From the
The human neutrophil lactoferrin (Lf), a cationic iron-binding
glycoprotein, has an inhibitor role on granulocyte macrophage
colony-stimulating factor (GM-CSF) production via interleukin-1 (IL-1).
The nuclear localization of Lf suggests that it may be involved in the
transcriptional regulation of GM-CSF gene expression. To explore this
possibility, the effect of Lf on GM-CSF gene expression was
investigated in various cell lines and in primary cultures of
fibroblasts. Down-regulation of GM-CSF mRNA level was observed in
Lf-transfected embryonic fibroblasts induced to produce GM-CSF by
IL-1
Human lactoferrin (Lf)
Various biological
functions are attributed to Lf although its exact biological role
remains unclear
(1) .
The expression of neutrophil Lf begins
at the myelocyte stage and appears to be a useful marker of terminal
myeloid differentiation. Therefore, reduced or absent Lf gene
expression is also found in conditions characterized by disordered
myeloid differentiation, such as acute leukemia and myelodysplastic
syndromes
(2) . Neutrophil Lf has been implicated in immune and
inflammatory responses and has been reported to be an inhibitor of
myelopoiesis, since it decreases granulocyte macrophage
colony-stimulating factor (GM-CSF) production/release
(3) . This
latter effect is secondary to the inhibited production of interleukin-1
(IL-1), an inducer of GM-CSF in physiological and pathological
conditions
(4, 5) .
Regulation of GM-CSF expression
involves a combination of both transcriptional and post-transcriptional
controls
(6) . Recently, a conserved AU-rich sequence in the
3`-untranslated region of mRNA encoding GM-CSF was found to be
responsible for selective mRNA degradation
(7) , and binding
proteins that recognize the AU-rich motif have been
described
(8) . Transcriptional regulation of GM-CSF plays an
important role in activated T-lymphocytes, in fibroblasts and in
endothelial cells. A region of 629 base pairs upstream of the
transcriptional starting site has been well characterized, as well as
some transcription factors that bind to this region
(6) .
The
molecular mechanism by which Lf controls GM-CSF production is still
unknown. It has been shown that in mice macrophages 10 nM Lf
is able to decrease the GM-CSF mRNA level after stimulation with fetal
calf serum
(9) .
Our aim was to find out how Lf can negatively
modulate GM-CSF production. A possible clue is provided by the ability
of Lf to interact with DNA, thus suggesting its possible involvement in
GM-CSF gene regulation as a nuclear factor. We previously demonstrated
that Lf, after binding to its specific receptor, can be translocated
into the nucleus of K562 cells, where it binds
DNA
(10, 36) . Moreover, Lf DNA-binding fragments have
been found in infant urine
(11) . Recently it has been shown that
Lf is able to specifically bind three consensus sequences supporting
the hypothesis that Lf is a nuclear factor
(12) .
We tested Lf
action on GM-CSF gene expression in two cellular systems: continuous
cell lines that constitutively produce GM-CSF and/or IL-1
In 5637 cells,
although Lf does not seem to down-regulate the high level of GM-CSF
mRNA, it is able to reduce of about 20% both the activity of the
endogenous promoter and the the amount of secreted protein.
The
significative mRNA reduction in PEU cells brought about by transfected
Lf and the low reduction in GM-CSF gene transcriptional rate in
Lf-treated 5637 cells seem to be due, at least in part, to a
transcriptional mechanism. In co-transfection experiments
down-regulation of GM-CSF promoter activity was significant when PEU
cells were treated with IL-1
GM-CSF mRNA expression is differently regulated in
5637 and PEU cells. In PEU cells, the low basal mRNA level can be
increased to relatively high level by induction with several agents. On
the contrary, in 5637 cells the constitutive high expression of GM-CSF
is not subjected to further increase. A possible explanation is the
endogenous production of IL-1
We observed a different quantitative effect of
Lf-treatment on endogenous and exogenous (pPF2000) GM-CSF promoter in
5637 cells. The endogenous GM-CSF promoter is strongly activated by
trans-acting factors. We hypothesize that the ratio between
trans-acting factors that strongly activate the GM-CSF promoter in 5637
cells and their related cis-acting elements is such to affect Lf
action. The addition of the exogenous promoter, as in co-transfection
experiment, is likely to significantly change this ratio, thus allowing
Lf to affect promoter activity. Furthermore, CAT activity does not take
into account post-transcriptional regulation of GM-CSF mRNA that is, of
course, differently regulated with respect to CAT mRNA.
