1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Veterans Affairs Medical Center and Emory University School of Medicine, Atlanta, Georgia, 30033; and 2 Division of Respiratory and Critical Care Medicine, Washington University School of Medicine, St. Louis, Missouri, 63110
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ABSTRACT |
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Lung injury is accompanied by increased
deposition of fibronectin (FN) matrices. Activated monocytic cells
recruited to sites of lung injury express integrin receptors for FN
that mediate their interaction with this matrix. One such integrin,
5
1, mediates many of the biological
effects of FN, and its expression may be important for immune cell
function at sites of lung injury. Herein, we examine the expression of
5
1 in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) in the human promonocytic cell
line U-937. We demonstrate that PMA enhanced the adherence of U-937
cells to FN by increasing the expression of both the
5-
and
1-subunit mRNAs and the surface expression of the
protein. In U-937 cells transfected with an
5 promoter-reporter gene, we found
that PMA induced the transcription of the
5 gene by acting on very specific
promoter sequences other than activator protein-1 in a protein kinase
C-dependent manner. Lipopolysaccharide had a similar effect. Modulation
of
5
1 expression may be important for
regulation of monocytic cell function in lung inflammation after injury.
protein kinase C; transcription factor; promoter; fibronectin; phorbol 12-myristate 13-acetate
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INTRODUCTION |
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LUNG INJURY IS CHARACTERIZED by increased vascular permeability and the activation of inflammatory and repair mechanisms involved in host defense (7, 42). One of these mechanisms is tissue remodeling, which involves the increased expression and deposition of extracellular matrix (ECM) molecules (38, 41). Fibronectin (FN) is one such ECM molecule that is rarely found in normal tissues, yet it is deposited in large quantities after injury (38). For example, increased FN deposition is seen in diseased lungs (6, 30, 39), glomerulosclerotic kidneys (4), injured cardiac muscle (20), brain tissue after stroke (27), and atherosclerotic vessels (46, 50).
Another mechanism elicited by lung injury is the accumulation of activated bloodborne monocytic cells (12). Monocytic cells express FN-binding receptors on their surface that are capable of signal transduction, and thus these cells can interact with newly deposited FN matrices (11, 23, 45). The consequences of monocytic cell-FN interactions in injured lungs are unknown; however, FN does affect various cell functions including cell adhesion and migration (1) and cell activation and cytokine expression in vitro (21, 24, 36, 38). The latter observations suggest that by binding to specific receptors on the surface of monocytic cells present in injured tissues, FN could affect their function and thus indirectly modulate inflammatory and tissue repair responses.
In view of the potential significance FN may have on the overall
response of the host lung to injury, a better understanding of the
factors that affect FN expression and deposition is warranted. A more
thorough understanding of the molecules and mechanisms involved in
regulation of cell recognition of extracellular FN matrices is also
necessary. This report explores the latter. FN exerts its effects
primarily by binding to a ubiquitous class of highly conserved
receptors termed integrins, a family of heterodimeric transmembrane
glycoproteins involved in cell-to-cell and cell-to-matrix interactions
(23). One of these integrins, 5
1,
mediates many of the biological effects of FN (43) and thus is the
focus of attention of our work.
It has been demonstrated (19) that differentiation of human U-937
promonocytic cells with phorbol 12-myristate 13-acetate (PMA) enhances
their ability to bind to FN substrates and that this stimulatory effect
is due to increased surface expression of
5
1 as determined by immunofluorescent
analysis. The mechanisms responsible for this stimulatory effect have
not been entirely elucidated. Herein, we use this system in an attempt
to begin to delineate the mechanisms involved in regulation of
5
1 expression in monocytic cells. Our
work demonstrates that by activating protein kinase C (PKC), PMA
induces transcription of the
5-integrin subunit gene, a
process that requires specific elements to be present within the
5 gene promoter. This results in
accumulation of
5 (and
1)-integrin
subunit mRNA followed by increased expression of the functional protein
heterodimer (i.e.,
5
1) at the cell surface and, ultimately, enhancement of the ability of cells to bind to
FN substrates.
