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,
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Arizona Respiratory Center and the Departments of
Pharmacology,
Medicine,
Pediatrics, and
¶ Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, AZ 85724; and
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Molecular Immunology Section, Children’s Hospital Research Foundation, University of Cincinnati, Cincinnati, OH, 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T, in the CD14 proximal
promoter. Homozygous carriers of the T allele have a significant
increase in soluble CD14, but a decreased total serum IgE. This
epidemiologic evidence led us to investigate the molecular basis for
the effects of CD14/-159CT on CD14
regulation in monocytes and hepatocytes, the two major cell types known
to express this gene in vivo. EMSA analysis showed that the
T allele results in decreased affinity of DNA/protein
interactions at a GC box that contains a binding site for Sp1, Sp2, and
Sp3 transcription factors. In reporter assays, the transcriptional
activity of the T allele was increased in monocytic Mono
Mac 6 cells, which express low levels of Sp3, a member of the Sp family
with inhibitory potential relative to activating Sp1 and Sp2. By
contrast, both alleles were transcribed equivalently in Sp3-rich
hepatocytic HepG2 cells. Our data indicate that the interplay between
CD14 promoter affinity and the [Sp3]:[Sp1 + Sp2] ratio
plays a critical mechanistic role in regulating transcription of the
two CD14 alleles. Variation in a key gene of innate
immunity may be important for the pathogenesis of allergy and
inflammatory disease through gene-by-gene and/or gene-by-environment
interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although CD14 exists as a single-copy gene, CD14 protein is found in two distinct forms: a 50- to 55-kDa glycosylphosphatidylinositol-anchored membrane molecule, membrane CD14 (mCD14),3 expressed primarily on the surface of monocyte/macrophages and neutrophils (6), and a soluble form lacking the glycosylphosphatidylinositol anchor (7). Soluble CD14 (sCD14) appears to derive from monocytes (8) as well as the liver (9, 10) and is found in normal serum at microgram concentrations (7). Both mCD14 and sCD14 are critical for LPS-dependent signal transduction. LPS-responsive cells that lack mCD14, such as endothelial cells, epithelial cells, and astrocytes, become sensitive to low concentrations of LPS in the presence of sCD14 (11).
Regulation of CD14 gene expression appears to be important in several disease states. Increased serum levels of sCD14 are associated with high mortality in Gram-negative septic shock (12). Consistent with these findings, CD14 knockout mice were found to be at least 10-fold more resistant to LPS-induced shock than wild-type controls (13). In addition, inflammatory infectious diseases such as atopic dermatitis (14), HIV infection (15), and malaria (16), as well as extensive tissue damage in polytrauma and severe burns (17), are marked by elevated sCD14 in the circulation.
This compelling epidemiologic evidence led us to investigate whether
CD14 expression is influenced by genetic variation.
Recently, our group has identified a single nucleotide polymorphism
(SNP) in the proximal CD14 promoter at position
-159 (18). CD14/-159CT is very
frequent among both Hispanic and non-Hispanic white populations, with
approximately one-half of all chromosomes carrying the C
allele and one-half carrying the T allele. Interestingly,
homozygous carriers of the T allele have a significant
increase in serum levels of sCD14 and a concomitant decrease in total
serum IgE (18), suggesting that CD14 may play a
role in the regulation of IgE synthesis and IgE-mediated diseases such
as allergy and asthma. In contrast, the association between
CD14/-159CT and risk for myocardial infarction recently
described in at least three different populations (19, 20, 21)
eloquently highlights the far-reaching effects that genetic variation
in CD14 may have on the pathogenesis of cardiovascular
diseases. In this context, the role of CD14 in inflammation
and/or its ability to bind and transport lipids, including cholesterol
(22), may be particularly relevant. Altogether, these
findings suggest that CD14/-159 may affect the regulation
of CD14 gene expression, thus modulating the impact that
CD14-mediated events have on innate and adaptive immune responses
(23).