Our results
show that IL-1
The
molecular mechanism underlying the observed effect of Lf on GM-CSF
promoter activity is still unknown. It is tempting to speculate that Lf
may interact directly with DNA elements that are located within
transcriptional control regions. Such a possibility is consistent with
the fact that Lf is able to bind DNA
(10, 11) , even in a
sequence-specific way
(12) . For instance, Lf could modulate the
activity of nuclear factors critical to GM-CSF gene activation.
Moreover, Lf is very prone to bind other
proteins
(33, 34) . Lf might be engaged in interactions
with factors that are involved in the regulation of GM-CSF
transcription. Protein-protein interactions seem to be involved in some
repression mechanisms operated through AP1
(35) . However, Lf
could participate less directly in transcriptional modulation; it might
somehow modify the intranuclear environment in a way that represses the
transcription of a set of genes (for example genes modulated by IL-1).
Further work will be necessary to identify more precisely the region
of the GM-CSF promoter responsive to the inhibitory effect of Lf and to
characterize the molecular mechanism in greater details.
Cells, at about 70% confluence,
were incubated or not with 0.1 µM Lf for 24 h, and GM-CSF
protein assay was performed in culture medium by the EASIA method (see
``Experimental Procedures'' for details). The reduction in
the protein concentration of Lf-treated cells compared with untreated
cells was also expressed as percent of reduction. The three experiments
were performed in duplicate.
We thank Dr. M. Musso for helpful advice, G. Bruzzone
for photographic assistance, Dr. O. Conneely for the gifts of the
p91023-B, and Dr. P. Fiorentini for the pPF2000 used. We also thank Dr.
R. Ravazzolo and Dr. B. Patrick for critically reviewing the
manuscript.
. In 5637 cell-line and in embryonic fibroblasts,
co-transfection experiments, in which an Lf expression vector was used
together with a vector carrying a reporter gene linked to the GM-CSF
promoter, revealed that Lf reduces the activity of the GM-CSF promoter.
This effect is marked in IL-1
-stimulated cells. These findings
suggest that Lf plays a negative role in GM-CSF expression at the
transcriptional level, perhaps through the mediation of IL-1
.
(
)
is a strongly
cationic, 80-kDa glycoprotein, secreted at a high concentration in milk
from glandular epithelia, and present, at a lower concentration, in
other exocrine secretions. It is present in small amounts (1-10
nM) in plasma, and is derived from neutrophils in which the
secondary granules synthesize and store Lf.
and
embryonic fibroblasts induced by IL-1
to produce GM-CSF. We report
here that Lf can inhibit GM-CSF mRNA in embryonic fibroblasts. In
IL-1
-stimulated cells (5637 and fibroblasts) Lf down-regulates
GM-CSF promoter activity.
Cells
The following cells were used: bladder
carcinoma cell line 5637, which constitutively produces GM-CSF and
IL-1
(13, 14) , histiocytic lymphoma cell line U937
producing IL-1
(15) , and T-lymphocyte cell line Mo, which
produces GM-CSF
(16) . Primary cultures of human embryonic lung
fibroblasts (PEU cells), obtained from Istituto Zooprofilattico
Sperimentale della Lombardia e dell'Emilia-Brescia, Italy, were
used in passages 10-25 in all experiments. Cell lines were
maintained in RPMI 1640 medium, and PEU cells were maintained in
Dulbecco's modified Eagle's medium (DMEM), supplemented
with 10% fetal calf serum, 2 mM glutamine, 1 mM
sodium pyruvate, and 0.05 mg/ml gentamycin. All induction experiments
were performed at 70% confluence for adherent cells or at a
concentration of 5
10
cells/ml for nonadherent
cells. Cells were incubated with different amounts of iron-saturated Lf
(Sigma), ranging from 1 pM to 0.1 µM at 37
°C, for the times indicated. Cells were utilized for total RNA
preparation. Cellular conditioned medium was used for GM-CSF protein
assay.