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METHODS |
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Reagents. All reagents were purchased from Sigma (St. Louis,
MO) or Fisher Scientific (Philadelphia, PA) unless otherwise specified.
FN was isolated from bovine plasma by affinity chromatography on
gelatin-Sepharose as described previously (40). The purity and
molecular weight of FN were verified by SDS-PAGE. The monoclonal antibodies clone P1D6 (anti-5), clone P1E6
(anti-
2), and clone P4C10 (anti-
1) were
purchased from GIBCO BRL (Life Technologies, Gaithersburg, MD), and
monoclonal antibody anti-
2 was from Chemicon (Temecula, CA).
Cell culture and treatment. Human monocytic/macrophage cells from histiocytoma U-937 (American Type Culture Collection) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic (100 U/ml of penicillin G sodium, 100 U/ml of streptomycin sulfate, and 0.25 µg/ml of amphotericin B) and incubated in a humidified 5% CO2 incubator at 37°C. For transfection studies, cells were washed twice and replated in 10 ml of Cellgro complete serum-free medium (MediaTech, Herndon, VA). The cells were treated with various concentrations of PMA (0-250 nM; Sigma). All experiments were repeated at least three times.
To avoid any confounding effects from lipopolysaccharide (LPS) contamination, all treatment materials and culture media were screened with a Limulus-based endotoxin assay with a sensitivity of 0.06 ng/ml (Endotect-Schwarz/Mann Biotech, Cleveland, OH). Reagents were found to remain endotoxin free throughout all experiments.
Adhesion assay. U-937 cells were treated with PMA (25 nM, 18 h,
37°C), washed, and submitted to a cell adhesion assay on plates coated with FN (0-50 µg/ml) as previously described (40). After 1 h, nonadherent cells were washed, and adherent cells were quantified with a colorimetric assay that detects the intracellular enzyme hexosaminidase as described by Landegren (31). To determine the role of
5
1 on cell adhesion to FN, some cells
were pretreated with either the anti-human
5 antibody
P1D6 (100 µg/ml, 37°C, 30 min) or a control IgG before the cells
were placed on FN-coated plates.
Immunoprecipitation analysis. U-937 cells were treated with 25 nM PMA for 18 h at 37°C. Afterward, the cells were washed, and
their surfaces were iodinated with lactoperoxidase and glucose oxidase
(33). The 5
1-integrin receptor was
immunoprecipitated from Triton X-100 extracts of the U-937 cells with
the use of the anti-
5-subunit antibody with protein
G-Sepharose as previously described (40). The precipitates were washed,
and antigen-antibody complexes were released by boiling in sample
buffer for 5 min. Supernatants were collected and subjected to
electrophoresis in a 7% polyacrylamide gel followed by
autoradiography. Densitometric scanning was performed for quantitative analysis.
Fluorescence-activated cell sorting. For fluorescence-activated
cell sorting (FACS), cells were immunostained with the
anti-5 or anti-
1 antibodies as previously
described (40). Afterward, the cells were fixed with 1%
paraformaldehyde and submitted to FACS in a Becton Dickinson scanner
with the Lysis II program (version 1.1; Becton Dickinson,
Bedford, MA).
RNA isolation and Northern blotting. Total cellular RNA was
extracted from U-937 cells with the method of Chomczynski and Sacchi
(9). Briefly, U-937 cells were suspended in RPMI 1640 medium
supplemented with 10% heat-inactivated FBS at 5 × 106 cells/ml and treated with 25 nM PMA for 2, 4, 6, and 8 h. After treatment, cells were harvested, supernatants were
removed, and cells were resuspended in 4 M guanidine
thiocyanate containing 2-mercaptoethanol. Total RNA was extracted with
phenol-chloroform-isoamyl alcohol followed by precipitation with cold
isopropanol. RNA was resuspended in 10 mM EDTA-0.5% SDS and
quantitated by measuring absorbance at an optical density of 260 nm. Before electrophoresis, RNA was dissolved in an equal
volume of RNA cocktail (1× formaldehyde gel running buffer,
17.5% formaldehyde, and 50% formamide) containing 0.01 µg/ml of
ethidium bromide, electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde, and electroblotted onto a nylon membrane (32, 48).
