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B and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced by Mycobacterium avium1
Departments of
*
Microbiology and
Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B sites, one IFN-regulatory factor site
and one AP-1 site. Site-directed mutagenesis revealed that the NF-B
and Sp1 sites but not the IFN-regulatory factor site or the AP-1 site
contributed to the basal level and the induction of luciferase activity
during M. avium infection. Binding of Sp1/Sp3 and
NF-B (p50/p65) was confirmed by EMSA. Further studies showed that
three copies of Sp1 elements or NF-B elements are not sufficient to
confer M. avium induction on a heterologous promoter. By
contrast, overexpression of NF-B p65 caused a strong increase in
transcription from an intact TLR2 promoter, whereas it
caused only a partial increase in promoter activity when cotransfected
with the TLR2 promoter with one of the Sp1 sites mutated.
Sp1 and NF-B were the minimum mammalian transcription factors
required for effective TLR2 transcriptional activity when
transfected into Drosophila Schneider cells. Together,
these data provide genetic and biochemical evidence for NF-B as well
as Sp1 in regulating TLR2
transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-mediated
pathways as well as the activation of nonreceptor tyrosine kinases,
protein kinase C, and several members of the mitogen-activated protein
kinase family. The observation that the interaction between bacterial
LPS and macrophages initiates a signaling cascade via Toll-like
receptor
(TLR)3 2 or
TLR4 has resulted in attention now focused on this family of
pattern recognition receptors (4, 5).
The prototypical Toll protein of Drosophila controls
dorsal-ventral patterning in larvae and is required for antifungal
resistance of adult flies (6). Toll is a type 1
transmembrane receptor with leucine-rich repeats in its extracellular
portion and a cytoplasmic domain with sequence homology with several
mammalian proteins including both chains of the IL-1
receptor (7), the IL-18 receptor (8), Ig
IL-1R-related molecule (9), and MyD88 (10).
Medzhitov et al. (11) first reported that an active form
of human TLR4 conferred to monocytes the ability to produce
proinflammatory cytokines IL-1, IL-6, and IL-8 and to express the
costimulatory receptor B7. Positional cloning analysis revealed that
C3H/HeJ and C57BL/10ScCr mice, which are unresponsive to LPS, have
separate genetic defects of TLR4, thus identifying
TLR4 as the LPS receptor (12, 13). Ten human
Toll homologues have been described (14). The two best
characterized Toll-like receptors, TLR2 and TLR4,
have been shown to have different physiological roles. TLR4
is required for responses to LPS derived from Gram-negative bacteria,
whereas TLR2 is involved in responses to Gram-positive
bacteria, mycobacteria, and yeast (15). Both
TLR2 and TLR4 activate NF-B via adapters MyD88
and TNFR-associated factor and two putative serine/threonine kinases
IL-1R kinase and IL-1R kinase-2 (16, 17, 18, 19).
We have previously reported that TLR2 mRNA is induced after
infection of murine macrophages with Mycobacterium avium
(20). To further understand the molecular mechanism that
controls the induction of TLR2, we cloned and analyzed the
murine TLR2 promoter. The results of the present study have
identified that the two NF-B elements and two Sp1 elements that
appear to be necessary for maximal induction of TLR2
transcription in murine macrophages after infection with M.
avium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine macrophage cell line J774A.1 (ATCC TIB-67) was
obtained from American Type Culture Collection (Manassas, VA) and
cultured in IMDM (Life Technologies, Gaithersburg, MD) supplemented
with 10% FBS (HyClone, Logan, UT), and 1% penicillin and
streptomycin. The Drosophila melanogaster Schneider cell
line (ATCC CRL-1963) was maintained in Drosophila cell
medium (Life Technologies) supplemented with FBS (10% v/v) and
antibiotics. M. avium (ATCC 35713) was grown in Middlebrook
7H9 broth supplemented with oleic acid-albumin-dextrose complex (Difco,
Detroit, MI) in 5% CO2. Bacteria were frozen in
1-ml aliquots in 10% glycerol at -80°C at the concentration of
2 x 108 CFU/ml. Frozen aliquots were thawed
and briefly sonicated before each use. Abs reacting with Sp1 (PEP2),
Sp3 (D-20-G), Egr-1 (sc-110), NF-B p50 (C-19),
p65 (C-20), p52 (K-27), c-Rel (sc-70-G), and Rel B (sc-226) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Gene cloning
The mouse TLR2 putative 5'-proximal promoter was identified by three rounds of chromosome walking in a murine genomic DNA library using the GenomeWalker kit from Clontech (Palo Alto, CA) and nested mouse TLR2-specific primers. For the initial round of chromosome walking, mouse TLR2-specific primers were designed from the published mouse TLR2 cDNA sequence (accession number AF124741). Subsequent rounds of chromosome walking used primers designed from the sequence of the previous round. After each round of chromosome walking, the resulting PCR products were cloned and sequenced. Three rounds of chromosome walking were required because we found that the 5'-untranslated region of the TLR2 gene was contained in two exons separated by two introns. We obtained 1.3 kb 5'-proximal DNA sequence. This sequence was analyzed for binding sites for transcription factors using the MatInspector program (http://www.gsf.de/c/s.dll/matsearch.pl).
