Monocyte Membrane Type 1-Matrix Metalloproteinase

PROSTAGLANDIN-DEPENDENT REGULATION AND ROLE IN METALLOPROTEINASE-2 ACTIVATION*

Uma T. ShankavaramDagger §, Wan-Ching LaiDagger , Sarah Netzel-Arnett, Paul R. ManganDagger , Jeanette A. ArdansDagger , Nancy Caterina, William G. Stetler-Stevenson||, Henning Birkedal-Hansen, and Larry M. WahlDagger **

From the Dagger  Immunopathology Section and  Matrix Metalloproteinase Unit, National Institute of Dental and Craniofacial Research and the || Extracellular Matrix Pathology Section, Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 19, 2000, and in revised form, March 19, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E2 and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular tissue remodeling is critical in the reorganization of connective tissue under both normal and pathological conditions including cell migration, angiogenesis, wound healing, inflammation, and invasion (1). Several matrix metalloproteinases (MMPs)1 take part in extracellular matrix remodeling (2), but the molecular mechanism of how these MMPs act is largely unknown (3). Critical events in the physiologic mechanism of proteinase activation, which is regulated at several levels, include cell type, site, and timing. MMPs are produced as zymogens (pro-MMPs) that require proteolytic activation through the elimination of the N-terminal propeptide (4). Once the enzymes are active, they are susceptible to inhibition by a family of specific tissue inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). TIMPs act to inhibit metalloproteinase activity by forming a complex with active MMPs (5). The activation and inhibition of MMPs is tightly regulated under normal physiological conditions; however, in a number of pathological situations the strict regulatory mechanisms are lost, leading to tumor metastasis and connective tissue diseases such as arthritis.

The growing family of MMPs at present consists of more than 20 members including the recently identified membrane type (MT1)-MMP. MMPs subclassified as type IV collagenases/gelatinases include MMP-2 (72 kDa; gelatinase A) and MMP-9 (92 kDa; gelatinase B). These MMPs are capable of degrading denatured and intact type IV collagen, a major component of basement membranes, as well as other extracellular matrix substrates. MMP-2 is unique in that the propeptide of this enzyme has no apparent cleavage site for the proteinases that activate other MMPs. MT1-MMP is believed to mediate the activation of MMP-2 on the cell surface. The expression of MT1-MMP and activation of MMP-2 has been associated with tumor invasion and metastasis (6, 7). Thus, production of MT1-MMP is likely a major rate-limiting component of MMP-2 activation; therefore the MT1-MMP/MMP-2 system is an attractive target for the prognosis and prevention of tumor progression.

Degradation of basement membranes and stromal extracellular matrix is crucial for invasion and metastasis of malignant cells (8-10). The expression of MMPs in tumors is regulated in a paracrine manner by growth factors and cytokines secreted by tumor infiltrating inflammatory cells, as well as by tumor or stromal cells. Recent studies have suggested continuous cross-talk between tumor cells, stromal cells, and inflammatory cells during the invasion process. Monocytes/macrophages found at sites of extracellular remodeling have been implicated in connective tissue destruction mediated through MMPs (11-16). Because of the spatial distribution of tissue monocytes, also known as tumor-associated macrophages, between invading tumors and surrounding tumors, we have analyzed (a) the effect of monocyte activation on the expression of MT1-MMP, (b) the mechanism of monocyte MT1-MMP production, (c) the functional analysis of monocyte-derived MT1-MMP in activating MMP-2 secreted from stromal cells, fibroblasts, or melanoma cells, and (d) the role of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in regulating the conversion or inhibition of MMP-2 to its active forms by MT1-MMP-expressing monocytes.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells, Culture Conditions, and Reagents-- Human peripheral blood monocytes were isolated by elutriation (17). Monocytes were enriched >90% as determined by morphology, nonspecific esterase staining, and flow cytometry. Purified monocytes were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM; BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine (Mediatech, Washington, D. C.), and 10 µg/ml gentamycin sulfate (BioWhittaker, Walkersville, MD). Indomethacin (Sigma), SQ 22536 (Biomol), Rp-cAMPs (Biomol), PGE2 (Sigma), or Bt2cAMP (Sigma) were added as indicated. Antibody against human MT1-MMP was obtained from Chemicon, Temecula, CA. Human recombinant TIMP-2 was obtained from Fuji Chemical Industries, Toyama, Japan. Progelatinase A (pro-MMP-2) was expressed in a vaccinia virus expression system and purified as previously described (18). Human bone marrow-derived fibroblasts (HBMFs; obtained from Drs. Larry Fisher and Berthold Fohr, National Institute of Dental and Craniofacial Research, NIH), normal male-derived fibroblasts (NMDFs), and human melanoma cell line (A2058) (American Type Culture Collection, Rockville, MD) were cultured in DMEM supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) on polystyrene tissue culture dishes (Falcon 2059; Falcon Labware, Oxnard, CA). These cells were then cultured in serum-free medium for 24 h, and the media supernatants were added to control or LPS-treated monocytes. The results are representative of observations obtained in three or more experiments using monocytes from different donors.

