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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4353-4365
Autocrine Signals Control CCAAT/Enhancer Binding Protein
 Expression, Localization, and Activity in Macrophages
By
Mark Baer,
Simon C. Williams,
Allan Dillner,
Richard C. Schwartz, and
Peter F. Johnson
From the ABL-Basic Research Program, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
MD; and the Department of Microbiology, Michigan State University, East
Lansing.
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ABSTRACT |
The transcription factor CCAAT/enhancer binding protein  (C/EBP, or NF-IL6) is expressed in macrophages, where it
participates in lipopolysaccharide (LPS)-mediated induction of
proinflammatory cytokine genes such as interleukin-6 (IL-6) and
IL-1. We have identified activities in conditioned medium from a
macrophage tumor cell line that regulates the expression, localization,
and transcriptional activity of C/EBP. One factor was shown to be tumor necrosis factor- (TNF-), which increased C/EBP
expression by a posttranscriptional mechanism. A second activity,
designated autocrine macrophage factor (AMF), elicited a change in
C/EBP localization from a punctate nuclear staining pattern to
diffuse nuclear distribution. The punctate form of C/EBP correlated
with increased susceptibility of this protein to cleavage by an
endogenous protease during nuclear extract preparation. Conditioned
medium stimulated the ability of C/EBP to transactivate a reporter
gene and activated the expression of two cytokine genes that are
putative targets of C/EBP. These observations suggest that diffuse
distribution of C/EBP in the nucleus corresponds to an activated
form of this protein. AMF activity could not be mimicked by an
extensive set of recombinant cytokines and growth factors and therefore
may represent a novel extracellular factor.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
PROINFLAMMATORY CYTOKINES such as
interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-
(TNF-) are produced by activated macrophages and are able to recruit
other cells involved in the inflammatory and immune
responses.1,2 One of the transcriptional regulators
implicated in cytokine expression is NF-IL6,3 also named
LAP, IL6-DBP, AGP/EBP, CRP2, and NF-M4 and now
commonly referred to as CCAAT/enhancer binding protein (C/EBP).5 C/EBP is a member of the C/EBP family of
basic-leucine zipper (bZIP) proteins that is expressed in
many tissues,4,6 including mature myelomonocytic
cells.7,8
Several studies indicate that C/EBP regulates transcription of
cytokine genes in monocyte/macrophages and other cells. The human
C/EBP homolog, NF-IL6, was originally identified as a nuclear factor
that bound to an IL-1 responsive element in the IL-6 promoter. C/EBP
is capable of transactivating the IL-6 promoter and cooperates with
NF- B to mediate lipopolysaccharide (LPS)-induced transcription of
the IL-6 gene.7,9,10 In addition, C/EBP and NF-B
synergistically activate the IL-8 promoter.9,11 C/EBP
binding sites have also been identified, and in some cases were shown
to function as regulatory elements, in the promoters of genes
TNF-,12 IL-1,13,14 granulocyte
colony-stimulating factor (G-CSF),3 monocyte
chemoattractant protein-1 (MCP-1),15 macrophage
inflammatory protein (MIP)1,16 and chicken
myelomonocytic growth factor (cMGF).17
Results of overexpressing or ablating C/EBP also support the notion
that this transcription factor is an important regulator of
inflammatory cytokine genes in activated macrophages. Ectopic expression of C/EBP in the lymphoblastic cell line, P388, is sufficient to confer the ability to activate transcription of IL-6 and
MCP-1 in response to LPS, whereas the parental cell line lacks this
capability. Conversely, inhibition of endogenous C/EBP expression by
antisense RNA interference blocks LPS induction of IL-6 and IL-1
expression in P388D1(IL1) macrophages.18 In addition,
primary macrophages from C/EBP-deficient mice are unable to induce
G-CSF expression in response to LPS and display defective bactericidal
and tumoricidal activities.19
Because of the potentially critical role of C/EBP in cytokine gene
induction, the expression of constitutive macrophage-specific markers
such as lysozyme and the avian mim-1
gene20,21 and in controlling other macrophage
functions,19 we have sought to understand the mechanisms
that modulate the synthesis and function of C/EBP in these cells.
Here we report the results of studies, which indicate that both the
expression and activity of C/EBP in macrophages is regulated by
autocrine signals.
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MATERIALS AND METHODS |
Cells and cell culture.
P388D1(IL1) [ATCC TIB 6322], IC-21 [ATCC TIB
18623], T4.324 and ANA-125 are
murine macrophage cell lines. P388D1(IL1) displays certain
characteristics of activated macrophages, including increased production of IL-1. Unless otherwise indicated, P388D1(IL1), IC-21, and
T4.3 cells were grown in RPMI-1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal clone I serum (FCS)
(Hyclone, Logan, UT), glutamine, kanamycin, and
pen/strep. ANA-1 cells were grown in Dulbecco's modified Eagle's
medium (DMEM) (BioWhittaker) containing the same
supplements. Escherichia coli LPS was obtained from Sigma (St
Louis, MO; serotype 026:B6). Biologically active recombinant cytokines and growth factors and the anti-TNF-
monoclonal antibody were obtained from the NCI Preclinical Repository
(Frederick, MD). Anti-IL-6 antibody was obtained from R & D
Systems (Minneapolis, MN; #AB406-NA).
Preparation and fractionation of conditioned medium.
