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Glomerular Bioengineering Unit, Department of Medicine, University College London Medical School, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cellular redox state is a crucial factor that controls a wide range of
cell function (5). It is mainly due to the fact that a variety of
signaling molecules are redox sensitive. For example, reactive oxygen
species trigger phosphorylation of various kinases and activate
transcription factors, including activating protein-1 and nuclear
factor-
B (6). We recently found that oxidants, well-known mediators
for tissue injury and inflammation, induce spreading of macrophages in
culture. Under pathologic milieu, oxidant stress may be an important
trigger for in vivo transition of macrophages to the spreading form.
A previous report (7) suggested a role of Egr-1 in the spreading of macrophages. The expression of egr-1 gene is regulated by serum response elements (SREs)3 in its promoter region (8). The activity of SRE is up-regulated by ternary complex factors that are phosphorylated by the mitogen-activated protein (MAP) kinase family of molecules (9). It has been shown that MAP kinases and SRE are redox sensitive and are activated in response to oxidant stress in certain cell types (10, 11). Based on these, the present study was conducted to identify intracellular events required for the oxidant-initiated spreading of macrophages, especially highlighting the roles of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38 MAP kinase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The normal alveolar rat macrophage NR8383 (12) was purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in DMEM/Ham’s F-12 (DMEM-F12; Life Technologies, Gaithersburg, MD) supplemented with 100 U/ml penicillin G, 100 µg/ml of streptomycin, 0.25 µg/ml amphotericin B, and 10 to 15% FCS.
For induction of spreading, NR8383 macrophages seeded in 24-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) were stimulated with menadione (2-methyl-1,4-naphthoquinone; 25 to 100 µM; Sigma, St. Louis, MO) or 300 µM hydrogen peroxide (Sigma). Menadione is a quinone compound that generates superoxide (O2-) via one-electron-transfer reactions and produces H2O2 and hydroxy radical (OH•) (13). Menadione, 50 to 100 µM, was generally used as an inducer of spreading. Morphologic examination was performed after 1 h using phase-contrast microscopy. The percentage of spreading cells was counted in each well, and the mean value of four wells was used to compare data in different groups. More than 60 cells per well were randomly examined for the evaluation. In some experiments, macrophages were pretreated with antioxidant N-acetyl-L-cysteine (10 mM; Sigma) for 2 h and then stimulated by menadione.
To examine roles of ERKs and p38 MAP kinase in the oxidant-triggered spreading, NR8383 macrophages were pretreated with PD098059 (10–75 µM; a gift from Dr. A. R. Saltiel, Parke-Davis Pharmaceutical Research, Ann Arbor, MI) (14) or SB203580 (10 µM; Calbiochem-Novabiochem, Nottingham, U.K.) for 2 h and then stimulated by menadione. All experiments were performed in quadruplicate.
Transfection
The role of ERKs in the oxidant-triggered cell spreading was investigated by transient transfection. Using a modified calcium phosphate coprecipitation method (15), NR8383 macrophages were transfected with pCI-ßGal (a gift from Promega, Madison, WI) together with pCEP4Erk1 + pCEP4Erk2 or pCEP4Erk1(K71R) + pCEP4Erk2(K52R), as described below. pCI-ßGal introduces a ß-galactosidase gene under the control of the immediate-early enhancer/promoter of human cytomegalovirus. pCEP4Erk1 and pCEP4Erk2 code for wt ERK1 and ERK2. pCEP4Erk1(K71R) and pCEP4Erk2(K52R) encode dominant-negative mutants of ERK1 and ERK2, respectively. When overexpressed, these mutants effectively suppress the function of endogenous ERK1 and ERK2 (16). NR8383 cells (2–6 x 106) were suspended in 1 ml of calcium phosphate-DNA complex containing 2 µg pCI-ßGal + 3 µg pCEP4Erk1 + 3 µg pCEP4Erk2, or 2 µg pCI-ßGal + 3 µg pCEP4Erk1(K71R) + 3 µg pCEP4Erk2(K52R). After incubation for 20 min, growth medium (10–15% FCS/DMEM-F12; 9 ml) was added, incubated for additional 2 h, and treated with chloroquine at a concentration of 50 µg/ml. After 2 h incubation, cells were washed and exposed to 15% glycerol in PBS for 90 s. Then, the cells were washed and seeded in 12-well plates. After incubation for 48 h, cells were reseeded in 6-well plates or 10-cm tissue culture plates, stimulated by 100 µM menadione for 1 h, and subjected to 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) assay. To test contribution of p38 MAP kinase, NR8383 macrophages were transfected with pCI-ßGal (2 µg) together with empty vector pcDNA3 (6 µg) or pcDNA3-p38(TY) (6 µg) encoding a dominant negative mutant of p38 (17).