These findings warranted studies aimed at providing a molecular basis
for the effects of CD14/-159CT on the regulation of
CD14 gene expression. We developed a reporter system to
assess the transcriptional activity of the two allelic variants of the
CD14 proximal promoter. We found a monocyte-specific
increase in transcription from the T allele that was
associated with a decreased affinity in DNA/Sp protein interactions at
a GC box that includes the polymorphic nucleotide.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic Mono Mac 6 cell line was generously provided by Dr. H. W. Ziegler-Heitbrock (University of Munich, Munich, Germany) and was cultured as described (24). Human hepatocytic HepG2 cells (HB8065) were obtained from the American Type Culture Collection (Manassas, VA). Primary human monocytes were isolated by countercurrent elutriation from normal volunteers as previously described (25). All cell culture reagents were endotoxin free as determined by the Limulus amebocyte lysate assay (limits of detection 3–6 pg/ml; BioWhittaker, Walkersville, MD).
Construction of CD14 reporter vectors
Fig. 1
A shows the
sequence of the CD14 5'-flanking region that contains the
proximal promoter (26, 27) and CD14/-159CT
(Fig 1A, bold and underlined). The -159C/luc and
-159T/luc reporter constructs contain a 304-bp insert
(position -232/+72; GenBank accession no. U00699) and were created by
PCR amplification of genomic DNA from individuals homozygous for either
C or T at position -159 using as primers
5'-GTGCCAACAGATGAGGTTCA (position -513/-493) and
5'-CGCAGCGGAAATCTTCATC (position +274/+292). The PCR products were
digested with NarI/SacI, shuttled through
pBluescript KS+ (Stratagene, La Jolla, CA) via
ClaI/SacI, and then directionally cloned into the
KpnI and SacI sites of the promoterless,
enhancerless luciferase reporter plasmid, pGL3-Basic (Promega, Madison,
WI). Constructs were verified by sequence analysis and were prepared
for transfection using the EndoFree Plasmid Mega kit (Qiagen,
Valencia, CA).
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Mono Mac 6 cells (5 x 106 cells/500 µl of serum-free RPMI 1640) were transiently transfected with -159C/luc, -159T/luc, or pGL3-Basic (20 µg) and a control Renilla luciferase reporter plasmid (pRL-CMV, 25 ng; Promega) using electroporation. Transfection was performed using a T820 Electro Square Porator (BTX, San Diego, CA) at 2.0 kV with three 99-µs pulses. Cells were then incubated at room temperature for 15 min before the addition of growth medium with 10% human serum (Sigma-Aldrich, St. Louis, MO). Following a 6-h incubation, luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega) on a TD 20/20 Luminometer (Promega). Protein concentration of cell extracts was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Results were normalized for Renilla activity and protein concentration, and were expressed as relative luciferase activity (RLA). In selected experiments, transfected Mono Mac 6 cells were stimulated with LPS from Salmonella minnesota (100 ng/ml; Sigma-Aldrich) for 6 h before measuring luciferase activity.
HepG2 cells (8 x 105/ml in DMEM) were transiently transfected with -159C/luc or -159T/luc (1 µg) and pRL-CMV (1.25 ng) using Lipofectamine 2000 (6 µl; Life Technologies, Rockville, MD). Following a 24-h incubation in growth medium, luciferase activity was measured as described above.
Oligonucleotides and probes for EMSA
EMSA analysis was performed using 22-bp oligonucleotides (22C or
22T) corresponding to the -167/-146 region in the CD14
promoter that encompasses CD14/-159 (Fig. 1
B).
Mutant oligonucleotides were generated by introducing three
nonoverlapping blocks of 5-bp transversions into wild-type 22C (Fig. 1C). In addition, we used oligonucleotides containing
binding sites for Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC) and STAT6
(5'-CTGTTCTTGGGAAGTC; consensus sequences are underlined).
Single-stranded oligonucleotides were obtained from Life Technologies.