Transient Transfections
5637 cell line and PEU
cells were plated at 70% confluence 24 h before transfection in 10-cm
diameter plastic dishes in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM glutamine,
and 0.05 mg/ml gentamycin. Cells were transfected by the calcium
phosphate coprecipitate method
(17) . Transfections were
performed with 10 µg of the p91023-B expression vector
(16) containing Lf cDNA(
)
(pLf), kindly
provided by Dr. O. Conneely (Baylor College of Medicine-Houston-Texas)
or with an equimolar concentration of a control plasmid pBluescript
(pBS). Cells, 18 h later, were reincubated for a further 18 h with or
without 15 units/ml IL-1
before harvesting. In other experiments,
cells were co-transfected with 10 µg of pLf and 6 µg of pPF2000
containing the promoter region (position -2010 to +26) of
the GM-CSF gene cloned in pBLCAT3 vector.
(
)
Transfection experiments included 4 µg of
pRSV-
-galactosidase, as an internal standard. In control
experiments, pBS in equimolar concentration was used instead pLf. For
all plasmids, at least two different preparations were used. All
experiments were performed in duplicate. Harvested cells were utilized
for total RNA extraction and for cytoplasmatic extract preparation.
Conditioned medium of transfected-cell was utilized for GM-CSF protein
assay.
RNA Analysis
Total cellular RNA was purified by
the guanidium isothiocyanate/cesium chloride method
(18) and,
from transfected cells, according to Chomczynski and
Sacchi
(19) . The integrity of RNA was assayed by evaluation of
the 28 S/18 S RNA ratio on ethidium bromide-stained agarose gel.
Purified RNA was size-fractionated by formaldehyde/agarose gel
electrophoresis and transferred onto a nylon filter
(20) . The
blots were hybridized with Lf cDNA (2.0-kilobase pair
EcoRI-TthIII fragment in p91023-B), GM-CSF cDNA,
(0.8-kilobase pair XhoI fragment in pXM), IL-1 cDNA
(1.3-kilobase pair PstI fragment in pSP64) kindly provided by
Dr. S. C. Clark (Genetics Institute, Cambridge, MA), and
-actin
cDNA (1.8-kilobase pair HindIII fragment in pEMBL8);
all of the probes were labeled by random priming.
-actin probe was
used to compare the level of specific hybridization with the amount of
RNA separated in each assay. The amount of each mRNA were quantitated
by densitometric scanning of autoradiographs. The experiments,
performed in duplicate, were repeated at least three times.
GM-CSF Assay
Assay of GM-CSF protein in cellular
conditioned medium was performed by a solid-phase immunoenzymetric
assay (EASIA, Medgenix Diagnostics, Fleurus, Belgium). This assay is
based on oligoclonal system in which several monoclonal antibodies
directed against distinct epitopes of GM-CSF are used. The use of
several distinct monoclonal antibodies avoids hyperspecificity and
allows high sensitive assay with extended standard range and short
incubation time. This assay allows to detect a minimum protein
concentration of 3 pg/ml and cross-reactions with M-CSF, G-CSF,
IL-1, IL-1
, IL-2, IL-3, IL-4, interferon-
,
interferon-
, interferon-
, tumor necrosis factor-
, and
tumor necrosis factor-
are insignificant.
Lf Immunoblot
Transfected 5637 cells were washed
twice, scraped with cold phosphate-buffered saline and then solubilized
at 4 °C for 1 h using a lysis containing 20 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM aprotinin, and 1
µg/ml leupeptin. Concentrated samples (about 100 µg of
proteins), suspended in gel loading buffer, were incubated for 15 min
at 25 °C and subjected to SDS-polyacrylamide gel
electrophoresis
(21) . The resolved proteins were transferred to
nitrocellulose filters using the Western blot procedure (22). Lf was
immunodetected as described previously
(10) , except that 5%
nonfat carnation dried milk was used instead of 3% bovine serum albumin
to block membranes, and 2% nonfat carnation dried milk was used during
antibodies incubation.