5 (1.2 kb) and
1 (1.6 kb) cDNA (3) probes
were labeled with [32P]dCTP (ICN) with a
random-primer DNA labeling kit (Stratagene, La Jolla, CA) and purified
through push columns (Stratagene). The membrane was hybridized with the
labeled probe (1-10 × 106
counts · min
1 · ml
1)
at 42°C in a rotisserie oven for 24 h, washed twice with 2× SSC (0.3 M sodium chloride and 0.03 M sodium citrate, pH 7.0) at
55°C, twice with 2× SSC containing 0.1% SDS at 55°C, and
twice with 0.1× SSC at 55°C. Blots were exposed to X-ray film
for 24 h at
70°C with intensifying screens. Densitometric
scanning was performed with a laser densitometer (Molecular Dynamics,
Sunnyvale, CA). Actin controls were used to verify even loading of
gels, and densitometry numbers were corrected for actin control.
Transfection of U-937 cells. All promoter sequences were taken
from the p5-CAT reporter vectors as described previously (3). Briefly, the promoters from the p
5-CAT constructs (
923,
178,
92,
41,
27, and
1 bp) were
digested with the appropriate restriction endonucleases, purified by
agarose gel electrophoresis, and ligated into the pGL2 luciferase (Luc)
reporter vector (Promega, Madison, WI) with T4 DNA ligase. All
p
5-Luc promoter constructs were sequenced to ensure that no
mutations occurred during construction.
U-937 cells were maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS and 1% antibiotic-antimycotic and incubated in a
humidified 5% CO2 incubator at 37°C. Cells were transfected with the 0.9-kb promoter construct [p5(
923
bp)-Luc] or 5'-deletion constructs of the human
5 promoter DNA containing sequences
from positions
923 to +23, which were connected to a Luc
reporter gene (3). Cells were collected and transferred to Cellgro
complete serum-free medium supplemented with 10 mM dextrose and 0.1 mM
dithiothreitol (DTT) to a final concentration of 6 × 107 cells/ml along with 40 µg of
5 promoter/Luc DNA and 20 µg of
-galactosidase DNA. The cells were then electroporated with 400 V
and 1,075 µF. Electroporated cells were pooled, divided into aliquots
in 24-well plates, and incubated with 25 nM of PMA for 18 h at
37°C. Cells were then harvested, washed with PBS, and resuspended
in cell lysis buffer (25 mM Tris phosphate, pH 7.8, 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid,
10% glycerol, and 1% Triton X-100). Luc activity was assessed with a
Dynatech ML 3000 microtiter plate luminometer. Results were recorded as
relative Luc units standardized for transfection efficiency with
-galactosidase activity.
DNA mobility shift assay. U-937 cells were grown as described in Transfection of U-937 cells. Cells were treated with 25 nM PMA for 18 h at 37°C. Afterward, they were washed with ice-cold PBS, and nuclear binding proteins were extracted with the method of Dignam et al. (18). Proteins were extracted in buffer containing 0.42 M NaCl, 1.5 M MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 1% aprotinin, 5 µg/ml of pepstatin A, and 20 mM HEPES, pH 7.9. Protein concentrations were determined by the Bradford (5) method with Coomassie blue G-250 (Bio-Rad, Hercules, CA).
Double-stranded consensus oligonucleotides (20 µg) to activator
protein (AP)-1 (c-jun;
5'-CGCTTGATGAGTTCAGCCGGAA-3') were radiolabeled with
[-33P]ATP with T4 polynucleotide kinase
enzyme. Nuclear protein (2.5-10 µg) was incubated with
radiolabeled AP-1 (150,000 counts · min
1 · ng
1)
for 30 min at room temperature in a reaction mixture containing 15 mM
HEPES, 90 mM KCl, 1 mM EDTA, 1 M DTT, 5% glycerol, and 0.1 µg/reaction of poly(dI-dC). DNA-protein complexes were separated on
6% native polyacrylamide gels (20:1 acrylamide-to-bis-acrylamide ratio) in low ionic strength buffer (22.25 mM Tris borate, 22.25 mM
boric acid, and 500 mM EDTA) for 2-3 h at 4°C at 10 V/cm2. Gels were fixed in a 10% acid-10% methanol
solution for 10 min, dried under vacuum, and exposed to X-ray film.