RNA Analysis-- Total RNA was isolated with TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) according to the instructions of the manufacturer. MT1-MMP cDNA (a generous gift of Dr. Motoharu Seiki) was used as a probe for identification of MT1-MMP mRNA. MT1-MMP and GAPDH cDNA were labeled using Roche Molecular Biochemicals random-primed DNA labeling kit. Prehybridization and hybridization of both MT1-MMP and GAPDH probes were carried out at 65 °C in QuikHyb from Stratagene. The membranes were washed 4 times in 2× SSC, 0.1% SDS for 20 min at 65 °C.

Cell Protein Isolation and Western Blot Analysis-- Cell membranes were prepared from purified human monocytes treated under various experimental conditions. The cells were washed in phosphate-buffered saline containing protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 100 µg/ml 4-(2-aminoethyl)-benzene sulfonylfluoride-HCl; Calbiochem, San Diego, CA). The cell pellets were suspended in 250 mM sucrose solution containing protease inhibitors and sonicated (Kontes ultrasonic cell disrupter; Vineland, NJ). The nuclear debris and unbroken cells were sedimented at 420 × g for 15 min in a refrigerated microfuge. The resulting supernatant was centrifuged at 20,800 × g for 30 min to pellet the membrane proteins. The protein concentration of the membrane fraction was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were analyzed on 8-16% SDS polyacrylamide Tris-glycine gels (Novex, San Diego, CA) under reducing conditions. Western analysis was performed as described (19) using an antibody against MT1-MMP (Chemicon International Inc., Temecula, CA). The bands were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).

Zymogram Analysis of MMP-2-- Conditioned media (20 µl) were mixed with 5 µl of sample buffer to give a final concentration of 0.1 M Tris-HCl (pH 6.8), 2% (w/v) SDS, 0.02% bromphenol blue, and 10% glycerol. The proteins were resolved on a discontinuous 10% polyacrylamide Tris-glycine gel containing 0.1% (w/v) gelatin. Following electrophoresis, the gels were equilibrated in 2.5% Triton X-100 solution for 30 min at room temperature and subsequently incubated at 37 °C in collagenase buffer (50 mM Tris-HCl (pH 7.5)/200 mM NaCl/5 mM CaCl2) for 2 h. The gels were stained with Coomassie Blue (0.25% Coomassie Blue, 45.4% methanol, and 9.2% glacial acetic acid) to visualize the gelatinolytic bands corresponding to MMP-2.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of LPS on Monocyte MT1-MMP RNA and Protein-- MT1-MMP is known to have substrate specificity for diverse extracellular matrices leading to physiological and pathological processes accompanying tissue remodeling. In addition to its direct proteolysis of extracellular matrix components, MT1-MMP plays a pivotal role in the activation of pro-MMP-2 (6, 20). In this study we examined whether activation of monocytes influenced the expression of MT1-MMP. For this purpose, we treated monocytes with different concentrations of LPS as indicated in Fig. 1. Northern analysis of mRNA revealed that control monocytes had little or no detectable levels of MT1-MMP mRNA (Fig. 1A). In contrast, stimulation with LPS resulted in a dose-dependent induction of MT1-MMP mRNA. Unlike other MMPs that are secreted as zymogens and are activated in the extracellular compartment, MT1-MMP is membrane-bound and is processed into an active enzyme prior to cell surface localization (21, 22). To determine the levels of membrane-associated MT1-MMP, Western analysis was performed using the membrane preparations from control and LPS-treated monocytes. As shown in Fig. 1B, LPS induced a dose-dependent increase of MT1-MMP protein levels with no detectable MT1-MMP on control monocytes.


