane Lafleur et al [6] have shown that TIMP-2, in addition to being involved in the first step in the MT1-MMP activation of MMP-2, can also take part in the second autoactivation step and function as an activator by promoting conversion of the 64 kDa intermediate to the fully active 62 kDa form In this study, we did not find any difference in the levels of TIMP-2 when cells were attached to plastic compared with monomeric 2D collagen (Fig 4B), hence TIMP-2 is not the cause of the observed difference in activation level of MMP-2 Our results rule out the two first alternatives, (a) and (b), as an explanation for the increased activation when cells are attached to monomeric 2D collagen However, alternative (c), reduced expression of an inhibitor of the second step of the activation, seemed to be an explanation We have shown that TIMP-1 is a regulator of the second step in the activation of proMMP-2 using both recombinant MT1-MMP and isolated cell membranes rich in MT1-MMP (Fig 7) This is consistent with previous observations that TIMP-1 inhibits autoactivation of several MMPs such as: the Ca2+-induced intramolecular autoactivation of proMMP-9 covalently linked to the core protein of a chondroitin sulfate proteoglycan [37]; the APMAinduced autoactivation of MMP-9 to the 80 kDa inactive intermediate and the 68 kDa active species, where TIMP-1 prevented the formation of the latter species [15,38,39]; APMA-induced autoactivation of proMMP2 [15]; APMA-induced autoactivation of proMMP-3 and N-terminally truncated proMMP-3 [15,40]; and APMA-induced autoactivation of proMMP-1 and proMMP-8 [16,17] At the cellular level, we have shown that exogenously added TIMP-1 increased the intermediate 64 kDa form of MMP-2 (Fig 8A), indicating that the decreased level of TIMP-1 is the main Collagen I-modulated MMP expression cause of increased activation of MMP-2 when cells were attached to monomeric 2D collagen Altogether, our results show that endogenously produced TIMP-1 can act as a modulator of the MT1-MMP-induced activation of proMMP-2 The interaction between cells and fibrillar or monomeric collagen, respectively, showed opposite effects on proMMP-2 activation This effect was independent of the endogenous level of S100A4 in the two cell lines By contrast, the expression of TIMP-1 was dependent on the cell endogenous level of S100A4 When cells were attached to plastic and fibrillar 3D collagen, those with a high endogenous level of S100A4 produced approximately twice as much TIMP-1 as those with a reduced S100A4 level However, when cells were attached to monomeric 2D collagen, the production of TIMP-1 from cells with a high level of S100A4 was reduced to approximately the same amount as from cells with a low S100A4 level This suggests that the interaction between cells and monomeric 2D collagen causes a block in the S100A4-induced pathway that upregulates TIMP-1 expression Taken together, our results show that osteosarcoma cells interact with two types of collagen I found in vivo, and the form of the collagen determines the cells synthesis and activation of MMP-2 as well as the synthesis of MT1-MMP and TIMP-1 Previously, it has been shown that the reduced level of S100A4 in the II-11b cells compared with pHb-1 cells resulted in a large reduction of in vivo and in vitro invasive capacity, as well as in vitro motility [12,13] The reduction in S100A4 also resulted in a decrease in the expression of MT1-MMP, TIMP-1 and MMP-2 at both the mRNA and protein levels, in addition to a decreased amount of activated MMP-2 [13,14] MMPs and TIMPs are associated with cell invasion and metastasis, although their role is dual [41–43] Both MMPs and TIMPs, as well as the in vivo substrates of a given MMP, can prevent or facilitate the invasion and metastasis process, depending on the time and localization of their expression The N-terminal part of TIMPs is involved in binding to the active site of MMPs and hence prevents their action, whereas the C-terminal part can bind to proteins in the cell membrane and modulate cell growth and viability independent of MMPs [44,45] One of the aims of current research on MMPs and TIMPs in cancer is to establish the localization and timeframe for their expression, as well as the identification of the in vivo substrates of individual MMPs It is thus important to discover how each ECM component in the microenvironment of a given cancer type affects expression and activation of MMPs and TIMPs Our investigation shows that two FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5281 Collagen I-modulated MMP expression R Elenjord et al forms of the main ECM component in the microenvironment of osteosarcoma cells differently affect their expression of MMPs and TIMPs as well as their activation of MMP-2 In