Primer extension analysis
Poly(A)+ RNA was isolated using poly(A) Tract System 1000 (Promega, Madison, WI). Determination of the transcription initiation sites of the mouse TLR2 gene was performed using a primer extension kit from Promega. Briefly, 1 and 4 µg poly(A)+ RNA were annealed to the 32P-end-labeled oligonucleotide 5'-TCAGAAACTATGATTGCGGACA-3' at 58°C for 1 h and then cooled for 10 min at room temperature. Reverse transcription was performed with avian myeloblastosis virus reverse transcription at 41°C for 30 min and then stopped by addition of 20 µl loading buffer. Extended products were separated on a 6% polyacrylamide, 8 M urea sequencing gel along with sequencing products of the corresponding region of the TLR2 gene obtained using the above primer.
Plasmid construction and purification
A series of deletion clones containing 1367, 847, 521, 326, 207,
and 139 bp proximal DNA sequence were created by PCR amplification
using the Expand High Fidelity PCR system (Roche Molecular
Biochemicals, Indianapolis, IN) and the cloned TLR2 genomic
DNA obtained from the chromosome walking as templates. The PCR
fragments were digested with MluI and BglII and
cloned into pGL3 basic (Promega) digested with the same restriction
enzymes. The DNA sequences of the constructs were verified by DNA
sequencing. For transient transfection, plasmid DNA was isolated and
purified using the High Pure Plasmid Purification Kit (Roche Molecular
Biochemicals). pCMV-NF-B p65 was generously provided by Dr. K.
Vousden (National Cancer Institute, Frederick, MD). pLuc-Sp1.1,
pLuc-Sp1.2, pLuc-NF-B1, and pLuc-NF-B2 were generated by cloning
three copies of Sp1 element (numbers 1 and 2) or NF-B elements
(numbers 1 and 2) into pLuc-MCS (Stratagene) digested with
HindIII and XhoI. pPac3.1 and pPac-Sp1 were
generous gifts from Dr. A. J.Courey (UCLA). The pPac-NF-B p65
was generated by cloning the p65 insert from the pFlag2-NF-B p65
provided by Dr. A. Baldwin (University of North Carolina, Chapel Hill,
NC) into the BamHI site of pPac3.1.
Site-directed mutagenesis
Mutations of transcription factor binding sites located in the
326-bp TLR2 promoter were made using the Quick-change
site-directed mutagenesis system (Stratagene, La Jolla, CA). The
mutated primers are indicated in Table I
. All of the mutant clones were verified by DNA sequencing.
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J774A.1 cells or Schneider cells were transfected using lipofectAMINE reagent (Life Technologies). Cells were initially plated overnight at a concentration of 2.5 x 105 cells/well in 24-well tissue culture plates. The next day, 0.4 µg reporter plasmid and 0.02 µg Renilla TK luciferase vector (Promega) were mixed with 3 µl lipofectAMINE plus reagent and 2 µl lipofectAMINE in 50 µl serum-free IMDM for 30 min at room temperature. The transfection mixtures were added dropwise to each well containing 200 µl serum-free IMDM. The cells were incubated for 3–5 h at 37°C, after which the transfection reaction was removed and replaced with serum containing IMDM. After overnight transfection, the cells were infected with M. avium at a bacteria/macrophage ratio of 8:1. Luciferase activity was analyzed 24 h later using the dual luciferase reporter assay (Promega). For those transfections without infection, luciferase activity was analyzed 24 h after transfection. The efficiency of transfection, determined by Renilla luciferase activity in the lysates, was used to normalize the activity of firefly luciferase activity. For Schneider cells, the transfection efficiency was normalized against protein concentration.
