From the Departments of §§ Pediatrics, and
¶ Biochemistry, Microbiology and Immunology, University of Ottawa,
Ottawa, Ontario K1H 8L1, Canada, the Division of Virology
and Molecular Immunology, Research Institute, Children's Hospital of
Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, the ** Division
of Infectious Diseases, Department of Medicine, Vancouver General
Hospital, University of British Columbia, Vancouver, British Columbia
V5Z 3J5, Canada, and
Health Canada,
Therapeutic Products Programme, Research Services Division,
Ottawa, Ontario K1A 0L2, Canada
Received for publication, December 12, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Interleukin-10 (IL-10), a pleiotropic
cytokine that inhibits inflammatory and cell-mediated immune responses,
is produced by a wide variety of cell types including T and B cells and
monocytes/macrophages. Regulation of pro- and anti-inflammatory
cytokines has been suggested to involve distinct signaling pathways. In
this study, we investigated the regulation of the human IL-10 (hIL-10)
promoter in the human monocytic cell line THP-1 following activation
with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences
revealed that DNA sequences located between base pairs An appropriate balance between pro- and anti-inflammatory
influences in the immune response is critical in the resolution of many
pathological conditions. Interleukin-10
(IL-10),1 a cytokine that
inhibits inflammatory and cell-mediated immune responses (1), has
enormous potential for the treatment of inflammatory and autoimmune
disorders. Human IL-10 (hIL-10), a nonglycosylated 178-amino acid
polypeptide, is encoded by a gene located on chromosome 1q and has more
than 73% amino acid sequence homology with murine IL-10 (mIL-10)
(1-3). IL-10 is a pleiotropic molecule that is produced by a wide
variety of cell types, including CD4+ Th0 and Th2 cells,
CD8+ T cells (4), B cells (1, 5, 6), and
monocytes/macrophages (7). The major biological effects of IL-10
include inhibition of antigen-presenting cell-dependent
cytokine synthesis by Th1 cells, costimulation of mast cell growth, and
costimulation of thymocyte growth in the presence of IL-2 and/or IL-4
(1, 8). IL-10 inhibits antigen-driven activity of both Th1 and Th2
subsets (1, 4, 8), although it facilitates the induction of Th2 cell
types. IL-10 exhibits stimulatory effects on B cell growth and
differentiation (6, 9, 10) and acts as an autocrine growth factor for
Ly-1+ B cells, which are important in murine models of autoimmune
disease (11).
The potent action of IL-10 on macrophages, particularly at the level of
monokine production (1, 7, 8), supports an important role for IL-10 not
only in the regulation of T cell responses (1, 4, 8) but also in acute
inflammatory and autoimmune responses (12-14). In mice, IL-10
administration has been shown to inhibit a number of immunological
effects, including delayed type hypersensitivity, alterations in
vascular permeability, and increases in footpad cytokine production
(15). Conversely, IL-10 transgenic mice were shown to be unable to
limit the growth of immunogenic tumor cells (16). In contrast, IL-10
knockout mice demonstrate a state of chronic inflammation (12), severe disease in experimental allergic encephalomyelitis (13), and bronchopulmonary aspergillosis (14), suggesting that IL-10 plays a
beneficial role in controlling the harmful inflammatory response in
these conditions. In humans, high levels of IL-10 have been shown to be
produced in patients with HIV infection (17-19) and in septic shock
(20). There is also evidence to suggest that polymorphism in the hIL-10
promoter region is associated with altered IL-10 expression in
autoimmune diseases including multiple sclerosis, rheumatoid arthritis,
and systemic lupus erythematosus (21).
The molecular mechanisms underlying the regulation of cytokine
synthesis in mononuclear phagocytes are not fully known. LPS is perhaps
the best characterized monocytic mitogen, which, following interaction
with its receptor CD14, induces first proinflammatory (IL-1, TNF- Regulation of gene expression for several pro- and anti-inflammatory
cytokines has been studied. Transcription factors including Rel, C/RBP,
AP-1, and NF- Cell Lines, Cell Culture, and Reagents--
THP-1, a
promonocytic cell line, was obtained from the American Type Culture
Collection (ATCC; Manassas, VA). 5-15% of these cells express CD14 on
their surface. THP-1 cells transfected with a plasmid containing CD14
cDNA sequences (THP-1/CD14) were kindly provided by Dr. Richard
Ulevitch (The Scripps Research Institute, La Jolla, CA) (Fig. 1). Cells
were cultured in Iscove's modified Dulbecco's medium (Sigma)
supplemented with 10% fetal bovine serum (FBS), 100 units/ml
penicillin, 100 µg/ml gentamicin, 10 mM HEPES, and 2 mM glutamine. PD98059 (Calbiochem), an inhibitor of
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase-1 kinase, selectively blocks the activity of ERK MAP
kinase and has no effect on the activity of other serine threonine
protein kinases including Raf-1, p38, and JNK MAP kinases, protein
kinase C, and protein kinase A (24, 38). The pyridinyl imidazoles SB202190 and SB203580 (Calbiochem), potent inhibitors of p38 and p38 Cell Stimulation, Collection of Culture Supernatants, and
Measurement of IL-10 by ELISA--
THP-1 cells were cultured at
concentrations of 0.5 × 106 cells/ml in 24-well
culture plates (Falcon, Becton-Dickinson, Franklin Lakes, NJ). Cells
were left unstimulated or were treated for 48 h with various
agents for different periods of time, following which they were
stimulated with 1 µg/ml LPS. The supernatants were frozen
at RNA Isolation and Semiquantitative Reverse Transcriptase-based
Polymerase Chain Reaction (PCR) for IL-10--
Total RNA was extracted
as described (40) using a monophase solution containing guanidine
thiocyanate and phenol (Tri Reagent solution; Molecular Research
Center, Inc., Cincinnati, OH). Total RNA (1 µg) was reverse
transcribed by using Moloney murine leukemia virus reverse
transcriptase (PerkinElmer Life Sciences). Equal aliquots (5 µl) of
cDNA equivalent to 100 ng of RNA were subsequently amplified for
IL-10 and Flow Cytometric Analysis--
Cells were subjected to flow
cytometric analysis as described (17, 40). Briefly, cells were stained
with 3 µl of fluorescein isothiocyanate-labeled anti-CD14 monoclonal
antibodies (Becton Dickinson) along with isotype (IgG2b)-matched
control antibodies (Becton Dickinson). The gates were set in accordance
with gates obtained with the isotype-matched control antibodies.