Nuclear Run-on Assay
Evaluation of GM-CSF
transcriptional rate was performed in 5637 cells. Briefly, cells
untreated or treated with 0.1 µM Lf for 18 h were lysed
with 10 mM Tris-HCl, pH 7.4, in the presence of 10 mM
NaCl, 3 mM MgCl, 0.5% Nonidet P-40, 2.75
mM dithiothreitol, and 20 units/ml RNasin. Pelleted nuclei
were resuspended in 10 mM Tris-HCl, pH 7.5, containing 5
mM MgCl
, 0.5 MD-sorbitol, 2.5%
Ficoll, 0.008% spermidine, 1 mM dithiothreitol, and 50%
glycerol. Elongation of nascent radiolabeled RNA was performed at 27
°C for 35 min in 40 mM Tris-HCl, pH 8.3, in the presence
of 200 µCi [
-
P]UTP, 0.63 mM
ATP, 0.31 mM GTP, 0.31 mM CTP, 150 mM
NH
Cl, 7.5 mM MgCl
, and 200
units/µl RNasin. The transcription reaction was terminated by the
addition of 40 units of DNase-RNase-free at 37 °C for 10 min. The
extracted RNA was dissolved in 10 mM Tris-HCl, pH 8.0,
containing 1 mM EDTA. Samples were normalized by radioactivity
quantification in a liquid scintillation
counter. Plasmids
containing the GM-CSF cDNA, IL-1
cDNA,
-actin cDNA, and pBS,
linearized by appropriate enzymatic digestion, were denatured and
immobilized on nylon filters in a slot-blot apparatus. The filters were
prehybridized at 42 °C for at least 10 h; equal amount of
transcribed RNAs (2.5
10
cpm/ml) were then added,
and the hybridization was performed for 72 h. After appropriate
washing, filters were exposed to film.
-actin cDNA was used as a
positive control, and pBS was used as a negative control. After
radioactive counting of transcripts with reference to
-actin, the
reduction in GM-CSF and IL-1
transcriptional rates of Lf-treated
cells compared with untreated cells was expressed as percent of
reduction. This experiment was repeated 3 times.
Chloramphenicol Acetyltransferase (CAT)
Assay
Transfected cells were washed twice with
phosphate-buffered saline, scraped in 0.25 M Tris-HCl, pH 7.9,
and lysed by three sequential cycles of freeze-thawing. Cell debris was
removed by centrifugation at 15,000 g for 10 min.
Supernatants were assayed for protein concentration using the Bradford
reagent according the manufacturer's instructions (Bio-Rad). The
amounts of cell extract used for CAT assay were normalized for
-galactosidase activity, and the assay was carried out as
described
(23) . After separation by thin-layer chromatography
(TLC), reaction products were detected by autoradiography and
quantified by liquid scintillation counting of thin-layer
chromatography plate areas containing
[
C]chloramphenicol and its acetylated
derivatives. CAT activity is expressed as a percentage of
chloramphenicol conversion.
RESULTS
Lf Does Not Affect Constitutive Expression of
GM-CSF
The 5637, Mo, and U937 cell lines incubated with
increasing concentrations of Lf from 1 pM to 0.1
µM for different times (from a few hours to some days) did
not show significant modifications of IL-1 and GM-CSF mRNA levels.
A representative Northern blot analysis performed from 5637 cells
showed that the mRNA levels of GM-CSF and IL-1
were not
significantly modified by treatment with 0.1 µM Lf for
some hours (Fig. 1). As shown in , 5637 cells
secreted variable amounts of GM-CSF in culture-conditioned medium. This
variability was dependent on culture conditions, although the cells
were used at about 70% confluence. Nevertheless, a constant but
moderate reduction (about 20%) in GM-CSF protein concentration was
observed after 24 h with 0.1 µM Lf treatment. Lf synthesis
in 5637 cells, which do not contain endogenous Lf, was obtained by
transfecting the pLf expression vector. We were able to detect Lf mRNA
(Fig. 2, lane1) as well as the Lf protein by
SDS-polyacrylamide gel electrophoresis (Fig. 3, lane3). From these experiments it appears that transfection
with pLf was effective for expression of Lf mRNA and protein, while
IL-1
and GM-CSF mRNA levels were not significantly modified by Lf
(Fig. 2). In culture medium of pLf transfected-cells, the level
of GM-CSF protein was 20% lower than that of the control cells.
Figure 1:
Level of
GM-CSF and IL-1 mRNA in Lf-treated 5637 cell line assay.
Subconfluent cultures of 5637, were treated with 0.1 µM
human purified Lf (+) for the times indicated. Cytoplasmatic RNA
(10 µg) was subjected to Northern analysis. Sequential
hybridizations were performed with
-
P-labeled GM-CSF,
IL-1
, and
-actin probes. See ``Experimental
Procedures'' for details of cell culture and mRNA
analysis.
Figure 2:
Level of GM-CSF and IL-1 mRNA in
Lf-transfected 5637 cell line. Cells were transfected with 10 µg of
Lf expression vector pLf (lane1) and with an
equimolar concentration of the control plasmid pBS (lane2). mRNA was analyzed as described in Fig. 1.