Conditioned medium (CM) was collected from confluent P388D1(IL1) cells
grown for 3 to 5 days in RPMI-1640 with 5% FCS. CM was concentrated
either in an Amicon stirred cell concentrator using 10,000 or 30,000 MW
cutoff membranes and then filtered with a 0.45-µm syringe filter
(Nalgene, Milwaukee, WI), or in Centriprep 30 or
Centriprep 10 centrifugal concentrators (Grace, Beverly, MD). CM was concentrated 10-fold and added to fresh cells
at a 2× dose (eg, 40 mL of conditioned medium was concentrated to
4 mL and added to a 15-cm plate of cells containing 20 mL fresh medium). As a control in each experiment, unconditioned medium was
concentrated 10-fold and added to cells at a 2× dose.
Immunofluorescent staining.
C/EBP localization was determined in P388D1(IL1), IC-21, and T4.3
macrophages cultured on glass coverslips in the presence of the
appropriate factors. Cells were fixed in methanol for 10 minutes,
washed with phosphate-buffered saline (PBS), and incubated with rabbit
anti-C/EBP peptide antiserum26 (diluted 1:100 in PBS)
for 1 hour at room temperature. The cells were washed three times with PBS, incubated for 1 hour with goat antirabbit
IgG conjugated with rhodamine (Boerhinger Mannheim Biochemicals,
Indianapolis, IN; 1:100 dilution) at room temperature,
washed three times with PBS, once with H2O,
and mounted on glass slides using Gel Mount (Biomedia,
Foster City, CA). In some cases, nuclei were stained with
4 ,6-diamidino-phenylindole:2HCl (DAPI) for 10 minutes before mounting.
Nuclear extracts.
Nuclear extracts were prepared by either hypotonic lysis or detergent
lysis procedures. Hypotonic lysis method27: cells were
scraped, washed once with PBS, resuspended in hypotonic lysis buffer
(buffer A: 20 mmol/L HEPES pH 7.9, 1 mmol/L EDTA, 10 mmol/L NaCl, 1 mmol/L dithiothreitol [DTT], 0.4 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 0.1 µg/mL leupeptin, 5 µg/mL antipain),
incubated on ice for 20 minutes and lysed by 8 to 10 passages through a 26-gauge syringe needle. Nuclei were pelleted by centrifugation at
14,000g for 20 seconds. Proteins were extracted from nuclei by
incubation with buffer C (420 mmol/L NaCl, 1 mmol/L EDTA, 20 mmol/L
HEPES pH 7.9, 25% glycerol, 1 mmol/L DTT, 0.4 mmol/L PMSF, 0.1 µg/mL
leupeptin, 5 µg/mL antipain) at 4°C for 20 minutes with vigorous
shaking. Nuclear debris was pelleted by centrifugation at
14,000g for 5 minutes and the supernatant was collected and stored at  80°C. Detergent lysis method: the procedure was
identical to the hypotonic lysis protocol except that the lysis buffer
contained 0.25% (vol/vol) Nonidet P-40 and the cells were incubated in
this buffer for 10 minutes before passage through a syringe.
Bacterially expressed proteins.
Full-length C/EBP and a truncated protein containing only the
DNA-binding domain (DBD) were expressed in Escherichia coli and
purified as described.28 A liver inhibitory protein (LIP) expression vector was constructed by inserting an Nco
I-HindIII fragment from pMEX-C/EBP28 into the
bacterial vector pT5.26 LIP was expressed in E coli
and extracted from cells as described29 and was used for
Western blot analysis without further purification.
Electrophoretic mobility shift assays (EMSA).
DNA-binding reactions were incubated for 20 minutes at room temperature
in a 25-µL reaction containing 100 mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/L EDTA, 4% (vol/vol) glycerol, 5% (wt/vol) Ficoll, .06%
bromophenol blue, 0.25 µg bovine serum albumin (BSA), 1 µg poly
dI-dC, 32P-labeled C/EBP binding site
oligonucleotide,18 and 6 to 10 µg nuclear extract.
C/EBP:DNA complexes were separated from free probe by
electrophoresis on 6% polyacrylamide gels in 0.5× TBE (45 mmol/L
Tris Base, 45 mmol/L boric acid, 0.25 mmol/L EDTA) at 160 V for 2 hours.30,31 Gels were dried onto Whatman 3MM paper (Whatman, Maidstone, UK) and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY). For antibody supershift assays, 1 µL of rabbit antiserum was incubated with the protein extract on ice for 30 minutes before addition to the binding reaction. The N-terminal C/EBP peptide antibody has been described.26 The
C-terminal antiserum was raised against a peptide from the carboxy
terminus of rat C/EBP (NH2-CKQLPEPLLASAGH-COOH).
Western blot analysis.
For the experiment of Fig 1B, extracts were mixed with an equal volume
of 2× protein sample buffer,32 heated to 100°C,
and loaded on precast 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Novex, San Diego, CA).
For the experiment of Fig 6, nuclear extracts were prepared by the
detergent lysis method described above except that 1× protein
sample buffer32 was added to the nuclear pellets instead of
extraction buffer. The samples were loaded on 15% SDS-PAGE gels. For
the experiment of Fig 7B, cells were harvested, washed with PBS, and
lysed in 1x protein sample buffer. The extracts were heated and an
aliquot loaded on a 12% SDS-PAGE gel. Gels were transferred to
Immobilon-P membranes (Millipore, Bedford, MA), blocked
with 2% BSA and probed with the appropriate antibody. The Western blot
of Fig 7B was blocked with 5% dry milk and probed with anti-FLAG M2
monoclonal antibody (Eastman Kodak Co). All blots were developed using
the enhanced chemiluminescence (ECL) detection system (Amersham,
Arlington Heights, IL).
RNA isolation and Northern blot analysis.