Population data were acquired on a Becton Dickinson FACScan flow
cytometer, and figures were generated using the WinMDI software package
(J. Trotter, Scripps Institute, San Diego, CA).
Gel Mobility Shift Assays--
Cells (107) were
harvested in Tris-EDTA-saline (TES) buffer, pH 7.8, and centrifuged at
200 × g for 5 min. The cells were lysed for 10 min at
4 °C with buffer A (10 mM HEPES, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, pH
7.9) containing 0.1% Nonidet P-40. The lysates were centrifuged at
20,000 × g for 10 min. The pellet containing the
nuclei was suspended in buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol) at 4 °C for 15 min. The
supernatant containing the nuclear proteins was collected and frozen at
Construction of Luciferase Reporter Gene Vectors--
A series
of hIL-10 promoter fragments (see Fig. 3; fragment Transient Transfection of Cells and Measurement of Luciferase
Activity--
Transfection of THP-1 and CD14-transfected THP-1
(THP-1/CD14) cells with plasmids containing various IL-10 promoter
fragments was performed using LipofectAMINE Reagent (Life Technologies, Inc.) following the manufacturer's instructions. 10 µg of the test
plasmid and 5 µg of pSV- Immunoprecipitation and Western Blot Analysis--
Cell pellets
were lysed for 30 min with lysis buffer (50 mM HEPES, pH
7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium vanadate, and 1 mM EGTA, pH 7.7).
Total protein lysates (2 mg) were precleared with protein A-Sepharose
4B beads (Amersham Pharmacia Biotech) for 1 h at 4 °C followed
by incubation for 2 h at 4 °C with protein A-Sepharose beads
and antibodies as indicated in the figure legends. Anti-p38 and
anti-p42/44 rabbit polyclonal antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Immune complexes were washed
three times with the lysis buffer, boiled for 5 min in
SDS-polyacrylamide gel electrophoresis buffer, and subjected to
electrophoresis on 8% polyacrylamide SDS gels. Proteins were transferred to ImmobilonTM-P membranes (Millipore Corp.,
Bedford, MA), and the membranes were probed for phosphorylated p38 and
p42/44 proteins using anti-phosphotyrosine 4G10 (UBL) antibodies. The
immunoblots were developed by ECL (Amersham Pharmacia Biotech) as per
the manufacturer's instructions.
Statistical Analysis--
All transfection studies were
performed in triplicate dishes in 3-5 separate experiments. The
results are expressed as mean ± S.D.
THP-1 Cells Produce IL-10 in Response to LPS--
To understand
LPS-induced IL-10 regulation in human monocytes, we employed two
promonocytic cell lines, THP-1 and THP-1/CD14. CD14 was found to be
expressed on ~10-15% of THP-1 cells, and this number increased to
more than 50% after stimulation with LPS (Fig.
1). Since production of cytokines by
LPS-stimulated monocytic cells lines is critically dependent on the
level of CD14 expression, we employed THP-1 cells transfected with CD14 (THP-1/CD14) for analysis of the regulation of IL-10 production. All
THP-1/CD14 cells constitutively expressed very high levels of CD14 on
their surfaces compared with untransfected cells (15%) (Fig. 1).
Stimulation of THP-1/CD14 cells with LPS induced IL-10 expression as
determined by enzyme-linked immunosorbent assay (Fig.
2A) and reverse
transcriptase-based PCR analysis (Fig. 2B, C).
Maximal levels of IL-10 mRNA were detected within 4 h after stimulation of THP-1 (Fig. 2B) and THP-1/CD14 (Fig.
2C) cells. Production of IL-10 protein was elevated in
THP-1/CD14 cells compared with THP-1 cells, an observation that
correlated with CD14 surface expression. Production of IL-10 was
dependent on the concentration of LPS used for cell stimulation, and 1 µg of LPS produced maximal levels of IL-10 (data not shown).
Determination of DNA Sequences in the IL-10 Promoter Region
Required for IL-10 Transcription--
Inducible genes, including
cytokine genes, contain DNA sequences within their promoter region that
are responsible for regulating transcription. The human IL-10 promoter
was recently cloned and characterized (41). To understand the
regulation of IL-10 gene transcription in LPS-stimulated THP-1 cells,
we used PCR to clone the hIL-10 promoter fragment encompassing
nucleotide residues from
To determine the DNA sequences in the hIL-10 promoter that are required
for IL-10 transcription, a series of hIL-10 promoter fragments (from 5'
Sp1 Binding Site in the hIL-10 Promoter Is Sufficient to Induce
IL-10 Production--
A computer-aided analysis of the hIL-10 promoter
sequence between Selective Role of p38 MAP Kinase in IL-10 Production by
LPS-stimulated THP-1 Cells--
It has been suggested that p38 MAP
kinase plays a vital role in hIL-10 synthesis following stimulation of
human monocytes with LPS (31). MAPKs are serine/threonine protein
kinases, which include the p42/44 ERKs, the p54 and p46 JNK1 and
-2/stress-activated protein kinase, and p38 MAPK (24). These three
families of MAPK form three parallel signaling cascades activated by
distinct and sometimes overlapping sets of stimuli (24). LPS-induced
cell signaling is known to involve activation of p38, p42/44 ERK, and JNK MAPK (25-27). We hypothesized, therefore, that the MAPK, and in
particular the p38 MAP kinase, regulate IL-10 synthesis through activation of Sp1 transcription factor in human monocytic cells.
To investigate the role of MAPK in the regulation of hIL-10 production
in LPS-stimulated THP-1/CD14 cells, we examined the activation of ERK
and p38 kinases. Cells were stimulated with LPS for 15 min and
subjected to immunoprecipitation with anti-p38 and anti-ERK antibodies,
followed by Western blot analysis with anti-phosphotyrosine antibodies.