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Fig. 1.   Effect of LPS on monocyte MT1-MMP expression. Human peripheral blood monocytes prepared by elutriation were cultured (20 × 106 cells per 4 ml of DMEM) for 18 or 48 h in the absence or presence of LPS. Total cellular RNA was prepared from monocytes treated with the indicated dose of LPS for 18 h and analyzed for MT1-MMP mRNA and GAPDH as a reference for equal loading (panel A). Panel B shows the immunoblot analysis of MT1-MMP protein expression. Cell membranes were prepared from monocytes treated with the indicated doses of LPS for 48 h. An equal amount of membrane protein (100 µg) from each sample was analyzed in a 10% SDS polyacrylamide gel electrophoresis. Western analysis was performed using an antibody against MT1-MMP. The protein bands were visualized by enhanced chemiluminescence detection.

Effect of PGE2 on Monocyte MT1-MMP Expression-- The induction of MMPs in monocytes by agents such as concanavalin A, LPS, or secreted protein, acidic and rich in cysteine, has been shown to occur through a prostaglandin-dependent pathway (13, 16, 23-26). To determine whether monocyte MT1-MMP production is regulated through this pathway, monocytes were pretreated with indomethacin for 1 h and then stimulated with LPS in the absence or presence of PGE2. Similar to the data in Fig. 1, LPS stimulation resulted in an induction of monocyte MT1-MMP mRNA and protein whereas control cells had no detectable MT1-MMP (Fig. 2, A and B). Indomethacin, at a concentration (10-6 M) that significantly inhibits the induction of monocyte PGE2 by LPS, suppressed the levels of MT1-MMP mRNA and protein in stimulated cells. The addition of PGE2 reversed the indomethacin-mediated suppression of MT1-MMP mRNA and protein. These findings demonstrate that LPS induces MT1-MMP, in large part, through a prostaglandindependent mechanism. Moreover, this induction requires a primary signal, in addition to PGE2, because PGE2 in the absence of LPS failed to induce MT1-MMP as shown either at the mRNA or protein level.


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Fig. 2.   Prostaglandin-dependent regulation of monocyte MT1-MMP. Purified monocytes were pretreated with indomethacin (10-6 M) for 1 h, and LPS (100 ng/ml) and PGE2 were added to some of the cultures as indicated. Panel A is representative of the Northern analysis of MT1-MMP. The total RNA was prepared from monocytes after 18 h of culture and subjected to mRNA analysis for MT1-MMP and GAPDH as described under "Materials and Methods." Panel B shows the Western blot of monocyte membranes (100 µg/lane), collected after 48 h of culture, using an antibody against MT1-MMP.