Total RNA was isolated from cells using the procedure of Chomczynski
and Sacchi33 and 10 to 15 µg was analyzed on Northern blots. Hybridization probes were labeled using a random priming kit
(United States Biochemical, Cleveland, OH). The C/EBP
probe was a 400-bp Nco I fragment from the rat C/EBP
expression vector pMEX-CRP2.28 The -actin probe was a
2-kb HindIII fragment excised from the plasmid
2000.34 Probes for IL-6, IL-1, and MCP-1 have been
described.18 Hybridization signals were quantitated using
an Ambis Radioanalytical Scanner (Ambis Corp, San Diego, CA).
Transfection assays.
Nonadherent ANA-1 cells were transfected using DEAE dextran sulfate as
follows. Cells were transfected in batch (2 × 106
cells/60-mm dish) using 2 µg reporter plasmid and 1 µg of either pMEX or the C/EBP expression vector pMEX-C/EBP [previously named pMEX-CRP228] per dish. The C/EBP reporter plasmid
(DE1)4-alb-LUC has been described.28 The
control luciferase reporter, "pGL2 promoter," was obtained from
Promega (Madison, WI). Plasmid DNAs were prepared by a
polyethylene glycol (PEG) precipitation method or by
using commercial kits (Qiagen, Chatsworth, CA). The cells
and DNA were incubated with 0.5 mg/mL DEAE dextran in DMEM/50 mmol/L
Tris (pH 8.0) for 75 minutes at 37°C on a rotator. Dimethyl
sulfoxide (DMSO) was then added to a final concentration of 10% and
incubated for 2 minutes. The cells were diluted 10-fold in serum-free
DMEM, pelleted, washed twice in DMEM, and plated in DMEM with 10% FCS. After 48 hours, the cells were collected, lysed, and analyzed for
luciferase activity using the Enhanced Luciferase Assay kit (Analytical
Luminescent Laboratory, San Diego, CA). Where
appropriate, CM or cytokines were added 16 hours before harvesting. The
protein concentration of each lysate was also measured (BioRad) and
used to normalize luciferase activity.
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RESULTS |
Cell culture conditions regulate the susceptibility of
C/EBP to proteolysis.
P388D1(IL1) is a macrophage-like tumor cell that was selected for
elevated production of IL-1. This cell line also expresses high levels
of C/EBP.18 In the course of analyzing
C/EBP-specific DNA-binding complexes in P388D1(IL1) nuclear
extracts, we observed that the pattern of binding species could be
altered dramatically by changing the cell growth medium
(Fig 1). Extracts prepared from cells grown for 3 days in the same medium displayed two
slow-migrating complexes in the EMSA using a consensus C/EBP site probe
(Fig 1, 0 hour). However, 1 hour after washing the cells and refeeding, the slow-migrating species were no longer detected and a fast-migrating complex appeared. This pattern of C/EBP complexes persisted for 12 hours. By 24 hours, the slow-migrating forms were again apparent. The
transition to the fast-migrating form was also seen in cells incubated
in PBS for 1 hour (data not shown), demonstrating that exposure to
serum factors is not required for this effect.
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| Fig 1.
Altered C/EBP isoforms in macrophage
extracts. (A) EMSA of C/EBP binding activities in P388D1(IL1)
macrophage nuclear extracts. Cells were cultured for 3 days without
medium change (0 hour), washed with PBS, and fed with fresh medium.
Nuclear extracts were prepared at the indicated times (1 to 24 hours)
and analyzed by EMSA using a consensus C/EBP binding site probe. (B)
Western blot analysis of truncated C/EBP polypeptides. Analysis of
C/EBP proteins in P388D1(IL1) nuclear extracts. A total of 15 µg
of each nuclear extract from the time course shown in (A) were analyzed
by Western blotting using an antiserum specific for the basic region
(panCRP28). Bacterially expressed forms of C/EBP
(full-length [p34], LIP [p20], and the DNA-binding domain [DBD];
lanes 8 through 10) were included as standards. Asterisks indicate weak
bands that correspond to p34-C/EBP and p20-C/EBP.
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The C/EBP species in these extracts were characterized by Western
blotting. Extracts from the time course experiment of Fig 1A were
analyzed by immunoblotting using an antibody (panCRP) specific for the
basic region. Samples in which the fast-migrating EMSA complex was
observed contained only a 14-kD immunoreactive polypeptide (Fig 1B, lanes 2 through 6). Extracts exhibiting the slower
EMSA species showed proteins of 34 kD and 20 kD, as well as a reduced
amount of the 14-kD form (lanes 1 and 7). The 34-kD protein comigrated
with bacterial C/EBP (lane 8) and the 14-kD form was similar in size
to a recombinant protein containing only the C-terminal bZIP region
(DBD, lane 10). Interestingly, the 20-kD product was indistinguishable
in size from bacterially expressed LIP (lane 9), a truncated C/EBP
isoform observed in other cells that was proposed to arise from
translation initiation at an internal methionine codon.35
The 20-kD protein in P388D1(IL1) extracts is a proteolytic cleavage
product derived from p34-C/EBP and is not a translational isoform
(see below and data not shown).
We also performed antibody supershift experiments using peptide
antisera specific for the N or C termini of C/EBP
(Fig 2A). The two
slower-migrating species reacted with both antibodies, demonstrating
that these complexes contain full-length C/EBP (p34-C/EBP). The
upper and lower slow-migrating bands represent homo- and heterodimeric
forms of C/EBP, respectively28 (M.B. and P.F.J.,
unpublished data). The fast-migrating complex was not
affected by the N-terminal antibody, but its formation was inhibited by
the C-terminal C/EBP antiserum. Therefore, the fast-migrating form
of C/EBP represents a truncated protein, p14-C/EBP, which encompasses the C-terminal DNA-binding (bZIP) region.
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| Fig 2.
