Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor spreading. Multiple myeloma (MM) is a plasma cell malignancy primarily localized within the bone marrow and characterized by its capacity to destroy bone matrix and to disseminate. We have reported recently that human myeloma cells were able to induce the conversion of pro-MMP-2 produced by the tumoral environment in its activated form. In the current study, we have investigated the mechanism involved in this process. We demonstrate that a soluble MMP constitutively produced by myeloma cells was responsible for pro-MMP-2 activation. Furthermore, we show that the soluble MMP, MMP-7, also known as matrilysin, was able to activate the MMP-2 produced in its latent form by bone marrow stromal cells. Finally, we demonstrate that myeloma cells constitutively produce MMP-7 with expected proteolytic activity. Our results suggest that MMP-7 produced by myeloma cells could participate in bone destruction and tumor spreading in MM, on one hand by its own proteolytic activity and on the other hand by its capacity to activate pro-MMP-2. These findings strengthen the idea that inhibition of MMP activity could represent an interesting therapeutic approach in MM.

The matrix-degrading metalloproteinases (MMPs)3 are a family of secreted zinc- and calcium-dependent proteolytic enzymes that hydrolyze extracellular matrix macromolecules such as collagens, laminin, fibronectin, and proteoglycans (for review, see Refs. 1, 2). MMP activity is thought to be important in the initial events leading to matrix degradation in both physiological and pathological conditions. In fact, MMPs have been implicated in normal processes such as embryonic development, angiogenesis, or tissue remodeling in wound healing. Of note, an excessive activity of these proteinases has been associated with several pathological conditions, i.e., tissue destruction during rheumatoid arthritis and tumor invasion or metastasis. Currently, 16 distinct MMPs have been identified. They are classified into five groups according to their structures and substrate specificities: 1) collagenases, 2) gelatinases, 3) stromelysins, 4) membrane-type MMPs or MT-MMPs, and 5) others, including MMP-7, i.e., matrilysin. These enzymes share a catalytic domain with the HEXGH motif responsible for ligating zinc, which is essential for catalytic function (1, 2). Almost all MMPs are secreted in catalytically latent forms (or pro-MMPs) that are subsequently activated in the pericellular and extracellular environment. MMP activity is highly regulated at many levels, and extracellular MMP activation is considered as a critical step of regulation of their activity. The means by which the pro-MMPs become activated are essential for the understanding of their role in biological processes. During MMP activation, the highly conserved amino-terminal domain (or propeptide) that is responsible for maintaining latency in zymogens is removed by a proteolytic mechanism (3). Pro-MMPs activation could be physiologically achieved by various proteinases belonging to MMP family as well as to other enzyme classes. Moreover, activity of MMPs can also be regulated by tissue inhibitors of MMPs, which form binary noncovalent complexes with MMPs (1, 2).

Evidence that MMPs play a functional role in pathological processes is now well documented. The gelatinases MMP-2 and MMP-9 that degrade collagen IV, the major constituent of basement membranes, have been especially involved in tumor invasion and metastasis (4, 5, 6). In a previous report, we have shown that an important increase of MMP activity occurs in the bone marrow environment of patients with multiple myeloma (MM), a plasma cell malignancy (7). MM is characterized by the proliferation of malignant plasma cells within the bone marrow, and is generally associated with a high capacity to destroy bone and to disseminate into the skeleton (8). We have demonstrated that myeloma cells on one hand constitutively secreted MMP-9, and on the other hand were able to induce the activation of the latent MMP-2 (pro-MMP-2) secreted by the bone marrow environment. These observations strongly suggest that the excessive MMP activity in the bone marrow participates in the osteolytic process and tumor progression in MM. In this study, we have focused on the potential mechanisms leading to pro-MMP-2 activation by myeloma cells. MMP-2 is unique among the MMPs concerning its activation, in that it fails to be activated after treatment with serine proteinases such as plasmin like other MMPs (9, 10). In contrast, some MMPs have been reported to activate MMP-2. First, Sato et al. have cloned a new MMP with a transmembrane domain MT1-MMP, which was a specific pro-MMP-2 activator (11). Since this report, three other MT-MMPs have been described as potential MMP-2 activators (12, 13, 14). Second, the involvement of MMP-7 or matrilysin in MMP-2 activation was first reported (15), but subsequently questioned (16).