Effect of Adenylyl Cyclase and Protein Kinase A Inhibitors on MT1-MMP Induction-- PGE2 is known to stimulate adenylyl cyclase, which results in the generation of cAMP and activation of protein kinase A. To further establish that this was a major pathway in the regulation of MT1-MMP in monocytes, inhibitors of adenylyl cyclase and protein kinase A, SQ 22536, and Rp-cAMPs, respectively, were tested for their effect on MT1-MMP induction. SQ 22536 caused a dose-dependent inhibition of MT1-MMP (Fig. 3). This suppression was reversed by adding Bt2cAMP, thereby bypassing the requirement for adenylyl cyclase in the generation of cAMP. Additional evidence for the involvement of cAMP and the subsequent increase in downstream signaling proteins, such as protein kinase A, was the dose-dependent inhibitory effect of Rp-cAMPs, a protein kinase A inhibitor, on MT1-MMP induction (Fig. 4, A and B).


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Fig. 3.   Effect of SQ 22536 on monocyte MT1-MMP. Purified monocytes (20 × 106 per 4 ml of DMEM) were pretreated with SQ 22536, an adenylyl cyclase inhibitor, at the indicated concentrations for 1 h. LPS (100 ng/ml) and Bt2cAMP were than added as indicated. Monocyte membranes were collected after 48 h, and 100 µg of protein was loaded in each lane and analyzed by Western blot with an antibody against MT1-MMP.


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Fig. 4.   Effect of Rp-cAMPs on monocyte MT1-MMP. Purified monocytes were incubated with the indicated concentrations of Rp-cAMPs, a protein kinase A inhibitor, for 1 h. LPS (100 ng/ml) was then added as indicated. Panel A represents the Northern analysis of MT1-MMP. The total RNA was prepared from monocytes after 18 h of culture and analyzed for mRNA of MT1-MMP and GAPDH as described under "Materials and Methods." Panel B shows the Western blot of monocyte membranes (100 µg/lane), collected after 48 h of culture, using an antibody against MT1-MMP.

Effect of Monocytes on MMP-2 Activation from HBMF, NMDF, and Cells-- To determine whether the monocytes could modulate MMP-2 activation, serum-deprived conditioned media from HBMF, NMDF, or A2058 cells were added to the monocyte cultures in the absence or presence of LPS. The incubation was carried out at 37 °C for 48 h. MMP-2, as in the case of the other MMPs, is initially secreted in a latent form, which undergoes extracellular activation to yield an intermediate and fully active form. Zymogram analysis can be used to demonstrate pro, intermediate, and active forms. Because the incubation of the zymogram with Triton X-100 involves conditions that renature the MMP-2 proteins, all forms of MMP-2 exhibit gelatinolytic activity when placed in collagenase buffer. Zymogram analysis of the supernatants revealed that although monocytes secrete significant amounts of MMP-9 when stimulated (data not shown) they do not produce detectable MMP-2 under these conditions (Fig. 5, lanes 1 and 2). In contrast, the supernatants from serum-starved HBMFs (lane 3), NMDFs (lane 6), and A2058 cells (lane 9) contained MMP-2 that was primarily in the latent form. When supernatants from these cell lines were added to monocytes that had been treated with LPS, the latent MMP-2 from these cell lines was converted to intermediate and fully active forms (lanes 5, 8, and 11). This was in contrast to control monocytes that failed to activate MMP-2. These results demonstrate that activated monocytes may play an important role in the conversion of latent MMP-2 produced by other cells to the fully active form.


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Fig. 5.   Activation of MMP-2 from cell lines by human monocytes. Monocytes (5 × 106 in 0.5 ml/well) were cultured for 18 h in the presence or absence (Control) of LPS and then serum-free conditioned media (0.5 ml) from HBMF, NMDF, or A2058 cells were added to the monocyte cultures or cell-free culture wells (-). Media were harvested 48 h later, and 20 µl of sample was subjected to zymogram analysis. The bands corresponding to P, I, and F are the pro, intermediate, and fully active forms respectively, of MMP-2.