The results show the LPS-induced tyrosine phosphorylation of p38 and
ERK kinases. To control for protein loading, the blots were stripped
and reprobed with anti-p38 and anti-ERK antibodies (Fig.
7). To better understand the role of MAP
kinases in LPS-mediated signaling, specific inhibitors of p38 (SB202190
and SB203580) and p42/44 ERK kinases (PD98059) were employed. SB202190
and SB203580 are selective and potent inhibitors of p38 and p38
To determine the role of p38 and ERK MAP kinases in IL-10 production,
THP-1/CD14 cells were treated with specific inhibitors for these MAP
kinases for 2 h prior to stimulation with LPS. Cell supernatants
were harvested at 48 h for analysis of IL-10 production, since
previous experiments had shown that cytokine production peaked 48 h after LPS stimulation (data not shown). The results show that
treatment of THP-1/CD14 cells with PD98059 at doses as high as 75 µM did not affect IL-10 production in a statistically significant manner (Fig. 8). Doses higher
than 50 µM for PD98059 were not used in subsequent
experiments, since these concentrations were cytotoxic as determined by
trypan blue exclusion (data not shown). In contrast, treatment of
THP-1/CD14 cells with SB202190 and SB203580 completely inhibited
LPS-induced IL-10 production in a dose-dependent manner
(Fig. 8). These results clearly demonstrate that LPS-induced IL-10
production is regulated by p38 MAP kinases in THP-1/CD14 cells.
The Inhibitor of p38 MAP Kinase Abrogates Luciferase Activity of
the Sp1-containing hIL-10 Promoter Construct--
To further
investigate the role of p38 MAP kinase in the activation of Sp1 leading
to IL-10 gene transcription, THP-1 and THP-1/CD14 cells were
transfected with pGL3B containing a series of successive 5' deletions
derived from
To confirm that p38 MAP kinase activates Sp1 transcription factor,
THP-1 and THP-1/CD14 cells were transfected with a plasmid containing a
wild type or mutant Sp1 sequence and cultured in the presence or
absence of SB202190. Treatment with SB202190 completely abrogated the
luciferase activity observed in LPS-stimulated cells transfected with
the plasmid containing the wild type Sp1 sequence (Fig.
10). Similarly, treatment of
LPS-stimulated cells transfected with plasmids containing either a
mutated Sp1 sequence or an EBNA-2-like transcription factor sequence
with p38 inhibitor SB202190 did not result in any change in luciferase
activity (Fig. 10). The results shown are a mean of four experiments
performed with both THP-1 (data not shown) and THP-1/CD14 cells (Fig.
10).
Sp1 Binding to the IL-10 Promoter in LPS-stimulated Cells Is
Regulated by p38 MAP Kinase--
To further understand which signaling
events downstream of MAP kinases may be involved in IL-10
transcription, we investigated the activation of p38 MAP kinase
substrates. The above results suggest that the p38 MAP kinase and the
Sp1 play a role in the regulation of hIL-10 transcription. Therefore,
we investigated whether LPS stimulation of THP-1 cells induced the
binding of Sp1 to the Sp1 binding site in the hIL-10 promoter. Cells
were stimulated with LPS over a period of time ranging from 0 to 240 min, and the nuclear extracts were analyzed in a gel shift assay for
binding to Sp1 oligonucleotide probes. The results revealed that the
maximum binding of Sp1 to the Sp1 oligonucleotide sequence of the
hIL-10 promoter occurred 30-45 min following stimulation of THP-1/CD14
cells with LPS (Fig. 11A).
We observed three distinct Sp1 DNA-protein complex bands, namely A, B,
and C, in a gel shift assay that were completely blocked by competition
with cold Sp1 oligonucleotides, indicating their specificities. It
should be pointed out that bands A and B were always induced by LPS.
However, induction of band C was not observed at all time points. The
possible reasons for inconsistent induction of band C are not known.
Similar results were obtained with LPS-stimulated THP-1 cells (data not shown). To determine whether p38 MAP kinase delivers a signal via the
activation of Sp1 transcription factor, we investigated whether
SB202190, an inhibitor of p38 MAP kinase, inhibits binding of Sp1 to
the Sp1-binding site of the IL-10 promoter. Incubation of THP-1/CD14
cells with SB202190 for 2 h prior to stimulation with LPS resulted
in the inhibition of Sp1 binding to the oligonucleotide containing the
Sp1 sequence (Fig. 11B). As above, p38 inhibitors significantly reduced Sp1 binding to its oligonucleotides in bands A
and B. In contrast, PD98059 did not affect Sp1 binding in
LPS-stimulated cells (data not shown). These results suggest that p38
MAP kinase may promote IL-10 expression by activating Sp1.
Bacterial endotoxin (LPS) is responsible for many of the cellular
responses to Gram-negative bacterial infections (43). These responses
may be induced after the association of LPS with the LPS-binding plasma
protein and the binding of this complex with the CD14 receptor
expressed on cells of monocytic lineage (44). LPS stimulates a variety
of cytokines including proinflammatory (IL-1, IL-6, TNF- To better understand the LPS-induced signaling pathway in the
regulation of IL-10 synthesis, we employed two types of THP-1 cell
lines that differ with respect to the level of CD14 receptor expression
on their surfaces. CD14 was expressed on 5-15% of the THP-1 cells. To
enhance LPS-mediated response in these cells, THP-1/CD14 cells were
used; these cells constitutively expressed CD14. Hence, transfection of
THP-1/CD14 cells with 5'-deletion mutants of hIL-10 promoter linked to
the luciferase gene consistently revealed higher luciferase activity
compared with THP-1 cells transfected with the same constructs. It
should be pointed out that LPS stimulation of THP-1 cells induces
cytokine production in a manner similar to that observed with normal
human monocytes. However, THP-1 cells, unlike normal human monocytes,
are free from negative feedback regulation mediated by endogenously
produced IL-10 (data not shown).