In this study, we demonstrate that a soluble MMP constitutively produced by myeloma cells is responsible for the activation of pro-MMP-2. Furthermore, we point out the ability of rMMP-7 to convert BMSC pro-MMP-2 to its activated form. Finally, we show for the first time the constitutive production of MMP-7 by myeloma cells. These findings suggest the involvement of tumoral MMP-7 in the activation of bone marrow environmental pro-MMP-2 and its participation in disease progression.

The protease inhibitors PMSF and E-64, the phorbol ester PMA, and the substrates β-casein and gelatin were purchased from Sigma (St. Louis, MO). Human rIL-6 was kindly provided by Novartis Pharma (Basle, Switzerland). The mAbs anti-MMP-2 and anti-MMP-7 were purchased from Oncogene Research Products (Cambridge, MA) and R&D Systems (Abingdon, U.K.), respectively. Human rMMP-7 was a generous gift of Dr. R. Martin (Roche Bioscience, Palo Alto, CA). This rMMP-7 was isolated from CHO cells transfected by pro-MMP-7 cDNA and chemically activated by p-chloromercuribenzoate before dialysis (17).

The human myeloma cell lines (HMCL) RPMI-8226, and U266, and the human colon adenocarcinoma cell line HT-29 were obtained from American Type Culture Collection (Manassas, VA). The HMCL NCI-H929 was purchased from DSM (Braunschweig, Germany). The HMCL XG-6 and MDN were established in our laboratory (18, 19). MDN was recently immortalized from the peripheral blood of a patient with a secondary plasma cell leukemia. Cells lines were maintained in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 5 × 10−5 M 2-ME. For the HMCL XG-6 and MDN, 3 ng/ml of rIL-6 was added to the culture.

Human stromal cells were isolated from bone marrow aspirates. After centrifugation over Ficoll-Hypaque gradient, mononuclear cells were plated in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 5 × 10−5 M 2-ME, and allowed to attach for 3 days, after which the medium is replaced by fresh medium. After 2 or 3 wk, a confluent adherent cell monolayer of BMSC was obtained; then after two passages using trypsin/EDTA solution, they were used for the study.

Human myeloma cells were obtained from bone marrow aspirates of patients with MM or peripheral blood of patients with plasma cell leukemia. Mononuclear cells were isolated by Ficoll-Hypaque density centrifugation; then the percentage of plasma cells was determined by morphology in May-Grünwald-Giemsa. Only samples with a plasmocytosis superior to 10% were used for the subsequent purification. Adherent cells were removed by allowing mononuclear cells to adhere to a plastic flask for 90 min in RPMI 1640 containing 5% FCS at 37°C in 5% CO2 humidified atmosphere. The purification of myeloma cells was performed by an immunomagnetic method (MACS system) using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) coated with the mAb B-B4. We have previously shown the ability of the mAb B-B4 to be restricted to myeloma cells in the bone marrow (20). The purity of myeloma cells was evaluated by standard morphology (May-Grünwald-Giemsa-stained cytospins), and only cell populations with a purity greater than 99.5% were used for experiments.

For experiments, BMSC (1.5 × 104 cells/well) and purified myeloma cells (1.25 × 106cells/ml) were seeded in 96-well plates alone or together in coculture experiments, in serum-free RPMI 1640. For coculture experiments, myeloma cells were added directly to the culture or placed in a Transwell insert. Conditioned media were harvested after 48 h.