Effect of Antibody against MT1-MMP on MMP-2 Activation-- Although MT1-MMP production was detected in HBMF, NMDF, and A2058 cells (data not shown), MMP-2 was not activated by these cells. Therefore, we evaluated whether the significant activation of MMP-2 by monocytes was specifically related to the MT1-MMP on the surface of the monocytes. Monocytes were cultured for 18 h with LPS to induce MT1-MMP. The media was then replaced with serum-free conditioned medium from HBMF cells, and an antibody against MT1-MMP or normal rabbit IgG was added to some of the cultures at a dilution of 1:100. As shown in Fig. 6, antibodies against MT1-MMP inhibited (lane 7) the conversion of HBMF latent or pro-MMP-2 to its intermediate and active forms by LPS-treated monocytes (lane5). In contrast, control normal rabbit IgG failed to affect this conversion (lane 9). These findings confirm that MT1-MMP on monocytes is involved in the activation of MMP-2.


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Fig. 6.   Human monocyte MT1-MMP mediates MMP-2 activation. Freshly prepared human peripheral blood monocytes were cultured at 5 × 106 cells/ml of DMEM. The cultures were incubated either in the absence or presence of LPS (100 ng/ml). After 18 h the culture medium was replaced with serum-free conditioned medium from HBMF cells, and an antibody against MT1-MMP or IgG from normal rabbit serum was added to some of the cultures. The culture incubation was carried out for an additional 48 h. The samples were subjected to zymogram analysis, and the pro (P), intermediate (I), and fully active (F) forms of MMP-2 were visualized after Coomassie Blue staining.

Effect of TIMP-2 on the Activation of MMP-2 by Monocytes-- It has been hypothesized that a complex formed between MT1-MMP and TIMP-2 acts as a receptor for pro-MMP-2. The formation of this trimolecular complex may be essential for the cell-mediated activation of pro-MMP-2, which presumably interacts with a free adjacent MT1-MMP (27-29). Immunoblot analysis with antibodies against TIMP-2 showed that monocytes produced very low levels of TIMP-2, whereas considerable amounts TIMP-2 were found in the media of the cell lines tested (data not shown). These findings indicate that the low levels of TIMP-2 expressed by monocytes may allow free MT1-MMP to bind to the MMP-2·TIMP-2 complex produced by other cell types, thus activating MMP-2. To determine whether this was a possibility, various concentrations of human recombinant TIMP-2 were added to the monocytes. As shown in Fig. 7, activation of HBMF MMP-2 by LPS-treated monocytes was inhibited in a dose-dependent manner with increasing concentrations of recombinant TIMP-2 (1 to 100 ng/ml). These results suggest that the conversion of pro-MMP-2 to the active form was inhibited when all of the free MT1-MMP on the monocyte surface was bound to TIMP-2. To determine whether this hypothesis was correct and that TIMP-2 is also involved in the activation of MMP-2, various concentrations of human recombinant TIMP-2 were added with purified-recombinant pro-MMP-2 (5 nM) to control and LPS-activated monocytes. Zymogram analysis revealed that a TIMP-2 dose-dependent activation of MMP-2 occurred in LPS-stimulated monocytes but not control cells (Fig. 8). Maximal activation was evident at approximately equal molar concentrations (5 nM) of TIMP-2 and pro-MMP-2. TIMP-2 at higher concentrations (15 to 20 nM) inhibited the activation of MMP-2. In contrast to monocytes, similar experiments carried out with human skin fibroblasts failed to result in activation of the pro-MMP-2 (data not shown). These findings support the concept that activation of MMP-2 involves binding of the MMP2·TIMP-2 complex to MT1-MMP with subsequent activation by free adjacent MT1-MMPs.


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Fig. 7.   Inhibition of HBMF MMP-2 activation by TIMP-2. Monocytes (5 × 106/ml) were cultured with or without LPS- and HBMF-conditioned medium, as described in Fig. 6, in the presence or absence of TIMP-2 at various concentrations as indicated. The samples were subjected to gelatin zymography, and the pro (P), intermediate (I), and fully active (F) forms of MMP-2 were visualized after Coomassie Blue staining.


