Phorbol ester-induced U-937 differentiation: effects on integrin alpha 5 gene transcription

Bonnie K. Boles1, Jeffrey Ritzenthaler1, Thomas Birkenmeier2, and Jesse Roman1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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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, alpha 5beta 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 alpha 5beta 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 alpha 5- and beta 1-subunit mRNAs and the surface expression of the protein. In U-937 cells transfected with an alpha 5 promoter-reporter gene, we found that PMA induced the transcription of the alpha 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 alpha 5beta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha 5beta 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 alpha 5beta 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 alpha 5beta 1 expression in monocytic cells. Our work demonstrates that by activating protein kinase C (PKC), PMA induces transcription of the alpha 5-integrin subunit gene, a process that requires specific elements to be present within the alpha 5 gene promoter. This results in accumulation of alpha 5 (and beta 1)-integrin subunit mRNA followed by increased expression of the functional protein heterodimer (i.e., alpha 5beta 1) at the cell surface and, ultimately, enhancement of the ability of cells to bind to FN substrates.


    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-alpha 5), clone P1E6 (anti-alpha 2), and clone P4C10 (anti-beta 1) were purchased from GIBCO BRL (Life Technologies, Gaithersburg, MD), and monoclonal antibody anti-beta 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 alpha 5beta 1 on cell adhesion to FN, some cells were pretreated with either the anti-human alpha 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 alpha 5beta 1-integrin receptor was immunoprecipitated from Triton X-100 extracts of the U-937 cells with the use of the anti-alpha 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-alpha 5 or anti-beta 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). alpha 5 (1.2 kb) and beta 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 palpha 5-CAT reporter vectors as described previously (3). Briefly, the promoters from the palpha 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 palpha 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 [palpha 5(-923 bp)-Luc] or 5'-deletion constructs of the human alpha 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 alpha 5 promoter/Luc DNA and 20 µg of beta -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 beta -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 [gamma -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 × 10-7 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, alpha 5 expression was evaluated in cells exposed to 5 ug/ml of LPS and processed as described above for PMA-treated cells.


    RESULTS
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INTRODUCTION
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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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Fig. 1.   Phorbol 12-myristate 13-acetate (PMA) stimulates adhesion of U-937 cells to fibronectin (FN) via alpha 5beta 1. A: U-937 cells were exposed to PMA (25 nM, 37°C, 18 h) and submitted to a cell adhesion assay on plates coated with indicated concentrations of FN. Note that PMA enhanced U-937 cell adhesion to FN by >2.5-fold over control untreated U-937 cells. Peak effect was observed at a FN concentration of 5 µg/ml. B: PMA-treated cells were layered on FN-coated plates (50 µg/ml) and allowed to adhere for 1 h in presence and absence of anti-alpha 5 antibody P1D6 (100 µg/ml). Afterward, cells were washed, and adherent cells were quantified. Note that anti-alpha 5 antibody P1D6 blocked adhesion of untreated and PMA-treated U-937 cells to FN, indicating that alpha 5beta 1 was responsible for their adhesion to FN. Anti-beta 2 antibody had no effect.

To determine whether alpha 5beta 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-alpha 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 alpha 5beta 1 was indeed responsible for their adhesion to this substrate. An antibody against the beta 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 alpha 5beta 1 FN receptor. To test whether enhanced binding of PMA-treated U-937 cells paralleled an increase in cell surface expression of the alpha 5beta 1 receptor, we analyzed the surface expression of both the alpha 5- and beta 1-subunits of this receptor using immunoprecipitation analysis. Cells were treated with PMA for 18 h, immunoprecipitated with anti-alpha 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 alpha 5- and beta 1-subunits, which migrate at the expected molecular masses of 150 and 130 kDa, respectively. PMA treatment resulted in increased expression of both alpha 5 (77% increase compared with control cells as determined by densitometric analysis of the bands) and beta 1 (47% increase compared with control cells) protein. Neither control IgG nor the alpha 2-integrin subunit was affected by PMA stimulation. In fact, the alpha 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 alpha 5 and beta 1 in cells exposed to PMA compared with that of untreated cells (Fig. 2B).