Functional activity of MMP-2 was evaluated by gelatin zymography, as previously described by Heussen et al. (21). Briefly, conditioned media were mixed with SDS/sample buffer without reducing agent. After SDS-PAGE on 7.5% polyacrylamide gels containing gelatin at 1 mg/ml, SDS was removed from the gel by an incubation in 2.5% Triton X-100 for 1 h at room temperature. Then the gels were incubated in a buffer containing 50 mM Tris-HCl, 5 mM CaCl2, pH 7.6, for 24 h at 37°C, and stained with Coomassie blue R 250 (0.25%). Proteolytic activities of latent MMP-2 (pro-MMP-2) and activated MMP-2 were evidenced as clear bands against the blue background of stained gelatin with a molecular mass of 72 and 62 kDa, respectively.

Functional activity of MMP-7 was evaluated by β-casein zymography on 12.5% polyacrylamide gels containing β-casein at 0.5 mg/ml, as described for gelatin zymography, except a step of prerunning the casein-embedded gel (40 mA) before loading samples, as proposed by Fernandez-Resa et al. (22). Proteolytic activities of latent MMP-7 and activated MMP-7 (rMMP-7) were evidenced as bands with a molecular mass of 28 and 20 kDa, respectively.

Conditioned media were subjected to SDS-PAGE for MMP-2 and MMP-7 detection on 7.5% or 12.5% polyacrylamide gel, respectively. Then proteins were electrically transferred onto polyvinylidene difluoride (PVDF) membranes, and after blocking, membranes were incubated with anti-MMP-2 mAb or anti-MMP-7 mAb. Blots were developed using a classical chemiluminescent detection system for MMP-2 (ECL; Boehringer Mannheim, Indianapolis, IN) and a more sensitive chemiluminescent method for MMP-7 (Supersignal Ultra; Interchim, Montluçon, France).

Total cellular RNA was prepared from 2 × 106 purified myeloma cells using Trizol reagent (Life Technologies, Cergy-Pontoise, France). All RNA were randomly reverse transcribed with 400 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies), according to the manufacturer’s protocol. MMP-7 cDNAs were then amplified by nested PCR. The first PCR was performed with the two MMP-7-specific following primer pairs: first (sense primer), 5′-GTTTAGAAGCCAAACTCAAGG-3′ and (antisense primer) 5′-CCATTTCCATAGGTTGGATACATC-3′; second (sense primer), 5′-AGATGTGGAGTGCCAGATGT-3′ and (antisense primer) 5′-TAGACTGCTACCATCCGTCC-3′. PCR was performed in a thermal cycler (PCR Express; Hybaid, Middlesex, U.K.) for 35 cycles of denaturation at 94°C for 1 min, followed by annealing at 60°C (for each PCR) for 1.5 min and extension at 72°C for 1 min. A β-actin gene amplification was also performed with the following primers: (sense primer) 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ and (antisense primer) 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′, for 35 cycles of denaturation at 94°C for 1 min by annealing at 60°C (for each PCR) for 1 min and extension at 72°C for 1 min. PCR products were electrophoresed on 1.5% agarose gel, followed by staining with ethidium bromide. A 100-bp DNA ladder (Life Technologies) was used as an ethidium bromide-stainable marker. MMP-7 mRNA were detectable as a band of 365 bp and β-actin mRNA as a band of 802 bp.

Using coculture experiments between stromal cells obtained from a long-term culture of BMSC and highly purified freshly explanted myeloma cells, we have previously shown by gelatin zymography that BMSC constitutively produced a protease with gelatinolytic activity at 72 kDa (Fig. 1,A), corresponding to MMP-2 in its latent form (pro-MMP-2) (Fig. 1,B). In contrast, myeloma cells do not secrete any MMP-2, but produce MMP-9 evidenced by a clear band at 92 kDa on gelatin zymogram. Interestingly, beside the gelatinolytic activity observed for each type of cells cultured alone, an additional band of 62 kDa was detected in all coculture experiments. The immunoblot analysis performed with an anti-MMP-2 mAb recognizing both latent and active MMP-2 confirms that this additional band corresponds to the active form of MMP-2 (Fig. 1 B). These results clearly show that myeloma cells are able, at least partially, to activate the latent form of MMP-2, i.e., pro-MMP-2 produced by BMSC.