Nuclear extracts and EMSA
Nuclear extracts were prepared as described by Hussain et al.
(21). Protein concentration was determined by the
bicinchoninic acid protein assay (Pierce, Rockford, IL). Nuclear
extracts were either assayed immediately or stored at -70°C until
further use. The oligonucleotides used in the EMSA assay are: NF-B
(number 1) 5'-TGA CCT GGG GAC ATC CCC TTC CCT-3'; NF-B (number 2)
5'-ACA CCT GGG GAA TTC CCA CAC G-3'; Sp1 (number 1) 5'-CGC ACC GGG GGC
GGT GCT GGC GA-3'; Sp1 (number 2) 5'-ACC CCT GTG GGC GGC GCT TGC C-3'.
Oligonucleotides were end-labeled with
[
-32P]ATP using T4 polynucleotide kinase.
The binding reactions were conducted using the gel shift assay core
system from Promega. Binding reactions contained 2–5 µg nuclear
extract and 10–50,000 cpm radiolabeled oligonucleotide in 10 mM
Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5
mM DTT, 50 mM NaCl, 1 µg polydeoxyinosylate-polydeoxycytidylate, and
4% glycerol. For DNA competition experiments, a 50-fold excess of
unlabeled competitor oligonucleotides was included in the binding
reaction. For Ab supershift assays, reactions were incubated with 1
µl Ab for 20 min before addition of radiolabeled probe. Binding
reactions were electrophoresed at 4°C in 5–6% native polyacrylamide
gels using 0.5x Tris-borate buffer followed by autoradiography.
RNase protection assay
Total RNA was extracted using the RNAqueous kit (Ambion, Austin, TX). RNase protection assays were performed using reagents from BD PharMingen (San Diego, CA) as previously described by Chen et al. (22). Probe templates for mouse TLR2 and G3PDH were developed in this laboratory and have been previously described (20). A total of 4–10 µg RNA was used for each RNase protection assay. Reactions were run on 6% polyacrylamide, 8 M urea sequencing gels followed by autoradiography. Data for TLR2 were normalized against the G3PDH housekeeping gene.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially designed primers for chromosome walking that covered
the translation start site based on information that the entire coding
region for the mouse TLR2 was located in a single exon
(15). However, two additional exons containing
5'-untranslated sequences were found when we compared the published
mouse TLR2 cDNA sequences (accession numbers AF124741,
AF185284, and AF165189) with the sequence we obtained after chromosome
walking. The gene structure is shown in Fig. 1, and the sequence of 1367 bp of the
mouse TLR2 promoter (accession number AF252535) is shown in
Fig. 2. The potential multiple
transcription factor binding sites were predicted using the
MatInspector program. Primer extension analysis was conducted to
determine the transcription start site. Four specific extension
products were found (data not shown). The first three are indicated in
Fig. 2.
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To investigate the DNA sequences responsible for promoter
activity, deletion constructs were cloned into the luciferase reporter
vector pGL3-Basic and transiently transfected into J774A.1 cells. The
results in Fig. 3 show that the highest
level of luciferase activity was associated with a 326-bp fragment
containing the region from -294 to +32. Fragments larger than the 326
bp resulted in less luciferase activity, suggesting that the region
from -1335 to -294 contains negative regulatory elements. The region
from -294 to +32 with the maximal promoter activity does not contain a
TATA box but has multiple possible transcription factor binding sites
including two NF-B sites, two Sp1 sites, and one each for
IFN-regulatory factor (IRF), AP-1, and Elk1 (Fig. 2
). Transient
transfection of the constructs containing 326 bp and above
TLR2 promoter sequences in J774A.1 cells all showed an
increase in luciferase activity by 2.3-fold after infection with
M. avium (Fig. 3). The fold increase in the 326-bp construct
was similar to that achieved in the 1367-bp promoter. The level of
induction of TLR2 promoter activity by M. avium
was reduced in the transfections with the 207-bp fragment and further
with the 139-bp construct. This suggests that the region from -294 to
-175, which contains an IRF site and a NF-B site, is required for
full induction of TLR2 promoter activity by M.
avium. Previously we showed that infection of mouse peritoneal
macrophages with M. avium up-regulated the expression of
TLR2 mRNA (20). Fig. 4 confirmed that TLR2 mRNA
could be induced by infection with M. avium and by LPS
treatment when using the J774A.1 cell line (Fig. 4).
