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Originally published online as doi:10.1189/jlb.0506328 on February 27, 2007

Published online before print February 27, 2007
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(Journal of Leukocyte Biology. 2007;81:1466-1476.)
© 2007 by Society for Leukocyte Biology

Native and fragmented fibronectin oppositely modulate monocyte secretion of MMP-9

Barak Marom*,{dagger},1, Michal A. Rahat*,1,2, Nitza Lahat*, Lea Weiss-Cerem{dagger}, Amalia Kinarty* and Haim Bitterman{dagger}

* Immunology Research Unit and
{dagger} Ischemia-Shock Research Laboratory, Carmel Medical Center, Rappaport Family Institute for Research in the Medical Sciences, and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel

2 Correspondence: Immunology Research Unit, Carmel Medical Center, 7 Michal St., Haifa, 34362, Israel. E-mail: rahat_miki{at}clalit.org.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes remodel the extracellular matrix (ECM) by secreting proteins composing the ECM such as fibronectin (FN) and degrading proteases such as matrix metalloproteinase-9 (MMP-9), which cleaves FN into fragments. The effects of FN and its fragmented products on the expression of monocyte MMP-9 are controversial and largely unknown. We showed that in human monocytes, the proinflammatory cytokine TNF-{alpha} induced MMP-9 secretion and increased fragmentation of FN into distinct fragments. When primary monocytes or the U937 monocytic cell line were incubated on a plastic substrate, plastic-coated with native FN, and plastic-coated with fragmented FN (frag-FN), native FN inhibited TNF-{alpha}-induced proMMP-9 secretion by twofold (P<0.01) compared with plastic or frag-FN. Exploration of the dynamics of inflammation by incubating cells sequentially on the three substrates showed that frag-FN opposed the inhibitory effect of native FN. Inhibition of proMMP-9 by native FN was exerted at the translational level, as no change in MMP-9 mRNA, intracellular protein accumulation, or proteomic degradation was observed, and when degradation was blocked, no de novo translation of MMP-9 could be measured. We also showed that the reduction of MMP-9 secretion by native FN was responsible for attenuated migration of U937 cells (P<0.05). We suggest that in the inflammatory tissue, intact, native FN has a homeostatic role in harnessing MMP-9 activity. However, as fragmented products accumulate locally, they alleviate the inhibition and enable faster migration of the monocytes through the degraded ECM.

Key Words: human • macrophages • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes, which are recruited to inflammatory sites, must first extravasate the vasculature by crossing the endothelial basement membrane (BM) and transversing the extracellular matrix (ECM). They migrate toward gradients of signals, which are provided by bacterial products, cytokines, chemokines, ECM proteins, and proteins deposited in the ECM and orient themselves according to the summated effect of these signals [1 , 2 ]. To enable their movement across the ECM, monocytes degrade its proteins by secreting proteases, including matrix metalloproteinases (MMPs) [3 ]. This family of specialized proteases can collectively cleave all the components of the BM and ECM and participate in its remodeling [4 5 6 ]. Among the different MMPs, MMP-9, which is induced strongly by proinflammatory cytokines (e.g., TNF-{alpha}), is associated specifically with monocyte/macrophage migration. To avoid excessive ECM degradation and tissue damage, MMP-9, as other MMPs, is regulated tightly at the transcriptional level [7 , 8 ], secreted as a zymogen (92 kDa), which requires processing to yield the activated form (84 kDa) [9 10 11 ], and is inhibited by endogenous inhibitors (e.g., tissue inhibitor of metalloproteinases) [10 , 12 , 13 ]. As a result of MMP activity, fragmented products of the ECM proteins and other degraded proteins, which were entrapped previously in the ECM, are released to the microenvironment, adding their distinct, chemotactic signals, which may be different from those of the native molecules [3 , 14 ].

One of the substrates for MMP-9 is the ECM glycoprotein fibronectin (FN) [9 ], which is produced by many cell types, including monocytes and macrophages [15 16 17 ], and is composed of two nearly identical subunits with a molecular weight of ~240 kDa. These two subunits are encoded by the same gene, which as a result of alternative splicing, gives rise to 20 variants of the FN molecule [18 , 19 ]. FN mediates cell attachment to the ECM via binding of an RGD motif in its binding domain to several integrins, in particular, {alpha}5ß1 and {alpha}4ß1, which were identified as the major receptors of FN. These integrins provide a link between the ECM and the cell cytoskeleton and offer a mechanism through which the ECM regulates different aspects of cell behavior such as morphology, migration, differentiation, and proliferation [18 , 20 , 21 ]. In addition, areas that are sensitive to proteolytic cleavage by different proteases are located between the various domains of FN and may give rise to discrete fragments [18 ]. These FN fragments may have biological activities different from those of the native, parental FN molecule and were shown to increase secretion of proinflammatory cytokines and several proteases as well as modulate cell migration and proliferation [22 23 24 25 ]. Thus, fragmentation of FN may provide additional environmental means to regulate the immune response.

Monocytes contribute to ECM remodeling by degrading its proteins via secretion of proteases such as MMP-9 on the one hand and by depositing ECM proteins such as FN on the other hand and were therefore chosen for this study. A feedback loop exists between MMP-9 and FN, as MMP-9 degrades FN, and binding of FN up-regulates the expression of MMP-9 [26 27 28 ]. However, the distinct effects of the fragmented products of FN on the expression and secretion of MMP-9 in different cell types were scarcely studied, and conflicting results were obtained. For example, the 70-kDa amino terminal fragment of FN was shown to reduce MMP-9 activity in macrophages [29 ], but the 45-kDa amino-terminal fragment had no effect on MMP-9 in chondrocytes and cartilage explants [30 ]. In addition, the recombinant extracellular domain A or CS-1 peptide from the cell-binding domain of FN induced MMP-9 in cartilage explants and in T cells [31 , 32 ]. In the current study, we ask whether FN or its fragments affect monocyte MMP-9 expression. We demonstrate that in an inflammatory microenvironment, FN is degraded into a mixture of fragments, which post-transcriptionally increase the secretion of TNF-{alpha}-induced MMP-9 from monocytes and consequently, enhance their migration, thus antagonizing the inhibitory effect of native FN molecules on MMP-9 production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human monocytes were isolated from peripheral blood donated by healthy volunteers. Blood (20 ml) was taken in the presence of 0.2% EDTA, and PBMC were separated by density centrifugation on Ficol-Hypaque (Uni-Sep NovaMed, Israel). After three washes in PBS, the PBMC were counted and plated in 24-well plates with DMEM and 20% FCS at a concentration of 2 x 106 cells/well, and after 2 h of incubation, the nonadherent cells were washed out extensively. Purity of the monocytes was routinely ≥80 ± 0.7%, as was evaluated by labeling the cells with anti-CD14 and anti-CD11b and analyzing the surface expression by flow cytometry. The Carmel Medical Center Helsinki committee (Israel) approved this part of the study. In addition, 2 x 106 cells/well of the human monocytic cell line U937 were plated in 24-well plates in DMEM with 10% FCS and antibiotics. To avoid possible masking of signals initiated by the exogenous stimuli, both cell types were incubated with DMEM without FCS before their exposure to the experimental conditions. Cells were incubated for 48 h, with or without the addition of TNF-{alpha} (20 ng/ml; Peprotech-Cytolab, Rehovot, Israel). In several experiments, cells were incubated on a plastic substrate, plastic-coated with native FN (200 µg; Biological Industries, Kibbutz Beit-Haemek, Israel), and plastic-coated with FN, which was fragmented by commercial MMPs previously (20 ng/ml) for 24 h and then washed extensively to remove all residual MMPs prior to the addition of the cells. In all experiments, cell viability was determined using the XTT kit (Biological Industries).