(A) Antibody supershift analysis of the fast
and slow-migrating C/EBP complexes. P388D1(IL1) nuclear extracts
from cells grown without medium change (Extract #1) or from freshly fed
cells (Extract #2) were incubated with normal rabbit serum or peptide
antibodies directed against the C/EBP N terminus (C/EBP-N) or C
terminus (C/EBP-C) before adding the binding site probe. Purified
recombinant C/EBP was included as a control. The two slow-migrating
complexes in extract #1 (p34-C/EBP) that are supershifted by the
N-terminal antibody represent C/EBP homodimers (faint upper band)
and a heterodimeric complex formed with an unidentified partner (lower
band). (B) Truncated C/EBP species are dependent on the cell lysis
procedure. Two hours before harvesting, P388D1(IL1) cells were washed
with PBS and fed with fresh medium. The harvested cells were divided
into two pools and nuclear extracts were prepared by either the
hypotonic lysis or detergent lysis procedures (see Materials and
Methods) and C/EBP species analyzed by EMSA.
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Subsequent experiments showed that p14-C/EBP is generated by
proteolysis of p34-C/EBP during nuclear extract preparation. A pool
of cells harvested 2 hours after addition of fresh medium was divided
into two aliquots, and nuclear extracts were prepared using either the
standard hypotonic cell lysis procedure or a detergent (Nonidet P-40)
lysis method. EMSA analysis of these extracts (Fig 2B) showed that the
hypotonic nuclear extract contained p14-C/EBP, confirming the
results of Fig 1. However, the extract prepared using detergent lysis
produced only the two slow-migrating (p34-C/EBP) complexes. This
experiment proves that C/EBP protein in these cells is intact and
that the faster-migrating complexes are generated by proteolysis of
p34-C/EBP during the hypotonic cell lysis procedure. We have found
that a calpain-like protease is responsible for cleaving C/EBP to
the 14- and 20-kD forms and that the activity of this protease does not
appear to be affected by the growth medium (M.B., A.J. Lincoln, E. Sterneck, and P.F.J., unpublished results).
It should be emphasized that although truncated C/EBP species do not
occur in the cell and are generated during hypotonic cell lysis, the
susceptibility of C/EBP to proteolytic cleavage was consistently
observed in extracts from cells exposed to fresh medium, but not from
cells grown for several days without medium change. These findings
suggest that C/EBP undergoes a regulated transition in the cell
that, under the appropriate experimental conditions, is manifested as
either sensitivity or resistance to an endogenous protease.
Proteolytic sensitivity of C/EBP is controlled by a
secreted factor.
Because p14-C/EBP appeared in response to fresh growth medium, we
postulated that the accumulation of a secreted factor might regulate
the conversion of C/EBP to a proteolytically resistant state. To
test this possibility, CM was harvested from 3-day old P388D1(IL1)
cultures, fractionated by ultrafiltration, and added to freshly fed
P388D1(IL1) cells. After culturing the cells for 8 hours, nuclear
extracts were prepared using the hypotonic lysis method and analyzed by
EMSA. As expected, nuclear extracts from untreated cells showed the
proteolyzed form of C/EBP (Fig 3, lane
1). However, addition of the >30-kD MW fraction from P388D1(IL1) CM
caused C/EBP to become more resistant to proteolysis (lane 5). The
0-to 10-kD and 10- to 30-kD MW fractions and unconditioned medium did
not exhibit this activity (lanes 2 through 4). As expected for a
protein factor, heating the >30-kD MW fraction for 10 minutes at
65°C abolished its activity (data not shown). We conclude that a
secreted protein, which we provisionally term autocrine macrophage factor or AMF, is responsible for eliciting the protease-resistant state of C/EBP in P388D1(IL1) macrophages.
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| Fig 3.
An autocrine factor in CM regulates C/EBP resistance
to proteolysis. CM from 3-day-old cultures of P388D1(IL1) cells was
fractionated by sequential ultrafiltration, with the flow-through
fraction from the 30-kD cutoff membrane applied to the 10-kD cutoff
filter. Fractions were applied to freshly fed cells, the cells were
cultured for 8 hours, and nuclear extracts were prepared by the
hypotonic lysis procedure. C/EBP binding activities were analyzed by
EMSA.
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CM elicits changes in C/EBP subnuclear localization
and expression.
We next examined whether the resistance of C/EBP to proteolysis
induced by CM was associated with altered cellular localization. Indirect immunofluorescence analysis of P388D1(IL1), cells grown in the
presence or absence of CM (hereafter, CM refers to the >30-kD MW
fraction of P388D1(IL1) conditioned medium) showed striking differences
in C/EBP localization within the nucleus. Freshly fed cells
exhibited punctate areas of C/EBP staining in the nucleus (Fig 4A, K, and M) that were
coincident with sites of strong DAPI staining (Fig 4B, L, and N). In
contrast, CM-treated cells showed a diffuse or speckled staining
pattern throughout most of the nucleus (Fig 4C, O, and Q). High
magnification images of single cells showed that the diffuse staining
was distinct from areas of intense DAPI staining; indeed, C/EBP
appeared to be excluded from these regions in CM-treated cells (compare
panels O and P, Q and R). These results demonstrate that a factor in CM
promotes redistribution of C/EBP within the nucleus. The transition
to diffuse localization appears to be responsible for the resistance of
C/EBP to proteolytic digestion, either because its altered location
in the nucleus renders it physically inaccessible to proteases or
because changes in C/EBP phosphorylation or protein-protein interactions inhibit proteolytic digestion.
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| Fig 4.