FIGURE 1.

Activation of BMSC pro-MMP-2 by human myeloma cells. A, Gelatin zymography analysis. BMSC were seeded in 96-well plates at the concentration of 1.5 × 104 cells/100 μl/well. A total of 1.25 × 105 cells/well of highly purified human myeloma cells (MM) was added to BMSC. Supernatants were harvested after 48 h of coculture. Lane 1, Monoculture of BMSC; lane 2, coculture of BMSC and MM; lane 3, monoculture of MM. B, Immunoblot analysis. The same samples were resolved by 7.5% SDS-PAGE under reduction, then transferred on PVDF membrane and blotted with mAb anti-MMP-2.

FIGURE 1.

Activation of BMSC pro-MMP-2 by human myeloma cells. A, Gelatin zymography analysis. BMSC were seeded in 96-well plates at the concentration of 1.5 × 104 cells/100 μl/well. A total of 1.25 × 105 cells/well of highly purified human myeloma cells (MM) was added to BMSC. Supernatants were harvested after 48 h of coculture. Lane 1, Monoculture of BMSC; lane 2, coculture of BMSC and MM; lane 3, monoculture of MM. B, Immunoblot analysis. The same samples were resolved by 7.5% SDS-PAGE under reduction, then transferred on PVDF membrane and blotted with mAb anti-MMP-2.

Close modal

Activation of MMPs occurs by proteolytic cleavage of the typical propeptide domain that protects MMP catalytic site. To define the mechanism involved in the activation of MMP-2 in our model, we have performed BMSC/myeloma cells coculture experiments in the presence or not of protease inhibitors. Western blot analysis of coculture supernatants shows that neither an inhibitor of serine proteases (PMSF, 1 mM) nor an inhibitor of cysteine proteases (E-64, 10 μM) was able to block MMP-2 activation (Fig. 2). In contrast, the metalloproteinase inhibitor 1-10 phenanthroline used at 5 mM completely prevented the appearance of the 62-kDa band of MMP-2 in coculture experiments. These results clearly demonstrate that a metalloproteinase produced by myeloma cells is responsible for the activation of MMP-2.

FIGURE 2.

Pro-MMP-2 activation in presence of protease inhibitors. BMSC and myeloma cells (MM) were incubated without protease inhibitor or with the serine protease inhibitor PMSF (1 mM), or with the cysteine protease inhibitor E-64 (10 μM) or the metalloproteinase inhibitor 1–10 phenanthroline (5 mM) for 48 h, then analyzed by immunoblotting.

FIGURE 2.

Pro-MMP-2 activation in presence of protease inhibitors. BMSC and myeloma cells (MM) were incubated without protease inhibitor or with the serine protease inhibitor PMSF (1 mM), or with the cysteine protease inhibitor E-64 (10 μM) or the metalloproteinase inhibitor 1–10 phenanthroline (5 mM) for 48 h, then analyzed by immunoblotting.

Close modal

To determine whether the contact of myeloma cells with BMSC was necessary to induce MMP-2 activation or not, two series of experiments were performed. First, we performed two types of cocultures with direct contact or not between myeloma cells and BMSC. To prevent direct cellular contact, we placed myeloma cells in Transwell inserts before adding to BMSC. We compared pro-MMP-2 activation in these two culture conditions by gelatin zymography. As shown in Fig. 3,A, MMP-2 activation similarly occurred in both conditions. Second, the incubation of BMSC-conditioned media with myeloma cell-conditioned media allowed MMP-2 activation, as shown in Fig. 3 B. Also, we conclude that in our model, MMP-2 activation involves a soluble protease secreted constitutively by myeloma cells that is neither a serine nor a cysteine protease. These data strongly suggest the involvement of a soluble MMP in this activation process.