Radiolabeled DNA-protein complexes were extracted from the gels and
quantitated by scintillation counter.
Other methods. Calphostin C (Sigma), a potent specific
inhibitor of PKC, was used to evaluate the role of PKC in the PMA
stimulation of U-937 cells (29). Activation of this agent is dependent
on exposure to light; thus in control experiments, cells were exposed to "inactive" calphostin C, which indicates treatment in the
absence of light. Cells were pretreated for 30 min with calphostin C (1 × 107 M) in the presence (active) or absence
(inactive) of light, then stimulated overnight with PMA again in the
presence or absence of light.
To test for the role of the transcription factor AP-1, AP-1 oligonucleotides (20 µg) (Oligos Etc., Guilford, CT) were cotransfected with plasmid DNA to compete for nuclear factors present on stimulation with PMA. Complementary oligonucleotides were annealed in 200 mM NaCl by being heated to 94°C for 7 min and slowly cooled to 4°C.
In some experiments, 5 expression
was evaluated in cells exposed to 5 ug/ml of LPS and processed as
described above for PMA-treated cells.
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RESULTS |
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PMA stimulates the adhesion of U-937 cells to FN. Because a
previous report (19) had demonstrated that PMA enhances the adhesion of
U-937 cells to FN, we set out to confirm these findings in our system.
U-937 cells were treated with PMA for 18 h (25 nM, 37°C, 5%
CO2), washed, and allowed to adhere to plates coated with
increasing concentrations of FN (0-50 µg/ml). As expected, PMA
treatment stimulated the adhesion of cells to FN 2.5-fold compared with
that of untreated cells (Fig. 1A).
Notice that PMA-treated cells demonstrated enhanced adhesion at all
concentrations of FN. The adhesion plateaued at a FN concentration of 5 µg/ml for control cells and at ~15 µg/ml for PMA-treated cells.
Cell adhesion to BSA-coated plates was poor and was not altered by PMA
(data not shown).
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To determine whether 5
1 was responsible
for the observed constitutive and PMA-stimulated adhesion of U-937
cells to FN (50 µg/ml), experiments were performed with cells
pretreated with the neutralizing anti-
5 antibody clone
P1D6 (Fig. 1B). Note that this antibody blocked the adhesion of
untreated and PMA-treated U-937 cells to FN, indicating that
5
1 was indeed responsible for their
adhesion to this substrate. An antibody against the
2-integrin subunit (Fig. 1B) and a control
murine IgG (data not shown) had no effect on cell adhesion (data not shown).
PMA treatment is associated with increased surface expression of
the
5
1
FN receptor. To test whether enhanced binding of PMA-treated U-937
cells paralleled an increase in cell surface expression of the
5
1 receptor, we analyzed the surface expression of both the
5- and
1-subunits
of this receptor using immunoprecipitation analysis. Cells were treated
with PMA for 18 h, immunoprecipitated with anti-
5 (clone
P1D6) antibodies, and submitted to SDS-PAGE followed by
autoradiography and densitometric analysis. Figure
2A demonstrates that untreated
cells show constitutive expression of both
5- and
1-subunits, which migrate at the expected molecular
masses of 150 and 130 kDa, respectively. PMA treatment resulted in increased expression of both
5 (77%
increase compared with control cells as determined by densitometric
analysis of the bands) and
1 (47% increase compared
with control cells) protein. Neither control IgG nor the
2-integrin subunit was affected by PMA stimulation. In
fact, the
2-subunit band is not visible in the control
cells and only faintly visible in the PMA-treated cells. Consistent
with the above data, FACS revealed a rightward shift in the curves for
both
5 and
1 in cells exposed to PMA compared with that of untreated cells (Fig. 2B).
