Download Transcriptional Regulation of the Human Toll

Transcriptional Regulation of the Human
Toll-Like Receptor 2 Gene in Monocytes and
Macrophages
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of August 3, 2017.
Viola Haehnel, Lucia Schwarzfischer, Matthew J. Fenton and
Michael Rehli
J Immunol 2002; 168:5629-5637; ;
doi: 10.4049/jimmunol.168.11.5629
http://www.jimmunol.org/content/168/11/5629
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References
The Journal of Immunology
Transcriptional Regulation of the Human Toll-Like Receptor 2
Gene in Monocytes and Macrophages1
Viola Haehnel,* Lucia Schwarzfischer,* Matthew J. Fenton,† and Michael Rehli2*
I
n vertebrate organisms the innate branch of the immune system greatly relies on the recognition of conserved microbial
structures (pathogen-associated molecular patterns (PAMPs)3).
Recent studies have demonstrated an import role of members of
the Toll-like receptor (TLR) family in the initiation of intracellular
signaling pathways through the recognition of PAMPs (1–3). Only
five years ago, the prototypic Toll protein of Drosophila—a transmembrane receptor which was initially shown to control dorsalventral patterning in the embryo—was also found to be required
for antifungal responses in adult flies (4). Since then, 10 mammalian Toll homologs have been identified that contain a leucine-rich
ectodomain and a conserved cytoplasmic domain shared with both
chains of the IL-1R, the IL-18R, SIGRR, and MyD88, the socalled Toll/IL-1R homologous region. Both genetic and biochemical data support a common signaling pathway that finally leads to
the activation of NF-␬B (1–3).
Several studies using either TLR knockout mice or TLR mutant
mouse strains have shown that mammalian TLR proteins are able
to recognize specific microbial structures. Signaling cascades activated by LPS, the abundant glycolipid of the outer membrane of
Gram-negative bacteria, are initiated through TLR4 (5–7). Another
PAMP, bacterially derived CpG DNA, is recognized through a
*Department of Hematology and Oncology, University of Regensburg, Regensburg,
Germany; and †Pulmonary Center, Boston University School of Medicine, Boston,
MA 02118
Received for publication October 29, 2001. Accepted for publication March 21, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant Re1310/2-1 from the Deutsche Forschungsgemeinschaft (to M.R.). Sequence data from this article have been submitted to GenBank (accession nos. AF424049 –AF424053).
2
Address correspondence and reprint requests to Dr. Michael Rehli, Department of
Hematology and Oncology, University Hospital, 93042 Regensburg, Germany. Email address: [email protected]
3
Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern;
MALP, macrophage-activating lipopeptide; TLR, Toll-like receptor.
Copyright © 2002 by The American Association of Immunologists
TLR9-dependent mechanism (8). TLR5 recognizes bacterial
flagellin from both Gram-positive and Gram-negative organisms
(9). The protein product of the TLR2 gene has been implicated in
signal transduction events induced by several microbial products
(e.g., peptidoglycans, lipopeptides, and lipoarabinomannans) (10 –
17). In contrast to other TLR proteins, TLR2 seems to recognize
microbial patterns as a heterodimer, e.g., with TLR6 or maybe
TLR1 (18). In mice, destructive mutations of TLR2 impede a normal response to lipoproteins and cause a high susceptibility to
Gram-positive infection. Murine TLR2⫺/⫺ macrophages show
normal responses to LPS stimulation (14). Although the genetic
evidence described above strongly supports the contention that
TLR4 is the predominant, if not exclusive, receptor for LPS, an
additional role of TLR2 as LPS receptor is still debated. TLR2
does appear to mediate cellular activation by purified LPS from
Leptospira interrogans and Porphyromonas gingivalis, which are
structurally different from enteric LPS (19, 20).
Expression of TLR2 in humans is restricted to a small number of
cell types, including predominantly myelomonocytic cells (monocytes, macrophages, dendritic cells, and granulocytes) (21, 22). Both
the basal level of TLR2 expression and its inducible regulation may
influence responses to microbial infection. Accordingly, we have
sought to characterize the human TLR2 promoter and to analyze those
factors that govern TLR2 gene expression in human monocytes/macrophages. Interestingly, the expression of human and murine TLR2
genes is controlled by distinct, nonconserved regulatory elements. We
have found that promoter sequences of both species show no significant homology and, in contrast to its murine counterpart, the human
TLR2 promoter does not respond to microbial pattern-activated
NF-␬B in monocytes and macrophages.
Materials and Methods
Chemicals
All chemical reagents used were purchased from Sigma-Aldrich (Berlin,
Germany) unless otherwise noted. Protease inhibitors are from Roche Biochemicals (Basel, Switzerland). Oligonucleotides were synthesized by TIB
0022-1767/02/$02.00
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This report investigates the molecular basis for tissue-restricted and regulated expression of the pattern recognition receptor
Toll-like receptor (TLR)2 in human monocytes and macrophages. To define the proximal promoter, the full 5ⴕ-sequence and
transcriptional start sites of TLR2 mRNA were determined. The human TLR2 gene was found to consist of two 5ⴕ noncoding exons
followed by a third coding exon. Alternative splicing of exon II was detected primarily in human blood monocytes. The proximal
promoter, exon I, and part of intron I were found to be located in a CpG island. Although CpG methylation of the proximal human
TLR2 promoter in cell lines correlated with TLR2 repression, the promoter was unmethylated in primary cells, indicating that
CpG methylation does not contribute to the cell-type specific expression of human TLR2 in normal tissues. The promoter sequence
contains putative binding sites for several transcription factors, including Sp1 and Ets family members. Reporter gene analysis
revealed a minimal promoter of 220 bp that was found to be regulated by Sp1, Sp3, and possibly PU.1. Interestingly, no sequence
homology was detected between human and murine TLR2 promoter regions. In contrast to murine TLR2, expression of human
TLR2 in monocytes/macrophages is not induced by the proinflammatory stimuli LPS or macrophage-activating lipopeptide-2, and
reporter activity of the promoter was not enhanced by stimuli-induced NF-␬B activation in THP-1 or MonoMac-6 cells. Our
findings provide an initial definition of the human TLR2 promoter and reveal profound differences in the regulation of an
important pattern recognition molecule in humans and mice. The Journal of Immunology, 2002, 168: 5629 –5637.