Zymography
Expression and secretion of MMP-9 were analyzed by zymography, which enables detection of the zymogen and the activated forms of MMP-9. Primary monocytes (2x106) or U937 cells were incubated in the experimental conditions, supernatants were collected, and cellular extracts were obtained from the cell line by harvesting the cells with RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml PMSF, 30 µg/ml aprotinin, 5 µg/ml leupeptin). Protein concentrations were determined by Bradford reagent, and equal amounts of nonreduced protein (30 µg) or equal volumes of the nonreduced, conditioned media were loaded onto an 8% SDS-PAGE containing 1% gelatin. After electrophoretic separation, gels were renatured by incubation in 2.5% Triton X-100 for 30 min, and gelatinase activity was revealed by overnight incubation in a developing buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35), followed by staining with 0.5% Coomassie blue G-250. The clear bands, which were obtained against the blue background, indicated the presence of MMP-9 in comparison with the molecular weight markers and MMP-2/MMP-9 zymography standards as positive control (Chemicon, Temecula, CA, USA). The OD of the bands was quantified using the bio-imaging system (Dinco and Renium, Jerusalem, Israel) and TINA software (Raytest, Straubenhardt, Germany).

ELISA
MMP-9 amounts were determined using the commercial ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. This ELISA enabled measurement of active and latent forms of MMP-9.

Quantitative real-time PCR analyses
Total RNA was extracted from 2 x 106 U937 cells exposed to the experimental conditions using TriReagentTM (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer’s instructions. Total RNA (5 µg) was transcribed to cDNA at 37°C for 1 h using 200 µM deoxynucleotides (Sigma Chemical Co., St. Louis, MO, USA), 5 µM random hexamers (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 20 U RNAguard (Amersham Pharmacia Biotech), and 200 U/µl Moloney murine leukemia virus-RT (US Biochemicals, Cleveland, OH, USA). MMP-9 mRNA expression levels were determined by quantitative real-time PCR on the cDNA samples using the TaqMan Assay on Demand kit and the ABI-PRISM 7000 (Applied Biosystems, Foster City, CA, USA). Analysis was carried out in triplicates in a volume of 20 µl (2 min at 50°C, 10 min at 95°C, and a total of 40 cycles, each of 15 s at 95°C and 1 min at 60°C) for MMP-9 and the endogenous reference gene RPLP0, and the comparative threshold cycle method was used. In each experiment, the nonstimulated RNA sample was used as a calibrator to allow comparison of relative quantity between the samples.

Western blots analyses
At the end of the incubation in the experimental conditions, supernatants from U937 cells were collected and concentrated 100-fold by VivaSpin2 (Vivascience, Lincoln, UK), and equal volumes were loaded on a 10% SDS-PAGE. After electrophoretic separation, the proteins were transferred and fixed onto cellulose nitrate membranes (Schleicher and Schuell, Dassel, Germany) in transfer buffer (25 mM Tris, 180 mM glycine, 20% methanol, pH 8.3). The membranes were incubated for 1 h in blocking buffer (20% skimmed milk, 1% BSA, 0.01% Tween-20, 10 mM Tris, pH 8.0, 150 mM NaCl) at room temperature and then probed for 1 h at room temperature with the diluted (1:1000) rabbit polyclonal anti-FN (Sigma Chemical Co.). After washing three times in 1x TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20), the membranes were incubated with HRP-conjugated donkey antimouse IgG (Jackson ImmunoReasearch Laboratories, West Grove, PA, USA), diluted 1:2000 in blocking buffer for an additional 1 h at room temperature, and then washed again. The ECL system (Amersham Pharmacia Biotech) was used for detection. The OD of the bands was quantified using the bio-imaging system (Dinco and Renium) and TINA software (Raytest).

Flow cytometry analysis
U937 cells (2x106) were cultured for 48 h, with or without the addition of TNF-{alpha}, and then were labeled with one of the mAb directed against the ß1, {alpha}4- or {alpha}5-integrin chains (Chemicon), and PE-conjugated goat antimouse (Chemicon). After washing, the cells were fixed in 0.1% formaldehyde, and cells were analyzed using a Coulter-XL flow cytometer (Coulter Electronics, UK). Dead cells were excluded from the analysis by their forward and sideway light-scattering properties.

Migration assay
Migration assays were performed in a modified Boyden chamber transwell (3 µm pores, Falcon). Briefly, a total of 5 x 105 U937 cells was added to the upper chamber, and the chemoattractant MCP-1 (100 ng/ml; R&D Systems) was added to the lower chamber. Basal migration and MCP-1-directed migration were determined after 48 h by adding a fixed volume of fluorescent beads (200 µl PKH26 reference beads, Sigma Chemical Co.), collecting the cells that migrated to the lower chamber and determining their ratio in the sample relative to the total number of cells using flow cytometry.

Statistical analyses
All values are presented as means ± SE. The data were analyzed using repeated measures ANOVA. The Student Newman-Keuls multiple comparisons test was used to evaluate significance between experimental groups, and P values exceeding 0.05 were not considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} induces secretion of MMP-9 and accumulation of FN fragments
As preliminary experiments (data not shown) indicated that differences in the amounts of MMP-9 secreted to the supernatants became visible only after incubation of 48 h, all the following experiments were conducted at that time. To avoid possible masking of signals initiated by exogenous stimuli, the cells were serum-starved for 48 h. These conditions, combined with the presence of TNF-{alpha} (20 ng/ml), did not cause cell death of the primary monocytes or the U937 monocytic cell line.

To assess the extent of endogenous secretion of FN from the U937 cells and primary monocytes, we concentrated the supernatants by 100-fold and tenfold, respectively, followed by Western blot analysis, which revealed three discrete bands at 45 kDa, 70 kDa, and 120 kDa, in addition to the native FN molecule at 240 kDa (Fig. 1A ). Addition of TNF-{alpha} increased the amounts of the 45- and 70-kDa fragments by approximately twofold (P<0.05) in both types of monocytes but had no effect on the accumulation of the 120-kDa fragment or the 240-kDa native FN itself (Fig. 1B and 1C) , suggesting the relative importance of these fragments in the inflammatory microenvironment.