To examine a role of SRE in the oxidant-triggered spreading of macrophages, a dominant-negative mutant of Elk-1 was used (18). Elk-1 forms complexes with serum response factor (SRF), and binding of Elk-1-SRF complexes to SRE leads to induction of target genes. If the dominant-interfering form of Elk-1 is overexpressed, activity of SRE is suppressed (18). NR8383 macrophages were transiently transfected with pCI-ßGal (3 µg) together with an empty plasmid pCMV5 (6 µg) or pDN-Elk (6 µg) encoding a mutated Elk-1 with deletion of the carboxyl-terminal activation domain. After 48 h, cells were exposed to menadione and subjected to X-gal assay. All transfection experiments were performed in quadruplicate.
X-gal assay
X-gal assay was performed as described before (19). In brief, cells were fixed in 0.5% glutaraldehyde, 2 mM MgCl2, and 1.25 mM EGTA in PBS for 10 min at room temperature and incubated at 37°C for 2 h in X-gal solution containing 1 mg/ml X-gal (Sigma), 20 mM K3Fe(CN)6, 20 mM K4Fe(CN)6·3H2O, 2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% Nonidet P-40 in PBS (pH 7.4). Percentage of spreading blue cells against total number of blue cells was calculated in each well, and the mean value of four wells was used to compare data in different groups.
Western blot analysis
Phosphorylated forms of ERKs and p38 were detected by Western blot analysis. NR8383 macrophages (1–2 x 106/well) cultured in six-well plates were stimulated by 75 µM menadione for 15 to 60 min. After the exposure, cells were rinsed by PBS, lysed with 300 µl of sample buffer (4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% ß-mercaptoethanol in 250 mM Tris-HCl; pH 6.8) and boiled for 5 min. Samples were passaged several times through 23-gauge needles. After centrifugation, supernatants were electrophoresed in 10% acrylamide gels and transferred onto nitrocellulose membranes. Analyses were performed using PhosphoPlus MAPK Ab Kit and PhosphoPlus p38 MAP Kinase Ab Kit (New England Biolabs, Hitchin, U.K.) following protocols provided by the manufacturer.
Activity of JNK was evaluated by phosphorylation of c-Jun, using SAPK/JNK Assay Kit (New England Biolabs). In brief, after exposure to menadione, cells were lysed with 300 µl of lysis buffer and passaged several times through needles. After centrifugation, each supernatant containing 50 µg of total protein was incubated with 1 µg of c-Jun fusion protein beads at 4°C overnight. After centrifugation, the pellets were washed, suspended in 50 µl of kinase buffer supplemented with 100 µM ATP, and incubated for 30 min at 30°C. Then, 50 µl of sample buffer was added to each, boiled for 5 min, and centrifuged. Supernatants were then subjected to electrophoresis and immunoblot analysis following the protocol provided by the kit.
Northern blot analysis
NR8383 macrophages were exposed to 50 µM menadione for 0.5, 1, and 2 h. Total RNA was extracted by a single-step method (20) and subjected to Northern blot analysis, as described before (21). In brief, RNA samples were electrophoresed on 1.2% agarose gels containing 10% formaldehyde and transferred onto nitrocellulose membranes. A mouse egr-1 cDNA (22), a human c-fos cDNA (23), and a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were labeled with [32P]dCTP using the random priming method. The membranes were hybridized with probes at 65°C overnight in a solution containing 4x SSC (600 mM sodium chloride, 60 mM sodium citrate), 5x Denhardt’s solution, 10% dextran sulfate, 50 µg/ml herring sperm DNA, and 50 µg/ml poly(A), washed at 50°C, and exposed to Kodak XAR films at -80°C.
Statistical analysis
Data were expressed as means ± SE. Statistical analysis was performed using the nonparametric Mann-Whitney U test to compare data in different groups. We used a value of p < 0.05 to indicate a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the effect of oxidants on macrophage spreading,
NR8383 cells were plated on tissue culture plastic and incubated in the
absence or presence of menadione, which generates reactive oxygen
intermediates. Within 1 h, the majority of cells adhered on
plastic and showed round appearance, with bright cytoplasm and clear
margin (Fig. 1
A,
left). After exposure to menadione, macrophages altered
their shape within 30 min and exhibited the "spreading form"
characterized by a flat and ameboid shape and dark cytoplasm with
vacuolation (1, 24) (Fig. 1A, right). After
1 h, approximately 60% of stimulated macrophages showed
spreading. The effect of menadione was dose dependent; i.e.,
percentages of spreading cells were 9.6% by 25 µM, 25.5% by 50
µM, 45.9% by 75 µM, and 55.5% by 100 µM, compared with
untreated control (4.8%) (Fig. 1B). This effect was
completely abrogated in the presence of antioxidant
N-acetyl-L-cysteine (Fig. 1C). The
stimulatory effect of oxidant stress on the cell spreading was further
confirmed using H2O2. As shown in Figure 1D, 300 µM H2O2 induced spreading
of macrophages (53.5 ± 4.1%, mean ± SE, p
< 0.05) similarly to the effect of menadione.