5630
Molbiol (Berlin, Germany). Antisera for supershift analyses were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cells
RNA preparation, RT-PCR, and Northern analysis
Total RNA was isolated from different cell types by the guanidine thiocyanate/acid phenol method (23). Two micrograms of total RNA from either
cell type was reverse transcribed using oligo(dT) primer and Superscript II
(Life Technologies). Primer positions and sizes for the amplified fragments
of TLR2 and ␤-actin are indicated (see Fig. 2). PCR conditions were optimized to assure that the amplification was still exponential at the indicated cycle numbers. The amplified products were sequenced to confirm
their identity. For Northern analysis, total RNA (10 ␮g/lane) was separated
by electrophoresis on 1% agarose/formaldehyde gels and transferred to
nylon membranes (Magna NT; Micron Separations, Westboro, MA). Hybridization was performed using a 32P-labeled cDNA probe of a 1500-bp
restriction fragment of human TLR2 (random primed labeling by Hartmann Analytics, Braunschweig, Germany). Autoradiography was performed at ⫺70°C and bands were scanned with a personal densitometer
(Molecular Dynamics, Sunnyvale, CA).
RNA ligase-mediated RACE-PCR
Ten micrograms of total RNA from purified human blood monocytes was
used for cDNA synthesis with the FirstChoice RLM-RACE kit (Ambion,
Austin, TX). The following TLR2-specific primers were used to amplify
full-length 5⬘ cDNA fragments of human TLR2: hTLR2-OUT (5⬘-AA
GATCCTGAGCTGCCCTTGC-3⬘), hTLR2-IN (5⬘-CCAAGACCCACAC
CATCCAC-3⬘). PCR products were cloned into pCR2.1-TOPO (TOPO
Cloning kit; Invitrogen) and inserts from 13 individual plasmid-containing
bacterial colonies were amplified by PCR and directly sequenced.
Plasmid construction and purification
A 2.8-kb genomic fragment of the human TLR2 promoter was amplified
from a BAC clone (Incyte Genomics, Palo Alto, CA) containing the human
TLR2 gene using the Expand High Fidelity PCR system (Roche Biochemicals) and the primers 5⬘-CGGACATACGGACATCTGTGC-3⬘ (sense) and
5⬘-CTGGGAGAACTCCGAGCAGTC-3⬘ (antisense). Primer sequences
were derived from a TLR2-containing BAC sequence deposited in the
GenBank database (GenBank accession no. AC013303). The obtained
PCR fragment was inserted into the plasmid vector pCR2.1-TOPO (TOPO
Cloning kit; Invitrogen) and completely sequenced. An EcoRI-digested,
Klenow-treated, and BglII-digested 2.7-kb genomic PCR fragment of the
human TLR2 proximal promoter was subcloned into the BglII and Klenowtreated HindIII restriction sites of pGL3-B (Promega, Madison, WI) and
sequenced. Deletions of this construct were generated using the internal
XbaI (⫺2070), HindIII (⫺370), PvuII (⫺220), PmaC I (⫺64), or SmaI
(⫹24) restriction sites of the TLR2 promoter. The (⫺45) deletion was
PCR-amplified from the 2.7-kb construct using primer ⌬SP-S (5⬘AGTCGCTAGCGTTCCCGCACCCCAGA-3⬘) and vector primer GL-2
and subcloned into BglII/NcoI sites of pGL3-B. Mutations of two putative
Sp1 sites were done by PCR-mediated mutagenesis using primers S1-S
(5⬘-GTGGAAGGTTAGGTTCCCGCACCCC-3⬘), S1-AS (5⬘-AACCTA
ACCTTCCACGGGGCAC-3⬘), S2-S (5⬘-GCTCACGGTCAAGGTTAG
GAGCC-3⬘), and S2-AS (5⬘-CCCAGCAGGCTCCTAACCTTGACC-3⬘).
Mutation of a putative Ets/PU.1 site in the (⫺64) construct was done using
primer PU-S (5⬘-TCTTACGCGTGCTAGGTGCCCCGACGAAGGGGC
GG-3⬘). Changed nucleotides in primer sequences are in boldface and italics. Mutated PCR fragments were back-cloned into pGL3-B. For transient
transfections, plasmids were isolated and purified using the Endofree Plasmid kit (Qiagen, Hilden, Germany).
DNA sequence analysis
The cDNA sequencing was done by Dye Deoxy Terminator Cycle Sequencing (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions and sequences were analyzed on the PE Applied
Biosystems DNA Sequencing System (model 373A).