Figure 1
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  		Figure 1. Effect of TNF-{alpha} on the production of FN fragments. FN was detected using polyclonal anti-FN in concentrated supernatants obtained from U937 cells (x100) or primary monocytes (x10) after incubation for 48 h, with or without TNF-{alpha} (20 ng/ml). (A) Representative Western blots showing the native (240 kDa) and fragmented products of FN. P.C., Commercial native FN as positive control. The graphs show densitometric analysis of each of the fragments and the 240-kDa, native FN molecule secreted from (B) the U937 cell line (n=7). (C) Primary monocytes (n=6). *, P < 0.05, compared with nonstimulated cells. TNF-{alpha} increased the secretion of the 45-kDa and 70-kDa fragments but not of the 120-kDa fragment or the 240-kDa, native FN.

 
In preliminary experiments, 20 ng/ml TNF-{alpha} induced maximal amounts of proMMP-9 secretion without reducing cell viability (data not shown). We next showed that in primary monocytes, proMMP-9 was visible by zymography, even without stimulation (Fig. 2A and 2B ), and that the extent of its induction by TNF-{alpha} was 2 ± 0.3-fold (P<0.01). Only minute amounts of proMMP-9 were secreted from the nonstimulated, U937 monocytic cell line (Fig. 2A and 2C) , and addition of TNF-{alpha} induced the secretion of the enzyme by 13-fold (P<0.001). This was confirmed further by ELISA of the secreted MMP-9, which detected negligible amounts of the enzyme in the supernatants of nonstimulated U937 cells (0.037±0.037 ng/ml) and an induction by TNF-{alpha} (9.3±2.6 ng/ml, P<0.01). Human primary monocytes are known to constitutively secrete MMP-9, because of cell-matrix interactions, which are mediated by engagement of integrins [33 ], or activation caused by their purification procedure [34 ]. Thus, although enhancing effects of TNF-{alpha} on MMP-9 secretion exist in the U937 cell line and the primary monocytes, the relative enhancement compared with basal secretion was higher in the U937 cells.


Figure 2
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  		Figure 2. Effect of TNF-{alpha} and FN on secretion of proMMP-9. Supernatants were obtained from U937 cells or primary monocytes incubated for 48 h, with or without TNF-{alpha} (20 ng/ml), on plastic or plastic coated with native FN (200 µg) or FN fragmented by gelatinases (20 ng/ml). (A) Representative zymography gels. PC, Positive control of commercial MMPs. (B) Densitometric analysis of proMMP-9 secretion from primary monocytes (n=4). (C) Densitometric analysis of proMMP-9 secretion from the U937 cell line (n=7). *, P < 0.05, and ***, P < 0.001, compared with nonstimulated cells (control); $$$, P < 0.001 compared with nonstimulated cells incubated on Native FN; §, P < 0.05, and §§, P < 0.01, compared with TNF-{alpha}-stimulated cells incubated on plastic; {dagger}{dagger}, P < 0.01, compared with TNF-stimulated cells incubated on native FN. Native FN inhibited the accumulation of proMMP-9 in both monocytic cells, and fragmented FN (Frag-FN) was not different than the plastic substrate.

 
Frag-FN and native FN affect secretion of proMMP-9 differently
Because of the small amounts of FN secreted from the U937 monocytes, we used exogenous native or frag-FN, which represented the amounts of FN in the tissue, to study their effects on MMP-9. We used the commercially available native FN, which contains only a small amount of frag-FN products, to coat the plastic wells. To obtain frag-FN, we coated the plastic wells with native FN and then incubated them with commercial MMP-9 and MMP-2 for 24 h, followed by extensive washes to assure removal of all residual MMPs, as was assessed by zymography (data not shown), but not of the matrix, which was deposited on the bottom of the well. MMPs were used to simulate as closely as possible the autocrine circuit by which MMP-9, via FN, may influence its own secretion from monocytes in an otherwise fixed, proinflammatory environment. Of note, incubation of FN with commercial MMP-9 and MMP-2 or with elastase resulted in a similar band pattern, which resembled the pattern of discrete bands observed previously to be secreted from primary monocytes and U937 cells (Fig. 1) . Viability of the monocytes was not changed, and incubation of the primary cells or the U937 cells, with or without the addition of TNF-{alpha} on plastic, native FN (1.06±0.06- and 0.95±0.02-fold from the plastic control) or frag-FN (1.01±0.06- and 0.99±0.01-fold from the plastic control), did not result in cell death but did affect the secretion of proMMP-9 (Fig. 2) . Incubation of primary monocytes on plastic with the addition of TNF-{alpha} enhanced the secretion of proMMP-9 by 2.2 ± 0.4-fold (P<0.05). In the absence of TNF-{alpha}, incubation of the primary monocytes on native FN or frag-FN did not change proMMP-9 secretion. However, when TNF-{alpha} was added, native FN inhibited proMMP-9 secretion by twofold (P<0.05) and frag-FN, by only 1.3-fold, relative to the incubation on plastic (Fig. 2B) . Incubation of the U937 cells on plastic with the addition of TNF-{alpha} induced the secretion of proMMP-9 by 15 ± 1.5-fold (P<0.001). In the absence of TNF-{alpha}, incubation of the U937 monocytic cell line on native FN increased the secretion of proMMP-9 by 1.5 ± 0.07-fold relative to plastic (P<0.001) and to frag-FN (P<0.001; Fig. 2C ). As native FN in the absence of TNF-{alpha} had only a minor effect, which was not confirmed by ELISA experiments and was not repeated in the primary monocytes, we did not pursue it further. In contrast, in the presence of TNF-{alpha}, the secretion of proMMP-9 from U937 cells incubated on native FN was inhibited by 1.9-fold (P<0.01), relative to the cells incubated on plastic or frag-FN (Fig. 2C) . In addition, evaluation of the amounts of secreted MMP-9 by ELISA confirmed that in nonstimulated U937 cells, MMP-9 secretion was negligible after incubation on the three substrates. Addition of TNF-{alpha} to cells incubated on plastic or frag-FN increased the secretion of MMP-9 to 9.3 ± 2.6 ng/ml and 9.05 ± 3 ng/ml, respectively, whereas incubation on native FN resulted in the accumulation of only 3.5 ± 2.1 ng/ml of the enzyme. Thus, the inhibitory effect of native FN on the secretion of TNF-{alpha}-induced proMMP-9 was repeated in both cell types, and the frag-FN demonstrated a similar and opposite effect.

