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 C-beta *

Bei Xie, Amale Laouar, and Eliezer HubermanDagger

From the Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta -deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha 5beta 1 integrin. HL-525 cells, which constitutively display high levels of surface alpha 5beta 1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha 5beta 1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Matrix metalloproteinases (MMPs)1 compose a family of structurally and functionally homologous extracellular proteinases, which govern the degradation of basement membrane and the underlying interstitial stroma (1). Under physiological and pathological conditions, macrophages participate in modulating the extracellular matrix (ECM) turnover. Macrophages may participate either directly by secreting MMPs and their specific inhibitors (2-6), or indirectly by releasing cytokines such as interleukin-1 and tumor necrosis factor-alpha , which stimulate resident fibroblasts or synovial cells to secrete MMPs (7, 8). The 92-kDa type IV collagenase/gelatinase (gelatinase B, MMP-9), which cleaves basement membrane collagen types IV and V, different types of gelatin, fibronectin (FN), and elastin (9-12), is the major MMP produced by human macrophages. Its proteolytic activity is thought to be necessary for a variety of monocyte/macrophage functions, such as extravasation, migration, and tissue remodeling during chronic inflammatory conditions (5, 6, 13). A number of previous studies have shown that the MMP-9 production by macrophages is closely associated with cellular differentiation (5, 6, 14). While low levels of MMP-9 proenzyme are secreted from human peripheral blood monocytes, its production is markedly up-regulated in alveolar macrophages as well as in macrophages derived from in vitro differentiation. To date, the underlying regulatory mechanisms for differentiation-dependent MMP-9 gene expression remain poorly defined.

Cell adhesion and spreading on ECM are hallmarks of macrophage differentiation (15). Among the ECM components, FN has been recognized as the key element in promoting cell adhesion and various functions of monocytes and macrophages. Both cell types adhere preferentially to FN-coated surfaces in comparison to laminin and other ECM components (16-19). Adherence to FN promotes migration and phagocytosis of these cells and modulates the expression of inflammatory cytokines such as interleukin-1, tumor necrosis factor-alpha , and macrophage-colony stimulating factor (M-CSF) (16, 20). These functions of FN are mediated by two surface receptors, alpha 5beta 1 and alpha 4beta 1 integrins, both of which are present on monocytes and macrophages (21). The alpha 5beta 1 integrin recognizes the cell-binding domain of the FN molecule that contains the arginyl-glycyl-aspartyl-serine (RGDS) sequence, whereas the alpha 4beta 1 integrin acts as the receptor for the CS-1 region of FN (22, 23).

In human peripheral blood monocytes, macrophage differentiation may be promoted by M-CSF or by phorbol 12-myristate 13-acetate (PMA) (24-26). Similarly, treatment with PMA and related reagents can induce a macrophage phenotype in the human HL-60 myeloid leukemia cells (27-29). In both cell types, activation of protein kinase C (PKC) is essential for the differentiation process (30-32). In HL-60 cells, several studies have established a correlation between macrophage differentiation and gene expression of PKC-beta , the most abundant PKC isozyme in these cells (33, 34). We have described previously a PMA-resistant HL-60 cell variant, HL-525, which harbors a defect in PKC-beta gene expression (32). Treatment with all-trans-retinoic acid, which enhances the PKC-beta expression, reverses the PMA resistance of HL-525 cells (35). The HL-60 cell variant described by Macfarlane and Manzel (36) is also deficient in PKC-beta expression and may be rendered PMA-susceptible by treatment with 1,25-dihydroxyvitamin D3, which similarly increases the expression of PKC-beta . Recently, we have shown that restoration of the PKC-beta expression in HL-525 cells by PKC-beta gene transfection restored the PMA responsiveness, suggesting the essential role of PKC-beta in PMA-induced HL-60 cell differentiation (37).