Transient DNA transfections
MonoMac-6 cells were transfected using DEAE-dextran. A total of 5 ⫻
105 MonoMac-6 cells per milliliter were seeded into tissue culture flasks
the day before transfection. On the next day, 6 ml cell suspension was
washed twice with suspension TBS (STBS) solution (25 mM Tris䡠Cl, pH
7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5
mM MgCl2) and pelleted. One microgram of reporter plasmid and 62.5 ng
renilla control vector were mixed with DEAE-dextran (400 ␮g/ml) in 130
␮l STBS buffer and immediately added to the pelleted MonoMac-6 cells.
The cells were incubated at 37°C for 20 min, washed twice with STBS,
resuspended, and cultured in complete RPMI medium. Where indicated,
LPS (100 ng/ml) or MALP-2 (400 U/ml) was added to the cultures 4 h
before harvesting. The cell lines THP-1 and HeLa were transfected as
previously described (24). The transfected cell lines were cultivated for
48 h and harvested, and cell lysates were assayed for firefly and renilla
luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) on a Sirius luminometer (Berthold, Nashua, NH). Firefly luciferase
activity of individual transfections was normalized against renilla luciferase activity. Drosophila S2 Schneider cells were transfected using Effectene reagent (Qiagen) according to the manufacturer’s instructions.
Briefly, 4 ⫻ 105 Schneider cells were cells were cotransfected using 10 ␮l
Effectene reagent and 1.5 ␮g total DNA (1 ␮g of reporter plasmid, 0.25 ␮g
of individual expression plasmids). Duplicate transfections were harvested
after 48 h and cell lysates were assayed for firefly luciferase activity using
the Luciferase Reporter Assay System (Promega). Firefly luciferase activity of individual transfections was normalized against protein concentration
measured using a BCA assay (Sigma-Aldrich).
Nuclear extracts and EMSA
Nuclear extracts were prepared with a variation of the method of Osborn et
al. (17). All buffers used contained 1 mM Na3VO4 and a mixture of protease inhibitors (2 ␮g/ml aprotinin, 0.5 ␮g/ml leupeptin, 1 ␮g/ml pepstatin,
20 ␮g/ml bestatin, 5 ␮g/ml E46, 50 ␮g/ml antipain, 100 ␮g/ml chymostatin). Double-stranded oligonucleotides were labeled with [␣-32P]deoxycytidine 5⬘-triphosphate using Klenow DNA polymerase. A double-stranded
oligonucleotide containing the proximal Sp1 motif from the human TLR2
promoter (Tlr2-Sp) as well as a consensus Sp1 motif (5⬘-CGATTCG
ATCGGGGCGGGGCGAGC-3⬘) were used as cold competitor. The binding reaction contained 2.5 ␮g of nuclear extract protein, 0.5 ␮g of poly(dI/
C), 20 mM HEPES (pH 7.9), 60 mM KCl, 1 mM DTT, 1 mM EDTA (pH
8), 5% glycerol, and 20 nmol of probe DNA in a final volume of 10 ␮l.
Antisera used in supershift analyses were added after 15 min and samples
were loaded onto polyacrylamide gels after standing at room temperature
for a total of 30 min. Buffers and running conditions used have been described. Gels were fixed in 5% acetic acid, dried, and autoradiographed.
DNA preparation and bisulfite sequencing
Genomic DNA from various cell types was prepared using the Blood and
Cell Culture DNA Midi kit from Qiagen. Modification of DNA with sodium bisulfite (25) was performed as follows. A total of 5 ␮g genomic
DNA in 50 ␮l 10 mM Tris䡠Cl, pH 8.0, 1 mM EDTA were denatured with
5.5 ␮l NaOH (3 M) at 37°C for 15 min. After the addition of 540 ␮l sodium
bisulfite (3.8 M) and 15 ␮l hydrochinone (0.4 M), samples were mixed,
divided into six aliquots, and covered with mineral oil. Incubation was
performed in a PCR cycler (five cycles at 95°C for 3 min and 55°C for 57
min). Samples were recombined after treatment and DNA was recovered
using Wizard (Promega) in 100 ␮l H2O and desulfonated by the addition
of 11 ␮l NaOH (3 M) and subsequent incubation at 37°C for 15 min. The
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PBMCs were separated by leukapheresis of healthy donors, followed by
density gradient centrifugation over Ficoll/Hypaque (Amersham Biosciences, Freiburg, Germany). Monocytes were isolated from PBMC by
countercurrent centrifugal elutriation in a J6 M-E centrifuge (Beckman,
München, Germany) as previously described. Monocytes were ⬎90% pure
as determined by morphology and expression of CD14 Ag. Isolated monocytes were cultured in low-endotoxin RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with vitamins, antibiotics, pyruvate, nonessential amino acids (all from Life Technologies, Eggenstein, Germany),
5 ⫻ 10⫺8 M 2-ME, and 2% human pooled AB-group serum on tissue
culture plates for the indicated time periods. The human monocytic cell line
THP-1 was grown in RPMI 1640 medium supplemented with vitamins,
antibiotics, pyruvate, and nonessential amino acids, plus 10% FCS (Life
Technologies). The human monocytic cell line MonoMac-6 was grown in
complete RPMI medium as above, with the addition of 1% OPI media
supplement. The human cervical carcinoma cell line HeLa was maintained
in DMEM plus 10% FCS. Where indicated, cells were treated with 100
ng/ml purified LPS from Salmonella abortus equi (a gift from C. Galanos,
Max-Planck-Institut, Freiburg, Germany) or with 400 U/ml of the mycoplasmal lipopeptide macrophage-activating lipopeptide (MALP)-2 (a gift
from P. Mühlradt, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany). The latter was kept as a stock solution of 1 mg/ml
(4 ⫻ 108 U/ml) in water-2-propanol (1:1, v/v) at 4°C and was diluted as
described. Drosophila S2 Schneider cells (a gift from Dr. W. Falk, Internal
Medicine I, University Hospital, Regensburg, Germany) were cultivated in
Schneider’s Drosophila medium (Invitrogen, Karlsruhe, Germany).