Fragmented and native FN affect secretion of proMMP-9 oppositely
To evaluate the effects of native and frag-FN on proMMP-9 secretion further and as it was technically impossible to generate a genuine mixture of native and frag-FN in different ratios, as they were deposited on the surface of the dish, we incubated the U937 cells on one substrate for 48 h and then transferred the cells and the medium containing the already secreted proMMP-9 to wells coated with another substrate for an additional 24 h. After 72 h, native FN inhibited the secretion of TNF-{alpha}-induced proMMMP-9 by 1.8-fold (P<0.05) relative to plastic and by 1.6-fold (P<0.05) relative to frag-FN, similar to its effect after 48 h, although more proMMP-9 was accumulated in the supernatants at that time (Fig. 3 ). Incubation of U937 cells on frag-FN or on plastic followed by incubation on native FN resulted in proMMP-9 levels similar to those obtained following 72 h incubation on native FN and were significantly different than those measured after 72 h incubation on plastic or on frag-FN (P<0.05). However, incubation of the cells on native FN followed by incubation on frag-FN resulted in proMMP-9 levels, which were similar to those after 72 h of incubation on frag-FN and were higher than those observed after incubation on native FN followed by incubation on plastic (P<0.05). Thus, native FN reduces proMMP-9 induction by TNF-{alpha}, and frag-FN may hamper the effects of native FN. The plastic substrate also consists of small amounts of native and frag-FN molecules secreted by the inflammatory monocytes (Fig. 1) . This mixture supports TNF-{alpha} induction of MMP-9 and is inhibited by native FN, much like the frag-FN. However, it may not be enough to support enhanced proMMP-9 production following preincubation on native FN.


Figure 3
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  		Figure 3. Frag-FN acts oppositely to native FN. U937 cells were incubated for 48 h with TNF-{alpha} (20 ng/ml) on plastic or on plastic coated with native FN (200 µg) or FN fragmented by gelatinases (20 ng/ml). After 48 h of incubation, the cells and supernatants were transferred to one of the three substrates, as depicted in the graph, for an additional 24 h. At the end of the 72 h, supernatants were collected and analyzed by zymography, and proMMP-9 levels were quantified by densitometry (n=7). *, P < 0.05, compared with 72 h incubation on plastic; §, P < 0.05, compared with 72 h incubation on native FN; #, P < 0.05, compared with 72 h incubation on Frag-FN; {dagger}, P < 0.05, compared with 48 h incubation on native FN, followed by 24 h incubation on Frag-FN. Native FN and frag-FN demonstrated opposing effects on proMMP-9 secretion.

 
The effects of native and frag-FN are mediated by integrin receptors, but only frag-FN signals through Rho kinase
As integrin receptors are known to mediate the binding of FN to cells, we first characterized their surface expression on U937 cells. Flow cytometry shows that the mean fluorescence values of the ß1 (52±9.6), {alpha}5 (22±4), or {alpha}4 (23±5.7) integrin chains, composing the {alpha}4ß1 and {alpha}5ß1 integrins, were not changed as a result of incubation on the three different substrates or the addition of TNF-{alpha}. To explore if these integrins mediate the effects of FN on proMMP-9, we incubated U937 cells with blocking antibodies directed against each of these chains. As shown before, in the absence of TNF-{alpha}, only small amounts of proMMP-9 were secreted, and no effect of the antibodies could be observed (Fig. 4 ). However, when U937 cells were incubated with TNF-{alpha} and each of the blocking antibodies, secretion of proMMP-9 was inhibited by ~2.3-fold on the plastic and frag-FN substrates (P<0.05 for the ß1 chain and P<0.001 for the {alpha}4 and {alpha}5 chains) and by 1.7-fold on the native FN substrate (P<0.05). We further investigated which signaling pathways are involved in mediating the opposing effects of native and frag-FN. To this end, U937 cells were incubated with TNF-{alpha} on the three substrates, different inhibitors of known signaling pathways were added to the culture medium, and their effect on the secretion of proMMP-9 was evaluated by zymography. Figure 5A shows that the JNK inhibitor II, which inhibits the three isoforms of JNK, reduced the secretion of proMMP-9 from cells incubated on all three substrates in a dose-dependent manner (P<0.001), suggesting that JNK is involved in mediating the inducing effects of TNF-{alpha} on proMMP-9. In addition, the Rho kinase inhibitor Y27632 dose-dependently inhibited by up to 2.5-fold the TNF-{alpha}-induced proMMP-9 secretion from U937 cells, which were incubated on plastic or on frag-FN (P<0.001) but not on native FN (Fig. 5B) . Other inhibitors that were used, such as the ERK1/2 inhibitor PD98050, the p38 inhibitor SB203580, the PKC inhibitor chelerythrine, and the NF-{kappa}B inhibitor SN50, showed no significant effect on the amounts of secreted proMMP-9 after incubation on all three substrates (Fig. 5C 5D 5E 5F) .


Figure 4
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  		Figure 4. Effect of integrins on the secretion of proMMP-9. U937 cells were incubated for 48 h, with or without TNF-{alpha} (20 ng/ml) and blocking mAb directed against the ß1, {alpha}4, and {alpha}5 integrin chains (5 µl/ml each) on plastic or on plastic coated with native FN or frag-FN. The amounts of secreted proMMP-9 in the supernatants were evaluated by zymography analysis (n=4). *, ***, P < 0.05, P < 0.001, compared with TNF-{alpha}-induced cells incubated on plastic; §, §§, P < 0.05, P < 0.001, compared with TNF-{alpha}-induced cells incubated on native FN; $$, $$$, P < 0.01, P < 0.001, compared with TNF-{alpha}-induced cells incubated on frag-FN. All the antibodies inhibited the secretion of proMMP-9 by U937, which were incubated on the three substrates.

 

Figure 5
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  		Figure 5. Effect of inhibitors of different signaling pathways on the secretion of MMP-9. U937 cells were incubated for 48 h with TNF-{alpha} (20 ng/ml) on plastic or on plastic coated with native or frag-FN. In some of the wells, the following signaling inhibitors were added in increasing amounts, as indicated in the graphs: (A) JNK inhibitor II, which inhibits all three isoforms of JNK, (B) Rho kinase inhibitor Y27632, (C) ERK1/2 inhibitor PD98050, (D) p38 MAPK inhibitor SB203580, (E) protein kinase C (PKC) inhibitor chelerythrine, (F) NF-{kappa}B translocation inhibitor SN50 (a single dose of 25 µg/ml). Amounts of proMMP-9 in the supernatants were evaluated by zymography analysis (n=4). *, P < 0.05, **, P < 0.01, and ***, P < 0.001, compared with the TNF-{alpha}-induced cells on plastic; §§§, P < 0.001, compared with TNF-{alpha}-induced cells on native FN; $, P < 0.05, $$, P < 0.01, and $$$, P < 0.001, compared with TNF-{alpha}-induced cells on frag-FN. TNF-{alpha} induced secretion of proMMP-9 via the JNK pathway, regardless of the substrate on which the cells were plated. Rho kinase mediated, at least in part, the effects of plastic and frag-FN on proMMP-9 secretion, whereas the other pathways were not involved in the effects of the substrates on proMMP-9 secretion.