addition, it adds to the earlier investigations by showing that the structure of the surrounding matrix components will be of importance for the effect of S100A4 on MMP and TIMP regulation and thereby on the cell’s possibility to promote invasion and metastasis Materials and methods Materials DMEM containing Hams F12 medium, penicillin, streptomycin, geneticin disulfate salt (G418), gelatin 300 Bloom type A (from porcine skin), egg white cystatin (C-0408), BSA, Igepal CA-630, bactrerial collagenase and Sigma serum replacement were all from Sigma-Aldrich (St Louis, MO, USA) Fetal bovine serum was from Biochrom AG (Berlin, Germany), l-glutamine was from Gibco BRL Life Technologies (Paisley, UK), nonessential amino acids (100·) were from PAA Laboratories GmbH (Pasching, Austria), sterile rat-tail collagen I was from Roche Diagnostics GmbH (Basel, Switzerland), poly(vinylidene difluoride) membranes were from Millipore (Bedford, MA, USA), Alexa Fluor 568-labelled phalloidin (A12380), Magic Mark Western Standard and 4–12% NuPAGE Bis ⁄ Tris gels were from Invitrogen Life Technologies (Carlsbad, CA, USA), Biotrac TIMP-1 and MMP-2 ELISA were from Amersham Biosciences (Little Chalfont, UK) and E-64 (N-1645) and E-64d (N-1650) were from Bachem (Bubendorf, Switzerland) Western Blotting Luminol Reagent was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), EZ-Link Sulfo-NHS-LC-LC biotin, streptavidin agarose resins and halt protease inhibitor cocktail were from Pierce Biotechnology (Rockford, IL, USA) The following antibodies were used; GAPDH rabbit mAb and pan-actin rabbit polyclonal from Cell Signaling Technology (Danvers, MA, USA), MT6-MMP mouse mAb from R&D systems (Minneapolis, MN, USA), TIMP-2 and MT1-MMP rabbit polyclonal from Panomics (Redwood City, CA, USA), S100A4 rabbit polyclonal from Abcam (Cambridge, UK) and RECK mouse mAb from BD Biosciences (San Jose, CA, USA) Anti- mouse and anti-rabbit IgG horseradish peroxidase-linked antibodies were from Cell Signalling Technology TIMP-1 was from Oncogene Research Products (Boston, MA, USA), purified human recombinant proMMP-2 and MT1-MMP (catalytic domain) were from Calbiochem (San Diego, CA, USA) Solution cell proliferation assay (Cell Titer 96AQueous One) was from Promega (Madison, WI, USA) Paraformaldehyde was purchased from Merck (Darmstadt, Germany) and Triton X-100 from BDH Biochemicals Ltd (Poole, UK) 5282 Cell cultures The highly metastatic osteosarcoma cell line, OHS, was established from a bone tumour biopsy from a patient treated at the Norwegian Radium Hospital [46] The OHS cell line was transfected with a vector encoding a S100A4-specific ribozyme or, as a control, with the vector alone [12] The ribozyme-transfected clone was designated II-11b, and the control cell clone transfected with the vector alone was designated pHb-1 The II-11b cell line had a reduced level of S100A4 and a decreased metastatic capacity, whereas the pHb-1 cell line maintained the S100A4 expression level and metastatic properties of the parental OHS cell line [12] Transfectants were subcultivated in a : mixture of DMEM and Hams F12 medium (basal medium) containing 10% fetal bovine serum, 400 lgỈmL)1 geneticin, 2.0 nm l-glutamine, nonessential amino acids (100· dilution), penicillin (100 ImL)1) and streptomycin (100 lgỈmL)1) The cells were kept in a humidified 5% CO2 atmosphere at 37 °C Preparation of wells for cell experiments Cells (6.0 · 104) were, in addition to plastic, attached to monomeric 2D collagen I and on top and inside fibrillar lattices of 3D collagen I, all in 0.33 cm2 wells Ten microlitres of 0.16 mgỈmL)1 collagen I in 0.2% acetic acid was spread into the wells to prepare monomeric collagen I The wells were dried for h, followed by washing in serum-free medium (culture medium in which fetal bovine serum was replaced by 2% serum replacement) and left with 50 lL serum-free medium for 20 3D fibrillar collagen I gels were prepared by adding 50 lL neutralized collagen I solution (7 : : : of each mgỈmL)1 collagen I in 0.2% acetic acid, 10· serum-free medium, 1.0 m Hepes, pH 7.3, and 0.33 m NaOH, respectively) to the wells After h of polymerization at 37 °C, wells were equilibrated with serum-free medium for 20 and the medium was removed before cells were added in a new aliquot of medium For cell attachment inside 3D collagen I gels, cells were mixed with 50 lL neutralized collagen I solution and left for h while polymerization took place Cell viability assay To determine the viability of the cells, trypsinized cells were suspended in serum-containing medium In order to remove serum, cells were washed three times with serum-free medium prior to seeding on the different plate surfaces as described above A 100-lL cell suspension was added to plastic and monomeric 