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* Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany; and
Pulmonary Center, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (1, 2, 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, 6, 7). Another PAMP, bacterially derived CpG DNA, is recognized through a 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, 11, 12, 13, 14, 15, 16, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Molbiol (Berlin, Germany). Antisera for supershift analyses were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cells
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 x 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 x 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).
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 µg/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).
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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'-AAGATCCTGAGCTGCCCTTGC-3'), hTLR2-IN (5'-CCAAGACCCACACCATCCAC-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
Klenow-treated 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'-AACCTAACCTTCCACGGGGCAC-3'), S2-S
(5'-GCTCACGGTCAAGGTTAGGAGCC-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'-TCTTACGCGTGCTAGGTGCCCCGACGAAGGGGCGG-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 x 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·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 µg/ml) in 130 µl 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 x 105 Schneider cells were cells were cotransfected using 10 µl Effectene reagent and 1.5 µg total DNA (1 µg of reporter plasmid, 0.25 µg 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 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1
µg/ml pepstatin, 20 µg/ml bestatin, 5 µg/ml E46, 50 µg/ml
antipain, 100 µg/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'-CGATTCGATCGGGGCGGGGCGAGC-3') were used as cold competitor. The
binding reaction contained 2.5 µg of nuclear extract protein, 0.5
µg 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 µl. 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 µg genomic DNA in 50 µl 10 mM Tris·Cl, pH 8.0, 1 mM EDTA were denatured with 5.5 µl NaOH (3 M) at 37°C for 15 min. After the addition of 540 µl sodium bisulfite (3.8 M) and 15 µl 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 µl H2O and desulfonated by the addition of 11 µl NaOH (3 M) and subsequent incubation at 37°C for 15 min. The DNA was then precipitated using ammonium acetate and ethanol and resuspended in 50 µl 10 mM Tris·Cl, pH 8.0, 1 mM EDTA. A total of 5 µl of DNA was amplified in individual nested PCR for both strands using the following primers: sense strand (first round), Ss1 (5'-GTTTTAAGAAAATATTGGTTGGG-3') and Sas1 (5'-CAAAAACTAAAAACCCAAATACAAC-3'); sense strand (first round), Ss2 (5'-TGGGTATTTAGTTTTTTTTGTGG-3') and Sas2 (5'-AAAAAACTCCRAACAATCACC-3'); antisense strand (first round), ASs1 (5'-CTTTCCCTATAATTACCAATCCC-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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A. Alternative splicing of the human TLR2
The majority of 5' fragments amplified from monocytes by
RLM-RACE-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
differentiation 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.
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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 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, 32, 33, 34).
Several GC-rich sequences and putative binding sites for Sp1 family
transcription factors are present in the proximal TLR2 promoter. As
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 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 additional 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 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 Sp1-mediated activation
of the proximal TLR2 promoter construct.
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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 IL-1, TNF, GM-CSF, and
LPS (35). The activity of its promoter is induced by LPS
and the induction is dependent on NF-B- and 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
LPS-stimulated 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-2-treated 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.
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 GC-rich 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, 32, 33, 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 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 high-level 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.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Michael Rehli, Department of Hematology and Oncology, University Hospital, 93042 Regensburg, Germany. E-mail address: Michael.Rehli{at}klinik.uni-regensburg.de
3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; MALP, macrophage-activating lipopeptide; TLR, Toll-like receptor.
Received for publication October 29, 2001. Accepted for publication March 21, 2002.
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B translocation. J. Biol. Chem. 276:22041. and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. Natl. Acad. Sci. USA 86:2336.B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression. J. Immunol. 166:4516.5 integrin gene expression in macrophages and osteoblasts. J. Biol. Chem. 275:8331.,25-dihydroxyvitamin D3. J. Immunol. 153:3276.[Abstract]
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