 
Native FN inhibits the translation of proMMP-9
The possibility that secreted proMMP-9 could be deposited in the ECM or degraded rapidly after its secretion was dismissed, as Western blot analysis of ECM proteins, which were scraped off the culture dish, did not reveal any band, and no degradation products of proMMP-9 could be detected (data not shown). As the main known regulation of MMP-9 is transcriptional, we expected changes in the MMP-9 mRNA levels upon incubation on native and frag-FN, which could explain the inhibitory effect of native FN. We measured the amounts of the steady-state MMP-9 mRNA using the quantitative real-time PCR technique (Fig. 6A ). TNF-{alpha} induced the transcription of MMP-9 by 35-fold (P<0.01), and in its presence, incubation on plastic, native FN, or frag-FN did not change the levels of MMP-9 mRNA. Therefore, we asked if native FN inhibited the secretion of proMMP-9 by causing its intracellular accumulation. Zymography analysis of cellular extracts obtained from U937 cells, which were incubated without TNF-{alpha} on plastic, native FN, and frag-FN, showed that only negligible amounts of proMMP-9 were visible (Fig. 6B) , whereas incubation in the presence of TNF-{alpha} on plastic or on frag-FN induced the accumulation of intracellular proMMP-9 by 7.5 ± 1.6-fold (P<0.001) and by 7 ± 1.3-fold (P<0.01), respectively. However, incubation of the cells on native FN in the presence of TNF-{alpha} reduced this accumulation by about twofold (P<0.05), relative to plastic or frag-FN. Quantification of intracellular MMP-9 by ELISA also showed that incubation on plastic or on frag-FN in the presence of TNF-{alpha} resulted in a small accumulation of MMP-9 (0.082±0.03 and 0.081±0.02 ng/ml, respectively), whereas incubation on native FN inhibited this accumulation by threefold (0.027±0.01 ng/ml). This magnitude of reduction of intracellular proMMP-9 was similar to the reduction in secreted amounts of the enzyme, and as no accumulation of intracellular proMMP-9 occurred, the possibility of interruption of the secretion process was negated. Of note, the zymography analysis of the cellular extracts revealed two bands of proMMP-9 at a molecular weight higher than the expected, activated form of MMP-9, which may represent the mature and the nonglycosylated forms of the enzyme [35 ]. We next tried to explain the inhibitory effect of native FN by enhanced degradation of proMMP-9 or by inhibited translation of its mRNA. To examine whether intracellular proMMP-9 is degraded rapidly, we used increasing amounts of the lysosome inhibitor bafilomycin A1, which prevents acidification of the lysosome and the proteolytic activity of its enzymes, and the proteome inhibitor MG132. Whereas bafilomycin A1 did not change the amounts of proMMP-9 and had no effect (data not shown), the proteasome inhibitor MG132 increased the intracellular accumulation of proMMP-9 dose-dependently by up to threefold (P<0.01; Fig. 6C ). However, this increase was of equal magnitude when cells were incubated on all three substrates, keeping the difference between MMP-9 levels constant and suggesting that degradation was not the mechanism involved in the effects of native FN. To directly examine the possibility that native FN inhibits translation of proMMP-9, we first incubated U937 cells on plastic with TNF-{alpha} to achieve high amounts of the enzyme and then washed the cells and further incubated them for an additional 24 h on the three substrates together with 200 nM MG132, which inhibited its degradation. Figure 6D shows that cellular extracts obtained after 24 h incubation on plastic or frag-FN with MG132 accumulated MMP-9 by about threefold (P<0.05), relative to the beginning of the incubation (Fig. 6B) , suggesting that the additional enzyme was the product of de novo synthesis. However, further incubation on native FN, even in the presence of MG132, did not increase the cellular accumulation of MMP-9, suggesting that no translation took place.


Figure 6
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  		Figure 6. Molecular mechanisms regulating the inhibitory effect of native FN on secretion of proMMP-9. U937 cells were incubated for 48 h on plastic or on plastic coated with native or frag-FN by gelatinases (20 ng/ml), with or without TNF-{alpha} (20 ng/ml). (A) Total RNA was extracted and reversed-transcribed, and MMP-9 mRNA was amplified by quantitative real-time PCR (n=6). RQ, Relative quantity. **, P < 0.01, compared with nonstimulated cells on plastic; §§, P < 0.01, compared with nonstimulated cells on native FN; {dagger}{dagger}, P < 0.01, compared with nonstimulated cells on frag-FN. TNF-{alpha} similarly induced MMP-9 mRNA, regardless of the substrate, ruling out transcriptional regulation. (B) Cellular proteins were extracted and analyzed by zymography. The two bands of proMMP-9 observed represent the mature and the nonglycosylated forms of the enzyme. P.C., Positive control of commercial MMPs; Pl, plastic; Na, native; Fg, frag-FN. Densitometric analysis of intracellular proMMP-9 (n=4) shows that native FN reduced the intracellular accumulation of proMMP-9. §, P < 0.05, compared with TNF-{alpha}-stimulated cells on plastic; $, P < 0.05, compared with TNF-{alpha}-stimulated cells on native FN. (C) TNF-{alpha}-stimulated cells were incubated with increasing amounts of the proteasome inhibitor MG132, and cellular extracts were analyzed by zymography (n=3). *, P < 0.05, and **, P < 0.01, compared with TNF-{alpha}-induced cells on plastic; §, P < 0.05, and §§, P < 0.01, compared with TNF-{alpha}-induced cells on native FN (Nat-FN); $, P < 0.05, and $$, P < 0.01, compared with TNF-{alpha}-induced cells on frag-FN. Degradation of proMMP-9 occurs in the proteasome, and incubation on the different substrates did not change its degradation rate. (D) U937 cells were incubated on plastic with TNF-{alpha} to increase MMP-9 intracellular levels and then transferred to the different substrates for an additional 24 h, together with 200 nm MG132 to block degradation of the enzyme. Cellular extracts were analyzed by zymography (n=6). *, P < 0.05, compared with TNF-{alpha}-induced cells on plastic after the first 24 h (0 h); §, P < 0.05, compared with TNF-{alpha}-induced cells on plastic together with TNF-{alpha} and 200 nM MG132 after an additional 24 h; $, P < 0.05, compared with TNF-{alpha}-induced cells on native FN together with TNF-{alpha} and 200 nM MG132 after an additional 24 h. When degradation of MMP-9 was blocked by MG132, the enzyme was accumulated over time, only upon incubation on plastic or frag-FN but not on native FN, indicating that native FN inhibits MMP-9 translation.