2D collagen, whereas 50 lL serumfree medium with or without cells was added to the 3D collagen gels After and 48 h incubation in 5% CO2 at 37 °C, cell viability reagent was added to the wells and the FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS R Elenjord et al absorbance at 490 nm was determined according to the instructions of the manufacturer’s protocol Prior addition of cell viability reagent to wells containing fibrillar 3D collagen, bacterial collagenase (15 lL of 18.6 mgỈmL)1 collagenase in 0.1 m Hepes pH 7,5 containing 0.9% NaCl) was added to dissolve the polymerized gel and after  25 at 37 °C, EDTA (10 mm) was used to stop the collagenase activity For each surface cells were attached to, standard curves were made to assure a linear response between cell number and absorbance Phalloidin staining of actin filaments Cells were cultured on glass covers slides, on monomeric 2D collagen I, on top of or inside fibrillar 3D collagen I for 24 h in serum-free media Cells were fixed in a final concentration of 4% paraformaldehyde for 10 on ice, washed with 1· NaCl ⁄ Pi and permeabilized by incubation with 0.1% Triton X-100 in 1· NaCl ⁄ Pi for 10 on ice After washing with 1· NaCl ⁄ Pi, cells were incubated with 1% BSA in 1· NaCl ⁄ Pi for 30 at room temperature before labelling with unit of Alexa Fluor 568-conjugated phalloidin in 1% BSA for 30 at room temperature Cells were then washed five times with 1· NaCl ⁄ Pi before images were obtained using a 40· water objective on a LSM 510 META confocal laser-scanning inverted microscope (Carl Zeiss International, Gottingen, Germany) ¨ Production of conditioned media for MMP and TIMP determination To determine the secretion of MMP-2 and TIMP-1 into the media, trypsinized cells were suspended in serum-containing medium In order to remove serum, cells were washed three times with serum-free medium prior to seeding on the different plate surfaces, as described above A 100 lL cell suspension was added to plastic and monomeric 2D collagen, whereas 50 lL serum-free medium with or without cells was added to the 3D collagen gels After 48 h incubation in 5% CO2 at 37 °C, the conditioned media and 3D gels were harvested Prior to freezing, the harvested media was centrifuged and taken to 10 mm CaCl2, 0.1 m Hepes, pH 7.3 To test whether TIMP-1 or legumain and other lysosomal cysteine proteinases were involved in proMMP-2 activation, cells were attached to plastic and 2D collagen I surfaces with or without inhibitors (25–300 ngỈwell)1 of TIMP-1, 10 lm E-64, 10 lm E-64d or lm egg white cystatin) Isolation of cell membranes Cells (1.4 · 107) were seeded in serum-free media on 66 cm2 Petri dishes, uncoated or coated with 2D monomeric collagen I (2 mL 0.16 mgỈmL)1 in 0.2% acetic acid), and kept the conditions described above Plasma membranes were Collagen I-modulated MMP expression prepared as previously described [18,47] Production and purification of plasma membranes from pHb-1 cells attached to plastic and treated with colchicine under serum free conditions were performed as previously described [18] Isolation of cell lysates To compare the level of S100A4 in the two cell lines cultivated on a plastic surface, confluent cells in a 75 cm2 were washed in NaCl ⁄ Pi, and released from the plastic with a rubber scraper, suspended in NaCl/Pi and pelleted at 4000 g Cells were then sonicated and the lysate centrifuged at 4000 g for at °C in order to remove cell debris The amount of cellular protein was detected by the Bradford method (Bio-Rad, Hercules, CA, USA), using BSA as a standard Activation of MMP-2 by cell membranes Membrane-mediated activation of human MMP-2 was performed by incubating the proenzyme (3 lgỈmL)1; 42 nm) with membrane protein (500 lgỈmL)1) from cells attached to plastic or 2D collagen I in 50 mm Tris ⁄ HCl, pH 8.0, mm CaCl2, 0,005% Brij 35 or 39.5 mm citric acid, pH 5,8, 121 mm Na2HPO4, 0.8% NaCl, 0.005% Brij 35 at 37 °C Aliquots were withdrawn after 0, 6, 12 and 24 h and analysed by gelatin zymography Activation experiments using cell membranes from colchicine stimulated pHb-1 cells were performed as described previously [14,18], with and without recombinant TIMP-1 present Activation of MMP-2 by recombinant MT1-MMP Activation of MMP-2 by MT1-MMP was performed by incubating human recombinant MMP-2 (3 lgỈmL)1; 42 nm) with human recombinant MT1-MMP catalytic domain (4 lgỈmL)1; 200 nm) in the absence and presence of recombinant TIMP-1 (0.588–2.352 lgỈmL)1; 21–84 nm) for 24 h at 35 °C in 50 mm Tris ⁄ HCl, pH 8.0, mm CaCl2, 0.005% Brij 35 Aliquots of MMP-2 corresponding to 400 pgỈwell)1 were applied to gelatin zymography Biotinylation of cell-surface proteins Cells (2.0 · 106) were seeded in serum-free media in six-well plates either uncoated or