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Fig. 2.   PMA treatment is associated with increased surface expression of alpha 5beta 1 FN receptor. Cell surface expression of alpha 5beta 1 was analyzed with immunoprecipitation (A) and fluorescence-activated cell sorting (FACS; B) as described in METHODS. A, top: PMA-treated (+PMA) U-937 cells (25 nM, 37°C, 18 h) and control untreated cells were submitted to immunoprecipitation analysis with P1D6. P1D6 immunoprecipitated 150 and 130-kDa proteins as expected for alpha 5- and beta 1-subunits, respectively, of alpha 5beta 1-integrin. Control antibody (Ab4) immunoprecipitated nonspecific high-molecular-mass bands. Anti-alpha 2 antibody did not immunoprecipitate significant amounts of this integrin subunit. Note that PMA appeared to induce a high-molecular-mass protein (~202 kDa) that was immunoprecipitated nonspecifically by IgG. A, bottom: relative expression of these proteins as measured by densitometric analysis of the bands. Note that PMA induced a 77% increase in alpha 5 expression and a 47% increase in beta 1 expression. B: PMA-treated cells were stained with anti-alpha 5 and anti-beta 1 antibodies and submitted to FACS. Note that curves for alpha 5 (left) and beta 1 (right) shifted right [indicating an increase in fluorescence intensity height (FL1-H)], suggesting an increase in protein expression.

PMA induction of alpha 5beta 1 protein expression was related to increased mRNA expression of the alpha 5- and beta 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 alpha 5beta 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 alpha 5 and beta 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 alpha 5- and beta 1-subunits (Fig. 3). Both alpha 5 and beta 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 alpha 5 mRNA expression showed a 79% increase above control level, whereas the peak beta 1 expression was 108% above control level.


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Fig. 3.   PMA induction of alpha 5beta 1 protein expression is related to increased mRNA expression of alpha 5- and beta 1-subunits. Top: Northern blot analysis of U-937 cells treated with PMA for 2, 4, 6, and 8 h showed increased accumulation of mRNA encoding for both alpha 5- and beta 1-subunits. Note increase as early as 2 h and peak at 4 h, with a slight increase remaining at 8 h. Bottom: densitometric units of gels showing a PMA-induced increase in alpha 5 expression of 79% above control value (left), whereas beta 1 peak increase is 108% above control value (right). Equal loading was verified with control alpha -actin (data not shown), and densitometric units were corrected for alpha -actin.

PMA induction of alpha 5beta 1 is regulated at the transcriptional level. The data presented above suggest that the effects of PMA on alpha 5beta 1 expression occur at the level of gene expression. To explore this issue, we transfected U-937 cells with palpha 5(-0.9kb)-Luc, a construct that contains a portion of the human alpha 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 alpha 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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Fig. 4.   PMA induction of alpha 5beta 1 is regulated at transcriptional level. A: PMA induces alpha 5 gene transcription in a time-dependent manner. U-937 cells were transfected with palpha 5(-0.9kb)-Luc, a construct that contains a portion of human of alpha 5 gene promoter connected to a luciferase (Luc) reporter gene. Cells were stimulated with PMA as described in Fig. 1 and then harvested at indicated time intervals; expression of gene was estimated by measuring Luc activity. Note that PMA induced transcription of alpha 5 gene promoter as early as 2 h, with a peak after 18 h of stimulation. Activity was decreased at 48 h (data not shown). B: dose-dependent response. U-937 cells were transfected with palpha 5(-0.9kb)-Luc, then stimulated with indicated concentrations of PMA. Peak in alpha 5 gene promoter activity was seen at 25 nM, with subsequent decreases at higher concentrations of PMA.

PMA induction of alpha 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 alpha 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 alpha 5 response.


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Fig. 5.   PMA induction of alpha 5-subunit gene transcription is mediated via protein kinase C activation. Cells transfected with palpha 5(-0.9kb)-Luc were pretreated with a selective protein kinase C inhibitor, calphostin C (CC), before stimulation with PMA. CC is active only in presence of light. Activated CC (CC*) alone had no effect on alpha 5 gene transcription. Note that CC* abolished stimulatory effect of PMA, whereas PMA in presence of inactive CC continued to upregulate alpha 5 gene transcription.