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To identify signaling pathways involved in the oxidant-initiated
macrophage spreading, a role of ERKs was investigated. NR8383
macrophages were treated with menadione for 15 min, and phosphorylation
of ERK1 and ERK2 was examined using phospho-specific Abs. Western blot
analysis showed that stimulation of macrophages with menadione rapidly
induced phosphorylation of ERK1 and ERK2 (Fig. 2A).
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B). Significant suppression was observed at concentrations
higher than 50 µM. Compared with control (42.7 ± 2.8%), the
percentages of spreading cells were reduced to 28.6 ± 1.6% (50
µM) and 26.0 ± 3.0% (75 µM).
The crucial role of ERKs was further confirmed by a transient
transfection assay. NR8383 cells were transfected with a reporter
plasmid pCI-ßGal together with pCEP4Erk1 + pCEP4Erk2 encoding wt ERKs
or pCEP4Erk1(K71R) + pCEP4Erk2(K52R) coding for dominant-interfering
forms of ERKs. After incubation for 48 h, cells were stimulated
with menadione and subjected to X-gal assay. Percentages of spreading
cells were evaluated using X-gal-positive cells. As shown in Figure 2C, transfection with dominant-negative mutants
significantly inhibited macrophage spreading. Compared with control
(100%), the relative percentage of spreading cells was reduced to
60.8 ± 9.4% (p < 0.05) by the
inhibition of ERKs.
Involvement of p38 MAP kinase and JNK
Pharmacologic and genetic inactivation of ERKs only partially
inhibited the oxidant-induced macrophage spreading. To further identify
molecules that cooperate with ERKs, involvement of p38 MAP kinase and
JNK was tested. NR8383 macrophages were treated with menadione for 15
to 60 min, and phosphorylation of p38 and c-Jun was examined. Western
blot analysis showed that treatment of macrophages with menadione
rapidly induced phosphorylation of p38, with a peak at 30 min (Fig. 3A). In contrast, JNK was
constitutively active in NR8383 cells, and its activation state was not
affected in response to the oxidant stress (Fig. 3B).
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C). Compared with control
(40.9 ± 3.3%), the percentage of spreading cells was reduced to
24.6 ± 2.1%. Pretreatment of the cells with SB203580 together
with PD098059 led to dramatic suppression of the macrophage spreading
(6.8 ± 0.9%).
The role of p38 was further confirmed by a transient transfection
assay. NR8383 cells were transfected with a reporter plasmid pCI-ßGal
together with an empty plasmid pcDNA3 or pcDNA3-p38(TY) that encodes a
dominant-negative mutant of p38. After incubation for 48 h, cells
were stimulated with menadione and subjected to X-gal assay. As shown
in Figure 3D, transfection with p38(TY) significantly
inhibited macrophage spreading. Compared with control (100%), the
relative percentage of spreading cells was reduced to 57.9 ±
7.5% by the inhibition of p38.
Involvement of SRE
ERKs and p38 are known to activate SRE via phosphorylation
of a ternary complex factor Elk-1 (9). To further identify molecular
events downstream of the MAP kinase activation, we examined the
involvement of SRE. Expression of egr-1 and c-fos
are regulated by SREs located in their 5'-flanking regions (8, 25).