Complementary oligonucleotide pairs were annealed and isolated as
previously described (28). When used as EMSA probes,
double-stranded oligonucleotides (50 ng) were end-labeled using
[
-32P]ATP (3000 Ci/mM; ICN Pharmaceuticals,
Costa Mesa, CA) and T4 polynucleotide kinase (Life Technologies).
Free radioactivity was removed by gravity chromatography over Sephadex
G25 columns (Boehringer Mannheim, Indianapolis, IN).
Nuclear extract preparation
Mono Mac 6 cells (107 cells) were washed
with ice-cold PBS, resuspended in 1 ml of ice-cold hypotonic buffer
(29) with protease inhibitors (1 mM PMSF, and 3 µg/ml
each of leupeptin, aprotinin, antipain, and pepstatin) and phosphatase
inhibitors (5 mM 
-glycerol phosphate, 1 mM benzamidine, 1 mM
orthovanadate, and 1 mM sodium fluoride), and were then incubated for
15 min on ice. Tubes were subsequently centrifuged for 30 s at
16,000 x g at 4°C. The nuclear pellets were washed
twice with ice-cold hypotonic buffer and were then treated with 100
µl of extraction buffer (29) with protease and
phosphatase inhibitors as above for 20 min on ice. Nuclear debris was
pelleted by centrifugation for 10 min at 16,000 x g at
4°C. The protein concentration of the nuclear extracts was determined
using the bicinchoninic acid assay (Pierce). Aliquots of the isolated
nuclear extracts were snap frozen in liquid nitrogen and stored at
-70°C until used. Nuclear extracts from HeLa cells were obtained
from Geneka (Montreal, Canada). Nuclear extracts from HepG2 were
prepared as previously described (28) with the addition of
0.1% Nonidet P-40 to the lysis buffer. Nuclear extracts from freshly
isolated monocytes were prepared following the Andrews and Faller
modification (30) of the procedure described by Dignam
(31).
EMSA
After preliminary data indicated that Sp family proteins bind differentially to the 22C and 22T oligonucleotides, EMSA conditions were optimized for Sp protein/DNA interactions as follows: nuclear extracts (5–10 µg) were incubated for 20 min on ice with binding buffer (20 mM HEPES-HCl (pH 7.6), 100 mM NaCl, 12.5% glycerol, 14 mM 2-ME, 1 mM EDTA, 0.5% Nonidet P-40, and poly(dI-dC) x poly(dI-dC) (1 µg; Amersham Pharmacia Biotech, Piscataway, NJ) in the presence or absence of oligonucleotide competitors. A 32P-labeled probe (30 fM, 100,000–150,000 cpm/lane) was then added to the binding reaction (final volume, 25 µl) for an additional 20-min incubation on ice. The binding reactions were run on a 3.2% polyacrylamide gel (acrylamide:bisacrylamide ratio of 37.5:1; Bio-Rad, Hercules, CA) in 0.25x Tris-borate-EDTA buffer (25 mM Tris, 25 mM boric acid, and 0.5 mM EDTA, pH 8) at 200 V for 2–3 h at 4°C. Gels were dried before exposure to autoradiographic film. Autoradiographs were quantitated by densitometric analysis using Molecular Analyst software (Bio-Rad).
For Ab supershift assays, polyclonal antisera (4 µg) were added to
nuclear extracts for 20 min on ice before incubation with the probe.