Sp1 is a ubiquitous transcription factor that regulates the
constitutive activity of many genes studied. Sp1 plays a vital role in
the regulation of transcription from TATA-less promoters that commonly
encode housekeeping genes (45). Sp1 activity and cellular content have
been shown to be regulated during development (46, 47), cellular
proliferation (48), apoptosis (49), and other cellular processes (50,
51). Sp1 has been shown to be involved in mediating responses to
various stimuli including induction of the TGF- How Sp1 mediates its responses is presently not fully understood. Sp1
is a well characterized protein composed of 778 amino acids. The
amino-terminal portion of the molecule contains two glutamine-rich
domains, each of which is associated with serine/threonine-rich regions
(55). These domains are involved in transcriptional activation. The
C-terminal region of the molecule contains the zinc finger
DNA-recognition domain (55). Most of the gene regulation mediated by
Sp1 requires either post-translational modifications of Sp1, such as
phosphorylation and glycosylation (46, 56, 57) or alterations in the
abundance of Sp1 protein (51). In addition, there are several
coactivators of Sp1 such as CRSP, Rb, and hTAFII 130, which allow Sp1
to stimulate transcription very effectively (45, 58). Translational
modification of any of these coactivators may modulate the ability of
Sp1 to regulate transcription. Molecular mechanisms by which Sp1
regulates IL-10 transcription remain to be investigated.
It was surprising to find that only one transcription factor (Sp1)
seems to play a prominent role in IL-10 regulation. This is in contrast
to most of the cellular genes, and especially cytokine genes, that are
regulated by multiple transcription factors. In view of established
models of multiple transcription factor involvement, it seems unlikely
that other transcription factors are not involved in the regulation of
the hIL-10 gene. Our studies do not rule out the involvement of other
transcription factors that may cooperate with Sp1 in hIL-10
transcription. It is likely that the transient transfection assay used
in the current study may not reveal the involvement of other
transcription factors. These factors may remain masked in our
experimental system of transient transfection, a system that is known
to generate a high plasmid copy number or the accumulation of aberrant
chromatin structure in these cells (59). To circumvent this
possibility, a stable transfection approach may be required. It is also
possible that other transcription factors interacting with the hIL-10
promoter region beyond the We have also investigated the upstream signaling events that lead to
Sp1 activation. We primarily investigated the role of MAP kinases in
this process and in IL-10 production via stimulation of CD14 receptors
in THP-1 cells. LPS has been shown to activate p38, p42/44 ERK, and JNK
MAP kinases (24). These three types of MAP kinases can be activated
individually or simultaneously, thereby suggesting their independent
signaling roles (24). The data presented in this study show the
selective involvement of p38 in IL-10 production in THP-1 cells.
Expression of the luciferase gene linked to the hIL-10 promoter was
mediated via p38 MAP kinase activation. A specific inhibitor of the
p42/44 ERK MAP kinases did not affect luciferase activity (data not
shown). In addition, we also demonstrated that LPS induces the
activation of Sp1 transcription factor in a time-dependent
manner. Maximum activity of Sp1 activation was observed at 30-60 min
poststimulation with LPS. Similarly, p38 inhibitors significantly
reduced Sp1 binding to its oligonucleotides. Furthermore, using Sp1
mutants of the hIL-10 promoter linked to the luciferase reporter gene,
we demonstrate for the first time that p38 MAP kinase plays a direct
role in Sp1 activation and in inducing Sp1 binding to the IL-10 promoter.
The LPS-induced signaling pathway leading to the activation of the p38
MAP kinase in monocytes/macrophages has been investigated. LPS
signaling through CD14 has been shown to involve Toll-like receptors
(TLR), specifically TLR-4, which associates with CD14 (60-62). LPS
interaction with CD14 promotes dimerization of the TLR and subsequent
recruitment of MyD88, a myeloid differentiation marker that functions
as an adaptor molecule (62, 63). MyD88 associates via its c-terminal
toll homology domain with TLR and via its N-terminal death domain with
a serine-threonine protein kinase, IL-1R associated kinase (63). Upon
interaction with MyD88, IL-1R-associated kinase is autophosphorylated
and binds to TRAF-6 (TNF-receptor associated factor-6). TRAF-6
subsequently activates TAK-1 and mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase kinase,
members of the MAP kinase signaling cascade. This interaction activates
NF- Activation of the p38 MAP kinase has been shown to play a critical role
in the regulation of several cellular and cytokine genes in T cells and
monocytes following their stimulation with various ligands. For
example, T cell activation with specific antigen or staphylococcal
antigens has been shown to induce p38 activation, resulting in TNF- In summary, our results clearly show for the first time the involvement
of the Sp1 transcription factor, and its activation via p38 MAP kinase,
in the regulation of hIL-10 gene transcription in human monocytic cell
lines. While this work was in progress, Brightbill et al.
(74) demonstrated the involvement of a nonconsensus Sp1-like sequence
in mIL-10 expression using RAW264.7, a murine macrophage cell line.
Although the hIL-10 gene bears >80% nucleotide sequence homology and
>73% amino acid sequence homology with mIL-10, the mIL-10 and hIL-10
genes and their promoters are distinct (1-3). In contrast to mIL-10,
hIL-10 cDNA clones contain the insertion of Alu
repetitive sequence elements in the 3'-untranslated region (2).
Furthermore, the Sp1 consensus sequence is not present in the mIL-10
promoter (74). In contrast to our findings, LPS stimulation of murine
monocytic cells did not result in Sp1 activation (74), indicating
perhaps differential regulation of IL-10 synthesis in murine and human
monocytic cells. Taken together, our results point to the key role of
Sp1 and its activation via p38 MAP kinase in the regulation of IL-10
transcription. These studies may provide a basis for the identification
of molecular players in IL-10 regulation that may help in designing
targeted drug therapy for inflammatory diseases.
652 and
571 are necessary for IL-10 transcription. A computer analysis of the
promoter sequence between base pairs
652 and
571 revealed the
existence of consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr
virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors.