 
Frag-FN increases migration of monocytes
We evaluated the effects of the plastic, native FN, and frag-FN on U937 cell migration and the specific involvement of MMP-9 in this process, by adding anti-MMP-9 or the recombinant protein. In the absence of TNF-{alpha} and therefore, in the absence of proMMP-9, only 6.9 ± 1.6% of the cells migrated, in comparison with the 42.9 ± 3.2% of the cells that migrated when TNF-{alpha} was added. Figure 7 shows that native FN inhibited the migration of U937 cells by 2.5-fold, relative to the incubation on plastic or frag-FN substrates (P<0.05). Addition of anti-MMP-9 inhibited migration of U937 cells, which were incubated on plastic (by 1.6-fold, P<0.05) or on frag-FN (by 1.9-fold, P<0.05) but had no effect on the already reduced migration of U937 cells incubated on native FN. Addition of recombinant MMP-9 did not significantly increase the migration of U937 incubated on plastic or frag-FN but increased the migration of cells incubated on native FN (P<0.05) to a level similar to those that were incubated on plastic or frag-FN.


Figure 7
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  		Figure 7. Involvement of proMMP-9 in monocyte migration. U937 cells were incubated in 24-transwell plates (3 µm pore size), which were not coated (plastic) or coated with native or frag-FN for 48 h with TNF-{alpha} (20 ng/ml). In some of the wells, anti-MMP-9 (20 ng/ml) or recombinant MMP-9 (recMMP-9; 20 ng/ml) was added. Cells that migrated to the lower chamber were collected, 200 µl PKH26 reference beads were added, and the mixture was counted by flow cytometry to determine the percentage of cells that migrated across the barrier from the total number of cells plated. *, P < 0.05 compared with TNF-{alpha}-induced cells on plastic; §, P < 0.05, and §§, P < 0.01, compared with TNF-{alpha}-induced cells on native FN; {dagger}, P < 0.05, compared with TNF-{alpha}-induced cells on frag-FN. #, P < 0.05 compared with TNF{alpha}-induced cells on plastic in the presence of anti-MMP-9. Native FN inhibited U937 cell migration, and addition of anti-MMP-9 or the recombinant enzyme implicated MMP-9 in the migration of these cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show a different and opposed influence of native and fragmented products of FN on the secretion of monocyte MMP-9. First, we show that in an inflammatory microenvironment, simulated by the addition of TNF-{alpha}, monocytes secrete elevated amounts of proMMP-9 and increase the portion of fragmented products of FN, which is secreted in only minute amounts (Figs. 1 and 2) . Thus, it may be suggested that a causal relationship may exist between the two phenomena and that the secreted TNF-{alpha}-induced enzyme degrades FN and increases its fragmented portion, demonstrating one arm of a positive-feedback loop. Indeed, incubation of exogenous FN with different degrading proteases such as MMPs or elastase resulted in fragments that were essentially similar to those obtained by degradation of the endogenous FN shown in Figure 1 , probably as a result of the use of the same sensitive sites on the FN molecule.

Monocytes were shown to produce and secrete FN [15 ], and tissue injury or inflammation was shown to increase its fragmentation, resulting in fragments of similar size to those we observed in the current study [36 37 38 ]. Previous studies that attempted to elucidate the in vitro effect of FN fragments on cell behavior used one isolated FN fragment, which was obtained commercially, often originating from the N terminus of the molecule [29 , 37 , 38 ]. However, such an approach does not take into consideration the fact that monocytes respond to the integrated sum of all signals provided by the mixture of different FN fragments. Hence, in the following experiments, we compared the differences in the regulatory effects on MMP-9 between the native FN and all of its fragments. The main finding was the inhibitory effect of native FN on TNF-{alpha}-induced, proMMP-9 secretion (Fig. 2) , which was similar in magnitude (twofold) in primary monocytes and the U937 monocytic cell line. This enabled us to focus on the U937 cells, as the study of the molecular mechanisms responsible for the inhibitory effect of native FN on proMMP-9 demanded a large number of cells.

Although native FN inhibited the TNF-{alpha}-induced secretion of proMMP-9, plastic and frag-FN enhanced it to a similar extent. We used a specific strategy to mimic the dynamic nature of inflammation, where the cells first encounter native FN and are exposed gradually to its accumulated, fragmented products. We exposed the cells to one substrate sequentially, collected the supernatants, which contained the cells and the enzyme already secreted, and transferred them to a new substrate, when "plastic" represents the minute amounts of FN secreted by the monocytes throughout the incubation period. We show that frag-FN actively oppose the inhibitory effects of native FN on proMMP-9 secretion (Fig. 3) and that plastic resembles frag-FN but cannot overcome the inhibitory effects of native FN. This leads us to speculate that frag-FN and native FN exert opposing, activating, and inhibitory signals, respectively, which regulate proMMP-9 secretion.

The different effects exerted by native and frag-FN suggest differences in their signaling pathways, where the main candidates to transduce the signals could be the integrins {alpha}4ß1 and {alpha}5ß1, which are the principal receptors binding FN. However, as the expression of these integrins was not altered, and blocking antibodies reduced the TNF-{alpha}-induced secretion of proMMP-9 from monocytes cultured on the three substrates (Fig. 4) , we suggest that these two integrins are involved in signaling of both FN types, that TNF-{alpha} is required to activate them, similar to its ability to activate the ß2-integrin receptors [39 ], and that native and frag-FN activate different signaling pathways downstream of the integrin receptors. Of note, these results do not exclude the possibility that integrin receptors other than {alpha}4ß1 and {alpha}5ß1 may take part in mediating the different effects of native and frag-FN. Through the use of different inhibitors of known signaling pathways, we show that TNF-{alpha} is required for proMMP-9 induction, regardless of the substrate on which the cells were incubated and that its effects are mediated through the JNK pathway (Fig. 5A) and not through the prominent NF-{kappa}B pathway (Fig. 5F) . This can be explained by the inhibitory cross-talk that exists between these two pathways [40 , 41 ]. The only difference in inducing proMMP-9 found between the substrates was that plastic and fragmented, but not native FN, mediates, at least part of, its effects via Rho kinase (Fig. 5B) . This is consistent with recent studies, which showed a correlation between activation of Rho kinase and phosphorylation of focal adhesion kinase (FAK) and increase in MMP-9 [42 , 43 ]. Thus, we can speculate that although adhesion to frag-FN activates FAK and Rho kinase via the integrin receptors, adhesion to native FN does not activate or even inhibits this pathway.