Effects of CM on subnuclear distribution and expression
of C/EBP in macrophage cell lines. C/EBP expression and
localization was assessed by indirect immunofluorescence in the
macrophage cell lines P388D1(IL1) (A through D, K through R), IC-21 (E,
F, I, and J), or T4.3 (G and H). Cells were grown in fresh medium for
16 hours in the absence (A, B, E, G, and K through N) or presence (C,
D, F, H, I, J, and O through R) of CM. C/EBP was visualized using a
peptide antibody specific for the N terminus of C/EBP. (B, D, L, N,
P, and R) Show DAPI staining patterns for the corresponding
immunofluorescent fields in the left-hand panels. Note that the
punctate DAPI pattern is unchanged by treatment with CM. (I and J) Show
CM-treated IC-21 cells stained with the C/EBP antibody in the
absence (I) or presence (J) of the synthetic peptide used to generate
the antiserum, and (K through R) are high magnification confocal images
of individual cells. Fluorescent images were recorded using a Nikon
Microphot-FXA microscope (Nikon, Tokyo, Japan) (A through
D) or a Zeiss LSM 310 confocal microscope (Zeiss,
Thornwood, NY) (E through R).
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C/EBP immunofluorescence studies were also performed on two other
murine macrophage cell lines, IC-21 and T4.3. In contrast to
P388D1(IL1) cells, only weak C/EBP staining was detected in the
nuclei of these cells before exposure to CM (Fig 4E and G). However,
after treatment with CM for 16 hours, strong C/EBP
immunofluorescence was observed in the nuclei of both cell lines (4F
and H). The pattern was predominantly diffuse, similar to that seen in
CM-treated P388D1(IL1) cells. The faint cytoplasmic fluorescence
detected in IC-21 and T4.3 cells was nonspecific, as the peptide used
to generate the C/EBP antiserum blocked nuclear, but not cytoplasmic staining (Fig 4I and J). Additionally, Western blot assays showed little or no C/EBP in the cytoplasmic fraction of either CM-treated or control cells (data not shown) and confirmed the increased nuclear
expression of C/EBP (see Table 1 and Fig
6). Thus, a factor secreted by P388D1(IL1) cells stimulates C/EBP
expression in IC-21 and T4.3 macrophages.
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Table 1.
Effects of Recombinant Cytokines and Growth Factors on
C/EBP Expression, Protease Sensitivity and Subnuclear Localization
in Two Macrophage Cell Lines
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In addition to its effects on C/EBP expression and localization, CM
altered the morphology of P388D1(IL1) cells. Control cells treated with
unconditioned medium displayed an elongated morphology and few vacuoles
(Fig 5A). In contrast, CM-treated cells
became rounded and less adherent and exhibited numerous vacuoles (Fig
5B). These changes in morphology may be indicative of activated
macrophages and were not observed in cells treated with individual
cytokines (see below). In heterogeneous populations of cells, the
vacuolized phenotype was only observed in cells that displayed diffuse
C/EBP staining (data not shown), suggesting that these two events
are coupled. Thus, an autocrine factor, possibly AMF itself, promotes
morphological changes in P388D1(IL1) macrophages.
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| Fig 5.
CM alters the morphology of P388D1(IL1) macrophages.
Cells were treated with UCM (A, C, and E) or CM (B, D, and F) for 16 hours and their morphology assessed. Fixed cells were visualized with
Nemarsky optics (A and B) on a Zeiss LSM 310 confocal microscope.
C/EBP localization (C and D) and DAPI staining (E and F) were
analyzed as described in the legend to Fig 4.
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AMF activity cannot be reconstituted by a panel of recombinant
cytokines and growth factors.
We next sought to determine if the activities in CM that elicit changes
in C/EBP expression and subnuclear localization could be attributed
to known cytokines or growth factors. Enzyme-linked immunosorbent assay
(ELISA) measurements of several cytokines in concentrated CM showed
detectable levels of TNF-, IL-6, and transforming growth factor-
(TGF-) (3.4, 6.1, and 4.4 ng/mL, respectively), but not IL-1,
IL-1, IL-4, or IL-10 (data not shown). Because these cytokines are
known to be involved in inflammatory responses, we tested these and
several other recombinant factors for their effects on C/EBP
expression and localization in P388D1(IL1) and IC-21 macrophages (Table
1). Freshly fed P388D1(IL1) cells were exposed to each factor for 16 hours, after which nuclear extracts were prepared by hypotonic cell
lysis and assayed for C/EBP binding activity by EMSA. None of the
agents tested imparted resistance to proteolysis (Table 1). In
addition, immunofluorescence staining showed that these factors did not
alter the punctate nuclear C/EBP staining pattern seen in control
cells, whereas CM-treated cells showed diffuse distribution.
We also tested the same panel of cytokines and growth factors on IC-21
cells. C/EBP protein levels in cytokine-stimulated cells were
assayed by Western blotting and compared with control and CM-treated
IC-21 cell extracts. While most of the factors had no effect, IL-1,
IL-1, IL-6, and TNF- stimulated C/EBP protein expression
several-fold (Table 1). Immunofluorescence analysis confirmed that
C/EBP is expressed in IC-21 cell nuclei after treatment with
IL-1, IL-1, IL-6, and TNF-. However, C/EBP expressed in
these cells exhibited the punctate staining pattern. Thus, while each
of the four factors induces C/EBP expression, none generated the
diffuse C/EBP staining observed in CM-treated P388D1(IL1), IC-21 and
T4.3 macrophages. Collectively, the data of Table 1 indicate that CM
contains a potentially novel activity that is distinct from all of the
cytokines tested to date.
Induction of C/EBP expression by CM is mediated by
TNF-.