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B and Sp1 sites are important for basal activity and
induction of maximal transcription activity after infection with
M. avium
To investigate the functional significance of the NF-B and Sp1
binding sites as well as the IRF and AP-1 sites, we used site-directed
mutagenesis to abolish each of these sites within the 326-bp mouse
TLR2 promoter region. The mutant primers are shown in Table I. Mutations of either NF-B site significantly inhibited the
transcription activity both at basal level and after infection of
J774A.1 cells with M. avium (Fig. 5). Mutations of both NF-B sites
completely abolished the induction of TLR2 promoter
activity. Similarly, mutations of either of the two Sp1 sites
dramatically decreased the basal promoter activity, whereas mutation of
both Sp1 sites also partially decreased the extent of induction after
infection with M. avium (at least 50% decrease). Deletion
of both Sp1 sites led to similar observations (data not shown).
Mutations of the IRF site or the AP-1 site did not affect promoter
activity. Combinations of both NF-B and Sp1 mutations completely
abolished promoter activity within the 326-bp fragment (data not
shown).
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B and Sp1 bind to elements within the 326-bp TLR2
promoter region
To determine the nature of nuclear factors that can interact with
the NF-B and Sp1 sites, EMSAs were performed with double-stranded
oligonucleotides corresponding to the two proximal putative NF-B
binding sites and Sp1 sites within the 326-bp region. Nuclear extracts
were prepared from J774.A1 cells. The results in Fig. 6 show that NF-B bound to both sites
and that the binding was increased after infection with M.
avium. Complex formation was inhibited by the addition of an
excess of unlabeled wild-type oligonucleotides but not by
oligonucleotides with mutated sequences. Both complexes were
supershifted or inhibited by the addition of Ab reacting with NF-B
p50 and p65 but not by Abs reacting with p52 or c-Rel and Rel B.
Similar results were achieved using nuclear extracts from mouse
peritoneal macrophages (data not shown). The proximal NF-B site also
bound Sp1 and Sp3, as shown in Fig. 6A. The binding of
Sp1/Sp3 to this NF-B site remained similar on infection (data not
shown).
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). Infection with M.
avium did not change the binding from 0 to 4 h after
infection. Complex formation was blocked by the addition of cold probe
but not with mutated oligonucleotides. Supershift assay revealed that
the upper band is a complex of Sp1 and Sp3 whereas the faster migrating
band represents binding of Sp3. Ab against Egr-1, another transcription
factor, which can bind to Sp1 element, did not affect the complex
formation.
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B element alone confers M.
avium responsiveness on a heterologous promoter
To investigate whether Sp1 or NF-B sites alone was sufficient
to confer M. avium responsiveness, we generated a
heterologous promoter by cloning three copies of each Sp1 site or
NF-B site into a pLuc-MCS vector that only contains a TATA element
in front of the luciferase reporter gene. Neither the vector alone nor
any of the insert preparations showed reactivity to M. avium
(Fig. 8).
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B p65 led to an increase in transcription
activity of TLR2 promoter
To further identify the role of NF-B in activating the
TLR2 promoter, we cotransfected the 326-bp TLR2
promoter with a NF-B p65 expression vector, the NF-B subunit with
the DNA activation domain. As shown in Fig. 9, overexpression of NF-B p65
increased the transcription activity of wild-type TLR2
promoter by 7- to 9-fold. By contrast, mutation of either of the
NF-B sites inhibited by 60–70% the transcriptional increase
induced by overexpression of NF-B p65, whereas mutation of Sp1 site
1 inhibited by 50% this increase in transcriptional activity. Mutation
of this site in combination with mutation of Sp1 site 2 did not lead to
a further decrease in transcriptional activity induced by
overexpression of NF-B p65. Finally, overexpression of NF-B p65
caused virtually no induction in the TLR2 promoter with
mutations of both NF-B sites, whereas mutation of Sp1 site 2 did not
by itself have a significant effect on the extent of induction
resulting from the overexpression of NF-B p65.