In the present study, we examined critical steps involved in the induction of MMP-9 gene expression during macrophage differentiation in HL-60 cells treated with PMA and in human peripheral blood monocytes treated with either PMA or M-CSF. By using these two cell systems, we demonstrate that FN-mediated cell adhesion and spreading are required for induction of MMP-9 gene expression during macrophage differentiation. This process is mainly mediated through the alpha 5beta 1 integrin signaling, with little contribution of the alpha 4beta 1 integrin. Furthermore, in HL-525 cells, which constitutively express high levels of surface alpha 5beta 1 integrin, we found that their adherence and spreading on FN were sufficient to induce MMP-9 gene expression and that PKC-beta is essential for the production of this proteinase during PMA-induced macrophage differentiation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Human plasma fibronectin (FN), gelatin, all-trans-retinoic acid (ATRA), cytochalasins B and D, preimmune IgG controls, and a murine monoclonal antibody (mAb) to human FN (FN-15, IgG1), which was dialyzed before use, were purchased from Sigma. Mouse mAbs to human beta 1 (K20, mouse IgG2a), alpha 4 (HP2/1, IgG1), and alpha 5 (SAM1, IgG2b) integrins were purchased from Immunotech (Westbrook, ME). mAb to human 92-kDa gelatinase (MMP-9) was purchased from Oncogene Science, Inc. (Cambridge, MA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Chemicals for Cancer Research (Eden Prairie, MN) and macrophage-colony stimulating factor (M-CSF) was from Biosource International (Camarillo, CA). H-7 and HA-1004 were from LC Laboratories (Woburn, MA).

Cells and Cell Culture-- The human HL-60 myeloid leukemia cell line was originally obtained from R. C. Gallo (National Cancer Institute). The differentiation-resistant HL-525 cell line was established by cloning HL-60 cells subcultured for 102 times in the presence of increasing concentrations (up to 3 µM) of PMA at 5-8-day intervals (30). These cells exhibited stable PMA-resistant phenotypes for at least 50-60 subcultures (200-300 generations). Human peripheral blood monocytes were isolated from heparinized whole venous blood by Ficoll-Paque density gradient (1.077 g/ml) centrifugation as described previously (38). The cells were cultured and maintained in Petri dishes in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 15% heat-inactivated fetal bovine serum (Intergen Co., Purchase, NY), penicillin (100 units/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM) (Life Technologies, Inc.) in a humidified atmosphere containing 8% CO2 at 37 °C. All treatments were carried out in tissue culture dishes or cluster plates in serum-supplemented RPMI 1640 medium. For HL-525 cells in some experiments, the cells were inoculated and treated in FN-coated wells. The wells were coated for 16 h at room temperature with 20 µg/ml of either FN or bovine serum albumin in phosphate-buffered saline. The nonspecific sites of FN-coated surfaces were blocked with 1% bovine serum albumin in phosphate-buffered saline for 30 min at 37 °C. The wells were then rinsed with 3 µM MnCl2 immediately before use.

Immunoblotting and Gelatin Zymography-- The cells were treated in serum-supplemented RPMI 1640 medium as indicated in the figure legends. After treatment, the cells were replaced with fresh serum-free medium containing the appropriate inhibitory reagent and incubated for 24-48 h prior to collection of conditioned media for analysis. Immunoblotting analysis was conducted as described previously (39). Mouse mAb to human MMP-9 at 2 µg/ml was used as a primary antibody, and goat anti-mouse IgG alkaline phosphatase conjugate (Bio-Rad) was used as a secondary antibody for color detection. Gelatin zymography was performed on 7.5% SDS-polyacrylamide gels impregnated with 1 mg/ml gelatin, as described previously (40). Gelatinolytic activity was visualized as clear zones with Coomassie Brilliant Blue R-250 staining. The amount of secreted MMP-9 proenzyme in treated cells relative to that of untreated cells was determined by using an HP ScanJet 4c Scanner (Hewlett-Packard).

Stable Transfection of Cells-- All transfections were performed by electroporation using a Bio-Rad Gene Pulser apparatus with capacitance extender in 0.4-cm gap electroporation cuvettes (Eppendorf Scientific, Madision, MI). The pMV7-RP58 plasmid (41, kindly provided by Dr. I. B. Weinstein, Columbia University, NY) contains both the full-length rat PKC-beta 1 cDNA and the bacterial neomycin phosphotransferase (neo) gene that confers resistance to the antibiotic G418 (Geneticin, Sigma). The pMV7 plasmid contains the neomycin gene only. For each transfection, 5 × 106 cells were mixed with 10 µg of supercoiled plasmid DNA and 0.2 ml of phosphate-buffered sucrose (272 mM sucrose, 7 mM Na2HPO4, pH 7.4) in a total volume of 0.5 ml. The cells were electroporated at 250 V and allowed to recover in 10 ml of serum-supplemented RPMI medium for 24 h prior to selection in medium containing 0.5 mg/ml G418. The G418-resistant transfectants were obtained by limited dilution in 24-well plates and tested for PKC-beta expression and PMA inducibility of cell adhesion and spreading as well as 92-kDa gelatinase production. The selected clones were maintained in G418-containing medium.