Possible mechanisms by which native FN inhibits the TNF-{alpha}-induced secretion of MMP-9 were evaluated. Although TNF-{alpha} induced MMP-9 transcription strongly (Fig. 6A ; ref. [44 ]), the lack of change in the level of MMP-9 mRNA following incubation on all three substrates indicated that transcriptional regulation could be ruled out. We are aware of only one study that showed increased levels of MMP-9 mRNA after incubation on FN in the Jurkat T cell line in the presence of the FN cell-binding peptide (CS-1) rather than the native FN molecule [31 ], further stressing the different effects of one selected fragment versus a mixture of many fragmented products. We then focused on identifying post-transcriptional mechanisms within the cells and their exact regulatory checkpoint. Enhanced extracellular degradation or deposition of MMP-9 in the ECM was ruled out (data not shown), and disruption of MMP-9 trafficking in the cell, a phenomenon that has recently been observed by us during exposure of TNF-{alpha}-stimulated U937 cells to hypoxia [45 ], was dismissed, as native FN reduced its intracellular amounts. This suggested that native FN accelerated proMMP-9 degradation or inhibited its translation. Degradation of proMMP-9 was inhibited dose-dependently by the proteasome inhibitor MG132 to a similar extent by all substrates (Fig. 6C) but not by the lysosomal inhibitor bafilomycin A1, demonstrating that although the enzyme’s turnover occurs in the proteasome rather than in the lysosome, native FN did not enhance its degradation. Last, by inhibiting MMP-9 degradation while allowing uninterrupted MMP-9 de novo synthesis, reduction of the intracellular enzyme was observed only in monocytes cultured on native FN, indicating a down-regulation of MMP-9 translation.

MMP-9 is strongly associated with cell migration, and we presumed that the changes in its amounts may have affected the migratory behavior of U937 cells. Indeed, the extent of the inhibition exerted by native FN on U937 cell migration (Fig. 7) was proportional to its inhibitory effect on proMMP-9 secretion, and the use of anti-MMP-9 and recombinant MMP-9 demonstrated further that migration depends on the presence of the enzyme. Although the 84-kDa-activated form of MMP-9 was not detected by zymography, the zymogen form was apparently sufficient to confer activity. Cultured cells are known to exhibit the zymogen form alone, as culture conditions promote the rapid dissociation of MMP-9 from the membrane or dilute soluble activators in the media, thus not allowing activation of the enzyme [35 ]. Nonetheless, activity of the proenzyme in the absence of its activated form exists in these conditions [35 ].

In conclusion, we have demonstrated that in proinflammatory conditions simulated by TNF-{alpha}, the ECM protein FN exerts opposite biological properties. The native FN molecule inhibits secretion of proMMP-9 and monocyte migration, whereas its fragmented form increases them. We propose that these antagonistic effects of native and frag-FN modulate and allow fine-tuning of the local concentrations of autocrine proMMP-9 during early and ongoing inflammation. At first, the monocyte, which is activated by proinflammatory signals such as TNF-{alpha}, induces the secretion of proMMP-9 as part of the protein machinery that is required for its migration. However, the quiescent ECM, represented in our system by native FN, reduces proMMP-9 levels and serves as a homeostasis control. As inflammation persists, the low levels of proMMP-9 gradually cleave native FN into fragments that accumulate locally, alleviating the inhibitory effect of native FN on MMP-9 secretion, thus turning on a positive-feedback loop, resulting in acceleration of cell migration. The exact mechanisms and signal transduction pathways that mediate the specific post-transcriptional effects of native versus frag-FN need to be explored further.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the Rappaport Family Institute for Research in the Medical Sciences and by Research grant 5343 from the Chief Scientist Office of the Israeli Ministry of Health.