FIGURE 3.

Implication of a soluble factor in pro-MMP-2 activation. A, BMSC were cultured with myeloma cells (MM) directly added to the culture (contact) or placed in a Transwell insert (without contact) for 48 h and analyzed by gelatin zymography. B, BMSC supernatants were incubated without or with MM supernatants for 48 h and analyzed by gelatin zymography.

FIGURE 3.

Implication of a soluble factor in pro-MMP-2 activation. A, BMSC were cultured with myeloma cells (MM) directly added to the culture (contact) or placed in a Transwell insert (without contact) for 48 h and analyzed by gelatin zymography. B, BMSC supernatants were incubated without or with MM supernatants for 48 h and analyzed by gelatin zymography.

Close modal

As MMP-7 effect on MMP-2 activation was suggested, we have tested the ability of this MMP to activate the pro-MMP-2 produced by BMSC. We have incubated for 24 h conditioned media of BMSC with increasing concentrations of active rMMP-7 (0.1–2 μM). Analysis by gelatin zymography of these samples has shown the appearance of a band at 62 kDa in presence of rMMP-7 (Fig. 4). The intensity of this band increased with the concentration of rMMP-7 added. These results indicate that MMP-7 is able to activate BMSC pro-MMP-2 and prompt us to determine whether myeloma cells produce MMP-7 or not.

FIGURE 4.

Pro-MMP-2 activation by rMMP-7. BMSC supernatants were incubated without and with decreasing concentrations of rMMP-7, then analyzed by gelatin zymography.

FIGURE 4.

Pro-MMP-2 activation by rMMP-7. BMSC supernatants were incubated without and with decreasing concentrations of rMMP-7, then analyzed by gelatin zymography.

Close modal

To determine whether MMP-7 could be responsible for MMP-2 activation in our coculture model, we looked for MMP-7 expression by myeloma cells using RT-PCR. Total RNAs were extracted from 2 × 106 freshly explanted and highly purified myeloma cells. Randomly primed cDNA were prepared as described in Materials and Methods, followed by specific MMP-7-nested PCR amplification. The human HT-29 colon adenocarcinoma cell line, which is known to produce MMP-7, was used as positive control, and a negative control was also included. As shown in Fig. 5,A, MMP-7 mRNA were detectable as a bromide ethidium-stainable band with the expected size of 365 bp in all cases. Southern hybridization with MMP-7 probe (23) was performed to confirm the specificity of PCR products (data not shown). Moreover, MMP-7 mRNA could also be detected in five over six HMCL, as shown in Fig. 5 B. β-actin message was detectable in all samples. In conclusion, we show that almost all myeloma cells, native or immortalized, express MMP-7 RNA.

FIGURE 5.

MMP-7 mRNA expression in human myeloma cells. A, RT-PCR analysis of purified myeloma cells. cDNA generated from purified myeloma cells from seven different patients (MM-1 to 7) and HT-29 cell line total RNA were subjected to specific MMP-7 and β-actin PCR amplification. The amplification products were electrophoresed on 1.5% agarose gels and detected as a band of 365 bp for MMP-7 and 802 bp for β-actin. B, RT-PCR analysis of HMCL. Specific MMP-7 and β-actin PCR amplifications were performed for HMCL NCI-H929, U266, RPMI-8226, MDN, and XG6, as previously described.

FIGURE 5.

MMP-7 mRNA expression in human myeloma cells. A, RT-PCR analysis of purified myeloma cells. cDNA generated from purified myeloma cells from seven different patients (MM-1 to 7) and HT-29 cell line total RNA were subjected to specific MMP-7 and β-actin PCR amplification. The amplification products were electrophoresed on 1.5% agarose gels and detected as a band of 365 bp for MMP-7 and 802 bp for β-actin. B, RT-PCR analysis of HMCL. Specific MMP-7 and β-actin PCR amplifications were performed for HMCL NCI-H929, U266, RPMI-8226, MDN, and XG6, as previously described.