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PMA induction of
5
1
protein expression was related to increased mRNA expression of
the
5- and
1-subunits. Having confirmed that PMA stimulation indeed enhances binding of
cells to FN and that this effect is at least partially mediated by
upregulation of
5
1 protein expression at
the cell surface, we set out to investigate the mechanisms responsible
for the stimulatory effects of PMA. We first examined the effect of PMA
on
5 and
1 mRNA expression. Northern blot
analysis of U-937 cells treated with PMA for 2, 4, 6, and 8 h
demonstrated increased accumulation of mRNAs encoding for both the
5- and
1-subunits (Fig.
3). Both
5 and
1 mRNA levels increased as early as 2 h, peaked 4 h
after PMA exposure, and remained slightly elevated afterward compared with control level. The peak
5 mRNA expression showed a
79% increase above control level, whereas the peak
1
expression was 108% above control level.
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PMA induction of
5
1
is regulated at the transcriptional level. The data presented above
suggest that the effects of PMA on
5
1
expression occur at the level of gene expression. To explore this
issue, we transfected U-937 cells with p
5(
0.9kb)-Luc, a
construct that contains a portion of the human
5 gene promoter connected to a Luc
reporter gene. After transfection, the cells were stimulated with PMA
as described in METHODS followed by
harvesting and Luc activity measurement. We observed that PMA indeed
induced the transcription of the
5
gene, with a mild increase detected as early as 2 h and the highest
increase observed at 18 h (Fig.
4A). The stimulatory effect of PMA
was dose dependent, with the highest stimulatory level observed at 25 nM (Fig. 4B) and decreased stimulation at higher
concentrations. Stimulation of cells with FN instead of PMA did not
affect transcription of the gene (data not shown).
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PMA induction of
5-subunit gene
transcription is mediated via PKC activation. In general, PMA acts
by entering the cells and activating PKC (8). However, PKC-independent
pathways of PMA-induced gene expression have been documented (17). To
determine whether the effects of PMA in our system were specifically
related to PKC activation, experiments were performed with transfected cells that were pretreated with a selective PKC inhibitor, calphostin C, before stimulation with PMA. Figure 5
demonstrates that activated calphostin C alone had no effect on
constitutive
5 gene transcription.
However, activated calphostin C abolished the stimulatory effect of
PMA. Cell viability was not affected by treatment with calphostin C as
determined by trypan blue exclusion analysis (data not shown). Inactive
calphostin C did not abolish the PMA-induced
5 response.
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Stimulatory effect of PMA on
5 gene transcription is
dependent on specific elements present within the
5 gene promoter other than
AP-1. To learn more about the transcriptional elements involved in
regulation of the expression of the
5 gene, U-937 cells were
transfected with the 923-bp
5
promoter construct [p
5(
938)-Luc] or
5'-deletion constructs starting at
178
[p
5(
178)-Luc],
92
[p
5(
92)-Luc],
41
[p
5(
41)-Luc],
27
[p
5(
27)-Luc], and
1
[p
5(
1)-Luc] bp downstream from the
transcriptional start site. Figure 6
depicts the results of these experiments. As before, PMA stimulated the
expression of the
923-bp promoter construct. The intensity of
the response diminished slightly with further deletions; however, there
was a drastic drop in the expression of the gene with plasmids
p
5(
41)-Luc, p
5(
27)-Luc, and p
5(
1-Luc), indicating the presence of an important regulatory element in promoter
sequences between
92 and
41 bp. This element appears important for both the PMA-induced response and the constitutive expression of the gene because little expression was detected in
unstimulated cells transfected with the smaller 5'-deletion constructs. A search with the HIBIO MacDNASIS PRO computer software (Hitachi Software Engineering America, San Bruno, CA) revealed no known
transcriptional elements in this region except for a putative AP-1 site
located
45 bp downstream from the transcriptional start site.
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Potential role of AP-1 in PMA induction of
5 gene transcription.