RNA Isolation and Northern Analysis-- Total RNA was purified by centrifugation through a cesium chloride cushion as described by Chirgwin et al. (42). Northern blot analysis was performed as described previously (43). Briefly, total RNA was electrophoresed on 1.2% agarose gels containing 2.2 M formaldehyde, transferred onto Magna Charge Nylon membranes (Micron Separations, Inc., Westborough, MA), and fixed to the membrane by UV irradiation. Human cDNA probes for MMP-9 (kindly provided by Dr. W. Stetler-Stevenson, National Institutes of Health, Bethesda, MD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (American Type Culture Collection, Rockville, MD) were labeled using a random primer kit (U. S. Biochemical Corp.) with [alpha -32P]dCTP (Amersham Pharmacia Biotech). The membranes were hybridized with the denatured probe at 60 °C for 18-24 h in hybridization buffer (0.5 M sodium phosphate, pH 7.2, 2 mM EDTA, 1% bovine serum albumin, 7% SDS) and washed at 60 °C for 30 min once in 1× SSPE (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 1 mM EDTA, 0.1% SDS) and once in 0.1× SSPE, 0.1% SDS. The blots were autoradiographed in the dark at -80 °C.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

MMP-9 Gene Expression Is Induced during Macrophage Differentiation-- Treatment with phorbol 12-myristate 13-acetate (PMA) for 1-2 days induces HL-60 cells to differentiate toward the macrophage lineage (27-29). To understand how MMP-9 gene expression is regulated during this process, we treated HL-60 cells with 3 nM PMA for 24 h. Secretion of the MMP-9 proenzyme into the culture medium was detected in PMA-treated cells but not in untreated cells (Fig. 1A). Time course studies revealed that induction of MMP-9 gene expression was a relatively late event, with high levels of MMP-9 steady state mRNA detected at 8 h and the peak not occurring until 24 h after the addition of PMA (Fig. 1B). We noted that the time course of PMA-induced MMP-9 gene expression paralleled that of cell adhesion and spreading, a hallmark of macrophage differentiation (15). To confirm our observation in normal blood cells, we isolated human peripheral blood monocytes and induced them to mature into macrophages by treatment with either 3 nM PMA or 250 units/ml M-CSF. Untreated monocytes produced small amounts of MMP-9 proenzyme (Fig. 2A). This result may be attributed to the fact that a fraction of monocytes spontaneously adhered to the tissue culture plates and differentiated into macrophages by the time conditioned media were collected for zymogram analysis (48 h after plating). Treatment with PMA or M-CSF promoted macrophage differentiation and, accordingly, enhanced MMP-9 secretion by 7- or 5-fold, respectively (Fig. 2A).


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Fig. 1.   Induction of MMP-9 gene expression in HL-60 cells in response to PMA treatment. A, the cells were either untreated (Control) or treated with 3 nM PMA for 24 h. Proteins in conditioned media were separated on a 7.5% polyacrylamide gel (SDS-PAGE) under reducing conditions, followed by transferring to a nitrocellulose filter. The blot was then incubated with 2 µg/ml anti-MMP-9 mAb as described under "Experimental Procedures." The molecular markers are as follows: beta -galactosidase, 112 kDa; bovine serum albumin, 80 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 36 kDa. B, the cells were either untreated (C) or treated with 3 nM PMA for the indicated periods. Total RNA was isolated and RNA samples (10 µg/lane) resolved on a 1.2% agarose gel containing 2.2 M formaldehyde as described under "Experimental Procedures." The blot was hybridized sequentially with 32P-labeled cDNA probes for MMP-9 and GAPDH.


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Fig. 2.   Production of MMP-9 proenzyme in human peripheral blood monocytes and HL-60 cells treated with PMA or M-CSF in the presence or absence of H-7 or HA-1004. Freshly isolated human peripheral blood monocytes at 1 × 105 (A) or HL-60 cells at 7 × 104 (B) were seeded in each well of a 48-well plate in serum-supplemented RPMI 1640 medium. The cells were pretreated for 1 h with H-7 (20 µM) or HA-1004 (40 µM) before the addition of 3 nM PMA or 250 units/ml M-CSF (monocytes only). At 24 h, the cells were replaced with serum-free medium containing either H-7 or HA-1004 and incubated for an additional 24 h. The conditioned media were then harvested and directly analyzed by gelatin zymography as described under "Experimental Procedures." The gelatinolytic activity of MMP-9 proenzyme is visualized as a clear band against a dark background. The experiment shown is representative of at least four separate experiments performed with blood monocytes isolated from four healthy individuals.