THP-1 cells transfected with a plasmid containing mutant Sp1
abrogated the promoter activity, whereas plasmids containing the
sequences for PEA1, YY1, and EBNA-2-like transcription factors did not
influence hIL-10 promoter activity. To understand the events upstream
of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase
kinases, failed to modulate IL-10 production. Furthermore, SB203580
inhibited LPS-induced activation of Sp1, as well as the promoter
activity in cells transfected with a plasmid containing the Sp1
consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn
regulates transcription of the hIL-10 gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
etc.) and then anti-inflammatory (IL-10, sTNF-R, and IL-1R antagonist)
cytokines (7, 22). LPS-induced cell signaling is known to activate
protein-tyrosine kinases (23) and the MAP kinases p38, p42/44
extracellular signal-regulated kinase (ERK), and p54 (stress-activated
protein kinase/c-Jun N-terminal kinase (JNK)) (24-27). A recent report
has indicated that IL-10 production is dependent on protein-tyrosine
kinases and protein kinase C activation in a murine cell line (28). In
addition, factors that elevate cAMP have been suggested to be involved
in the regulation of monocytic IL-10 synthesis, primarily at the mRNA level (29, 30). Recently, it has been suggested that p38 MAPK
is involved in the regulation of IL-10 production (31).
B have been implicated in the regulation of
proinflammatory cytokine genes (32-37). However, very little is known
about the regulation of the hIL-10 gene and the involvement of MAP
kinases in this process. To gain insight into hIL-10 gene regulation,
we have employed promonocytic THP-1 cells that produce IL-10, IL-12,
and TNF-
following LPS stimulation as do normal human monocytes.
Using mutagenesis, we analyzed the promoter sequence of the hIL-10
gene, and we present evidence that an element located at
650 bp,
encompassing a Sp1 consensus sequence is involved in the transcription
of the hIL-10 gene. This was further demonstrated by introducing a
mutation in the Sp1 consensus sequence that abrogated IL-10 promoter
activity. Furthermore, the Sp1 transcription factor is induced by LPS
stimulation and is selectively regulated by the p38 MAP kinase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MAP kinases, have no significant effect on the activity of the ERK or
JNK MAP kinase subgroups (24, 39). LPS was purchased from Sigma.
70 °C and thawed at the time of analysis. IL-10 levels were measured by enzyme-linked immunosorbent assay by using two
different monoclonal antibodies that recognize distinct epitopes, as
described (17, 40).
-actin. The oligonucleotide primer sequences for IL-10 and
-actin (Stratagene, La Jolla, CA) are as follows: IL-10 sense
(5'-GCC TAA CAT GCT TCG AGA TC-3'); IL-10 antisense (5'-TGA TGT CTG GGT
CTT GGT TC-3');
-actin sense (5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT
CTA-3');
-actin antisense (5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA
GGG-3'). The amplification conditions for IL-10 and
-actin have been
described (40). The amplified products IL-10 (204 bp) and
-actin
(610 bp) were resolved by electrophoresis on 1.2% agarose gels and
visualized by ethidium bromide staining.
80 °C. Both buffers A and B contained the proteolytic inhibitors dithiothreitol, phenylmethylsulfonyl fluoride, and spermidine at 0.5 mM as well as 0.15 mM spermine and 5 µg/ml
each of aprotinin, leupeptin, and pepstatin. Nuclear proteins (5 µg)
were mixed for 20 min at room temperature with 32P-labeled
oligonucleotide probe Sp1, and the complexes were subjected to
nondenaturing 17% polyacrylamide gel electrophoresis for 90 min. The
gel was dried and exposed to x-ray film. The oligonucleotide sequences
for Sp1 are as follows: 5'-d(ATT CGA TCG GGG CGG GGC GAG C)-3' and
3'-d(TAA GCT AGC CCC GCC CCG CTC G)-5' (Promega Corp., Madison, WI).
890 to +120;
GenBankTM accession number X78437) were amplified from
genomic DNA by PCR. The primers with restriction sites used to amplify
the hIL-10 promoter fragments from genomic DNA are shown in Table
I. The amplification consisted of
denaturation at 95 °C for 2 min, 30 cycles of denaturation at
95 °C for 30 s, annealing at 58 °C for 1 min and extension
at 72 °C for 2 min, and final elongation at 72 °C for 10 min. The
amplified promoter products were subcloned into the PCRII-TOPO vector,
and the sequences were confirmed. The correct insertions were subcloned
into the XhoI polylinker site of pGL3B, the basic luciferase
reporter plasmid, and sequences were confirmed again. All DNA
sequencing was performed by the Biotechnology Research Institute
(University of Ottawa). A site-directed mutation of the Sp1-binding
sequence (cccgcc) was generated by PCR using mutagenic primers (Table
I) to substitute cytosine with guanine at
631 and cytosine with
adenine at
636 (see Fig. 6A). The fragment containing the
Sp1 mutation (
650 to +120 bp) was inserted into the pGL3B reporter
vector.
Primers for amplification of IL-10 promoter fragments and sizes of the
PCR products generated from genomic DNA
-galactosidase internal control vector
(Promega) were incubated for 45 min with 10 µl of LipofectAMINE reagent in 200 µl of OPTI-MEM I Reduced Serum Medium (Life
Technologies) to allow formation of DNA-liposome complexes. These
complexes were added to the cell suspension in each well, and cells
were cultured for 24 h. Following incubation, cells were
stimulated with 1 µg/ml LPS and were cultured for another 24 h.
Cells were harvested and then assayed for luciferase and
-galactosidase activity by using a luciferase assay kit and
-galactosidase assay kit purchased from Promega in a Bio Orbit 1250 Luminometer (Fisher).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Flow cytometric analysis of CD14
expression on THP-1 and THP-1/CD14 cells. THP-1 and
THP-1/CD14 cells were stimulated with LPS (1 µg/ml) for 24 h and
analyzed by flow cytometry for CD14 surface expression.
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Fig. 2.
LPS stimulation induces the synthesis of
IL-10. THP-1 and THP-1/CD14 cells were stimulated with LPS (1 µg/ml) for various times ranging from 2 to 48 h and analyzed by
enzyme-linked immunosorbent assay for IL-10 production (A).
THP-1 (B) and THP-1/CD14 (C) cells were
stimulated with LPS (1 µg/ml) for various times ranging from 2 to
24 h. Cells were harvested for mRNA isolation. IL-10
expression was determined by semiquantitative reverse
transcriptase-based PCR analysis using -actin as a standard
control.