Close modal

To confirm that myeloma cells secrete MMP-7, we analyzed purified myeloma cell-conditioned media by immunoblot analysis with anti-MMP-7 mAb. Active rMMP-7 and the conditioned medium of HT-29, a cell line known to produce MMP-7 in its latent form, have been included as positive controls. As shown in Fig. 6,A, a band of 28 kDa identical to the one obtained in HT-29 was observed in all myeloma cell-conditioned media. These results indicate that myeloma cells produce MMP-7 in its latent form, as the adenocarcinoma cell line HT-29. Moreover, we analyzed 48-h serum-free cell-conditioned media by casein zymography, the most suitable technique to detect MMP-7 activity. rMMP-7 used as positive control was detectable as a band with the expected molecular mass of 20 kDa (Fig. 6 B). A band of 28 kDa with caseinolytic activity was detected in myeloma cells as well as in HT-29-conditioned media corresponding to latent MMP-7. Of note, the 28-kDa protein could be converted in 20-kDa species corresponding to the activated MMP-7 by the organomercurial compound 4-aminophenylmercuric acetate (APMA) (data not shown). These data clearly demonstrate that myeloma cells produce MMP-7 with proteolytic (caseinolytic) activity.

FIGURE 6.

MMP-7 secretion by myeloma cells. A, Immunoblot analysis. Freshly explanted and purified myeloma cells from three patients (MM-A to C) and HT-29 colon adenocarcinoma cell line were incubated at the concentration of 2.5 × 106 cells/ml for 48 h; then culture supernatants were harvested, subjected to 12.5% SDS-PAGE, transferred on PVDF membrane, and hybridized with anti-MMP-7 mAb. rMMP-7 was also used as positive control. B, Casein zymography analysis. The same culture supernatants were subjected to casein zymography.

FIGURE 6.

MMP-7 secretion by myeloma cells. A, Immunoblot analysis. Freshly explanted and purified myeloma cells from three patients (MM-A to C) and HT-29 colon adenocarcinoma cell line were incubated at the concentration of 2.5 × 106 cells/ml for 48 h; then culture supernatants were harvested, subjected to 12.5% SDS-PAGE, transferred on PVDF membrane, and hybridized with anti-MMP-7 mAb. rMMP-7 was also used as positive control. B, Casein zymography analysis. The same culture supernatants were subjected to casein zymography.

Close modal

Numerous studies have demonstrated that the excessive degradation of basement membrane and connective tissue by matrix MMPs is critical for tumor cell invasion and dissemination. The increase of MMP activity could be linked to the MMP production by tumor cells themselves and/or to the recruitment of MMP present in tumoral environment. In a previous report, we have highlighted the involvement of MMPs in MM (7). We have pointed out the ability of myeloma cells to activate the pro-MMP-2 produced in its latent form by bone marrow environment in which the tumor develops. Active MMP-2 has been shown to contribute to the invasive phenotype in modulating cell attachment and cell migration by its proteolytic activity (4). These observations strongly suggest the participation of MMP-2 in tumor progression during MM. Because pro-MMP-2 secretion, especially in BMSC (7), is not induced by the cytokines that usually regulate other MMPs, the final step of activation appears to exert a more critical influence in controlling MMP-2 activity than for other MMPs. In this study, we have focused on the mechanism responsible for the MMP-2 activation induced by myeloma tumor and we show that the myeloma cells themselves produce MMP-7 or matrilysin, which could be involved in both MMP-2 activation and tumor progression.