PKC-beta Is Required for PMA-induced MMP-9 Gene Expression during Macrophage Differentiation-- Because activation of PKC is central to PMA- or M-CSF-induced macrophage differentiation (30-32), we were interested in defining the role of PKC in stimulation of MMP-9 gene expression during such a differentiation. We added either H-7 (20 µM) or HA-1004 (40 µM) to blood monocytes and HL-60 cells 1 h before and during PMA or M-CSF treatment (Fig. 2); we used M-CSF only in monocytes because HL-60 cells do not express the M-CSF receptor (c-fms proto-oncogene) (44). At 20 µM, H-7 inhibits PKC as well as cAMP- or cGMP-dependent kinase, whereas HA-1004 at 40 µM only affects the latter two (45). The doses we used for both inhibitors did not affect cell viability, as determined by trypan blue exclusion assay. While HA-1004 affected neither cell adhesion and spreading nor MMP-9 production, H-7 was a potent inhibitor of both cell adhesion and MMP-9 production in blood monocytes treated with either PMA or M-CSF (Fig. 2A) and in HL-60 cells treated with PMA (Fig. 2B). The inhibitory effect of H-7 on PMA-induced MMP-9 production in HL-60 cells was confirmed by Northern blot analysis (data not shown). These results suggest the involvement of PKC in induction of MMP-9 gene expression during macrophage differentiation. Interestingly, H-7 was equally effective in inhibiting the basal level secretion of MMP-9 proenzyme from untreated monocytes (Fig. 2A). It is noteworthy that in both monocytes and HL-60 cells, inhibition of the MMP-9 production invariably paralleled the inhibition of cell adhesion and spreading.

To define further the role of PKC in MMP-9 gene expression during macrophage differentiation, we examined expression of this enzyme in HL-525 cells, HL-60-derived variant cells that are deficient in PKC-beta expression and are resistant to PMA-induced macrophage differentiation (30, 34, 37). When treated with 30 nM PMA, these cells failed to produce the MMP-9 proenzyme, in contrast to the marked induction of this enzyme with 3 nM PMA in HL-60 cells (Fig. 3A). Treatment with all-trans-retinoic acid (ATRA) alone or in combination with PMA resulted in a more than 4-fold increase in PKC-beta gene expression (Fig. 3B). Combined treatment with PMA and ATRA induced MMP-9 gene expression and secretion of the proenzyme into the culture medium, whereas treatment with ATRA alone failed to do so (Fig. 3, A and B), suggesting that restoration of PKC-beta gene expression alone is not sufficient to induce MMP-9 gene expression; activation of PKC activity by PMA is also required. As a comparison, addition of ATRA to HL-60 cells had no apparent influence on PMA-induced MMP-9 production (Fig. 3A). The MMP-9 induction in HL-525 cells was concurrent with the appearance of cell attachment and spreading.


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Fig. 3.   Induction of PKC-beta gene expression in HL-525 cells and MMP-9 production in HL-525 and HL-60 cells in response to ATRA and PMA. A, gelatin zymographic analysis. HL-525 cells at 3 × 104 or HL-60 cells at 7 × 104 were seeded in each well of a 48-well plate. HL-525 cells were either untreated (Control) or treated for 24 h with 30 nM PMA or 1 µM ATRA. HL-60 cells were treated with 3 nM PMA or 1 µM ATRA. For the combined treatment, both cell types were treated for 24 h with ATRA and PMA at respective doses for 24 h. After incubation, the cells were replaced with serum-free medium and incubated for an additional 24 h before conditioned media were collected and analyzed for the presence of gelatinolytic activity of MMP-9 by gelatin zymography, as described. B, Northern blot analysis. HL-525 cells were incubated without (Control) or with 30 nM PMA or 1 µM ATRA for 24 h. For the combined treatment, the cells were treated with ATRA and PMA as described above. After incubation, the cells were harvested for total RNA extraction. RNA samples (20 µg/lane) were analyzed by Northern blotting as described. The blot was hybridized sequentially with 32P-labeled cDNA probes for PKC-beta , MMP-9, and GAPDH.