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B p65
We next determined whether Sp1 and NF-B are the minimum
mammalian transcription factors required for efficient transcription of
TLR2 promoter. The Drosophila Schneider cell line
was used because these cells lack expression of both NF-B and Sp1
(23, 24). We found that whereas Sp1 alone caused a minimum
induction of TLR2 promoter activity, NF-B alone did not
have a significant effect on the promoter activity. By contrast,
expression of both Sp1 and NF-B p65 led to a much higher level of
transcriptional activity of the TLR2 promoter construct
(Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B,
and single sites for AP-1, IRF, and Elk1. We showed that the two
NF-B elements in the mouse TLR2 promoter were required
for induction of TLR2 gene expression in macrophages after
infection with M. avium. In addition, we presented evidence
that Sp1 transcription factor is required for TLR
transcription at the basal level and for the increased TLR2
expression after infection with M. avium. During the
preparation of this article, Musikacharoen et al. (25)
reported cloning of the mouse TLR2 promoter. Although the
promoter sequences are identical, they identified two NF-B sites
(equivalent to NF-B sites 2 and 3 indicated in Fig. 2) as important
for the induction of TLR2 by LPS or TNF-
stimulation in
RAW264.7 cells. Our results differ in that we found that infection with
M. avium was maximally controlled by promoter sequences more
proximal to the transcription start site. This included two NF-B
sites and at least one SP1 site. Thus, Musikacharoen et al. observed
that another NF-B site (equivalent to NF-B number 3 in Fig. 2) is
also important for LPS reactivity. However, they failed to identify the
(number 1) NF-B site, which may explain why they did not observe a
complete loss of LPS responsiveness from a promoter construct with
double mutations of NF-B (numbers 2 and 3) sites. The difference
between the two studies could be due to the different signaling
pathways that are activated after LPS stimulation and infection with
M. avium. LPS activates cells via a
TLR4-dependent pathway, whereas response to M.
avium is mediated at least by TLR2. The two pathways
may not be exactly the same. TLR2 and TLR4 are differentially regulated. Infection of macrophages with M. avium resulted in the induction of TLR2 mRNA, while at the same time the expression of TLR4 mRNA decreased (20). Several different cell types also express TLR2 mRNA, including macrophages, fibroblasts, and T cells (18, 26, 27, 28); in contrast, TLR4 is expressed in myeloid cells (29). The promoter region of both the human and mouse TLR4 genes contains a site, which is not present in the mouse TLR2 promoter, that binds the PU.1 myeloid-specific transcription factor. This is consistent with the observation that the expression of TLR2 is not restricted to myeloid cells.
The observation that two NF-B sites were required for induction of
TLR2 transcription and the inducible binding of NF-B
p50/p65 to these two sites suggested a role for NF-B in regulating
TLR2 gene expression. Subsequent studies, by over expressing
NF-B p65 further showed that NF-B was sufficient to activate an
intact TLR2 promoter. Because TLR2 has been
implicated as signal transducer for M. avium and
TLR2 activation leads to activation of NF-B (20, 30, 31, 32, 33, 34), our results further suggest that there may be a
NF-B-dependent TLR2 autoregulation. In the present study,
NF-B sites also appear to be required for the basal level of
TLR2 transcription in J774A.1 macrophages. Similarly, the
regulation of basal transcription of ICAM-2 and P-selectin requires
intact NF-B elements (35, 36). Basal activity of the
two NF-B sites of the TLR2 promoter could be due to
binding of Sp1/Sp3 to the proximal NF-B site and the low level
constitutive binding of the NF-B p50/p65 to the number 2 NF-B
site (Fig. 6B).
Previously, we reported that TLR2 mRNA was induced after
infection of peritoneal macrophages from NF-B p50 knockout mice with
M. avium (20). This observation appears to be
in contrast to those reported here, because we identified the factors
that inducibly bind NF-B elements as p50/p65 heterodimer. To resolve
this apparent conflict, we also analyzed nuclear extracts of
macrophages from NF-B p50 knockout mice infected with M.
avium. We found that there was an inducible binding to the two
NF-B sites after infection with M. avium that could not
be shifted with Abs to Rel family members (data not shown). The
identity of this unknown factor remains to be determined. It is also
possible that the number 3 NF-B site that was identified by
Musikacharoen et al. may function redundantly to confer responsiveness
to M. avium because it served as a binding site for NF-B
p65/p65 in their study.