STRUCTURE AND REGULATION OF THE HUMAN TLR2 GENE
The Journal of Immunology
DNA was then precipitated using ammonium acetate and ethanol and resuspended in 50 ␮l 10 mM Tris䡠Cl, pH 8.0, 1 mM EDTA. A total of 5 ␮l
of DNA was amplified in individual nested PCR for both strands using the
following primers: sense strand (first round), Ss1 (5⬘-GTTTTAAGAAAA
TATTGGTTGGG-3⬘) and Sas1 (5⬘-CAAAAACTAAAAACCCAAATA
CAAC-3⬘); sense strand (first round), Ss2 (5⬘-TGGGTATTTAGTTTT
TTTTGTGG-3⬘) and Sas2 (5⬘-AAAAAACTCCRAACAATCACC-3⬘);
antisense strand (first round), ASs1 (5⬘-CTTTCCCTATAATTACCAA
TCCC-3⬘) and ASas1 (5⬘-TAGGAAGGGGTGTAGAGAGATT-3⬘); antisense strand (second round), ASs2 (5⬘-CAAACCCCCAACTCTCTTC-3⬘)
and ASas2 (5⬘-AGAATTTYGAGTAGTTATTTGAGAGAA-3⬘). Products
from the second round of nested PCR were precipitated and sequenced.
Unmethylated cytosine residues are converted into thymidine residues during the bisulfite treatment and subsequent PCR amplification, whereas
methylated cytosine residues are protected.
Results
Isolation of full-length TLR2 transcripts and structure of the
human TLR2 gene
Alternative splicing of the human TLR2
The majority of 5⬘ fragments amplified from monocytes by RLMRACE-PCR was found to lack the sequence of exon II, indicating
the presence of alternatively spliced forms of TLR2. To analyze
the presence of splicing forms in different TLR2-expressing cell
types, RT-PCR with specific primers flanking the region between
exons I and III of the human TLR2 gene was performed. As shown
in Fig. 2A, the obtained band pattern varied among cell types and
individuals. With the exception of monocytes, all cell types primarily expressed the longest transcript containing exon I, II, and
III. In monocytes three distinct products were detectable; however,
the relative distribution of transcripts was dependent on the individual donor. Sequencing of cloned fragments from monocytes
revealed five different splicing variants and the usage of two alternative splice acceptor sites and three splice donor sites in exon
II (see Fig. 1B). All splice junctions contained the expected GT
splice donor and AG splice acceptor. The shortest fragment completely lacked exon two, and the three intermediate fragments were
using acceptor site I/donor site I, acceptor site II/donor site II, or
acceptor site III/donor site II (see Fig. 1B). All splicing variants
contained the complete sequence of exon III and are predicted to
code for one TLR2 protein. Interestingly, splicing of TLR2 mRNAs rapidly changed upon adherence of freshly isolated monocytes. As shown for two individual donors in Fig. 2B, the ratio of
short and long splice variants is altered during monocyte differ-
entiation in vitro, with the long splice variant being predominant
after adherence of monocytes for only 3 h.
Activity of the proximal human TLR2 promoter in myeloid
THP-1 and nonmyeloid HeLa cells
Published Northern blot analyses suggest that human TLR2 is predominantly expressed in spleen, lung, and peripheral blood leukocytes (26, 27). Monocytes, monocyte-derived dendritic cells, and
granulocytes were identified as the major TLR2-expressing cells in
human blood (21, 22).
To further analyze mechanisms regulating myeloid expression
of TLR2, we cloned fragments of the 5⬘ proximal promoter region of the human TLR2 gene, ranging from 2.7 kb to 100 bp
upstream of the ATG-start codon, into a luciferase reporter plasmid (Fig. 3). Transient transfection analysis was performed in the
monocytic cell line THP-1 and nonmyeloid cell line HeLa (cervical carcinoma). Luciferase activities were normalized for transfection efficiency by cotransfection with a renilla construct, and
results for individual cell lines were compared relative to the activity of a CMV promoter-driven construct that was used as a
positive control. As shown in Fig. 3, deletion analysis localizes a
region directing maximal reporter gene expression in THP-1 cells
to ⬃220 bp proximal to the major transcriptional start site. Significant reporter activity was also measured in HeLa cells, which
do not express TLR2 (data not shown), indicating that additional
regulatory elements are involved in cell type-specific expression of
the human TLR2 gene.
The proximal promoter region of human TLR2 is distinct from
murine TLR2
The 5⬘ proximal region of human TLR2 lacks TATA boxes or
consensus initiator sequences. Instead it contains several GC-rich
regions that are often found in housekeeping genes and may determine transcriptional initiation (Fig. 1B). Within the ⫺220 bp
region, putative binding sites for Sp1 family transcription factors
and an E-box element were identified using computational analysis
(TRANSFAC database). In addition, purine-rich elements with a
5⬘-GGAA-3⬘ core on either strand were detected, which could
serve as binding sites for Ets family transcription factors. The sequence of the murine TLR2 promoter has been deposited in GenBank (accession no. AF252535), and a recent publication described the inducible regulation of murine TLR2 by NF-␬B and
STAT5 (28). Whereas the coding regions of human and murine
TLR2 share a high degree of homology (75%), sequence comparison using a ClustalW algorithm did not reveal a significant level
of homology (6 –10%) between the proximal promoter regions and
exons I and II of mouse and human TLR2 genes. This is in sharp
contrast to the strong homology (61–77%) of corresponding regions in human and murine TLR4 genes and indicates that TLR2
may be regulated differently in both species.