Hybridization was also performed with
-
P-labeled Lf
probe. See ``Experimental Procedures'' for details of
plasmids and transfection conditions.
Figure 3:
Immune-detection of Lf produced in 5637
cells. Samples of transfected 5637 cells were solubilized, and about
100 µg of proteins were subjected to SDS-polyacrylamide gel
electrophoresis and Western blot analysis. Lf was immunodetected with a
polyclonal anti-human Lf antibody. Lane1, 0.2 µg
of human purified Lf; lane2, cells transfected with
pBS; lane3, cells transfected with pLf. See
``Experimental Procedures'' for details of
immunodetection.
Lf in PEU Cells Can Inhibit GM-CSF Expression in Response
to IL-1
We then used PEU cells in which GM-CSF expression
is low and can be induced to higher levels by various
agents
(10) . The induction of GM-CSF expression by tumor
necrosis factor-, IL-1, and IL-1
was tested, and IL-1
(15 units/ml) induced the highest levels of GM-CSF mRNA (data not
shown). To test whether Lf was able to affect the expression of GM-CSF
in PEU cells stimulated by IL-1
, we transfected these cells with
pLf. Lf in transfected cells was detectable both as mRNA (Fig. 4,
lane3) and protein (data not shown). The level of
GM-CSF mRNA induced by IL-1
, in pLf-transfected cells, was
significantly lower than that found in the untransfected cells
(Fig. 4, lanes2 and 3). A low
secretion of GM-CSF protein was found in PEU cells culture medium
(about 0.1 ng/ml), and this protein amount showed a similar trend to
mRNA; IL-1
treatment induced a 2-3-fold increase in the
GM-CSF protein level, while Lf was able to inhibit such an effect since
the protein level was the same as in unstimulated cell culture medium
(data not shown).
Figure 4:
GM-CSF mRNA decrease induced by Lf in
IL-1-stimulated PEU cells. PEU cells transfected (lanes1 and 2) with pBS and with pLf (lane3) were untreated (lane1) and treated
(lanes2 and 3) with 15 units/ml of
IL-1
. mRNAs were analyzed as indicated in Fig. 1.
-
P-Labeled Lf and GM-CSF probes were used for
hybridization. 28 and 18 S rRNA were visualized by ethidium bromide gel
staining.
Activity of Endogenous GM-CSF Promoter in Lf-treated 5637
Cells
We investigated whether Lf could down-regulate the rate of
mRNA transcription in these cells. As shown in a representative nuclear
run-on assay (Fig. 5), GM-CSF transcript appeared more abundant
than IL-1 and even more so than
-actin. Lf-treated cells
showed a 20% reduction in GM-CSF transcription rate. We also observed
that transcription of IL-1
gene seems to be affected by Lf in a
similar way to GM-CSF.
Figure 5:
Activity of endogenous GM-CSF promoter in
Lf-treated 5637 cells. 5637 cells were treated without or with 0.1
µM Lf for 18 h. Nuclei were extracted, and the
transcriptional rate was measured as described under
``Experimental Procedures.'' We analyzed GM-CSF and IL-1
genes;
-actin was used as positive control and pBS as negative
control.
Lf Down-modulates GM-CSF Promoter Activity
We next
asked whether Lf could mediate a reduction in the transcriptional
activity of the GM-CSF promoter. pLf was cotransfected into PEU and
5637 cells together with pPF2000, in which the human GM-CSF promoter is
fused to the CAT reporter gene (Fig. 6). We first verified
(Fig. 7a) that CAT activity (expressed as mean ±
S.E. of four experiments), under the control of the GM-CSF promoter,
was increased about 3-fold in IL-1-treated PEU cells compared with
untreated cells (4.82 ± 0.18 versus 1.68 ±
0.18). In 5637 cells, in which IL-1
is endogenously
expressed
(14) , the CAT activity driven by the GM-CSF promoter
was high both in treated and in untreated cells (7.18 ± 0.87
versus 6.55 ± 1.84). Co-transfection with pLf
(Fig. 7b) caused a significant reduction in CAT activity
in both 5637 and PEU cells. A 7.4-fold reduction was achieved in PEU
cells stimulated by IL-1
, while only a 1.7-fold reduction was
observed in IL-1
-untreated cells (13.55 ± 2.14% versus 100% and 57.38 ± 1.07% versus 100%, respectively).