Polyclonal antisera specific for human Sp2 (rabbit K-20), Sp3 (goat
D-20), Sp4 (rabbit V-20), and Fra1 (rabbit R-20) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit
polyclonal antiserum to Sp1 was purchased from Geneka. Recombinant Sp1
protein was obtained from Promega. An AP2 EMSA kit (including AP2
oligonucleotide (5'-CCACAAACGACCGCCCGCGGGCGGT), AP2 mut
oligonucleotide (5'-CCACAAACGACCGATTGCGGGCGGT), JEG-3
nuclear extracts, binding buffer, and anti-AP2-
Ab) was obtained
from Geneka and was used for the detection of AP2/DNA interactions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transcriptional activity of the CD14/-159C and
T variants was compared by transiently transfecting the
-159C/luc and -159T/luc reporter constructs
into monocytic Mono Mac 6 cells. Luciferase activity in cell extracts
was assessed 6 h post-transfection and was expressed as fold
increase in the activity of the CD14 reporter constructs
compared with the empty vector, pGL3-Basic. The data presented in Fig. 2 indicate that, consistent with previous
reports (26), the wild-type CD14 promoter
(-159C/luc) had strong basal transcriptional activity.
Importantly, the CT SNP at -159
(-159T/luc) resulted in a 32% increase in constitutive
gene expression. This difference in activity was significant
(p < 0.0001) and consistent in 14 independent
transfections.
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Proteins of the Sp family bind the polymorphic GC box in the CD14 promoter
To understand the mechanism(s) responsible for the increased
transcriptional activity of the CD14/-159T allelic variant,
the sequence of the CD14 promoter encompassing the SNP was
analyzed for the presence of potential transcription factor binding
sites (33). A putative Sp protein-binding GC box and an
AP2 motif were predicted to span CD14/-159 (Fig. 1B). Therefore, EMSA analysis was performed to assess the
ability of AP2 and Sp proteins to bind the C and/or
T allelic variants using 22-bp oligonucleotides (22C and
22T, position -167/-146) that include both the GC box and the AP2
site. AP2 complexes were undetectable using nuclear extracts from
AP2-expressing JEG-3 cells (Ref. 34 and data not shown),
thus ruling out an interaction between the polymorphic region of the
CD14 promoter and AP2 proteins.
Transcription factors of the Sp family activate a wide subset of
mammalian genes containing GC box promoter elements (reviewed in Ref.
35). Currently, this family consists of four proteins
designated Sp1–4. The GC-rich region encompassing the -159
polymorphic site (Fig. 1B) represents a potential Sp protein
binding motif (36, 37, 38). Of note, the presence of a
T at -159 decreases the homology between the
CD14 promoter GC box and the Sp consensus sequence.
Sp protein/DNA interactions were investigated by EMSA. Fig. 3 (top) shows that
incubation of a 22-bp probe containing a high-affinity binding site for
Sp1 with nuclear extracts from HeLa cells that express high levels of
Sp proteins (39) resulted in the formation of three
specific complexes, all of which were disrupted by the addition of a
100-fold molar excess of unlabeled Sp1 oligonucleotide, but not by an
unrelated STAT6 oligonucleotide. Complex I appeared to contain Sp1 and
Sp2 because it was supershifted by Sp1- or Sp2-specific antisera.
Complexes II and III were identified by Ab supershifts as containing
two Sp3 species of different mobility (40). None of the
complexes contained Sp4 as assessed by supershifting using Sp4-specific
antiserum (data not shown). A control antiserum to Fra1 had no effect
on the migration of the complexes. Notably, all three specific
complexes generated by the Sp1 probe were competed by unlabeled 22C and
22T oligonucleotides, suggesting that Sp proteins may bind at, or in
the vicinity of, CD14/-159.
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, center and
bottom). Indeed, all three complexes were competed
specifically by an Sp1 oligonucleotide and contained Sp1 (Fig. 3, complex I), Sp2 (Fig. 3, complex I), and Sp3 (Fig. 3, complexes II and
III), as identified by supershift assays. In addition, recombinant Sp1
protein was found to interact with the 22C and 22T as well as Sp1
probes (data not shown). Thus, our results demonstrate that the
transcription factors Sp1, Sp2, and Sp3 can bind the CD14
promoter region that contains CD14/-159CT. Importantly,
the competition curves with 22C and 22T oligonucleotides provided the
first evidence to suggest a difference in affinity of the two allelic
variants for Sp family members.