Indeed, differences in TLR2 expression have been observed in
mice and humans. Northern blot analysis of various mouse tissues
by Matsuguchi et al. (29) revealed the highest expression of TLR2
in lung and spleen followed by thymus and brain. Tlr2 expression
was also observed in murine adipose tissue (30). However, no (or
very low) TLR2 expression was detected in mouse blood (29). In
human tissues, the strongest TLR2 expression was detected in peripheral blood leukocytes, followed by spleen and lung; no expression was detected in human thymus (26, 27). TLR2 expression in
T cells was observed only in mice, not in humans (22, 29). These
differential expression patterns are likely due to the differences in
TLR2 genomic sequences and regulatory context in mice and humans. As indicated in Fig. 1A, a copy of exon III ( psIII) is located
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As an initial step to characterize regulatory regions of the human
TLR2 gene, we determined the transcriptional start sites and the
full-length sequence of TLR2 transcripts in human monocytes,
which express relatively high levels of TLR2 mRNA. PCR fragments of full-length 5⬘ ends were obtained by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), a method
which allows the selective amplification of capped, full-length
transcripts. PCR fragments were cloned and sequenced as described in Materials and Methods. Thirteen individual fragments
were specific for human TLR2, extending the longest published
cDNA sequence (GenBank accession no. AF051152) for an additional 110 bp. The positions and relative abundance of transcription start sites are indicated in Fig. 1B.
The public human genome database was searched for sequenced
BAC clones containing the human TLR2 gene, and a 77-kb BAC
sequence was identified which contained the entire TLR2 gene
(GenBank accession no. AC013303). Comparison of genomic and
cDNA sequences revealed that the additional 5⬘ sequence corresponds to a novel first exon. The structure of the complete human
TLR2 gene is shown in Fig. 1A.
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STRUCTURE AND REGULATION OF THE HUMAN TLR2 GENE
⬃4 kb upstream of the human TLR2 gene. The presence of a noncoding pseudogene indicates that, in humans, a duplicate of the
coding exon III may have been placed into a different regulatory
context during evolution.
Sp1 and Sp3 bind and activate the proximal human TLR2
promoter
Binding sites for Sp1 family members are implicated in the regulation of several macrophage-specific genes (31–34). Several GCrich sequences and putative binding sites for Sp1 family transcription factors are present in the proximal TLR2 promoter. As shown
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FIGURE 1. The human TLR2
gene. A, Physical map of the human
TLR2 gene. Boxes mark the position
of exons I–III. In-frame start- and
stop-codons are indicated. The box
marked psIII indicates the position of
a region highly homologous to the
third exon of TLR2. B, Promoter sequence and intron flanking sequences
of exons I–III. Potential binding sites
in the proximal promoter for transcription factors are over- and underlined. Nucleotide sequences of exons
are in boldface and italics and transcription start sites are marked with
arrows. The number of fragments obtained for each start site are given
above the arrows. Start sites of the
published sequences are indicated
with their respective GenBank accession numbers. CpGs are in bold lettering. The numbers (1–20) below
CpGs in the proximal promoter region refer to the numbering in Table
I. Alternative splice donor and acceptor sites in exon II are indicated. All
splice junctions contain the expected
GT splice donor and AG splice acceptor. The predicted N-terminal
amino acid sequence in exon III is
also shown.
in Fig. 4, specific binding of Sp1 and Sp3 to the proximal putative
Sp1 site was observed in EMSA using THP-1 nuclear extracts as
well as HeLa cell nuclear extracts (data not shown). The oligonucleotide used in EMSA also contained a putative binding site for
members of the Ets family of transcription factors, which includes
PU.1, an important regulator of macrophage-specific genes. However, no specific binding of recombinant PU.1 was detectable in
EMSA, and the addition of PU.1 Ab in supershift experiments had
no effect on the observed band pattern (data not shown). The proximal and a putative distal Sp1 site were mutated to analyze their
functional significance in reporter assays. In monocytic THP-1
The Journal of Immunology
cells, mutation of the proximal site showed a marked decrease in
reporter activity compared with the wild-type promoter (Fig. 5A),
indicating that this element is required for full reporter activity.
Mutation of the putative distal site had no impact on reporter gene
activity. We also deleted and mutated the putative Ets/PU motif
immediately upstream of the proximal Sp1 site to determine
whether this element accounts for the residual activity in the ⫺64
promoter. As shown in Fig. 5B, mutation of this site had no effect
on the activity of the ⫺64 promoter. Also, deletion of both Sp1 and
putative Ets elements in the ⫺45 promoter resulted in a similar
decrease in reporter activity as observed using the ⫺64 promoter
with a mutated Sp1 site. The results indicate the presence of ad-
FIGURE 3. Deletion analysis of the human TLR2 promoter. Each deletion mutant
was transiently transfected into myeloid
THP-1 cells and nonmyeloid HeLa cells as
described in Materials and Methods. Luciferase activity is relative to a CMV promoterdriven positive control and values are the
mean ⫾ SD obtained from at least three independent experiments.