   FOOTNOTES
 
1 These authors are equally contributing senior authors. Back

Received May 15, 2006; revised January 24, 2007; accepted January 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Vaday, G. G., Lider, O. (2000) Extracellular matrix moieties, cytokines, and enzymes: dynamic effects on immune cell behavior and inflammation J. Leukoc. Biol. 67,149-159[Abstract]
  2. Schor, H., Vaday, G. G., Lider, O. (2000) Modulation of leukocyte behavior by an inflamed extracellular matrix Dev. Immunol. 7,227-238[Medline]
  3. Vaday, G. G., Franitza, S., Schor, H., Hecht, I., Brill, A., Cahalon, L., Hershkoviz, R., Lider, O. (2001) Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix J. Leukoc. Biol. 69,885-892[Abstract/Free Full Text]
  4. Nagase, H., Woessner, J. F., Jr (1999) Matrix metalloproteinases J. Biol. Chem. 274,21491-21494[Free Full Text]
  5. Nagase, H. (1997) Activation mechanisms of matrix metalloproteinases Biol. Chem. 378,151-160[Medline]
  6. Visse, R., Nagase, H. (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry Circ. Res. 92,827-839[Abstract/Free Full Text]
  7. Vincenti, M. P., Brinckerhoff, C. E. (2002) Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors Arthritis Res. 4,157-164[CrossRef][Medline]
  8. Lazure, C. (2002) The peptidase zymogen proregions: nature’s way of preventing undesired activation and proteolysis Curr. Pharm. Des. 8,511-531[CrossRef][Medline]
  9. Opdenakker, G., Van den Steen, P. E., Van, D. J. (2001) Gelatinase B: a tuner and amplifier of immune functions Trends Immunol. 22,571-579[CrossRef][Medline]
  10. Opdenakker, G., Van den Steen, P. E., Dubois, B., Nelissen, I., Van, C. E., Masure, S., Proost, P., Van, D. J. (2001) Gelatinase B functions as regulator and effector in leukocyte biology J. Leukoc. Biol. 69,851-859[Abstract/Free Full Text]
  11. Van den Steen, P. E., Dubois, B., Nelissen, I., Rudd, P. M., Dwek, R. A., Opdenakker, G. (2002) Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9) Crit. Rev. Biochem. Mol. Biol. 37,375-536[CrossRef][Medline]
  12. Gardner, J., Ghorpade, A. (2003) Tissue inhibitor of metalloproteinase (TIMP)-1: the TIMPed balance of matrix metalloproteinases in the central nervous system J. Neurosci. Res. 74,801-806[CrossRef][Medline]
  13. Brew, K., Dinakarpandian, D., Nagase, H. (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function Biochim. Biophys. Acta 1477,267-283[CrossRef][Medline]
  14. Madri, J. A., Graesser, D. (2000) Cell migration in the immune system: the evolving inter-related roles of adhesion molecules and proteinases Dev. Immunol. 7,103-116[Medline]
  15. Kitamura, N., Nishinarita, S., Takizawa, T., Tomita, Y., Horie, T. (2000) Cultured human monocytes secrete fibronectin in response to activation by proinflammatory cytokines Clin. Exp. Immunol. 120,66-70[CrossRef][Medline]
  16. Hershkoviz, R., Alon, R., Gilat, D., Lider, O. (1992) Activated T lymphocytes and macrophages secrete fibronectin which strongly supports cell adhesion Cell. Immunol. 141,352-361[CrossRef][Medline]
  17. Laouar, A., Collart, F. R., Chubb, C. B., Xie, B., Huberman, E. (1999) Interaction between a5b1 integrin and secreted fibronectin is involved in macrophage differentiation of human HL-60 myeloid leukemia cells J. Immunol. 162,407-414[Abstract/Free Full Text]
  18. Pankov, R., Yamada, K. M. (2002) Fibronectin at a glance J. Cell Sci. 115,3861-3863[Free Full Text]
  19. Kornblihtt, A. R., Pesce, C. G., Alonso, C. R., Cramer, P., Srebrow, A., Werbajh, S., Muro, A. F. (1996) The fibronectin gene as a model for splicing and transcription studies FASEB J. 10,248-257[Abstract]
  20. Wierzbicka-Patynowski, I., Schwarzbauer, J. E. (2003) The ins and outs of fibronectin matrix assembly J. Cell Sci. 116,3269-3276[Abstract/Free Full Text]
  21. Johansson, S., Svineng, G., Wennerberg, K., Armulik, A., Lohikangas, L. (1997) Fibronectin-integrin interactions Front. Biosci. 2,d126-d146[Medline]
  22. Barilla, M. L., Carsons, S. E. (2000) Fibronectin fragments and their role in inflammatory arthritis Semin. Arthritis Rheum. 29,252-265[CrossRef][Medline]
  23. Labat-Robert, J. (2002) Fibronectin in malignancy Semin. Cancer Biol. 12,187-195[CrossRef][Medline]
  24. Okamura, Y., Watari, M., Jerud, E. S., Young, D. W., Ishizaka, S. T., Rose, J., Chow, J. C., Strauss, J. F., III (2001) The extra domain A of fibronectin activates Toll-like receptor 4 J. Biol. Chem. 276,10229-10233[Abstract/Free Full Text]
  25. Schenk, S., Quaranta, V. (2003) Tales from the crypt[ic] sites of the extracellular matrix Trends Cell Biol. 13,366-375[CrossRef][Medline]
  26. Esparza, J., Vilardell, C., Calvo, J., Juan, M., Vives, J., Urbano-Marquez, A., Yague, J., Cid, M. C. (1999) Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways Blood 94,2754-2766[Abstract/Free Full Text]
  27. Xie, B., Laouar, A., Huberman, E. (1998) Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase Cb J. Biol. Chem. 273,11576-11582[Abstract/Free Full Text]
  28. Thant, A. A., Nawa, A., Kikkawa, F., Ichigotani, Y., Zhang, Y., Sein, T. T., Amin, A. R., Hamaguchi, M. (2000) Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells Clin. Exp. Metastasis 18,423-428[CrossRef][Medline]
  29. Penc, S. F., Blumenstock, F. A., Kaplan, J. E. (1998) A 70-kDa amino-terminal fibronectin fragment supports gelatin binding to macrophages and decreases gelatinase activity J. Leukoc. Biol. 64,351-357[Abstract]
  30. Stanton, H., Ung, L., Fosang, A. J. (2002) The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases Biochem. J. 364,181-190[CrossRef][Medline]
  31. Yakubenko, V. P., Lobb, R. R., Plow, E. F., Ugarova, T. P. (2000) Differential induction of gelatinase B (MMP-9) and gelatinase A (MMP-2) in T lymphocytes upon a4b1-mediated adhesion to VCAM-1 and the CS-1 peptide of fibronectin Exp. Cell Res. 260,73-84[CrossRef][Medline]
  32. Saito, S., Yamaji, N., Yasunaga, K., Saito, T., Matsumoto, S., Katoh, M., Kobayashi, S., Masuho, Y. (1999) The fibronectin extra domain A activates matrix metalloproteinase gene expression by an interleukin-1-dependent mechanism J. Biol. Chem. 274,30756-30763[Abstract/Free Full Text]
  33. Chase, A. J., Bond, M., Crook, M. F., Newby, A. C. (2002) Role of nuclear factor-{kappa} B activation in metalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo Arterioscler. Thromb. Vasc. Biol. 22,765-771[Abstract/Free Full Text]
  34. Rosengart, M. R., Arbabi, S., Garcia, I., Maier, R. V. (2000) Interactions of calcium/calmodulin-dependent protein kinases (CaMK) and extracellular-regulated kinase (ERK) in monocyte adherence and TNF{alpha} production Shock 13,183-189[Medline]
  35. Fridman, R., Toth, M., Chvyrkova, I., Meroueh, S. O., Mobashery, S. (2003) Cell surface association of matrix metalloproteinase-9 (gelatinase B) Cancer Metastasis Rev. 22,153-166[CrossRef][Medline]
  36. Trial, J., Baughn, R. E., Wygant, J. N., McIntyre, B. W., Birdsall, H. H., Youker, K. A., Evans, A., Entman, M. L., Rossen, R. D. (1999) Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration J. Clin. Invest. 104,419-430[Medline]
  37. Trial, J., Rossen, R. D., Rubio, J., Knowlton, A. A. (2004) Inflammation and ischemia: macrophages activated by fibronectin fragments enhance the survival of injured cardiac myocytes Exp. Biol. Med. (Maywood) 229,538-545[Abstract/Free Full Text]
  38. Birdsall, H. H., Porter, W. J., Trial, J., Rossen, R. D. (2005) Monocytes stimulated by 110-kDa fibronectin fragments suppress proliferation of anti-CD3-activated T cells J. Immunol. 175,3347-3353[Abstract/Free Full Text]
  39. Menegazzi, R., Busetto, S., Cramer, R., Dri, P., Patriarca, P. (2000) Role of intracellular chloride in the reversible activation of neutrophil ß 2 integrins: a lesson from TNF stimulation J. Immunol. 165,4606-4614[Abstract/Free Full Text]
  40. Giri, D. K., Pantazis, P., Aggarwal, B. B. (1999) Cellular resistance to vincristine suppresses NF-{kappa} B activation and apoptosis but enhances c-Jun-NH2-terminal protein kinase activation by tumor necrosis Apoptosis 4,291-301[Medline]
  41. Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., Franzoso, G. (2004) Linking JNK signaling to NF-{kappa}B: a key to survival J. Cell Sci. 117,5197-5208[Abstract/Free Full Text]
  42. Brest, P., Turchi, L., Le’Negrate, G., Berto, F., Moreilhon, C., Mari, B., Ponzio, G., Hofman, P. (2004) Escherichia coli cytotoxic necrotizing factor 1 inhibits intestinal epithelial wound healing in vitro after mechanical injury Infect. Immun. 72,5733-5740[Abstract/Free Full Text]
  43. Turner, N. A., O’Regan, D. J., Ball, S. G., Porter, K. E. (2005) Simvastatin inhibits MMP-9 secretion from human saphenous vein smooth muscle cells by inhibiting the RhoA/ROCK pathway and reducing MMP-9 mRNA levels FASEB J. 19,804-806[Abstract/Free Full Text]
  44. Vaday, G. G., Hershkoviz, R., Rahat, M. A., Lahat, N., Cahalon, L., Lider, O. (2000) Fibronectin-bound TNF-{alpha} stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis J. Leukoc. Biol. 68,737-747[Abstract/Free Full Text]
  45. Rahat, M. A., Marom, B., Bitterman, H., Weiss-Cerem, L., Kinarty, A., Lahat, N. (2006) Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association J. Leukoc. Biol. 79,706-718[Abstract/Free Full Text]




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