Stimulatory effect of PMA on alpha 5 gene transcription is dependent on specific elements present within the alpha 5 gene promoter other than AP-1. To learn more about the transcriptional elements involved in regulation of the expression of the alpha 5 gene, U-937 cells were transfected with the 923-bp alpha 5 promoter construct [palpha 5(-938)-Luc] or 5'-deletion constructs starting at -178 [palpha 5(-178)-Luc], -92 [palpha 5(-92)-Luc], -41 [palpha 5(-41)-Luc], -27 [palpha 5(-27)-Luc], and -1 [palpha 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 palpha 5(-41)-Luc, palpha 5(-27)-Luc, and palpha 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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Fig. 6.   Transcriptional regulation of alpha 5 gene promoter by PMA. U-937 cells were transfected with palpha 5(-0.9kb)-Luc either as described in Fig. 5 or with 5'-deletion constructs palpha 5(-178)-Luc, palpha 5(-92)-Luc, palpha 5(-41)-Luc, palpha 5(-27)-Luc, and palpha 5(-1)-Luc. Transfected cells were stimulated with PMA as described in Fig. 1 and harvested at varying time intervals after stimulation and expression of the gene was estimated by measuring Luc activity. As before, PMA stimulated expression of -923-bp promoter construct. Intensity of response diminished slightly with further deletions; however, there was a drastic drop in expression of the gene with plasmids palpha 5(-41)-Luc, palpha 5(-27)-Luc, and palpha 5(-1)-Luc, indicating presence of an important regulatory element promoter sequences between -92 and -42 bp. Constitutive expression of the gene was also abolished by deletion sequences between -92 and -42 bp from transcriptional start site. C, untreated control cells.

Potential role of AP-1 in PMA induction of alpha 5 gene transcription. Having demonstrated a role for PKC in PMA-induction of alpha 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 alpha 5 gene promoter near the site of transcriptional initiation at position -45 (3), which is contained in the palpha 5(-92)-Luc construct. The expression of this construct is stimulated by PMA, whereas the palpha 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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Fig. 7.   Nuclear translocation of transcription factor activator protein (AP)-1 occurs in response to PMA. PMA-treated and untreated (control) cells were harvested, and nuclear proteins were extracted and submitted to DNA electrophoresis mobility gel shift assay on a 6% polyacrylamide gel. Lane 1, AP-1 probe alone (no shift noted); lanes 2 and 3, control and PMA-treated cells with bound (B) probe. Note increase in density band depicting bound probe in lane 3. Lane 4, bound probe decreases in cells cotreated with competing AP-1 oligonucleotide.

To determine the potential role of AP-1 in alpha 5 gene transcription, cells were cotransfected with the human alpha 5 promoter construct and with a competing AP-1 oligonucleotide before treatment with PMA. The rationale is that if PMA induction of alpha 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 alpha 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 alpha 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)-1beta promoter results in a decrease in the FN-induced upregulation of IL-1beta gene transcription (data not shown).


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Fig. 8.   Stimulatory effect of PMA on alpha 5 gene transcription is not mediated by induction of AP-1. U-937 cells were cotransfected with palpha 5(-0.9kb)-Luc and competing AP-1 oligonucleotides (20 µg) before treatment with PMA followed by determination of Luc activity. Seen again is enhanced transcription in response to PMA (25 nM). AP-1 alone did not significantly affect transcription, whereas cells cotransfected with AP-1 followed by stimulation with PMA showed same upregulation of alpha 5 gene transcription as control PMA-treated cells.

Induction of alpha 5 gene transcription by LPS. To examine the relevance of the pathways described, we studied the effects of LPS on alpha 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-alpha 5 antibody but not by an antibody to beta 2 (data not shown). Next, we showed that the exposure of transfected U-937 cells to LPS induced the transcription of the alpha 5 gene (Fig. 9B). This effect was blocked by the PKC inhibitor calphostin C but not by its inactive form.