FIGURE 4. Sp1/3 binding to a proximal binding site of the human
TLR2 promoter. Labeled TLR2-Sp oligonucleotide containing the putative
proximal Sp1 site as well as an adjacent putative Ets site was used in
EMSA with THP-1 nuclear proteins. Addition of unlabeled oligonucleotides for competition analysis or antisera against Sp-1 family transcription
factors is indicated above each lane. Arrows indicate Sp1/3-containing
complexes, SS indicates Ab supershifts, and ⴱ indicates unspecific
complexes.
ditional elements important for promoter activity downstream of
the proximal Sp1 site. To test the ability of Sp1 and Sp3 to transactivate the TLR2 promoter, cotransfection experiments were performed in Drosophila Schneider cells, which lack endogenous expression of Sp1 family transcription factors. As the promoter also
contains several putative binding sites for Ets family transcription
factors, an expression plasmid coding for PU.1, which is an important regulator of macrophage-specific genes, was also tested for
its ability to transactivate the TLR2 promoter in combination with
Sp1 family members. A representative reporter gene analysis is
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FIGURE 2. Detection of alternative spliced TLR2 transcripts. RT-PCR
with total RNA from monocytes (four different donors), in vitro differentiated macrophages, and myeloid cell lines MonoMac-6, THP-1, and U937
(A) or freshly isolated monocytes (0 h) and monocytes cultured for 3 h,
24 h, and 7 days (B) using primers located in exon I (87S: 5⬘-GTGACT
GCTCGGAGTTCTCCC-3⬘) and exon III (751AS: 5⬘-GTCCATATTTC
CCACTCTCAGG-3⬘). The amplified fragments were separated by agarose
gel electrophoresis along with molecular mass markers and stained with
ethidium bromide. The size and composition of each detected fragment is
indicated.
5633
5634
shown in Fig. 6. In contrast to the ⫹24 promoter, which was minimally activated by Sp1, Sp3, or PU.1, the ⫺220 promoter was
strongly induced by Sp1. Sp3 and PU.1 alone only weakly activated the ⫺220 promoter; however, in combination a significant
induction was observed. Both Sp3 and PU.1 further increased Sp1mediated activation of the proximal TLR2 promoter construct.
The human TLR2 promoter is not directly induced by microbial
pattern-activated NF-␬B
The regulation of pattern recognition receptors by inflammatory
cytokines or bacterial products, e.g., LPS, is of particular importance for innate immune mechanisms. In mice, TLR2 is rapidly
up-regulated by multiple proinflammatory stimuli, including IL1␤, TNF, GM-CSF, and LPS (35). The activity of its promoter is
induced by LPS and the induction is dependent on NF-␬B- and
FIGURE 6. Cotransfection analysis in Drosophila Schneider cells. Two
TLR2 promoter reporter constructs—TLR2-(⫺220) containing the minimal promoter and TLR2-(⫹24) containing only parts of exon I without
relevant promoter sequences—were cotransfected with expression plasmids for Sp1, Sp3, and PU.1 into Drosophila Schneider cells as described
Materials and Methods. Relative luciferase activities from a representative
experiment (of three) relative to the control transfection (without expression plasmids) are shown
STAT5-binding sites (28). Therefore, we investigated whether the
human TLR2 promoter would respond to two proinflammatory
stimuli: LPS (a known ligand for TLR4) and mycoplasmal lipopeptide MALP-2 (a known ligand for TLR2/TLR6). Initially,
transient transfections were performed in THP-1 cells with and
without stimulation for 4 h. Neither TLR2 construct was significantly induced by any of the two stimuli, whereas a NF-␬B-responsive control plasmid was markedly induced in response to
LPS or MALP-2 challenge (data not shown). We repeated the
same experimental setup with MonoMac-6 cells, which secrete
comparable cytokine levels upon activation as primary monocytes.
As shown in Fig. 7, the NF-␬B-inducible endothelial-leukocyte
adhesion molecule promoter was markedly induced in response to
LPS or MALP-2 challenge. However, neither the full-length nor
the ⫺220 construct was activated by either stimulus (Fig. 7). In
Northern blot analyses, endogenous TLR2 mRNA levels were also
not affected by LPS treatment of human THP-1 or MonoMac-6 cell
lines (data not shown). Published data on TLR2 expression in LPSstimulated human monocytes are conflicting. Some authors
claimed that LPS induces TLR2 expression in human monocytes
(21, 36), whereas others showed that TLR2 is not induced (22). To
reinvestigate this issue, Northern blot analysis was performed on
total RNA from freshly isolated monocytes and in vitro differentiated macrophages treated or untreated with LPS or MALP-2. As
shown in Fig. 8, TLR2 expression was up-regulated in monocytes
after 3 h of adherence in the presence of serum. No additional
induction of human TLR2 was observed in LPS- or MALP-2treated monocytes. During the differentiation of untreated cells,
TLR2 expression was down-regulated after 24 h. In stimulated
monocytes, higher levels of TLR2 expression were sustained. Little or no induction of TLR2 mRNA was observed in LPS- or
MALP-2-treated adherent monocyte-derived macrophages (Fig.
8B). Our observations are in line with the seemingly conflicting
published data. Muzio et al. (22) compared monocytes treated or
untreated with LPS after 3 h and also observed no induction of
TLR2 mRNA by LPS treatment. Yang et al. (21) detected an induction of TLR2 mRNA after 16 h, which probably corresponds to
the differences we observed after 24 h. Finally, Visintin et al. (36)
were detecting an increase of TLR2 mRNA after LPS treatment
compared with freshly isolated monocytes; however, they failed to
compare the induced mRNA levels with untreated adherent
monocytes.