To assess directly the involvement of PKC-beta in MMP-9 gene expression, HL-525 cells were transfected with an expression plasmid containing the full-length PKC-beta cDNA and the neomycin gene that confers resistance to Geneticin (G418). As a control, the cells were also transfected with a plasmid containing the neomycin gene only. Stable transfectants were selected by limited dilution and maintained in G418-containing medium. The PKC-beta gene expression in two PKC-beta transfectants, HL-525/beta 3-2 and HL-525/beta 3-30, were restored to the level comparable to that in HL-60 cells (Fig. 4A). Treatment of these clones with 30 nM PMA induced cell adhesion and spreading (data not shown), which was accompanied by MMP-9 gene expression and secretion of the MMP-9 proenzyme into the culture medium (Fig. 4, B and C). Trace amounts of the MMP-9 proenzyme were detected in untreated PKC-beta transfectants; addition of PMA resulted in a more than 10-fold increase in secretion of MMP-9 proenzyme in both PKC-beta clones (Fig. 4C). In contrast, no MMP-9 steady state mRNA or proenzyme was detected in the parental HL-525 or the neomycin transfectant (HL-525/neo), regardless of PMA treatment. Therefore, we conclude that activated PKC-beta plays a critical role in PMA-induced MMP-9 gene expression during HL-60 cell differentiation.


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Fig. 4.   Induction of MMP-9 gene expression by PMA in HL-525 cells stably transfected with a PKC-beta expression plasmid. HL-525 cells were transfected by electroporation either with the pMV7 plasmid containing the bacterial neomycin phosphotransferase gene or with the pMV7-RP58 plasmid containing both the full-length PKC-beta cDNA and the neomycin gene. Stable neomycin (HL-525/neo) and PKC-beta (HL-525/beta 3-2, HL-525/beta 3-30) transfectants were selected and maintained in serum-supplemented RPMI 1640 medium in the presence of 0.5 mg/ml G418. A, Northern blot analysis of total RNA samples (20 µg/lane) for PKC-beta steady state mRNA levels in parental HL-525, HL-525/neo, HL-525/beta 3-2, and HL-525/beta 3-30 cells. HL-60 cells were included for comparison. B, Northern blot analysis of total RNA samples (20 µg/lane) for MMP-9 steady state mRNA levels in parental HL-525, HL-525/neo, HL-525/beta 3-2, and HL-525/beta 3-30 cells. The cells were either untreated (-) or treated with (+) 30 nM PMA for 30 h prior to RNA isolation. GAPDH hybridization is used to demonstrate equal loading of the RNA samples. C, gelatin zymographic analysis of MMP-9 proenzyme secreted from HL-525, HL-525/neo, HL-525/beta 3-2, and HL-525/beta 3-30 cells. The cells (1.5 × 105 cells/ml) were either untreated (-) or treated (+) with 30 nM PMA for 24 h in serum-supplemented RPMI 1640 medium followed by further incubation in serum-free medium containing PMA. After 48 h, the culture media were harvested, concentrated 5:1 by Amicon microconcentrators, and analyzed by gelatin zymography.

Cell Adhesion and Spreading Mediated by Fibronectin and alpha 5beta 1 Integrin Are Required for Induction of MMP-9 Gene Expression during Macrophage Differentiation-- The 8-h lag phase in PMA-induced MMP-9 gene expression in HL-60 cells indicates that the induction is probably not directly mediated by activation of PKC but rather by cellular events downstream of PKC activation. We noted throughout our study that the induction of MMP-9 gene expression in both blood monocytes and HL-60 cells closely paralleled the timing and extent of cell adherence and spreading, an event which involves fibronectin (FN) and its surface integrin receptors.2 To determine whether FN-mediated cell adhesion and spreading have a causal role in MMP-9 gene expression, HL-60 cells were treated with PMA and peripheral blood monocytes with either PMA or M-CSF in the presence of neutralizing mAbs to FN and its integrin receptors. The anti-FN mAb, but not the preimmune IgG, blocked MMP-9 gene expression (Fig. 5A), as well as cell adhesion and spreading (data not shown). Accordingly, secretion of the MMP-9 proenzyme into culture media was diminished (Fig. 5B). Antibodies that neutralize the function of the RGDS-dependent FN receptor alpha 5beta 1 integrin demonstrated similar inhibitory efficacy (Fig. 5B). On the contrary, neutralizing anti-alpha 4 mAb had little effect, suggesting that RGDS-dependent cell adhesion and spreading are involved in this process. The antibody-mediated inhibition of MMP-9 gene expression observed in HL-60 cells was reproduced in blood monocytes in which both basal (untreated) and stimulated (with PMA or M-CSF) secretion of MMP-9 proenzyme were strongly inhibited by anti-FN, anti-alpha 5, and anti-beta 1 integrin mAbs but not by the preimmune IgG (Fig. 6). Again, anti-alpha 4 mAb was ineffective (data not shown). Doses of mAbs used in both HL-60 cells and blood monocytes (up to 70 µg/ml) did not affect cell viability, as determined by trypan blue exclusion assay. In fact, we noted that in HL-60 cells, cell proliferation inhibited by PMA treatment was partially resumed by the antibody treatment.