coated with monomeric 2D collagen (290 lL, 0.16 mgỈmL)1 collagen I in 0.2% acetic acid), and kept under the conditions described above Conditioned media were removed and wells were washed with cold NaCl ⁄ Pi (pH 8) To release cells, NaCl ⁄ Pi was added and cells were incubated at 37 °C Cells were suspended at a concentration of 25 · 106 cellsỈmL)1 in NaCl ⁄ Pi and 16 lL of 10 mm biotinreagent solution was added The reaction mixture was incubated at room temperature for FEBS Journal 276 (2009) 5275–5286 ª 2009 The Authors Journal compilation ª 2009 FEBS 5283 Collagen I-modulated MMP expression R Elenjord et al 30 Cells were washed with NaCl ⁄ Pi containing 100 mm glycine to remove excess biotin reagent and thereafter lysed (in 50 mm Tris ⁄ HCl pH 7.4, 150 mm NaCl, mm CaCl2, mm MgCl2, 0.5% Igepal, 1% inhibitor coctail) To remove biotinylated surface proteins, cell lysate was added to a streptavidin agarose resin and incubated for 30 After centrifugation, only unlabelled intracellular proteins were found in the supernatant Gelatin zymography Conditioned medium was mixed with loading buffer (333 mm Tris ⁄ HCl, pH 6.8, 11% SDS, 0.03% bromophenol blue and 50% glycerol) and loaded onto a 10% gelatin gel To determine the amount of gelatinase in the harvested 3D collagen I gels, an equal volume of gel and loading buffer were mixed and left for 30 at room temperature prior to centrifugation (4000 g for at °C) and the extract was applied to the zymography gel To determine whether gelatinases bind to monomeric 2D collagen, 10% dimethyl sulfoxide in serum-free media was added to wells after removal of conditioned medium, and the extract was added loading buffer and applied to the gelatin gel Gelatin zymography gels were run, washed and stained as described previously [14] Gelatinolytic activity was evident as transparent zones in the blue gels The area of the cleared zones was analysed using the genetools program from SynGene (Cambridge, UK) TIMP-1, i.e free TIMP-1 and TIMP-1 bound to MMPs The MMP-2 assay recognizes proMMP-2 and proMMP-2 bound to TIMP-2, but not the active form of MMP-2 Legumain activity Legumain was measured by recording the cleavage of the substrate Z-Ala-Ala-Asn-NHMec (Department of Biochemistry, University of Cambridge, UK), as previously described [48,49] Twenty microlitres of cell lysate were added to black 96-well microtiter plates (Costar) After the addition of 100 lL buffer and 50 lL substrate solution (10 lm final concentration), a kinetic measurement based on increase in fluorescence over 10 was performed Temperature was kept at 30 °C and all measurements were performed in triplicate Statistics Statistical analyses were performed using the student t-test for independent analysis Data are presented as mean ± SD (gelatin zymography, western blotting and ELISA data) A P-value < 0.05 was considered significant Analyses were based on three or more independent cell culture experiments Conditioned medium from each experiment was run in duplicate on gelatin zymography, ELISA and western blots Acknowledgements Western blot analysis After removal of conditioned media, cells were lysed in gel loading buffer containing 0.1 m dithiothreitol, and boiled for Samples were electrophoresed on acrylamide gradient gels (4–12%) and proteins were transferred to a poly(vinylidene difluoride) membrane by electroblotting After blocking nonspecific binding sites with non-fat milk (5% solution), blots were incubated for h at room temperature with primary antibodies against S100A4, MT1MMP or TIMP-2 After washing, the blots were incubated for h at room temperature with horseradish peroxidaseconjugated secondary antibodies diluted in blocking solution, and developed using a western blotting luminol reagent The membranes were washed, blocked and reprobed for the detection of actin or GAPDH The amount of protein in the detected spots were analysed on either the gelbase ⁄ gelblotÔ pro program from Ultra Violet Products (Cambridge, UK) or the genetools program ELISA The levels of TIMP-1 and MMP-2 were determined from serum-free conditioned media, according to manufacturer’s instructions The TIMP-1 assay recognizes total human 5284 This work was supported in part by grants from The Norwegian Cancer Society and the Erna and Olav Aakre Foundation for Cancer 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(1999) Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type matrix metalloproteinase activity in high and low invasive melanoma... cysteinerich protein with kazal motifs (RECK) is a cell membrane associated inhibitor of MMP-2 that is able to inhibit the second activation step of MMP-2 [9] The small calcium-binding protein S100A4. .. (1999) S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti -S100A4 ribozyme