Sp1 is a ubiquitous factor that regulates the constitutive expression
of many genes. Sp1 elements are often found in the enhancers or
promoters of NF-B-regulated genes, including those for HIV, ICAM-1,
and GM-CSF (23, 37, 38, 39, 40). Sp3 in contrast can either
activate or attenuate gene transcription (41, 42). It is
generally believed that Sp1 is part of the basal transcription
initiation machinery. Indeed, we found that mutation of Sp1 sites
suppressed the transcription of TLR2 at the basal level. Sp1
has also been reported to mediate the induction of several genes
(40, 43, 44, 45). Posttranslational modifications, such as
phosphorylation and glycosylation, have been proposed to be involved in
altering the transcriptional activity of Sp1. Here we found that Sp1
sites are necessary for maximal transcription induced by M.
avium infection but not sufficient alone to confer M.
avium responsiveness. Correspondingly, binding of Sp1/Sp3 to Sp1
sites did not increase after infection with M. avium. Our
observations are consistent with the report in which Sp1/Sp3 site was
found to be necessary for the LPS induction of the mouse TNFR gene
promoters in transfected RAW264.7 cells. The binding of Sp1/Sp3 was not
changed by LPS stimulation (45, 46). To reinforce our
observation concerning the involvement of Sp1 sites in the activation
of TLR2 transcription, we cotransfected different
TLR2 promoters with a NF-B p65 expression plasmid. We
found that overexpression of NF-B p65 caused a dramatic increase in
transcriptional activity of the wild-type TLR2 promoter, but
not the promoter with two mutated NF-B sites. In contrast, mutation
of Sp1 site 1 suppressed the induction resulting from the
overexpression of NKB p65 by 50%. In contrast, we found that only a
mutation of both Sp1 sites significantly decreased the induction of
TLR2 after infection with M. avium. The
difference may be due to the different effects of endogenous activation
of p65 in which only 2-to 3-fold induction of TLR2 promoter
activity after M. avium infection was observed and exogenous
expression of p65 in which a much greater increase in induction was
achieved. Nevertheless, in both cases at least one of the Sp1 sites is
required for the full induction of transcriptional activity of
TLR2. The accumulated data suggest that a minimal set of
gene-specific transcription factors for the expression of the
TLR2 gene could consist of NF-B and Sp1. To test this
hypothesis, Drosophila Schneider cells were used. These
cells have been used in other systems to demonstrate a role for Sp1 and
NF-B activity on a regulatory sequence (23, 47). The
wild-type TLR2 promoter fused with luciferase gene was
cotransfected into Schneider cells with plasmids expressing Sp1 and/or
NF-B p65. Whereas plasmid expressing NF-B p65 provided no
significant increase in the level of expression over background when
introduced alone, plasmid expressing Sp1 alone provided a minimal level
of the expression. By contrast, a synergistic increase in expression
was observed when both expression plasmids were introduced. Thus, we
conclude that Sp1 and NF-B p65 are the sufficient mammalian factors
required for expression of TLR2. In addition, the effect of
NF-B requires the presence of Sp1. These results and our observation
that the proximal NF-B site within the TLR2 promoter also
serves as a binding site for Sp1/Sp3 support the possibility that
Sp1/Sp3 could be interacting with NF-B to induce the TLR2
transcription. Sp1 and NF-B transcription factors have been shown to
physically interact with each other to activate gene transcription
(47). Because the TLR2 promoter lacks a TATA
box, Sp1 may be required for linkage with the transcriptional complex.
Sp1 may help to recruit NF-B to the basal transcription machinery
after infection with M. avium and thus may lead to the
activation of transcription of TLR2. For example, the
activation of the monocyte chemoattractant protein-1 gene by tumor
necrosis factor, in which the binding of Sp1 to monocyte
chemoattractant protein-1 promoter is critical to the NF-B assembly
and activation of the gene (48). The other possibility is
that binding of Sp1 and NF-B together cause chromatin remodeling,
which is required for the gene activation of TLR2. Studies
are under way to test these possibilities.