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Fig. 5.   Inhibition of PMA-induced MMP-9 gene expression and proenzyme production by anti-fibronectin and anti-alpha 5beta 1 integrin mAbs in HL-60 cells. A, HL-60 cells were either untreated (Control) or treated with 3 nM PMA in the absence or presence of 70 µg/ml anti-fibronectin (anti-FN) mAb or preimmune IgG. At 24 h, cells were harvested for total RNA extraction and RNA samples (10 µg/lane) examined by Northern blot analysis. B, HL-60 cells at 7 × 104 were plated in a 48-well plate in serum-supplemented RPMI 1640 medium. At the initiation of culture, the cells were either untreated (Control) or treated with 3 nM PMA in the absence or presence of 70 µg/ml preimmune IgG or neutralizing mAb to fibronectin (anti-FN), beta 1 integrin (anti-beta 1), alpha 5 integrin (anti-alpha 5), or alpha 4 integrin (anti-alpha 4). After incubation for 24 h, the cells were replaced with serum-free medium containing the appropriate antibody and incubated for additional 24 h. The supernatants were collected for gelatin zymographic analysis as described. The results are representative of three independent experiments.


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Fig. 6.   Inhibition of MMP-9 proenzyme production by anti-fibronectin and anti-alpha 5beta 1 integrin mAbs in human peripheral blood monocytes. Freshly isolated human peripheral blood monocytes at 1 × 105 were plated in a 48-well plate in serum-supplemented RPMI 1640 medium. The cells were either untreated or treated with PMA (3 nM) or M-CSF (250 units/ml) in the presence or absence of 70 µg/ml anti-fibronectin (anti-FN), anti-beta 1 integrin (anti-beta 1), or anti-alpha 5 integrin (anti-alpha 5) mAb, or with preimmune IgG as a control. After incubation for 24 h, the cells were washed, replaced with serum-free medium containing the appropriate antibody, and incubated for an additional 24 h. The culture fluids were harvested and directly applied to gelatin zymographic analysis. The results are representative of blood monocytes isolated from four different healthy donors.

We assessed the direct role of FN-mediated adherence and spreading in MMP-9 gene expression in HL-525 cells, which constitutively display a more than 10-fold increase in the surface levels of the alpha 5beta 1 integrin compared with the parental HL-60 cells.2 Unlike HL-60 cells, which do not differentiate on immobilized FN without stimulation, HL-525 cells readily attach, spread, and differentiate into macrophages on FN-coated surfaces.2 When MMP-9 gene expression was examined in HL-525 cells cultured on immobilized FN, we found that FN-mediated cell adherence and spreading (Fig. 7A) were sufficient to induce MMP-9 gene expression (Fig. 7B) with concomitant secretion of MMP-9 proenzyme (Fig. 7C). Incubation with the anti-FN mAb, but not with the preimmune IgG, abolished the FN-induced cell adhesion and spreading (Fig. 7A, c, d) with a concomitant loss of MMP-9 gene expression and production of the MMP-9 proenzyme (Fig. 7, B and C). Anti-alpha 5 and anti-beta 1 integrin mAbs were equally effective in inhibiting MMP-9 secretion, whereas the preimmune IgG had little or no effect (Fig. 7C). Although HL-60 cells were able to adhere to FN-coated surfaces in the presence of the divalent cation Mn2+ (which activates alpha 5beta 1 integrin) (46), the adherence was followed neither by cell spreading nor by an induction of MMP-9 gene expression.


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Fig. 7.   Effect of mAbs on cell adhesion and spreading, MMP-9 gene expression, and proenzyme production induced by immobilized fibronectin in HL-525 cells. A, manifestation of cell adhesion and spreading on immobilized FN. The cells (1.5 × 105 cells/ml) were cultured for 24 h either directly in plastic tissue culture wells (Control) or in wells precoated with 20 µg/ml FN in the absence or presence of 70 µg/ml anti-FN mAb or preimmune IgG in serum-supplemented RPMI 1640 medium. a, control; b, on FN; c, FN + anti-FN; d, FN + IgG. 320 ×. B, MMP-9 gene expression. The cells were treated as above. After 24-h incubation, the cells were harvested for total RNA isolation, and the RNA samples (20 µg/lane) were subject to Northern blot analysis as described. C, production of MMP-9 proenzyme. The cells (1.5 × 105 cells/ml) were plated either directly into tissue culture wells (Control) or in FN-coated wells in the presence or absence of 70 µg/ml preimmune IgG or mAb to FN (anti-FN), beta 1 integrin (anti-beta 1), or alpha 5 integrin (anti-alpha 5) in serum-supplemented RPMI 1640 medium. After 24 h, the culture was replaced with serum-free medium containing the appropriate antibody and incubated for additional 24 h before the conditioned media were collected and subjected to gelatin zymogram analysis. The results shown are representative of three independent experiments performed.