Our findings showing the involvement of a MMP in MMP-2 activation by myeloma cells are consistent with the fact that pro-MMP-2 is unique among the MMPs in that it fails to be activated after treatment with many proteinases that have been identified as putative activators of this family, as serine proteases plasmin, trypsin, or cathepsin G (9). Moreover, we show that physical contact between BMSC and myeloma cells was not required for MMP-2 activation. These data argue against the implication of MT-MMPs, the group of MMPs most recently described and defined by their localization at the cell surface and characterized as pro-MMP-2 activators (11, 12, 13). However, as production of soluble MT1-MMP has been reported in two human breast carcinoma cells (24, 25), we have looked for MT1-MMP expression by myeloma cells. But neither MT1-MMP mRNA were detected by Northern blot analysis in HMCL, nor MT1-MMP protein was precipitated by a MT1-MMP mAb in HMCL supernatants (data not shown). Altogether, these data do not support the involvement of MT-MMP in our model. In contrast, the ability of rMMP-7 to activate pro-MMP-2 produced by bone marrow environment is in agreement with the demonstration that MMP-7 could activate many MMPs (16, 26) and especially MMP-2 (15, 17). But it contrasts with the report of Imai et al. (16), in which no MMP-2 activation by MMP-7 has been detected. This discrepancy may be linked to the nature (recombinant vs purified) of MMP-7 and pro-MMP-2 or to the methods of detection of MMP-2 activity (gelatin zymography vs 14C gelatin degradation) used in these experiments performed in vitro.

We demonstrate by different approaches that myeloma cells express and secrete constitutively MMP-7. In fact, all primary myeloma cells tested by RT-PCR express MMP-7 (n = 10 patients) and five of six HMCL were positive. And the MMP-7 activity could be evidenced in myeloma cell-conditioned media by the sensitive technique of casein zymography as a band of 28-kDa corresponding to the latent form of MMP-7 and confirmed by Western blot analysis. This band could be converted to a 20-kDa band corresponding to the activated MMP-7 by organomercurial compounds such as 4-aminophenylmercuric acetate (APMA) (data not shown). It is intriguing that MMP-7 is only detected in its latent form in myeloma cell-conditioned media, but it is now well established that in vivo overexpression of a specific MMP in its latent form is sufficient to induce a corresponding protease hyperactivity, especially for MMP-7 (27). The mechanisms of MMP-7 activation are not well documented. MMP-3 and MMP-10 have been involved in this process (16, 28), and both of these MMPs have been evidenced in stromal cells (29). Also, we can speculate that MMP-7 activation could occur in our model through BMSC MMP-3 and/or MMP-10 production. This would be in keeping with the cascade of activation involved in MMP activity.

The MMP-7 cDNA was initially isolated from human malignant tissues (23), and its expression has been since associated with various types of cancers, including human adenocarcinomas of prostate (30), stomach (31), colon (32, 33), and breast (34). MMP-7 expression has also been detected in normal tissues, including secretory and ductal epithelial cells in the endometrium and in various exocrine glands (35, 36, 37). Moreover, recent works also reported MMP-7 expression in nonepithelial cells, i.e., endothelial cells (38), and in cultured mononuclear phagocytes at an intermediate stage of their differentiation (39). Our study provides the first evidence of MMP-7 production by nonadenocarcinoma tumoral cells.

Some quantitative differences seem to exist among the different myeloma tumors that we have studied, and it would be interesting to quantify MMP-7 production to accurately determine whether a correlation between MMP-7 production and disease aggressivity could be defined or not. It is noteworthy that the level of MMP-7 activity detected in myeloma cell supernatants is sufficient to induce MMP-2 activation. In fact, we quantified myeloma cell MMP-7 activity by comparing the intensity of the bands obtained by casein zymography with the one obtained with rMMP-7. This allowed us to evaluate the MMP-7 production by myeloma cells close to 1 μM (data not shown), which corresponds to the concentration of rMMP-7 able to induce MMP-2 activation. Finally, our coculture model allowed us to point out for the first time that MMP-7 could activate MMP-2 in physiological conditions, and these findings argue for the participation of MMP-7 in a MMP activation cascade in vivo.