FIGURE 7. TLR2 reporter activity in MonoMac-6 cells upon LPS or
MALP-2 stimulation. MonoMac-6 cells were transfected with the indicated
plasmids. Four hours before harvesting the cells, cells were treated with
LPS (100 ng) or sMALP-2 (400 U) or left untreated as described in Materials and Methods. Induction of luciferase activity is shown relative to
activities for untreated cells. Values are the mean ⫾ SD obtained from
three independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 5. Effect of Sp1 binding site mutations on TLR2 promoter
activity. Mutations of putative Sp1 (A and B) or Ets (B) binding sites as
well as a deletion of the proximal Ets/Sp1 motif were introduced in reporter
constructs TLR2-(⫺220) (A) and TLR2-(⫺64) (A and B) and transiently
transfected into monocytic THP-1 cells as described in Materials and
Methods. Luciferase activities are relative to the wild-type constructs and
values are the mean ⫾ SD obtained from four to six independent
experiments.
STRUCTURE AND REGULATION OF THE HUMAN TLR2 GENE
The Journal of Immunology
5635
Discussion
In normal tissues, CpG methylation is not involved in
tissue-specific TLR2 promoter activity
Sequence analysis of the human TLR2 gene revealed that the proximal promoter, exon I, and portions of intron I are located within
a CpG island (see Fig. 9). Methylation of CpG motifs has been
described as a repression mechanism active in X chromosome inactivation, genomic imprinting, and silencing of mobile elements
and has also been implicated in tissue-specific repression of genes
(37). To investigate a possible effect of CpG methylation in the
tissue-restricted expression of human TLR2, the methylation status
of the proximal TLR2 promoter was analyzed by bisulfite sequencing. DNA from various cell lines, primary cell types, and different
tissues was amplified and sequenced after bisulfate treatment.
Whereas the promoter was unmethylated in normal tissues and
primary cell types regardless of the transcriptional activity of the
gene, almost complete CpG methylation was detected in several
cell lines that do not express TLR2 (Table I). Therefore, CpG
methylation is probably not involved in the tissue-specific regulation of human TLR2 in normal cells. In tumors, e.g., leukemia cells
such as U937, CpG methylation of the proximal promoter may be
involved in down-regulation of TLR2 expression.
FIGURE 9. CpG content of the human TLR2 gene. A, Distribution of
CpG sites in a 10,000-bp fragment of the human TLR2 gene. Exons I and
II are represented as boxes, and an arrow marks the transcription start site.
B, CpG island of the promoter region exploded.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 8. Northern analysis of TLR2 expression in monocytes and
macrophages. Total RNA was isolated from freshly isolated human blood
monocytes (MO), monocytes adherent for 3 h or 24 h (A), and in vitro
differentiated macrophages either untreated or treated with LPS (100 ng) or
sMALP-2 (400 U) at 0 h (B) for the indicated time periods. The blots were
hybridized with a 32P-labeled TLR2 cDNA. The ethidium bromide staining
is shown as a control for mRNA loading.
In this study we investigated the transcriptional regulation of
TLR2 in human monocytes/macrophages. We determined the fulllength sequence and transcriptional start sites of human TLR2 and
identified alternatively spliced forms of human TLR2 mRNA in
human monocytes. Furthermore, we performed an initial characterization of trans-acting factors controlling the expression of human TLR2 and defined a minimal proximal promoter that confers
maximal reporter activity in human monocytic THP-1 cells. We
also show that human and murine TLR2 genes are regulated by
structurally different promoters, which may explain the different
expression patterns observed in both species.
As its murine homolog, the human TLR2 gene is composed of
three exons. The first and second exons are noncoding, and the
complete open reading frame is located on exon III. Alternative
splicing of exon II was primarily detected in human monocytes,
generating up to five different mRNA species. The shortest splicing form completely lacked exon II. The other four variants contained exon II, although it was spliced at different acceptor and
donor sites. Interestingly, the relative abundance of TLR2 splicing
forms varied among individuals tested, and the short form of TLR2
was detectable only in freshly isolated blood monocytes. Adherence of monocytes to tissue culture plates rapidly and selectively
induces transcription of the long slice variant. In theory, alternative
splicing does not change the putative open reading frame; all
mRNA isoforms are predicted to encode identical protein products.
However, it is possible that differences in the 5⬘ untranslated sequences influence the stability of the mRNA transcript or mRNA
secondary structure (e.g., hairpin loop formation), which may in
turn affect the extent of TLR2 protein translation. Further investigations will be needed to clarify these issues.
The 5⬘ regulatory region of TLR2 is contained in a CpG island.
Similar to many housekeeping genes and several tissue-restricted
genes, the proximal TLR2 promoter was completely unmethylated
in normal tissues, which excludes methylation of promoter CpGs
as a mechanism for regulating TLR2 expression in normal cells.
However, CpG methylation of the proximal promoter region was
detected in several tumor cell lines and correlated with undetectable TLR2 expression. Interestingly, TLR2 agonists have been
shown to deliver a proapoptotic signal (38, 39). Therefore, transcriptional inactivation of TLR2 expression by aberrant methylation of the TLR2 promoter may represent an advantage for tumor
cell survival. Further investigations shall clarify a role of TLR2
promoter methylation in growth and survival of tumors (e.g.,
leukemias).