890 to +120 bp relative to the +1
transcription site (Fig. 3). The
amplified promoter fragment was subcloned into the XhoI polylinker site of the luciferase reporter plasmid, pGL3B. THP-1/CD14 cells were transiently transfected with the IL-10-promoter/luciferase reporter construct (pIL-10Pr-GL3B). After 24 h of transfection, the cells were stimulated with LPS for varying periods of time ranging
from 6 to 36 h, following which relative luciferase activity was
assessed. The results show that luciferase activity could be detected
by 12 h and peaked at 24 h following stimulation with LPS
(Fig. 4A). The maximum
increase in luciferase activity ranged from 6- to 8-fold relative to
the unstimulated cells. The cells transfected with the promoterless
plasmid pGL3B did not show any increase in luciferase activity
following stimulation with LPS (Fig. 4B). Similar results
were obtained for THP-1 cells, although the increase in luciferase
activity was relatively lower than for the THP-1/CD14 cells transfected
with the pGL3B containing the IL-10 promoter (Fig. 4B).
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Fig. 3.
Nucleotide sequence of the 5'-flanking
promoter region of hIL-10 gene (GenBankTM accession number
X78437). The translation start codon (ATG) is
italicized. The putative cis-regulatory elements are marked
with solid lines
below/above according to their orientation,
respectively.
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Fig. 4.
Luciferase activity in LPS-stimulated
THP-1 and THP-1/CD14 cells transfected with a hIL-10
promoter/luciferase construct. A, time course of
luciferase gene expression. THP-1 cells (1.5 × 106)
were transiently cotransfected with 10 µg of either hIL-10
promoter/luciferase reporter pGL3B construct or pGL3B vector control
and with 5 µg of pSV- -galactosidase control plasmid. Cells were
grown for 24 h followed by treatment with 1 µg/ml of LPS for 6, 12, 18, 24, and 36 h. Luciferase and
-galactosidase activities
were determined for the cell lysates. B, THP-1 and
THP-1/CD14 cells (1.5 × 106) were transiently
cotransfected with 10 µg of either hIL-10 promoter construct or
vector control and with 5 µg of
-galactosidase control plasmid and
allowed to grow for 24 h. The transfected cells were treated with
1 µg/ml of LPS for 24 h followed by measurement of luciferase
and
-galactosidase activities. Cells transfected with vector pGL3B
alone served as a negative control. Luciferase activity was normalized
for
-galactosidase activity to give relative luciferase units
(RLU). The results shown are a mean ± S.D. of four
experiments performed in triplicate and normalized for
-galactosidase activity.
890 to 3' +120 bp relative to the +1 transcription site of the hIL-10
gene) were produced by generating successive deletions starting from
the 5'-end. Various hIL-10 promoter fragments were amplified from the
hIL-10 promoter region, sequenced, and inserted into the luciferase
expression plasmid (pGL3B). The exact size of the amplified product and
the location of consensus sequences for various transcription factors
identified within the hIL-10 promoter (Fig. 3) are depicted in Fig.
5 (left panel).
Examination of the DNA sequences within the hIL-10 promoter region
containing various deletions revealed that deletion of sequences from
890 to
652 bp had no effect on luciferase activity compared with the plasmid containing the complete promoter sequence. However, deletion of sequences from
571 bp and beyond completely abrogated luciferase activity compared with cells transformed with unmutagenized promoter sequences. Furthermore, the luciferase activities of these
constructs was comparable with the activity observed in unstimulated
cells and in cells transfected with the control plasmid (pGL3B) (Fig.
5). Similar results were obtained for both THP-1 (Fig. 5,
middle panel) and THP-1/CD14 (Fig. 5,
right panel) cells. The results shown are a mean
of four experiments performed with each of the THP-1 and THP-1/CD14
cells transfected with the hIL-10 promoter constructs containing
5'-deletions. These results suggest that DNA sequences located between
571 and
652 bp relative to the +1 transcription site are necessary
for hIL-10 transcription in THP-1 and THP-1/CD14 cells following LPS
stimulation.
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Fig. 5.
Transcriptional activities of hIL-10 promoter
in LPS-stimulated THP-1 and THP-1/CD14 cells. Left, the
positions of the potential regulatory elements in the hIL-10 promoter
region ( 890 to +120 bp relative to the start site). The
line diagram represents the extent of deletions
within the hIL-10 promoter region in the seven DNA constructs used in
the experiments. Putative binding sites for the transcription factors
glucocorticoid response element (GRE), cAMP,
granulocyte-macrophage colony-stimulating factor
(GM-CSF), Sp1, and signal transducers and activators of
transcription (STAT) are shown. THP-1 (middle)
and THP-1/CD14 (right) cells were cotransfected with 10 µg
of either a promoter construct or vector control and with 5 µg of
-galactosidase control plasmid. After 24 h, cells were
stimulated with LPS (1 µg/ml) for another 24 h. Cell lysates
from unstimulated and LPS-stimulated cells were assayed for luciferase
and
-galactosidase activities. Luciferase activity was normalized
for
-galactosidase activity to give relative luciferase units
(RLU). The results shown are a mean ± S.D. of four
experiments performed in triplicate.
652 and
571 bp revealed the existence of consensus
sequences for four transcription factors, including Sp1 (5'-cccgc-3' at
636 to
631 bp), PEA1 (5'-aggaag-3' at
622 to
617 bp), YY1 (5'-aaaatggaa-3' at
600 to
592), and EBNA-2-like factor
(5'-cttgggaactt-3' at
585 to
575) (Fig. 3). This suggests that any
one or more of the above mentioned transcription factors may be
involved in regulating transcription of the hIL-10 gene. To investigate
the role of the Sp1 transcription factor in hIL-10 gene transcription, we used PCR to introduce site-directed mutations in the Sp1 consensus sequence by substituting cytosine with guanine at position
631 bp and
cytosine with adenine at position
636 bp (Fig.
6A). The fragment containing
the Sp1 mutant sequence was cloned into pGL3B. To understand the role
of EBNA-2-like transcription factor, we amplified another fragment
spanning a distance from
589 to +120 bp and cloned it into pGL3B.