To examine further the induction of C/EBP expression by CM, we
determined the levels of C/EBP mRNA and protein in CM-stimulated IC-21 cells over a 24-h time course (Fig
6). Western blot analysis showed a low basal level of C/EBP
expression in this cell line, which increased as early as 4 hours after
addition of CM. Maximal C/EBP expression ( fivefold induction) was
attained by 24 hours. Untreated cells displayed only a weak induction
at the 24-hour time point. This modest but reproducible increase in
C/EBP levels in control cells suggests that IC-21 cells also secrete
an autocrine factor that stimulates C/EBP expression. Northern
analysis showed that C/EBP mRNA levels remained substantially
unchanged after CM stimulation, increasing less than twofold during the
time course. Thus, the CM-induced increase in C/EBP protein
expression is regulated, at least in part, at the posttranscriptional
level.
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| Fig 6.
TNF- in CM induces C/EBP expression. Kinetics of
C/EBP protein and mRNA expression were assessed in control and
CM-treated IC-21 macrophages. Cells were washed with PBS and given
fresh medium 2 hours before the start of the experiment. At time 0, the
cells were washed again and given fresh medium containing either
concentrated P388D1(IL1) CM or concentrated unconditioned medium
(control). In lanes 10 through 13, CM was preincubated with
anti-TNF- neutralizing antibody for 1 hour before addition to the
cells. Cells were harvested at the indicated times and divided into two
aliquots for protein and RNA analysis. Nuclear extracts were prepared
by detergent cell lysis and C/EBP protein was analyzed by
immunoblotting using an anti-C/EBP antibody. C/EBP mRNA was
analyzed by Northern blotting and levels were quantitated and
normalized to actin. The fold increase in C/EBP mRNA was determined
by comparison to the 0 hour time point.
|
|
The relatively high levels of TNF- in CM together with the ability
of recombinant TNF- to induce C/EBP expression in IC-21 cells
suggested that this cytokine might be responsible for the increased
C/EBP levels in CM-treated cells. Therefore, we examined the effect
of a neutralizing anti-TNF- antibody on CM-induced expression of
C/EBP in IC-21 cells. As shown in Fig 6, lanes 10 through 13, preincubating CM with an anti-TNF- antibody inhibited the induction
of C/EBP. This result indicates that TNF- is the major component
of CM responsible for upregulating C/EBP expression.
Because P388D1(IL1) macrophages secrete TNF- constitutively, we
wished to determine if the high level of C/EBP in these cells might
result from autocrine stimulation by TNF-. Removing the growth
medium from 2- or 3-day-old cultures and adding fresh medium did not
diminish C/EBP expression in P388D1(IL1) cells (Fig 1 and data not
shown). However, because this procedure may not have completely removed
TNF- from cell surface receptors, we used a more stringent washing
protocol. When cells from 2-day-old cultures were washed extensively
with PBS at 37°C and then placed in fresh medium, they displayed
reduced levels of C/EBP. Subsequent exposure of these cells to
TNF- for 4 hours caused a significant increase in C/EBP protein
expression (data not shown). Thus, autocrine signaling by TNF- may
be responsible for the high constitutive levels of C/EBP in
P388D1(IL1) cells.
CM stimulates C/EBP activity.
The relocalization of C/EBP in response to CM suggested that this
protein might undergo an associated change in its transcriptional activity. To test this possibility, we cotransfected a C/EBP-dependent promoter-reporter construct and a C/EBP expression vector into macrophages and compared reporter expression in the presence and absence of CM. The macrophage cell line ANA-1 was used because these
cells are more efficiently transfected than P388D1(IL1) or IC-21. The
reporter construct used [(DEI)4-alb-LUC] contains four
copies of a C/EBP binding site from the albumin gene inserted upstream
of the albumin minimal promoter and is strongly activated by
C/EBP.28 The (DEI)4-alb artificial promoter
contains no other known transcription factor binding sites.
ANA-1 cells were cotransfected with (DEI)4-alb-LUC and a
C/EBP expression vector, pMEX-C/EBP.28 Cotransfection
of the C/EBP vector increased luciferase expression 20-fold to
200-fold over that of the reporter alone (data not shown). Treatment of the transfected cells with unconditioned medium (UCM) did not affect
luciferase activity (Fig 7A). However, CM
stimulated reporter gene expression 3.8-fold, indicating that a factor
in CM enhances the transcriptional activity of C/EBP. CM therefore
activates C/EBP in addition to altering its nuclear distribution and
resistance to proteolysis. Expression of a reporter gene under the
control of the SV-40 early promoter ("pGL2 promoter") was not
stimulated by CM, demonstrating that the effect of CM is specific.
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| Fig 7.
CM stimulates the transcriptional potential of C/EBP.
(A) Effect of CM or cytokines on C/EBP transcriptional activity in
ANA-1 macrophages. Cells were transiently transfected with
(DEI)4-alb-LUC and pMEX-C/EBP (upper panel) or
pGL2-promoter (lower panel), a control promoter-luciferase construct.
The cells were treated with the indicated agents 16 hours before
harvesting. UCM and CM were preincubated for 1 hour at room temperature
with 10 µg/mL -IL-6 or 10 µg/mL TNF- neutralizing antibodies.
Recombinant human IL-6, murine TNF-, and LPS were used at
concentrations of 10 ng/mL, 20 ng/mL, and 10 µg/mL, respectively.
Luciferase activities were measured and normalized to protein
concentration in each lysate. The data represent the average of at
least three independent experiments. (B) C/EBP expression in
transfected ANA-1 cells. The cells were transfected with
pMEX-C/EBP-F, a derivative of the pMEX-C/EBP vector in which the
C/EBP leucine zipper was replaced by the FLAG epitope.48
Whole cell extracts were prepared and analyzed by Western blotting
using the anti-FLAG M2 monoclonal antibody. CRM, cross-reacting
material.
|
|
To determine whether the activation of C/EBP was attributable to
TNF- or IL-6 present in CM, we pretreated CM with neutralizing antibodies against these cytokines. Although anti-TNF- had no significant effect, anti-IL-6 inhibited much of the stimulatory activity of CM. Recombinant TNF- did not activate C/EBP and IL-6
had only a modest stimulatory effect. Indeed, in most individual experiments recombinant IL-6 failed to enhance C/EBP transactivation (note that the data of Fig 7A represent the average of several experiments). TNF- and IL-6 in combination also did not activate C/EBP. These results suggest that IL-6 is required, but not
sufficient for efficient activation of C/EBP. The stimulatory
activity of CM apparently requires both IL-6 and another component of
CM, most likely AMF.