Using these genes as indicators, activity of SRE was tested by Northern
blot analysis. After stimulation of macrophages with menadione,
expression of both egr-1 and c-fos was rapidly
induced within 30 min and peaked at 1 h (Fig. 4A). To examine whether
activation of SRE is required for the oxidant-triggered spreading, a
dominant-negative mutant of Elk-1 was used. Elk-1 forms complexes with
SRF, and binding of Elk-1-SRF complexes to SRE leads to induction of
target genes. If the dominant-interfering form of Elk-1 is
overexpressed, activity of SRE is abrogated (18). NR8383 macrophages
were transiently transfected with pCI-ßGal together with an empty
plasmid pCMV5 or pDN-Elk. After 48 h, cells were exposed to
menadione, and X-gal assay was performed. As shown in Figure 4B, introduction of the Elk-1 mutant significantly inhibited
macrophage spreading. The relative percentage of spreading cells was
reduced to 29.0 ± 10.7% compared with the value of control
(100%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oxidant stress is involved in various diseases, including inflammation (29), where monocytes/macrophages play an important role. It is known that oxygen radical species stimulate production of monocyte chemoattractant protein-1 and expression of receptors for leukocyte adhesion, leading to accumulation of circulating monocytes at affected sites (30, 31). Our current results, together with the previous reports, suggest potential roles of oxygen radical intermediates for local macrophage function. At the site of inflammation, oxidant stress may attract monocytes and facilitate their extravasation, migration, and differentiation to tissue macrophages. Of note, in the pathologic milieu, the monocyte/macrophage is an important source of local oxidant production. Oxygen radical intermediates produced by local macrophages may further facilitate accumulation and activation of macrophages.
Intracellular signaling pathways involved in macrophage spreading are poorly understood. Previous reports suggested possible roles of certain signaling molecules, including protein kinase C, cytosolic free calcium, phospholipase A2, and arachidonic acid (2, 3, 32, 33). To date, however, successive signaling cascades involved in the macrophage spreading are not identified. In the present investigation, we highlighted the role of the MAP kinase-SRE pathways. ERKs, JNK, and p38 MAP kinase are known to be redox sensitive and activated by oxidants in certain cell types (10, 34). SRE is also regarded as an oxidant-responsive regulatory element (11, 35). Consistent with these, our results showed that menadione phosphorylated ERKs and p38, leading to activation of SRE. Inhibition of either step partially suppressed the oxidant-mediated spreading of macrophages. When both pathways were concomitantly blocked, dramatic suppression of macrophage spreading was observed. Similar, substantial suppression was also achieved by inactivation of SRE. These results indicate that, in response to oxidant stress, the ERK pathway and the p38 pathway cooperatively incite macrophage spreading via activation of SRE.
Rho family members of GTP-binding proteins (Rho, Rac, and Cdc42) play a role in alteration in cytoskeleton organization, motility, and cell shape (36, 37). It has been reported that these GTP-binding proteins can be activated in response to oxidant stress (38) and participate in phorbol ester-induced spreading of macrophages (39). The activation of the Rho family molecules leads to phosphorylation of JNK (40), which may phosphorylate Elk-1 and activate SRE (41). Although, in NR8383 cells, the activity of JNK was not affected by the oxidant stress, the Rho-JNK-SRE pathway might, in part, participate in the oxidant-induced spreading in other cells of monocyte/macrophage lineage.
Molecular events downstream of SRE are currently undetermined. Since the spreading of macrophages is rapid, some immediate-early genes may be involved. A previous report has shown that expression of egr-1 is essential for phorbol ester-induced spreading of myeloid leukemia cells and normal myeloblasts (7). As is known, the expression of egr-1 is induced by oxidants and regulated by SREs in its 5'-flanking region (8, 42). We observed that menadione transiently induced the expression of egr-1 in macrophages preceding their spreading. However, our preliminary results showed that 1) inhibition of egr-1 expression by antisense oligodeoxynucleotides did not inhibit the oxidant-induced spreading of macrophages, and 2) transient transfection with a full-length egr-1 cDNA failed to induce macrophage spreading. Similarly, transfection with a c-fos cDNA, another oxidant inducible immediate-early gene controlled by SRE (25), did not result in macrophage spreading (our unpublished observation). These data imply a possibility that downstream molecule(s) other than egr-1 and c-fos may be involved in the oxidant-initiated macrophage spreading.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masanori Kitamura, Glomerular Bioengineering Unit, Department of Medicine, University College London Medical School, The Rayne Institute, 5 University Street, London WC1E 6JJ, U.K. E-mail address:
3 Abbreviations used in this paper: SRE, serum response element; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; SRF, serum response factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; wt, wild-type.
Received for publication January 22, 1998. Accepted for publication June 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin via deactivation of CArG box elements. Kidney Int. 53:690.[Medline]
and immunoglobulin G. Evidence for involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase. J. Clin. Invest. 92:1564.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ERKs), as described in
Materials and Methods. After incubation for 48 h,
cells were stimulated by menadione for 1 h and subjected to X-gal
assay. Percentages of spreading cells were evaluated using
X-gal-positive cells. Assays were performed in quadruplicate.
Percentage of spreading blue cells against total number of blue cells
was calculated in each well, and the mean value was used to compare
data in different groups. The result (mean ± SE) is shown as a
relative percentage against the value of control (100%). An asterisk
indicates a statistically significant difference (p
< 0.05) compared with the cells transfected with wt ERKs.