This fragment was devoid of Sp1, PEA1, and YY1 transcription
factor-binding sites. THP-1 and THP-1/CD14 cells transfected with a
plasmid containing an EBNA-2-like transcription factor sequence did not
show any increase in luciferase activity (Fig. 6B),
indicating that the EBNA-2-like transcription factor may not play any
role in hIL-10 gene transcription. However, THP-1 and THP-1/CD14 cells
transfected with a plasmid containing an Sp1 mutant sequence did not
show luciferase activity compared with the plasmid containing the wild
type Sp1 consensus sequence. The results shown are a mean of four
experiments performed with THP-1 (data not shown) and THP-1/CD14 cells
(Fig. 6B). These results suggest that Sp1 plays a
significant role in transcription of the hIL-10 gene. Since the plasmid
containing the Sp1 mutant sequence also contained consensus sequences
for PEA1, YY1, and EBNA-2-like transcription factors, these results
further suggest that PEA1, YY1, and EBNA-2-like factors may not be
involved in the induction of hIL-10 gene transcription by LPS.
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Fig. 6.
The effect of a mutant Sp1 binding site on
hIL-10 promoter activity in LPS-stimulated THP-1/CD14 cells.
A, a site-directed mutation of the Sp1 consensus sequence
within the hIL-10 promoter is shown with respect to the wild type Sp1
sequence. The substituted nucleotides at positions 631 and
634 of
the wild type consensus sequence are depicted in boldface
letters. The amplified fragment (
650 to +120 bp)
containing the mutations in the Sp1 sequence was cloned into pGL3B
vector. B, THP-1/CD14 cells were cotransfected with either
10 µg of wild type or mutant Sp1 construct and with 5 µg of
-galactosidase control vector. The transfected cells were treated
with 1 µg/ml of LPS for 24 h. Luciferase activities of both
unstimulated and LPS-stimulated cells were measured and are shown as a
mean ± S.D. of three experiments performed in triplicate and
normalized for
-galactosidase activity. Putative binding sites for
the transcription factors GRE, cAMP, and SP1 are shown in the
left panel. STAT, signal transducers and activators of
transcription; GM-CSF, granulocyte-macrophage
colony-stimulating factor.
MAP
kinases, respectively, and have no significant effect on the activity
of the ERK or JNK MAP kinase subgroups (39, 42). Similarly, PD98059 is
a potent and specific inhibitor of ERK. PD98059 mediates its effects by
binding to and inactivating the ERK MAP kinase without affecting the
activity of either p38 or JNK (38, 42). To determine whether SB202190, SB203580, and PD98059 specifically inhibit the phosphorylation of p38 and ERK kinases, respectively, THP-1 cells were treated with
these inhibitors at a concentration of 10 nM for 2 h,
followed by stimulation with LPS for 15 min. The results show that
SB202190 (Fig. 7A) and SB203580 (data not shown) inhibited
the phosphorylation of p38 and that PD98059 inhibited the
phosphorylation of ERK-2 MAP kinase (Fig. 7B). The
concentration of inhibitors used in above experiments did not affect
cell viability (data not shown).
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Fig. 7.
LPS stimulation induces p38 and p42/44 MAP
kinase activity in THP-1/CD14 cells. To determine the effects of
the p38 MAP kinase-specific inhibitor (SB202190) and the p42/44 MAP
kinase-specific inhibitor (PD98050) on LPS-induced activation of p38 or
p42/44 MAP kinase, respectively, cells were treated with SB202190 or
PD98059 for 2 h. Cells were then stimulated with LPS (1 µg/ml)
for 20 min, followed by centrifugation and lysis of cell pellets.
Proteins from the cell lysates were immunoprecipitated with anti-p38
(A) and anti-p42/44 (B) rabbit polyclonal
antibodies (Santa Cruz Biotechnology). The immune complexes were
subjected to SDS-polyacrylamide gel electrophoresis followed by
transfer of proteins onto the membranes. The membranes were blotted
with anti-phosphotyrosine antibodies ( py). To control for
protein loading, the membranes were stripped and reprobed with the same
antibody used for immunoprecipitation. Ab, a control
immunoprecipitation performed with antibody and Sepharose beads in the
absence of lysate. The experiment shown is representative of three
experiments.
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Fig. 8.
LPS-stimulated IL-10 production is
selectively inhibited by inhibitors of p38 MAP kinases. To
determine the effects of the p38 MAP kinase-specific inhibitors,
SB203980 and SB202190, and the p42/44 MAP kinase-specific inhibitor
PD98050 on LPS-induced IL-10 production, cells were treated with
inhibitors for 2 h prior to stimulation with LPS (1 µg/ml). The
supernatants were harvested after 48 h and analyzed by
enzyme-linked immunosorbent assay for IL-10 production. The experiment
shown is representative of three experiments.
890 to +120 bp of the hIL-10 promoter sequence. The
transfected cells were cultured for 2 h in the presence and in the
absence of p38 inhibitor, SB203580, prior to stimulation with LPS.
Luciferase activity was measured after 24 h. As observed above,
deletion of sequences spanning
652 to
890 bp from the hIL-10
promoter region revealed an 8-10-fold increase in the luciferase
activity in LPS-stimulated THP-1/CD14 cells, compared with the
unstimulated cells or cells transfected with the control plasmid (Fig.
9). Treatment of the same cells with p38
inhibitor SB202190 completely abrogated the luciferase activity (Fig.
9), suggesting the involvement of p38 in the regulation of Sp1
activity. As observed above, deletion of sequences between
571 and
+120 bp did not show any increase in luciferase activity that remained
comparable with the activity observed in unstimulated or in cells
transfected with the control plasmid (pGL3B). Similar results were
obtained with THP-1 cells transfected with the above mentioned plasmids
containing hIL-10 promoter sequences with 5'-end deletions and cultured
in the presence of p38 inhibitor (Fig. 9).
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Fig. 9.
Effect of the p38 inhibitor SB202190 on
LPS-induced hIL-10 promoter activation in THP-1/CD14 cells. Cells
(1.5 × 106) were transiently cotransfected with 10 µg of either hIL-10 wild type promoter or its deletion constructs and
with 5 µg of -galactosidase control vector. The transfected cells
were pretreated with 15 µM SB202190 for 2 h followed
by treatment with 1 µg/ml of LPS for 24 h. Unstimulated,
LPS-stimulated, and LPS + SB202190-treated cells were harvested, and
their lysates were assessed for luciferase and
-galactosidase
activities. The results shown are means ± S.D. of three
experiments performed in triplicate and normalized by
-galactosidase
activity.