To further dissect the impact of -159CT on
CD14 promoter/protein interactions, we performed mutational
analysis to assess how different regions of the GC box contribute to Sp
protein binding. Three mutants, each containing a block of five
transversions, were generated on the 22C backbone (Fig. 1C)
and were used as competitors in EMSA analysis. Fig. 4 shows that Mut2 (in which the inner
core of the GC box had been disrupted) was severely impaired in its
capacity to compete 22C probe binding to HeLa nuclear proteins. By
contrast, Mut1 (which includes CD14/-159) and Mut3 retained
substantial protein binding ability. We conclude that the core of the
GC box in the CD14 proximal promoter is essential for Sp
protein binding, whereas the polymorphic region immediately upstream
may contribute to the overall affinity of Sp protein/DNA
interactions.
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Nuclear extracts from HeLa cells represented a useful tool
to characterize Sp protein/CD14 promoter interactions
because they contain high levels of Sp1–3. However, to correlate the
functional data from transient transfections with the results provided
by EMSA analysis, we needed to test nuclear extracts from the monocytic
cell line used in reporter assays, Mono Mac 6. Fig. 5 (left) shows that
incubation of Mono Mac 6 nuclear extracts with an Sp1 probe resulted in
the formation of three specific complexes of mobility similar to that
observed using HeLa nuclear extracts (see Fig. 3), even though complex
III was barely detectable. All complexes were disrupted by the addition
of a 100-fold excess of unlabeled Sp1 oligonucleotide, but not by an
unrelated STAT6 oligonucleotide. Ab-mediated supershifting indicated
that complex I contained Sp1 and Sp2 proteins. Complex II and the faint
complex III were completely abrogated by anti-Sp3 Abs. When EMSA
analysis was performed using Mono Mac 6 nuclear extracts and a 22C
probe, a similar pattern of protein/DNA interactions was detected (Fig. 5, center).
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(right) shows that the 22C probe was able to bind Sp1, Sp2,
and Sp3, resulting in the formation of complexes I, II, and III. All
three complexes were specifically abrogated by Sp1 as well as by 22C
and 22T oligonucleotides, with competition and Ab supershift patterns
similar to those obtained with HeLa and Mono Mac 6 nuclear proteins.
Overall, our results confirm a similar pattern of
Sp1–3/CD14 promoter interactions for HeLa cells, Mono Mac 6
cells, and freshly isolated human monocytes, except for the low
expression of the high mobility form of Sp3 in Mono Mac 6 cells. Sp proteins have enhanced affinity for the -159C variant of the CD14 promoter GC box
The finding that Sp1, Sp2, and Sp3 bound both the 22C and 22T
oligonucleotides indicated that there was no qualitative difference in
the ability of Sp proteins to interact with the two allelic variants of
the CD14 promoter. In contrast, the 22C and 22T
oligonucleotides appeared to differ in their capacity to compete for Sp
protein binding (see competition lanes in Figs. 3 and 5). Therefore, we
sought to assess whether the presence of a C or a
T at -159 resulted in a difference in the
affinity of Sp proteins for the CD14 promoter. To this
purpose, nuclear extracts from HeLa cells were incubated with an Sp1
probe and an increasing molar excess of Sp1, 22C, or 22T
oligonucleotide competitor. The intensity of the resulting bands was
quantitated by scanning densitometry, and the percentage of binding
relative to uncompeted probe was plotted against the molar excess of
competitor added to each sample (Fig. 6, top). The mean ± SEM of three independent experiments
is shown for each competitor in each panel. As expected, an unlabeled
Sp1 motif competed Sp protein binding quite effectively even when added
at a 25-fold molar excess. By contrast, 22C and 22T bound less avidly
and, furthermore, markedly different affinities were consistently
observed for the two oligonucleotides. Indeed, a 100-fold molar excess
of 22T was required to obtain the same degree of competition detected
using 22C at 50-fold molar excess. These results indicate a preference
for Sp protein binding to the CD14/-159C allelic variant.