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Fig. 8.   Requirement for TIMP-2 in the activation of MMP-2 by human monocytes. Monocytes (5 × 106/ml) were cultured in the presence or absence (Control) of LPS (100 µg/ml) for 18 h and then human recombinant TIMP-2 and purified-recombinant pro-MMP-2 were added as indicated. The supernatants were harvested after an additional 24 h and analyzed by gelatin zymography. The pro (P), intermediate (I), and fully active (F) forms of MMP-2 were visualized after Coomassie Blue staining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present work demonstrates that activation of monocytes results in the expression of mRNA for MT1-MMP with the subsequent localization of the protein on the surface of the cell. It has been reported that monocytes secrete a number of MMPs in response to various stimulants (12, 30-32). The induction of these MMPs is thought to occur at the pre-translational level. Consistent with these findings, we have observed an increase in mRNA and protein levels of MT1-MMP in LPS-stimulated monocytes. Examination of MT1-MMP revealed that only stimulated monocytes expressed mRNA for MT1-MMP and had MT1-MMP on their membrane surface.

MMPs have been associated with several pathological conditions such as chronic inflammatory lesions and cancer metastasis (33-35). Therefore, regulation of signal transduction events that are associated with the production of inflammatory mediators, such as MMPs, has been of considerable interest. A PGE2-cAMP-dependent pathway leading to the production of MMP-1, MMP-9, and MMP-7 (matrilysin) by activated monocytes has been demonstrated previously (13, 16, 23-26). In the present study, we have demonstrated that LPS induces monocyte MT1-MMP and that the signaling pathway for MT1-MMP synthesis involves, in large part, a PGE2-dependent mechanism. This was demonstrated by the ability of exogenous PGE2 to reverse indomethacin-mediated inhibition of MT1-MMP. Additional evidence for the involvement of cAMP in this pathway is the suppression of MT1-MMP by adenylyl cyclase or protein kinase A inhibitors. Regulation of MT1-MMP through a PGE2-dependent mechanism has also been reported for the induction of MT1-MMP in fibroblasts stimulated with monensin (36). In the same study, the induction of fibroblast MT1-MMP by monensin was also inhibited by indomethacin, an effect that was reversed by PGE2.

Many lines of evidence link increased MMP levels with tumor invasiveness and metastatic potential (37-43). It is hypothesized that basement membrane degrading MMP-2 is involved in the invasion and metastasis of malignant tumors and in the angiogenesis required for tumor growth (35, 37). In many instances, enhanced MMP-2 production has been traced to the malignant cells themselves (37, 40). However, it has also been shown that some tumors produce little MMP-2, and the major source of this enzyme is from stromal cells and fibroblasts (8, 9). Regardless of the source of MMP-2, activation of this enzyme is critical for its proteolytic activity and indicates the invasive phenotype of the tumors. In the present study, we demonstrate that the MT1-MMP on activated monocytes may have an important role in the activation of MMP-2 produced by other cells. Zymogram analysis of conditioned media from control or activated monocytes failed to detect MMP-2. In contrast, the conditioned media collected from HBMF, NMDF, or A2058 cells contained substantial levels of latent MMP-2, which was converted to its intermediate and fully active forms when cultured with activated monocytes. Furthermore, the activation of MMP-2 was blocked by antibodies against MT1-MMP. These results suggest that communication between monocytes producing MT1-MMP with either tumor or tumor-associated cells expressing MMP-2 is essential for the activation of MMP-2, leading to the metastatic potential of the tumor cell. Our finding that only activated but not control monocytes were able to convert pro-MMP-2 to its active form indicates the significance of activated monocytes in this process. Although we have used LPS to stimulate monocytes in vivo it is likely that stimulatory molecules, such as cytokines, cancer cell-associated factors, or extracellular matrix components may exert the same effect.