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Fig. 10.
The effect of the p38 inhibitor SB202190 on
wild type and Sp1 mutant hIL-10 promoter activities in
LPS-stimulated THP-1/CD14 cells. The cells were transiently
cotransfected with 10 µg of either wild type or Sp1 mutant hIL-10
promoter construct and with 5 µg of -galactosidase control
plasmid. The transfected cells were pretreated with 15 µM
SB202190 for 2 h followed by treatment with 1 µg/ml of LPS for
24 h. Unstimulated, LPS-stimulated, and LPS + SB202190-treated
cells were harvested, and their lysates were assessed for luciferase
and
-galactosidase activities. The results shown are a means ± S.D. of three experiments performed in triplicate and normalized by
-galactosidase activity.
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Fig. 11.
LPS stimulation activates Sp1 transcription
factor in a time-dependent manner (A), and
Sp1 activation is inhibited by inhibitors of p38 MAP kinase
(B). A, THP-1/CD14 cells were
stimulated with LPS (1 µg/ml) for various times ranging from 15 min
to 4 h followed by centrifugation and collection of cell pellets.
B, to determine the effects of the p38 MAP kinase-specific
inhibitor, SB202190, on LPS-induced Sp1 activation, cells were treated
with SB202190 for 2 h prior to stimulation with LPS (1 µg/ml).
To perform the gel shift assay, nuclear extracts were harvested from
the cell pellets obtained at each time point. Nuclear extracts
containing 5 µg of proteins were incubated for 1 h with
32P-labeled oligonucleotides corresponding to the consensus
sequence for Sp1. To determine the specificity of Sp1 transcription
factor binding, the nuclear extracts were incubated with either
unlabeled oligonucleotides, corresponding to the consensus sequence for
Sp1, or with the control base pair-matched irrelevant oligonucleotide.
The complexes were subjected to electrophoresis followed by
autoradiography. Three distinct Sp1 DNA-protein complex bands, namely
A, B, and C, were completely blocked by competition with cold Sp1
oligonucleotides, indicating their specificities.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, etc.) and
anti-inflammatory cytokines (e.g. IL-10) (7, 31, 33, 43). It
is believed that LPS may stimulate the expression of proinflammatory
cytokines through a common signaling pathway during inflammation. There
is reasonably good evidence that production of TNF-
, IL-1
, and
IL-6 can be regulated by the transcription factor NF-
B in various
cell types (33-37). However, little is known concerning the regulation
of hIL-10. The lack of
B binding sites in the hIL-10 promoter makes
it unlikely that the NF-
B is involved in IL-10 regulation (41). In
this study, we investigated the regulation of the hIL-10 promoter in a
human promonocytic cell line, THP-1, following activation with LPS. Extensive deletion analysis of hIL-10 promoter sequences revealed that
an element encompassing the Sp1 transcription factor-binding site is
essential for IL-10 transcription. This was confirmed by transfecting
THP-1 cells with a plasmid containing a mutagenized Sp1 site, which was
unable to drive the expression of luciferase reporter. In addition, we
analyzed the events upstream of Sp1 activation. Our results clearly
demonstrate that p38 MAP kinase regulates the LPS-induced activation of
Sp1, which in turn regulates the transcription of the hIL-10 gene.
These data suggest that the molecular regulation of pro- and
anti-inflammatory cytokine genes is differentially regulated through
diverse transcription factors (7, 8, 33, 34).
receptor gene (51,
52), epidermal growth factor-mediated expression of the gastrin gene
(53), and cAMP-dependent induction of the
CYP11A gene (54).
890 bp region participate in regulating
the transcription of the hIL-10 gene. Further studies are required to
address this possibility. It is also likely that other transcription
factors may be involved in IL-10 gene regulation in different
IL-10-producing cell types and in response to distinct stimuli.
B (64, 65) complex in addition to the p38 MAP kinase (64). The
molecular mechanism by which the p38 MAP kinase activates Sp1 to induce IL-10 gene transcription remains to be investigated. Nonetheless, the
p38 MAP kinase has been implicated in the activation of Sp1 in
IL-1
-induced vascular endothelial cell growth factor gene expression
(66) and in hyperosmotic stress-regulated cellular utilization of the
serum- and glucocorticoid-inducible protein kinase (Sgk) (67).
production (25). It has also been demonstrated that p38 MAP kinase
regulates IL-1 (25), IL-6 (68), TNF-
(25), IL-10 (31), and
prostaglandin H synthase-2 (69) production in human monocytes through
the activation of the CD14 receptor. Functional roles for p38 have also
been described. The p38 MAP kinase is constitutively active in mouse
thymocytes, suggesting a role in T cell survival (70, 71). Antigen
receptor or Fas-mediated apoptosis of T and B cells is accompanied by
p38 activation (72, 73).
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ACKNOWLEDGEMENTS |
---|
Drs. Ken Dimock and Gina Graziani-Bowering are gratefully acknowledged for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Health, Ontario, Canada, the Research Institute, Children's Hospital of Eastern Ontario, and the Canadian Foundation for AIDS Research (A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Ontario HIV Treatment Network.
Supported by a fellowship from the Medical Research Council of
Canada and the Strategic Areas of Development from the University of
Ottawa, Ottawa, Ontario.
¶¶ To whom correspondence should be addressed: Division of Virology, Research Institute, Children's Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-738-3920; Fax: 613-738-4819; E-mail address: akumar@med. uottawa.ca.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M011157200
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ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; hIL, human interleukin; mIL, murine interleukin; EBNA-2, Epstein-Barr virus-specific nuclear antigen-2; ERK, extracellular signal-regulated kinase; JNK, c-Jun N terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MAPK, MAP kinase; TNF, tumor necrosis factor; bp, base pair(s); PCR, polymerase chain reaction; TLR, Toll-like receptor(s).
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REFERENCES |
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