In conclusion, our studies have demonstrated that four
cis-acting elements within mouse TLR2 promoter,
two NF-B sites and two Sp1 sites, are required for the maximal
transcription of the mouse TLR2 gene after infection with
M. avium. The proximal NF-B site also appears to bind
Sp1/Sp3 constitutively. Thus, NF-B and Sp1/Sp3 may cooperatively
determine the level of TLR2 expression. The cloning of mouse
TLR2 promoter provides a basis for further investigation of
the regulation of gene expression of TLR2 by various
infectious agents.
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Acknowledgments |
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B p65, pFlag2-NF-B p65, pPac3.1,
and pPacSp1. We also thank Dr. Amanda Simcox for generously providing
the Schneider cells and Drs. Donald Kuhn and Wangjian Zhong for helpful
discussion. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce S. Zwilling, Department of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. E-mail address: zwilling.1{at}osu.edu
3 Abbreviations used in this paper: TLR, Toll-like receptor; Sp1, stimulating factor 1; Sp3, stimulating factor 3; IRF, IFN-regulatory factor.
Received for publication July 11, 2001. Accepted for publication October 19, 2001.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B p50. Mol. Cell. Biol. 18:5678. Janus kinase-STAT signaling and gene induction by down-regulation of the IFN- receptor. J. Immunol. 163:2041.B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 12:3551.[Medline]
B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression. J. Immunol. 166:4516.
T cells bearing invariant V6/V1 induced by Escherichia coli infection in mice. J. Immunol. 165:931.) response elements in the human ICAM-2 promoter. J. Cell Sci. 112:4695.[Abstract]
B, and Sp1 are major determinants of the promoter activity of the human protein kinase CK2 gene. J. Biol. Chem. 275:18327. and tumor necrosis factor- in monocytic cells involves cooperation between interferon regulatory factor-1, NFB, and Sp1 transcription factors. J. Biol. Chem. 270:27920.This article has been cited by other articles:
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  Z. Cai, Z. Shi, A. Sanchez, T. Zhang, M. Liu, J. Yang, F. Wang, and D. Zhang Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells J. Biol. Chem., November 27, 2009; 284(48): 33088 - 33096. [Abstract] [Full Text] [PDF]
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 A. M. Noronha, Y. Liang, J. T. Hetzel, H. Hasturk, A. Kantarci, A. Stucchi, Y. Zhang, B. S. Nikolajczyk, F. A. Farraye, and L. M. Ganley-Leal Hyperactivated B cells in human inflammatory bowel disease J. Leukoc. Biol., October 1, 2009; 86(4): 1007 - 1016. [Abstract] [Full Text] [PDF]
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Z. Shi, Z. Cai, S. Wen, C. Chen, C. Gendron, A. Sanchez, K. Patterson, S. Fu, J. Yang, D. Wildman, et al. Transcriptional Regulation of the Novel Toll-like Receptor Tlr13 J. Biol. Chem., July 31, 2009; 284(31): 20540 - 20547. [Abstract] [Full Text] [PDF]
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 N. K. Phulwani, N. Esen, M. Md. Syed, and T. Kielian TLR2 Expression in Astrocytes Is Induced by TNF-{alpha}- and NF-{kappa}B-Dependent Pathways J. Immunol., September 15, 2008; 181(6): 3841 - 3849. [Abstract] [Full Text] [PDF]
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 Q. Wu, R. J. Martin, S. LaFasto, B. J. Efaw, J. G. Rino, R. J. Harbeck, and H. W. Chu Toll-like Receptor 2 Down-regulation in Established Mouse Allergic Lungs Contributes to Decreased Mycoplasma Clearance Am. J. Respir. Crit. Care Med., April 1, 2008; 177(7): 720 - 729. [Abstract] [Full Text] [PDF]
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C. M. Johnson and R. I. Tapping Microbial Products Stimulate Human Toll-like Receptor 2 Expression through Histone Modification Surrounding a Proximal NF-{kappa}B-binding Site J. Biol. Chem., October 26, 2007; 282(43): 31197 - 31205. [Abstract] [Full Text] [PDF]