Furthermore, we have found that MMP-7 secretion by myeloma cells is enhanced by the tumor promotor PMA (data not shown). These data are consistent with the fact that the promoter region of MMP-7 gene includes TATA, AP-1, and PEA-3 elements as several other MMP genes conferring PMA sensitivity (40). In contrast, we did not find any regulation of myeloma cell MMP-7 secretion either by IL-6, the major cytokine involved in MM biology (41), or by IL-1β or IL-10, two cytokines involved in cytokine network in MM (42, 43) (data not shown). These data are in accordance with the absence of IL-6 and IL-10 effects on mononuclear phagocytes (44) and on prostatic cells (45). The effect of IL-1 on MMP-7 production seems to vary with cellular types. In fact, this cytokine induced MMP-7 production in the prostatic cell line LNCap (45), but fails to modulate it in phagocytes (44) and in myeloma cells (the current study).

MMP-7 possesses potent and broad proteolytic activity against a variety of extracellular matrix substrates, including glycoproteins such as fibronectin, elastin, entactin, proteoglycans, and also casein, denatured collagens I, III, and IV (46, 47, 48). Several reports demonstrated that MMP-7 expression was correlated with the progression of carcinoma (32) and that its overexpression favored tumorigenicity and tumor cell invasion in an experimental animal model (27, 49, 50). Also, we can hypothesize that MMP-7 production by myeloma cells participates in tumor progression by favoring the spreading of myeloma cells inside and outside the bone marrow.

Moreover, MMP-7 could also activate MMP-1 or interstitial collagenase (16) involved in the initiation of bone resorption (51). As we have previously shown the ability of myeloma cells to induce MMP-1 secretion in the bone marrow environment, we can therefore assume that MMP-7 production by myeloma cells probably participates in bone matrix degradation. This hypothesis is well supported by the report showing that MMP-7 is overexpressed in human osteoarthritis (52). These observations underline the role of MMP-7 in the excessive extracellular bone matrix degradation and its probable contribution in the bone resorption that is a major characteristic of MM. The production of MMP-7 by myeloma cells and its probable involvement in activation of environmental MMPs are of major interest for the management of patients with MM with the purpose of limiting bone resorption and tumor spreading. In fact, MMP inhibitors are now available and have been shown efficient in two different types of pathological states in animal models: metastatic cancer and disease associated with articular cartilage and bone destruction (53, 54, 55). For example, the administration of the MMP inhibitor batimastat in Min mice, which spontaneously develop premalignant intestinal tumors, resulted in a decrease of Min tumor similar to that observed in mice lacking MMP-7 (56). This study demonstrates the therapeutical interest in inhibiting MMP-7 activity. These inhibitors have now reached the stage of clinical testing in human cancers, and these current trials should reveal whether this approach will become an interesting antineoplasic therapy in the future or not.

Taken together, our findings suggest that the inhibition of MMP activity could represent an interesting therapeutical approach in MM to limit two major deleterious effects observed in the evolution of this disease, i.e., bone destruction and myeloma cell spreading.

We thank Drs. R. Martin and H. E. Van Wart (Roche Bioscience, Palo Alto, CA) for providing us with rMMP-7, and Dr. R. Breathnach (Institut National de la Santé et de la Recherche Médicale U463, Nantes, France) for MMP-7 cDNA. We also thank M. Etrillard and M. Collette for technical assistance.

1

This work was supported by a grant from the Association pour le Recherche contre le Cancer (9659).

3

Abbreviations used in this paper: MMP, metalloproteinase; BMSC, bone marrow stromal cell; HMCL, human myeloma cell line; MM, multiple myeloma; MT-MMP, membrane-type metalloproteinase; PVDF, polyvinylidene difluoride.

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