Cytoskeletal Integrity Is Required for Induction of MMP-9 Gene Expression during Macrophage Differentiation-- Because the induction of MMP-9 gene expression requires FN-mediated cell adhesion and spreading, we decided to examine the importance of cytoskeletal integrity in this process. We added cytochalasin D or B, which disrupts cytoskeletal structure, to PMA-treated HL-60 cells or HL-525 cells cultured on FN (Fig. 8). Both cytochalasins prevented cell adhesion and spreading induced by PMA in HL-60 cells and by FN in HL-525 cells (data not shown). Accordingly, the MMP-9 production was substantially reduced in HL-60 cells and abolished in HL-525 cells by both cytochalasins, suggesting that cytoskeletal integrity is important for inducing MMP-9 gene expression during macrophage differentiation.


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Fig. 8.   Inhibitory effect of cytochalasin B or D on production of MMP-9 proenzyme in HL-60 cells treated with PMA and in HL-525 cells cultured on fibronectin. HL-60 (4 × 105 cells/ml) or HL-525 (1.5 × 105 cells/ml) cells were pretreated with 1 µM cytochalasin D (CD) or B (CB) for 1 h before and during PMA (3 nM) treatment of HL-60 cells or incubation of HL-525 cells in wells precoated with 20 µg/ml FN. After 24 h incubation, the cells were replaced with serum-free medium containing the cytochalasin and incubated for an additional 24 h prior to collection of culture media for gelatin zymographic analysis. The results shown are representative of three separate experiments performed with HL-60 cells and two with HL-525 cells.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Under physiological and pathological conditions, peripheral blood monocytes emigrate from circulation and mature into specialized tissue macrophages through a series of interactions with endothelial cells and the extracellular matrix (47, 48). One of the key changes during this differentiation is a marked increase in the expression of MMP genes, the MMP-9 gene in particular (5, 6). In our study, secretion of MMP-9 proenzyme, which was detected at low levels in untreated blood monocytes, was markedly enhanced by addition of PMA or M-CSF, both of which promoted cell adhesion and spreading and, subsequently, macrophage differentiation in monocytes (24-26). The basal level secretion of MMP-9 proenzyme by cultured blood monocytes may be attributed to a fraction of cells that spontaneously adhere to tissue culture plates and differentiate into macrophages during a 2-day incubation. This hypothesis is supported by the observation that MMP-9 production increases substantially when spontaneous cell adherence and differentiation are enhanced by extending the incubation time to 7 days (5, 14).

Throughout our study, we noted that induction of MMP-9 gene expression closely paralleled the timing and extent of cell adhesion and spreading. Reagents and conditions that promote cell adhesion and spreading induce or stimulate MMP-9 gene expression; this is particularly manifested in HL-60 cells treated with PMA, in blood monocytes treated with either PMA or M-CSF, and in HL-525 cells cultured in the presence of immobilized FN. On the other hand, reagents that block cell adhesion and spreading suppress MMP-9 gene expression; this is seen with the PKC inhibitor H-7 and with anti-FN and anti-alpha 5beta 1 integrin mAbs. Cytochalasins B and D also prevent cell adhesion and spreading and inhibit MMP-9 production, suggesting that not only cell adhesion and spreading but also cytoskeletal integrity are important for induction of this proteinase during macrophage differentiation.