Having demonstrated a role for PKC in PMA-induction of
5 gene transcription, we examined
the importance of the transcription factor AP-1 in this process. AP-1
was tested for two reasons. First, it is a potent transcription factor
that is induced in response to PKC activation by PMA (22). Second, an
AP-1 site has been identified in the
5 gene promoter near the site of
transcriptional initiation at position
45 (3), which is
contained in the p
5(
92)-Luc construct. The expression of this
construct is stimulated by PMA, whereas the p
5(
41)-Luc construct (which does not contain AP-1) is not.
A demonstration of the nuclear translocation of AP-1 in response to PMA
can be seen in Fig. 7, which shows a DNA
electrophoresis mobility gel shift assay prepared with nuclear extracts
of PMA-treated and untreated cells. Note that the intensity of the band
for AP-1 in the PMA-treated cells is higher than that in untreated
cells (control). The addition of an AP-1 competitor oligonucleotide diminished the response. This indicates that PMA induces nuclear translocation of the AP-1 transcription factor.
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To determine the potential role of AP-1 in
5 gene transcription, cells were
cotransfected with the human
5 promoter construct and with a competing AP-1 oligonucleotide before treatment with PMA. The rationale is that if PMA
induction of
5 is due to AP-1, the
introduction of "decoy" AP-1 oligonucleotides into the system
would diminish the response. We found that cotransfection of cells with
the AP-1 oligonucleotide did not affect the PMA induction of
5 transcription, indicating that
although translocation of AP-1 to the nucleus occurs in response to
PMA, this particular transcription factor is not involved in the
increased transcription of the
5
promoter in response to PMA (Fig. 8). In
other experiments, we have demonstrated that cotransfection of AP-1
oligonucleotides in U-937 cells transfected with the interleukin (IL)-1
promoter results in a decrease in the FN-induced upregulation of IL-1
gene transcription (data not shown).
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Induction of 5 gene
transcription by LPS. To examine the relevance of the pathways
described, we studied the effects of LPS on
5 gene transcription using the same
system. LPS is a component of the outer wall of gram-negative bacteria
that is considered responsible for many of the proinflammatory
mechanisms activated during gram-negative bacterial sepsis. First, we
demonstrated that cells exposed to LPS bound more often to FN-coated
substrates than to untreated cells (P < 0.001; Fig.
9A). As for PMA, this effect was
blocked by an anti-
5 antibody but not by an antibody to
2 (data not shown). Next, we showed that the exposure of
transfected U-937 cells to LPS induced the transcription of the
5 gene (Fig. 9B). This
effect was blocked by the PKC inhibitor calphostin C but not by its
inactive form.
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DISCUSSION |
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This report examines the mechanisms of expression of the FN receptor
5
1 in human promonocytic U-937 cells
exposed to PMA. We showed that PMA enhanced the adhesion of U-937 cells
to FN-coated substrates. This effect was associated with an increase in
the surface expression of
5
1 receptors as
determined by immunoprecipitation analysis and FACS. These receptors
appeared to be responsible for 100% of the enhanced cellular adhesion
to FN as evidenced by the ability of an anti-
5 antibody
to abolish the stimulatory effect of PMA. We then set out to
investigate the mechanisms by which PMA enhances the expression of
5
1. Using Northern blotting, we
demonstrated that PMA induces the accumulation of mRNAs encoding for
both the
5- and
1-subunits of the
5
1 receptor. This suggested that PMA
worked by enhancing the transcription of
5 and
1 genes and/or by decreasing the
degradation of the relevant mRNA. To test for the first possibility, we
examined the effects of PMA on transcription of p
5(
0.9kb)-Luc
introduced into U-937 cells. Using this system, we found that PMA
induces transcription of the gene in a dose- and time-dependent manner. Stimulation of transcription was blocked by a selective PKC inhibitor, indicating a role for this serine/threonine protein kinase in the
process. Finally, we demonstrated that induction of
5 by PMA requires specific sequences within the 51 bp of the
5 gene promoter region between
92 and
41 bp downstream of the transcriptional start
site. This region includes a consensus AP-1 site located at position
45 bp of the promoter. Altogether, these data indicate that PMA
induces expression of
5
1 receptors on the
surface of U-937 cells by activating PKC and inducing transcription of
the
5 gene, which in turn results
in accumulation of the relevant mRNA, synthesis of the
5
protein, and, presumably, assembly of
5- with
1-subunits followed by expression of the functional
heterodimer at the cell surface.