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 H. Izadi, A. T. Motameni, T. C. Bates, E. R. Olivera, V. Villar-Suarez, I. Joshi, R. Garg, B. A. Osborne, R. J. Davis, M. Rincon, et al. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages Infect. Immun., October 1, 2007; 75(10): 5027 - 5034. [Abstract] [Full Text] [PDF]
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 Q. M. Nhu, N. Cuesta, and S. N. Vogel Transcriptional regulation of lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) expression in murine macrophages: role of interferon regulatory factors 1 (IRF-1) and 2 (IRF-2) Innate Immunity, October 1, 2006; 12(5): 285 - 295. [Abstract] [PDF]
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Z. Guo, S. Garg, K. M. Hill, L. Jayashankar, M. R. Mooney, M. Hoelscher, J. M. Katz, J. M. Boss, and S. Sambhara A Distal Regulatory Region Is Required for Constitutive and IFN-{beta}-Induced Expression of Murine TLR9 Gene J. Immunol., December 1, 2005; 175(11): 7407 - 7418. [Abstract] [Full Text] [PDF]
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 A.H. Schoneveld, M.M. Oude Nijhuis, B. van Middelaar, J.D. Laman, D.P.V. de Kleijn, and G. Pasterkamp Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development Cardiovasc Res, April 1, 2005; 66(1): 162 - 169. [Abstract] [Full Text] [PDF]
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 S. Dunzendorfer, H.-K. Lee, and P. S. Tobias Flow-Dependent Regulation of Endothelial Toll-Like Receptor 2 Expression Through Inhibition of SP1 Activity Circ. Res., October 1, 2004; 95(7): 684 - 691. [Abstract] [Full Text] [PDF]
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F. Takeshita, K. Suzuki, S. Sasaki, N. Ishii, D. M. Klinman, and K. J. Ishii Transcriptional Regulation of the Human TLR9 Gene J. Immunol., August 15, 2004; 173(4): 2552 - 2561. [Abstract] [Full Text] [PDF]
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R. Haase, C. J. Kirschning, A. Sing, P. Schrottner, K. Fukase, S. Kusumoto, H. Wagner, J. Heesemann, and K. Ruckdeschel A Dominant Role of Toll-Like Receptor 4 in the Signaling of Apoptosis in Bacteria-Faced Macrophages J. Immunol., October 15, 2003; 171(8): 4294 - 4303. [Abstract] [Full Text] [PDF]
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T. K. Means, F. Hayashi, K. D. Smith, A. Aderem, and A. D. Luster The Toll-Like Receptor 5 Stimulus Bacterial Flagellin Induces Maturation and Chemokine Production in Human Dendritic Cells J. Immunol., May 15, 2003; 170(10): 5165 - 5175. [Abstract] [Full Text] [PDF]
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L. R. P. Ferreira, E. F. Abrantes, C. V. Rodrigues, B. Caetano, G. C. Cerqueira, A. C. Salim, L. F. L. Reis, and R. T. Gazzinelli Identification and characterization of a novel mouse gene encoding a Ras-associated guanine nucleotide exchange factor: expression in macrophages and myocarditis elicited by Trypanosoma cruzi parasites J. Leukoc. Biol., December 1, 2002; 72(6): 1215 - 1227. [Abstract] [Full Text] [PDF]
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T. Wang, W. P. Lafuse, K. Takeda, S. Akira, and B. S. Zwilling Rapid Chromatin Remodeling of Toll-Like Receptor 2 Promoter During Infection of Macrophages with Mycobacterium avium J. Immunol., July 15, 2002; 169(2): 795 - 801. [Abstract] [Full Text] [PDF]
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 A. G. Eliopoulos, C. Davies, S. S. M. Blake, P. Murray, S. Najafipour, P. N. Tsichlis, and L. S. Young The Oncogenic Protein Kinase Tpl-2/Cot Contributes to Epstein-Barr Virus-Encoded Latent Infection Membrane Protein 1-Induced NF-{kappa}B Signaling Downstream of TRAF2 J. Virol., March 27, 2002; 76(9): 4567 - 4579. [Abstract] [Full Text] [PDF]
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, Uninfected;
,
M. avium. Luciferase activity is relative to the
Renilla-TK luciferase activity, and values are the
means ± SD (SDM) obtained from at least three independent
experiments. RLU, relative light units.