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Fig. 9.   Lipopolysaccharide (LPS) stimulation of alpha 5 expression. A: U-937 cells treated with LPS (5 pg/ml, 18 h, 37°C) were allowed to adhere to FN-coated substrates (50 ug/ml) overnight. Note that LPS enhanced adhesion of cells to FN. B: U-937 cells transfected with palpha 5(-0.9kb)-Luc were exposed to LPS as described above and examined for expression of the gene. Note that LPS induced expression of alpha 5 above control levels. CC* had no effect on constitutive levels of alpha 5 but did inhibit LPS-induced response (CC* + LPS). Inactive CC did not block LPS response (CC + LPS).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This report examines the mechanisms of expression of the FN receptor alpha 5beta 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 alpha 5beta 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-alpha 5 antibody to abolish the stimulatory effect of PMA. We then set out to investigate the mechanisms by which PMA enhances the expression of alpha 5beta 1. Using Northern blotting, we demonstrated that PMA induces the accumulation of mRNAs encoding for both the alpha 5- and beta 1-subunits of the alpha 5beta 1 receptor. This suggested that PMA worked by enhancing the transcription of alpha 5 and beta 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 palpha 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 alpha 5 by PMA requires specific sequences within the 51 bp of the alpha 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 alpha 5beta 1 receptors on the surface of U-937 cells by activating PKC and inducing transcription of the alpha 5 gene, which in turn results in accumulation of the relevant mRNA, synthesis of the alpha 5 protein, and, presumably, assembly of alpha 5- with beta 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 alpha 5beta 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 alpha 4beta 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 alpha 5beta 1 are also consistent with those of Bellon et al. (2), who showed that myeloid cell lines have increased alpha 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 alpha 5 (77 and 79%, respectively) but different for beta 1 (47 and 108%, respectively). We believe this is related to differences in the regulation of gene transcription and protein processing pathways related to alpha 5- and beta 1-integrin subunits. In addition, in contrast to the alpha 5-subunits, beta 1-subunits are stored in granules in immune cells such as monocytes/macrophages (45). Because of this, PMA may induce the expression of alpha -integrin subunits other than alpha 5 that could be coupled with newly made or stored beta 1-subunits for surface expression. This may explain the discrepancy between the levels of mRNA and protein detected for beta 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 alpha 5beta 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 alpha 5beta 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 alpha 5beta 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 alpha 5beta 1 with its recognized signal-transducing ability affects any of these PMA-induced events attributed to accelerated cellular differentiation.

Mechanisms of PMA-induced alpha 5 gene transcription. Expression of alpha 5beta 1 is dependent on transcription and translation of the alpha 5-subunit followed by noncovalent association with the beta 1-subunit. This complex is then transferred to the cell surface. The regulatory elements that control integrin (including alpha 5) gene expression in monocytic cells are unclear (26). Thus far, characterization of the alpha 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 alpha 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 alpha 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 alpha 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 alpha 5 mRNA accumulation before the observed maximal increase in expression of the gene. This suggests that the accumulation of mRNA encoding for alpha 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 alpha 5-subunit mRNA degradation in response to transforming growth factor-beta 1 stimulation. Another observation is that higher doses of PMA did not result in further increases in alpha 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 palpha 5(-92)-Luc construct compared with the larger construct palpha 5(-178)-Luc, suggesting the presence of an important regulatory site(s) for constitutive expression of alpha 5 in this region. This region contains two AP-2 sites and 2 Sp-1 sites, but their role in control of alpha 5 expression is unknown.

Implications for altered alpha 5beta 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 alpha 5beta 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 alpha 5beta 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 alpha 5beta 1 and other integrins in lung macrophages (25). In this report, we show that LPS can induce the transcription of the alpha 5 gene in U-937 cells, a process that was associated with increased adhesion to FN substrates. By inducing the transcription of alpha 5 in these cells, LPS may facilitate the migration and invasion of monocytes/macrophages into infected tissues. Some cytokines such as IL-1alpha and tumor necrosis factor-alpha 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 alpha 5beta 1. Transforming growth factor-beta 1, for example, stimulates not only FN production in human lung fibroblasts but also increased alpha 5beta 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.


    ACKNOWLEDGEMENTS

We thank William Schuyler and Susan Roser for technical assistance.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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