PMA and U-937 cell adhesion to FN. Our findings help explain
the observations made by Ferreira et al. (19), who
demonstrated that PMA-treated U-937 cells show enhanced adhesion to FN
as a result of an increase in the surface expression of the
5
1 receptor. These investigators also
demonstrated that the stimulatory effect of PMA is not generalized to
all FN receptors because it reduced the expression of the
4
1-integrin, a receptor that binds to the
CS-1 site located in the carboxy-terminal end of the FN molecule (51).
Our findings regarding PMA induction of
5
1 are also consistent with those of
Bellon et al. (2), who showed that myeloid cell lines have increased
5 protein (by FACS analysis) in response to PMA.
Interestingly, we found that in PMA-treated U-937 cells, the increases
in mRNA and protein were similar for
5 (77 and 79%,
respectively) but different for
1 (47 and 108%, respectively). We believe this is related to differences in the regulation of gene transcription and protein processing pathways related to
5- and
1-integrin subunits. In
addition, in contrast to the
5-subunits,
1-subunits are stored in granules in immune cells such
as monocytes/macrophages (45). Because of this, PMA may induce the
expression of
-integrin subunits other than
5 that
could be coupled with newly made or stored
1-subunits
for surface expression. This may explain the discrepancy between the levels of mRNA and protein detected for
1.
Of note, others have demonstrated that the enhancement of FN adhesion
by PMA may be attributed not exclusively to increased receptor numbers
in all cells but also to increased receptor affinity, postreceptor
events, or alterations in receptor internalization and degradation. For
example, the work by Danilov and Juliano (14) demonstrated that Chinese
hamster ovary cells treated with PMA showed increased binding to FN,
yet the expression of 5
1 at the cell
surface was not increased nor was the receptor affinity for FN. They
postulated that in this particular cell line, a postreceptor event was
responsible for the observed increase in integrin-mediated adhesion to
FN of cells exposed to PMA. In other work, Dalton et al. (13)
demonstrated that
5
1 expression in
fibroblasts is controlled by adhesion to the extracellular substrate,
which prevents integrin internalization and degradation. The effect of
PMA on this process is unknown.
Independent of the mechanism(s) involved, it is clear that PMA induces
increased adhesion of U-937 cells to FN substrates. The increased
expression of 5
1 (and perhaps other
receptors) may explain the altered phenotype of U-937 cells when
treated with PMA. However, the effects of PMA are not solely on cell
morphology but also on biochemical events. In U-937 cells treated with
PMA, alterations in membrane antigen expression and a macrophage-like respiratory burst are induced (34). It is unclear whether increased expression of
5
1 with its recognized
signal-transducing ability affects any of these PMA-induced events
attributed to accelerated cellular differentiation.
Mechanisms of PMA-induced
5 gene transcription.
Expression of
5
1 is dependent on
transcription and translation of the
5-subunit followed
by noncovalent association with the
1-subunit. This complex is then transferred to the cell surface. The regulatory elements that control integrin (including
5) gene expression in monocytic
cells are unclear (26). Thus far, characterization of the
5 promoter gene has shown that
several elements required for transcription are located between
positions
178 and
27. The promoter contains consensus
binding sites for several transcription factors, including AP-1, the
Ets family of protooncogenes, Sp-1, and AP-2 (3). These
sequences are all included in our promoter construct. The role of these
elements in
5 gene transcription
remains undefined, particularly in monocytic cells, because most of the
data regarding this process has been obtained from fibroblasts or
malignant cell lines (15, 16, 28, 49). Our data with 5'-deletion
constructs of the
5 gene promoter
suggest that there is an important regulatory site located between
positions
92 and
41. The only known cis-acting element present in this region is AP-1. However, the cotransfection experiments indicate that AP-1 is not a necessary transcription factor
for PMA-induced enhancement of
5 expression. Therefore, we believe that an unidentified site present within this region is
important for constitutive as well as PMA-induced transcription of the
gene. Deletional mutagenesis analysis will be required to further
delineate this putative element. PMA is known to induce transcription
factors other than AP-1, such as the pituitary-specific factor
Pit-1/GHF-1 and a member of the Ets family of transcription factors, Ets-1 (10, 35).