Several studies have suggested activation of MMP-2 through physical cell-cell contact (27, 44, 45). Contrary to these reports, we have demonstrated that soluble pro-MMP-2 secreted by HBMF, NMDF, or A2058 cells into the conditioned medium could still be activated when incubated with monocyte MT1-MMP. Pro-MMP-2, unlike other MMPs, is usually found complexed with endogenous TIMP-2 (11, 46, 47). The presence of inhibitor bound to the latent enzyme suggests that the inhibitor may play some additional role. Indeed, several recent reports have suggested that the formation of a complex among pro-MMP-2, TIMP-2, and MT1-MMP is needed for MMP-2 activation and that TIMP-2 levels are critical in MMP-2 activation by MT1-MMP (27, 48-50). Based on these studies, it has been hypothesized that two molecules of MT1-MMP are required for activation of MMP-2. One of these molecules binds a complex of TIMP-2·MMP-2 whereas an adjacent free MT1-MMP activates MMP-2 (for review see Ref. 22). This model of activation may explain the possible mechanism of activation of pro-MMP-2 in our study. Although we observed substantial amounts of MT1-MMP and TIMP-2 protein on HBMF, NMDF, and A2058 cells (data not shown) these cells failed to activate the MMP-2. A possible explanation for this finding is that the excess TIMP-2 produced by these cells binds to all or most of the MT1-MMP on the surface of these cells, thus preventing activation of MMP-2 by MT1-MMP. In contrast, monocytes produce very little TIMP-2 or MMP-2. As a result, there is sufficient free MT1-MMP on the monocyte cell surface to bind the TIMP-2·MMP-2 complex derived from other cells with enough remaining free MT1-MMP to activate MMP-2. Thus, the ratio of TIMP-2 and MT1-MMP is critical for optimum activation of MMP-2. This hypothesis is supported by data shown in Figs. 7 and 8. The data in Fig. 7 demonstrate that excess TIMP-2 does indeed inhibit the activation of MMP-2 from HBMF by monocytes. In Fig. 8, the addition of human recombinant TIMP-2 with purified-recombinant pro-MMP-2 to LPS-stimulated monocytes demonstrated that TIMP-2 is required for activation of pro-MMP-2. Moreover, the optimal activation occurred when pro-MMP-2 and TIMP-2 were in equal molar concentrations. However, when TIMP-2 was in significant excess the activation of MMP-2 was inhibited, presumably because of the saturation of the available MT1-MMP molecules by TIMP-2.

In summary, we have demonstrated that activated monocytes produce functionally active MT1-MMP through a prostaglandin-cAMPdependent mechanism. The monocyte-derived MT1-MMP is involved in the activation of MMP-2 produced by other cells. These results suggest that the in vivo interaction between monocytes and MMP-2-secreting cells may play a crucial role in the regulation of normal developmental processes and disease states, such as tumor invasion. Thus, defining the mechanism and developing therapeutic interventions involved in the regulation of monocyte MT1-MMP may be important in controlling the immunopathology associated with various diseases.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: SRA International, Inc., Rockville, MD 20852.

** To whom correspondence should be addressed: Immunopathology Section, 30 Convent Dr., National Institute of Dental and Craniofacial Research, NIH, Bldg. 30, Rm. 325, Bethesda, MD 20892. Tel.: 301-496-9219; Fax: 301-402-1064; E-mail: lwahl@dir.nidcr.nih.gov.

Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M009562200

    ABBREVIATIONS

The abbreviations used are: MMP(s), matrix metalloproteinase(s); MT, membrane-type; TIMP(s), tissue inhibitors of metalloproteinases; LPS, lipopolysaccharide; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagle's medium; Bt2cAMP, dibutyryl cyclic AMP; HBMF(s), human bone marrow-derived fibroblasts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TIMP-2, tissue inhibitor of matrix metalloproteinases-2; NMDF, normal male-derived fibroblasts.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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