Expression of human TLR2 transcripts has primarily been detected in myeloid cells (monocytes, macrophages, dendritic cells,
and granulocytes) (21, 22). The 5⬘ proximal region of the human
TLR2 gene lacks a TATA box and instead contains several GCrich regions, which may be involved in transcriptional initiation. In
reporter assays, the TLR2 promoter was strongly active in THP-1
cells but also had significant activity in HeLa cells, which do not
express endogenous TLR2 message. This indicates that additional
regulatory elements are involved in the observed myeloid-specific
regulation of human TLR2. Our data suggest that Sp1 family transcription factors play an important role in the activation of the
proximal TLR2 promoter. Although ubiquitously expressed, Sp1
family transcription factors have been implicated in the regulation
of several myeloid-specific genes (31–34), most likely in collaboration with more tissue-restricted transcription factors. For example, the transcription factor PU.1 was shown to be required for the
optimal expression of a growing number of myeloid-specific
genes. Accordingly, PU.1 was able to collaborate with Sp3 and to
5636
STRUCTURE AND REGULATION OF THE HUMAN TLR2 GENE
Table I. CpG methylation of the proximal TLR2 promotera
CpG No.b
Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
mRNAc
MO (I)
MAK (I)
Dendritic cells (I)
B cells
T cells
MonoMac-6
THP-1
U937
HeLa (epithelial)
HaCaT (keratinocyte)
HepG2 (hepatocellular)
Mel Im (melanoma)
HMEC (endothelial)
0
0
0
0
0
0
0
4
4
4
4
0
0
0
0
0
0
0
0
0
4
4
4
4
0
4
0
0
0
0
0
0
0
4
4
4
4
0
0
0
0
0
0
0
0
0
4
4
4
2
0
0
0
0
0
0
0
0
0
4
4
4
4
0
0
0
0
0
0
0
0
0
2
4
1
2
0
0
0
0
0
0
0
0
0
2
4
2
4
0
0
0
0
0
0
0
0
0
2
4
2
2
0
0
0
0
0
0
0
0
0
2
4
1
2
0
0
0
0
0
0
0
0
0
2
4
2
2
0
0
0
0
0
0
0
0
0
2
4
2
2
0
0
0
0
0
0
0
0
0
2
4
2
2
0
0
0
0
0
0
0
0
0
2
4
4
4
0
4
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
0
0
0
0
0
0
0
2
4
4
4
0
0
⫹
⫹
⫹
⫺
⫺
⫹
⫹
⫹/⫺
⫺
⫺
⫺
⫹/⫺
⫹/⫺
a lesser extent with Sp1 to transactivate the TLR2 promoter in
Drosophila Schneider cells. Although the exact binding sites for
PU.1 still need to be determined, these initial experiments suggest
that PU.1 also participates in the transcriptional regulation of
TLR2 in humans.
The sequence of the murine TLR2 promoter has recently been
published by two groups. In contrast to human and murine TLR4,
which share homologous promoter regions, the promoters of human and murine TLR2 were not conserved during evolution. We
were unable to detect a significant level of homology of human and
murine 5⬘ upstream sequences or 5⬘ untranslated regions, indicating that the coding third exon has been placed into a different
genetic context in mice and humans. The different regulatory sequences provide the most likely explanation for the observed differences in TLR2 tissue distribution, e.g., T cell expression of
TLR2 in mice but not in humans.
The different regulation of TLR2 in myeloid cells of both species may be of particular importance. In mice, TLR2 expression is
low or undetectable in blood cells and is strongly induced by
proinflammatory cytokines or microbial patterns (e.g., LPS). Accordingly, binding sites for NF-␬B and STAT5 in the murine
TLR2 promoter have been implicated in the rapid induction of
TLR2 expression by proinflammatory stimuli in murine macrophages. However, in humans, the highest constitutive levels of
TLR2 expression have been observed in peripheral blood leukocytes. In human monocytes, TLR2 expression is further up-regulated after adherence to tissue culture plates, but no additional
induction was observed after short-term stimulation with bacterial
products LPS or MALP-2. TLR2 expression is down-regulated in
adherent monocytes after 24 h of culture, whereas monocytes stimulated with LPS or MALP-2 appear to express higher levels of
TLR2. Activation of purified human blood monocytes with TLR
agonists is known to inhibit their differentiation into mature macrophages, which is normally accompanied by down-regulation of
TLR2 expression. This presumably leads to sustained higher levels
of TLR2 mRNA in activated monocytes after 24 h of culture. In
addition, marginal to no induction of TLR2 was observed in monocyte-derived macrophages. This is in sharp contrast to the highlevel TLR2 induction in stimulated mouse macrophages (35).
In conclusion, our observations suggest that the human TLR2
gene is regulated by Sp1 family members, probably in collaboration with the Ets family transcription factor PU.1. Additional elements (e.g., tissue-specific enhancers and silencers) are likely to
contribute to the regulation of TLR2 and are the subject of further
studies. In addition, our study identifies fundamental differences in
the transcriptional regulation of TLR2 in mice and humans. The
observed differences in basal cell type-specific and -induced expression of TLR2 may significantly influence the immune response
of both species to bacterial challenges.
Acknowledgments
We are grateful to Dr. Guntram Suske and Dr. Susanne Müller for providing Sp1 family and PU.1 expression vectors for Drosophila Schneider
cells and to Dr. Peter Mühlradt for providing sMALP-2. We are also grateful to Dr. Daniel Salamon for his help with the CpG-methylation analysis.
We acknowledge the excellent technical assistance of Sabine Pape. We
thank Dr. Stefan Krause for suggestions and members of our laboratories
for helpful discussions.
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