One interesting observation we made is that PMA appeared to induce
5 mRNA accumulation before the observed maximal increase in expression of the gene. This suggests that the accumulation of mRNA
encoding for
5 occurs very early in cells treated with PMA and/or that its accumulation may be due to other mechanisms in
addition to increased transcription of the gene. Decreased RNA
degradation could be involved. This would be similar to a prior study
(37) that suggested a decrease in
5-subunit mRNA degradation in response to transforming growth factor-
1 stimulation. Another observation is that higher doses of PMA did not result in
further increases in
5 gene
transcription; this may be a result of downregulation of PKC, which may
occur by stimulation with higher doses of PMA (52). We also noted an
increase in constitutive expression of the p
5(
92)-Luc
construct compared with the larger construct p
5(
178)-Luc,
suggesting the presence of an important regulatory site(s) for
constitutive expression of
5 in this region. This region
contains two AP-2 sites and 2 Sp-1 sites, but their role in control of
5 expression is unknown.
Implications for altered
5
1
expression on the surface of monocytes during inflammation. The
function of FN is likely to be relevant to acute and chronic forms of
lung injury because increased FN expression is observed under both types of circumstances (6, 30, 38, 39, 41, 42). The effects of FN on cell activity in vivo are unclear, yet based on its in
vitro activity, several reasonable hypotheses can be proposed (38). By
binding to specific integrin receptors, FN may affect many immune and
nonimmune cell functions. For example, FN and FN fragments are
chemotactic to neutrophils, fibroblasts, and monocytes. In addition, FN
may serve as a temporary substrate for the adhesion, migration, and
proliferation of incoming immune cells, epithelial cells, and
fibroblasts in injured lungs. Finally, FN can affect proinflammatory
cytokine expression.
In view of the possible cellular effects of FN, regulation of the
expression of its receptor 5
1 in
monocytes may affect the course of the inflammatory response. The true
biological relevance of the observations reported herein remains
unclear, and the extrapolation of our findings in transformed cells to
monocytes/macrophages should be done with caution. Nevertheless, many
biological agents could affect
5
1
expression via pathways common to those stimulated by PMA in U-937
cells. LPS, for example, is a component of the outer wall of
gram-negative bacteria that can induce monocyte/macrophage activation
via PKC-dependent pathways (44). LPS has been shown to affect the
expression of
5
1 and other integrins in
lung macrophages (25). In this report, we show that LPS can induce the
transcription of the
5 gene in
U-937 cells, a process that was associated with increased adhesion to
FN substrates. By inducing the transcription of
5 in
these cells, LPS may facilitate the migration and invasion of
monocytes/macrophages into infected tissues. Some cytokines such as
IL-1
and tumor necrosis factor-
are also produced in high
quantities during lung injury, exert cellular effects via PKC (47), and
are capable of stimulating the expression of various integrins
including
5
1. Transforming growth
factor-
1, for example, stimulates not only FN production in human
lung fibroblasts but also increased
5
1
protein synthesis and mRNA levels (43).
A better understanding of the regulation of integrin expression would enhance our knowledge of the mechanisms by which integrins and ECMs affect the function of immune and nonimmune cells. This may lead to subsequent elucidation of the role of integrins and ECMs in lung injury and repair.
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ACKNOWLEDGEMENTS |
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We thank William Schuyler and Susan Roser for technical assistance.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-51639 (to J. Roman).
B. K. Boles was a recipient of the Fellowship Award from the Georgia Lung Association and a National Research Service Award from the National Institutes of Health.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Roman, Atlanta VA Medical Center (150-P), 1670 Clairmont Rd., Decatur, GA 30033 (E-mail: roman-rodriguez.jesse{at}atlanta.va.gov).
Received 15 January 1998; accepted in final form 23 November 1999.
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