From the Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833
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ABSTRACT |
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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)--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-
expression plasmid restored PKC-
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
5
1
integrin. HL-525 cells, which constitutively display high levels of
surface
5
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
5
1
integrin-mediated interaction of immature hematopoietic cells with FN
plays a critical role in modulating matrix-degrading activities during
macrophage differentiation.
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INTRODUCTION |
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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-, 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-, and macrophage-colony
stimulating factor (M-CSF) (16, 20). These functions of FN are mediated
by two surface receptors,
5
1 and
4
1 integrins, both of which are present
on monocytes and macrophages (21). The
5
1
integrin recognizes the cell-binding domain of the FN molecule that
contains the arginyl-glycyl-aspartyl-serine (RGDS) sequence, whereas
the
4
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-, 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-
gene expression (32). Treatment with
all-trans-retinoic acid, which enhances the PKC-
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-
expression and may be rendered PMA-susceptible by treatment
with 1,25-dihydroxyvitamin D3, which similarly increases
the expression of PKC-
. Recently, we have shown that restoration of
the PKC-
expression in HL-525 cells by PKC-
gene transfection
restored the PMA responsiveness, suggesting the essential role of
PKC-
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 5
1 integrin
signaling, with little contribution of the
4
1 integrin. Furthermore, in HL-525
cells, which constitutively express high levels of surface
5
1 integrin, we found that their adherence and spreading on FN were sufficient to induce
MMP-9 gene expression and that PKC-
is essential for the
production of this proteinase during PMA-induced macrophage
differentiation.
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EXPERIMENTAL PROCEDURES |
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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 1 (K20, mouse
IgG2a),
4 (HP2/1, IgG1), and
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-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-
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 [-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.
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RESULTS |
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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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PKC- 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.
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Cell Adhesion and Spreading Mediated by Fibronectin and
5
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
5
1
integrin demonstrated similar inhibitory efficacy (Fig. 5B). On the contrary, neutralizing anti-
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-
5, and
anti-
1 integrin mAbs but not by the preimmune IgG (Fig.
6). Again, anti-
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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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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DISCUSSION |
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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-5
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 5
1 integrin without
the involvement of the
4
1 integrin,
because MMP-9 production was blocked by anti-
5 and
anti-
1 mAbs, but not by the anti-
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
5 and
1 integrins
(49), whereas the same treatment decreases the surface level of the
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
5 integrin and a loss
of cell surface
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
5
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
5
1 integrin and
FN gene expression as well as activation of the surface
5
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-
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-
expression plasmid, suggesting that PKC-
is essential for PMA-induced MMP-9 gene expression during HL-60 differentiation. PKC-
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-
B may also be
critical for the differentiation-associated MMP-9 gene
expression, since both AP-1 and
NF-
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-
B-binding activity was induced by
PMA in HL-60 cells (57), and treatment with pyrrolidine
dithiocarbamate, a potent inhibitor of NF-
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-
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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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