By using inhibitory mAbs, we have shown that induction of the MMP-9 gene expression by PMA in HL-60 cells or by PMA or M-CSF in blood monocytes, and the basal level secretion of this enzyme in untreated monocytes, all require FN-mediated cell adhesion and spreading. This is further strengthened by our observation that adhesion and spreading of HL-525 cells on immobilized FN are sufficient to evoke MMP-9 gene expression. It appears that this process is mediated by the alpha 5beta 1 integrin without the involvement of the alpha 4beta 1 integrin, because MMP-9 production was blocked by anti-alpha 5 and anti-beta 1 mAbs, but not by the anti-alpha 4 MAb. In support of this finding, we have found that PMA treatment of HL-60 cells results in augmentation of the steady state mRNA and surface protein levels of both alpha 5 and beta 1 integrins (49), whereas the same treatment decreases the surface level of the alpha 4 integrin.2 These findings are in line with those reported by Ferreira et al. (50). In their study, PMA-treated U937 cells demonstrated an enhanced attachment to FN and to an RGDS-containing FN fragment; this enhancement paralleled an increase in the surface expression of the alpha 5 integrin and a loss of cell surface alpha 4 integrin. It should be noted, however, that unlike adherent cells such as fibroblasts and epithelial cells, integrins on the surface of unstimulated leukocytes and leukemic cell lines are not fully functional, a property which is vital for their function (48). In fact, PMA is well known to facilitate leukocyte adhesion to extracellular matrix proteins via different mechanisms without affecting the surface levels of integrins; these mechanisms include activation of surface integrins (51-53) and promotion of post-receptor events such as integrin/cytoskeletal interactions (54). However, the precise mechanisms through which PMA modulates the alpha 5beta 1 integrin function in HL-60 cells and blood monocytes have yet to be established. In addition, PMA was found to induce a 5-fold increase in the level of FN steady state mRNA in HL-60 cells followed by surface manifestation and extracellular deposition of the FN protein (49). Taken together, these data suggest that up-regulation of the alpha 5beta 1 integrin and FN gene expression as well as activation of the surface alpha 5beta 1 integrin are critical steps preceding the induction of MMP-9 gene expression during macrophage differentiation.

Protein kinase C (PKC) plays a central role in PMA- or M-CSF-induced macrophage differentiation in both HL-60 cells and human peripheral blood monocytes (30-32). HL-525 cells, which are deficient in PKC-beta activity (30, 34), thus provide us a useful cell system to define the role of this PKC isozyme in MMP-9 gene expression. PMA fails to induce MMP-9 gene expression as well as cell adhesion and spreading in HL-525 cells, and the PMA resistance is reversed by transfection with a PKC-beta expression plasmid, suggesting that PKC-beta is essential for PMA-induced MMP-9 gene expression during HL-60 differentiation. PKC-beta may act as an upstream signal to activate the Raf-1/mitogen-activated protein kinase cascade which, in turn, leads to activation of AP-1-binding activity by inducing c-jun and c-fos gene expression in HL-60 cells (55). In addition to AP-1 activity, activation of NF-kappa B may also be critical for the differentiation-associated MMP-9 gene expression, since both AP-1 and NF-kappa B sites were required and acted in concert in PMA-induced MMP-9 gene expression in HT-1080 cells (56). In support of this hypothesis, NF-kappa B-binding activity was induced by PMA in HL-60 cells (57), and treatment with pyrrolidine dithiocarbamate, a potent inhibitor of NF-kappa B (58), completely blocked cell adhesion and MMP-9 gene expression induced by PMA in HL-60 cells or by FN in HL-525 cells.3 Studies are currently underway to characterize the role of AP-1- and NF-kappa B-binding activities in regulating the FN/integrin-mediated MMP-9 gene expression during macrophage differentiation.

In summary, we have shown in this study that both PKC activities and FN/integrin-mediated cell adhesion and spreading are required for induction of MMP-9 gene expression during macrophage differentiation in HL-60 cells and human peripheral blood monocytes. Our results suggest that integrin-mediated interaction of blood monocytes with FN plays a critical role in modulating the matrix degrading activities, which are essential for monocyte migration and macrophage tissue remodeling during inflammatory reactions.

    FOOTNOTES

* This work was supported by the U. S. Department of Energy, Office of Biological and Environmental Research, under Contract W-31-109-ENG-38.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.

Dagger To whom communications and requests for reprints should be addressed: Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Ave., Argonne, IL 60439-4833. Tel.: 630-252-3819; Fax: 630-252-3853.

1 The abbreviations used are: MMP, matrix metalloproteinase; ATRA, all-trans-retinoic acid; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody; M-CSF, macrophage-colony stimulating factor; MMP-9, 92-kDa type IV collagenase/gelatinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RGDS, arginyl-glycyl-aspartyl-serine; ECM, extracellular matrix.

2 A. Laouar, C. B. H. Chubb, F. R. Collart, and E. Huberman, manuscript in preparation.

3 B. Xie and E. Huberman, unpublished results.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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