In the 5637 cell line, Lf caused a 7.3-fold reduction in CAT activity
in IL-1
-treated cells and a 6.1-fold reduction in untreated cells
(13.67 ± 3.95% versus 100% and 16.33 ± 7.31%
versus 100%, respectively). We show a representative CAT
assay, of 5637 (Fig. 6a) and PEU
(Fig. 6b) cells.
Figure 6:
Effect of Lf on GM-CSF promoter activity
in 5637 and PEU cells. The data show a representative CAT assay. 5637
(a) and PEU (b) cells were transfected with pLf
(+) or an equimolar amount of pBS (-) together with 6 µg
of pPF2000 (CAT reporter gene containing the GM-CSF promoter). The
cells were IL-1-stimulated (+) or unstimulated (-). See
``Experimental Procedures'' for details of plasmids,
transfection, and CAT assay.
Figure 7:
Kinetics of Lf inhibition of GM-CSF
promoter activity. Bars show the mean ± S.E. of the CAT
activity of four experiments performed in duplicate. a, to
compare CAT activities in two different cell types, percentage
chloramphenicol conversion was divided by -galactosidase activity
expressed as OD/1 h. Untreated cells (openbars),
IL-1
-treated cells (stripedbars). b,
CAT activity of the pBS transfected cells (openbars)
represented 100% activity; percentage CAT activity of pLf-transfected
cells (solidbars) was related to respective
pBS-transfected cells. Transfections were performed as described in
Fig. 6. See ``Experimental Procedures'' for details of
plasmids, transfection, and CAT assay.
DISCUSSION
This work was aimed at investigating the effect of Lf on the
expression of GM-CSF in various in vitro cultured cells. The
results presented here demonstrate that Lf is not able to down-regulate
the high levels of GM-CSF and IL-1 expressed in 5637, Mo, and U937
cell lines, but a 20% constant reduction of secreted GM-CSF protein was
observed in Lf-treated 5637 cells. By contrast, in PEU cells, Lf leads
to a reduction in GM-CSF mRNA level in IL-1
-treated cells. The
mRNA level, which is 3 times increased by IL-1
, was reduced to the
basal level by transfected Lf. Reduction in mRNA was followed by a
similar reduction in the secreted GM-CSF protein.
. In 5637 cells, Lf inhibition of the
exogenous GM-CSF promoter was significant even in the absence of
IL-1
treatment. This can be explained by the endogenous production
of IL-1
.
and IL-1
, which maintain high
level of the GM-CSF through a transcriptional and post-transcriptional
regulation
(4, 24) . In 5637 cells, mRNA half-life is
increased up to 4 h compared with an average of 0.5 h in tumor necrosis
factor-stimulated or unstimulated fibroblasts
(25) . This
different regulation in the two cell types may explain the different
effect of Lf on the overall mRNA accumulation. Therefore Northern blot
analysis is not so sensitive to indicate a 20% difference in mRNA
levels upon Lf-treatment. On the other hand the results obtained by run
on assay and by protein assay confirme a Lf action on GM-CSF
expression.
stimulates GM-CSF promoter activity in embryonic
fibroblasts, and provides additional information about the mechanism by
which IL-1 regulates GM-CSF expression. Previous results have shown
that, in endothelial cells, IL-1
increases GM-CSF expression at
the transcriptional level; furthermore, IL-1
activates GM-CSF
promoter by acting through a sequence close to the TATA box (26). In
WI-38 fibroblasts, IL-1 seems to have no effect on GM-CSF
transcription
(27) , and other results indicate that, in
fibroblasts, GM-CSF expression appears to be regulated both
transcriptionally and post-transcriptionally by
IL-1
(4, 24) . The cis and trans elements controlling
GM-CSF transcription have been partly identified. Of particular
interest is the AP1-like recognition site at -53 in human and
mouse promoter. It has been demonstrated that AP1, which is induced by
IL-1
(28, 29) , binds this
sequence
(30, 31, 32) . Our co-transfection
experiments revealed that Lf down-modulates GM-CSF promoter activity
both in PEU and 5637 cells. We believe that, in both cell types, the
effect of Lf on GM-CSF promoter is mediated by IL-1
.
Table:
GM-CSF protein concentration in
Lf-treated and untreated 5637 cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.