To verify that the increase in C/EBP-dependent transcription was not
due to enhanced expression from the pMEX-C/EBP vector, we measured
C/EBP expression in the absence and presence of CM. We used an
FLAG-tagged C/EBP gene (pMEX-C/EBP-F; A.J. Lincoln, Y. Monczak,
S.C.W. and P.F.J., unpublished results) for this
experiment to monitor C/EBP expression from the vector. ANA-1 cells
were transfected with pMEX-C/EBP-F, exposed to UCM, CM, IL-6,or
TNF- for 16 hours and then analyzed for C/EBP-F expression by
Western blotting (Fig 7B). None of the treatments significantly
increased C/EBP-F expression. These data support the notion that CM
stimulates C/EBP-mediated transcription by posttranslational
activation of the protein.
Cytokine gene expression is selectively activated by CM.
Because CM stimulated the ability of C/EBP to transactivate a
C/EBP-dependent promoter-reporter construct, we asked whether CM
treatment affected the expression of endogenous cytokine genes that are
believed to be targets of C/EBP. Previous studies showed that
C/EBP can stimulate transcription of IL-1, MCP-1, and
IL-6.7,9,10,13,14,18 To test whether CM induces expression
of these genes, P388D1(IL1) cells were treated for 16 hours with CM or
UCM and RNA was prepared. Northern blot analysis showed that CM
elicited the expression of mRNAs for IL-1 and MCP-1, but not IL-6 or
TNF- (Fig 8). In contrast, all of these
genes were strongly induced by LPS (data not shown). Thus, activation
of C/EBP by an autocrine factor is associated with increased
expression of two cytokine genes that are putative targets of this
transcriptional regulator.
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| Fig 8.
CM induces expression of endogenous cytokine genes.
P388D1(IL1) cells were grown in serum-free medium in the presence or
absence of CM for 16 hours. RNA was harvested and analyzed by Northern
blotting using probes for IL-6, MCP-1, IL-1, TNF-, and -actin.
The blot was hybridized sequentially with the five probes.
|
|
| |
DISCUSSION |
Macrophages secrete C/EBP-regulatory factors.
We report two activities secreted by P388D1(IL1) macrophages that
influence the synthesis, subnuclear localization, and transcriptional activity of C/EBP. One of these factors, identified as TNF-, stimulates C/EBP expression. TNF- was previously found to
increase C/EBP levels in fibroblasts,36 although
induction of C/EBP by TNF- in macrophages has not been reported.
TNF- was also shown to stimulate cytoplasmic-nuclear transport of
C/EBP in hepatoma cells.37 Thus, TNF- may either
elicit increased expression of C/EBP or promote its translocation to
the nucleus, depending on the cell type.
A second activity, which we refer to as AMF, has not been attributed to
any known cytokines or growth factors. AMF promotes redistribution of
C/EBP within the nucleus and stimulates the transcriptional activity
of C/EBP in transfected macrophages. These responses were not
elicited by TNF- or IL-6, both of which are present in P388D1(IL1)
CM, and an anti-TNF- antibody did not block the redistribution of
C/EBP by CM (data not shown). These findings demonstrate that
TNF- and AMF are independent activities. Our observations suggest
that C/EBP is regulated in macrophages by two kinds of autocrine
signals: one type (eg, TNF- and IL-6) activates C/EBP expression
and generates the punctate nuclear distribution pattern, while another
(AMF) alters C/EBP localization in the nucleus and stimulates its
transcriptional activity. In addition to increasing C/EBP
expression, IL-6 may also cooperate with AMF to enhance C/EBP
activity, as a neutralizing antibody against IL-6 inhibited much of the
stimulatory effect of CM.
C/EBP localization within the nucleus is altered by
CM.
Treatment of P388D1(IL1) cells with CM elicited a striking change in
C/EBP localization, causing a transition from punctate nuclear
distribution to diffuse nuclear staining (Fig 4). This is the first
report of a regulated change in C/EBP subnuclear distribution. The
punctate pattern was also observed in IC-21 cells stimulated with IL-1,
IL-6, or TNF-, whereas CM treatment led to diffuse C/EBP staining
in these cells (Table 1). The areas of intense C/EBP staining in
"punctate" cells colocalized with regions of strong DAPI
fluorescence. The pattern of DAPI staining did not change following CM
treatment, indicating that there were no gross alterations in nuclear
structure. However, C/EBP was no longer localized in punctate units
and, indeed, appeared to be excluded from the DAPI-staining bodies.
Other studies support the idea that the dispersed distribution of
C/EBP reflects its association with active genes. For example, Carter et al38 reported that areas of nuclear poly(A) RNA
concentration, called transcript domains, corresponded to regions of
low DNA density (ie, weakly staining with DAPI) and hypothesized that these regions may be sites of active transcription. Transcript domains
are distinct from the regions strongly labeled by DAPI, suggesting that
the latter represent transcriptionally silent domains of the genome. In
addition, Zeng et al39 found that RNA polymerase II is
distributed in a punctate nuclear pattern in poorly transcribing cells,
but displays dispersed localization in actively transcribing cells.
Although the relationship between the punctate structures identified by
RNA pol II staining and those described for C/EBP is presently
unclear, our data are consistent with the notion that a transition to
diffuse C/EBP localization in cells treated with CM reflects its
association with actively transcribed genes.
A factor in CM activates C/EBP.
The change in C/EBP localization elicited by CM is associated with
an increase in the transcriptional potential of this factor (Fig 7).
Recombinant TNF- did not activate C/EBP and IL-6 conferred only a
weak effect, whereas both cytokines stimulated C/EBP expression. We
favor the interpretation that changes in C/EBP subnuclear localization and activity are related events elicited by AMF. In
support of this idea, the punctate pattern correlated with the
inactivity of two putative C/EBP target genes, IL-1 and MCP-1,
both of which can be transactivated by C/EBP and contain C/EBP
binding sites in their promoter regions.13,14,18 In contrast, diffuse C/EBP distribution in CM-treated cells coincided with increased IL-1 and MCP-1 mRNA levels (Fig 8). Although we observe a correlation between diffuse nuclear localization and increased transcriptional activity of C/EBP, a relationship between these events can only be inferred at this point. A definitive test of
whether a single factor is responsible for both the
recompartmentalization and functional activation of C/EBP must await
the purification of these activities from CM.
C/EBP is known to exist in a latent state due to the presence of two
autoregulatory domains that inhibit its DNA-binding and transactivation
functions, respectively.28,40 The repressed protein can be
activated by deletion of the regulatory domains,28,40 expression in certain cell types,26,28 or the action of
specific protein kinases.40-43 It is possible that
functional activation of C/EBP by CM involves reversal of the
repression conferred by one or both inhibitory domains. The mechanism
by which CM stimulates C/EBP activity (ie, whether DNA-binding or
transcriptional potential is increased) is presently under
investigation.
Cleavage of C/EBP by a cellular protease.
C/EBP isolated from cells cultured in the absence of CM was
quantitatively cleaved by an endogenous protease when the cells were
lysed in hypotonic buffer. Proteolysis occurred rapidly, before the
nuclear isolation step. At present, it is unclear whether the protease
resistant state is due to relocalization of C/EBP within the nucleus
or whether protein:protein interactions or phosphorylation events
induced by CM (and resultant structural changes in the protein) mask
proteolytic cleavage sites in C/EBP. The efficient proteolysis of
C/EBP during nuclear extract preparation may be relevant to reports
of a 20-kD C/EBP species, LIP, which was proposed to arise from
translational initiation at an internal methionine codon.35
LIP has been detected in extracts from several cell
types.35,44,45 As shown in Fig 1B, one of the partially cleaved C/EBP polypeptides from P388D1(IL1) extracts is
indistinguishable from LIP, as judged by Western blot analysis. The
extreme sensitivity of C/EBP to site-specific proteolysis, even in
the presence of protease inhibitors, raises the possibility that
truncated forms of C/EBP arise from proteolytic cleavage and not
from alternative translational initiation. Regardless of whether in
other cells LIP is a translational or proteolytic product, as a
practical consideration, one should avoid hypotonic cell lysis
procedures when preparing nuclear extracts for analysis of DNA-binding
proteins, especially C/EBP, to minimize proteolysis.
Proposed biological role of AMF.
By analyzing the culture medium of LPS-stimulated IC-21 macrophages, we
have detected an activity whose effects on cytokine production that are
similar to those of AMF.46 These and other findings raise
the possibility that AMF is normally secreted by activated macrophages
and serves as an autocrine signal that stimulates transcription of
specific cytokines through posttranslational activation of C/EBP.
TNF- could also function as a positive autocrine signal in
LPS-stimulated macrophages by increasing C/EBP expression, in
addition to its ability to activate NF-B.47 Thus,
TNF- and AMF may act in concert to enhance C/EBP expression and
activity, respectively, in activated macrophages. Although AMF
production may normally be limited to activated macrophages, its
accumulation in the medium of nonstimulated P388D1(IL1) cells could
reflect deregulated expression of this factor in macrophage tumor
cells.
It is unknown whether the production of AMF or responsiveness to this
factor are exclusive properties of monocyte/macrophage cells. However,
we were unable to detect AMF-like activities in either HepG2
(hepatocarcinoma) or L (fibroblast) cell supernatants, using
proteolytic resistance of C/EBP in P388D1(IL1) cells as an assay
(M.B. and P.F.J., unpublished results). It is now of considerable interest to determine whether other cell types secrete or
respond to AMF and to purify and further characterize the biological functions of this factor.
| |
ACKNOWLEDGMENT |
We are indebted to H. Young for cDNAs and advice, C. Reynolds for
recombinant cytokines, J. Turpin for advice and critical comments, J. Resau for confocal microscopy, T. Copeland for peptide antisera, P. Donovan for assistance with indirect immunofluorescence, and J. Turpin,
H. Young, and E. Sterneck for critical reading of the manuscript. We
also thank C. Weinstock and H. Marusiodis for expert secretarial
assistance.
| |
FOOTNOTES |
Submitted February 1, 1998;
accepted July 15, 1998.
Supported in part by the National Cancer Institute, Department of
Health and Human Services, under contract with Advanced BioScience Laboratories. R.C.S. is supported by American
Cancer Society (ACS) Research Grant No. DB-110.
The contents of this publication do not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the US Government.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Peter F. Johnson, PhD,
ABL-Basic Research Program, National Cancer Institute-Frederick Cancer
Research and Development Center, PO Box B, Frederick, MD 21702-1201;
e-mail: johnsopf{at}mail.ncifcrf.gov.
| |
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A short polypeptide marker sequence useful for recombinant protein identification and purification.
Biotechnology
6:1205, 1988
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Abstract
Introduction