Of note, a similar pattern of competition was observed using nuclear
extracts from freshly isolated human monocytes (Fig. 6, center).
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An apparent paradox emerged from our results described above:
higher transcriptional activity of the CD14/-159T allele
was associated with DNA/protein interactions of lower affinity.
However, our results also demonstrated that the nuclear factors binding
to CD14/-159CT are members of the Sp family, which
includes proteins with different functional properties. Indeed, Sp1 has
been shown to function as a transcriptional activator, whereas Sp3 is
characterized usually as a repressor (reviewed in Ref.
35). Therefore, we reasoned that the higher
transcriptional activity of the T allele in monocytes may be
due to its decreased affinity for Sp3-containing complexes with
inhibitory potential. As a corollary, monocytes would be expected to
express low and possibly limiting levels of Sp3 relative to Sp1 and
Sp2. By the same token, an excess of Sp3 should overcome
affinity-related transcriptional differences between the two
CD14 alleles.
To test our hypothesis, we took advantage of the existence of another
major cell type that expresses CD14 in vivo. The hepatocytic cell line
HepG2, as well as primary hepatocytes, shows moderate but readily
detectable constitutive levels of CD14 transcription
(10, 41). In preliminary experiments, we used EMSA
analysis to determine the relative expression of Sp family members in
HepG2 cells. Fig. 7A shows
that HepG2 nuclear extracts formed three major complexes with an Sp1 or
22C probe. Complex I appeared to contain Sp1 and Sp2 because it was
supershifted by Sp1- and Sp2-specific antisera. Complexes II and III
contained Sp3 because the bands disappeared upon addition of an
Sp3-specific Ab. As expected, all complexes were specifically competed
by 22C and 22T oligonucleotides, but not by an unrelated
oligonucleotide (STAT6). Of note, binding studies using the two allelic
variants of the CD14 promoter and HepG2 nuclear extracts
again showed a marked decrease in the affinity of the T
allele (Fig. 6, bottom). Even more importantly, upon
densitometric analysis of the complexes formed by Sp factors, we noted
that the relative intensity of the Sp3-containing bands was
dramatically increased in HepG2 cell EMSAs relative to monocytic cells.
Indeed, the ratio of Sp3 (complexes II and III) to Sp1 and Sp2 (complex
I) was 2:1 in HepG2 cells, whereas an inverted 1:2 ratio was observed
in both Mono Mac 6 cells and freshly isolated monocytes (Fig. 7B).
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C, and the experiments provided information that
supports our hypothesis. On the one hand, the increased activity of the
T allele was lost in Sp3-rich HepG2 cells when compared with
Mono Mac 6 cells. Thus, an excess of Sp3 overrode the difference in
affinity created by the polymorphism. In addition, the overall
transcriptional activity of the CD14 reporter vectors was
diminished in HepG2 cells, probably because of Sp3-mediated repression
through other Sp binding motifs known to exist in the proximal
CD14 promoter (26). Taken together, these
findings suggest that the interplay between CD14 promoter
affinity and the [Sp3]:[Sp1 + Sp2] ratio plays a critical
mechanistic role in regulating CD14 transcription, and more
specifically, in determining the differential activity of the two
allelic variants of the CD14 promoter. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the central role of CD14 in innate immunity, the regulation
of CD14 gene expression has been extensively investigated. A
227-bp region upstream of the major transcription initiation site was
found to be sufficient to drive maximal reporter activity in transient
transfection assays (26). This region contains binding
sites for Sp1 and members of the CCAAT/enhancer-binding protein (C/EBP)
family of transcription factors (26, 42). Basal promoter
activity was strictly dependent on the integrity of Sp1 and C/EBP
motifs located at position -110 and -135, respectively. The same
C/EBP site was also involved in the TGF-/Vit D3-dependent activation
of CD14 expression (42). More recently, in vivo
studies with transgenic mice have shown that an 80-kb human genomic
fragment that includes the CD14 coding region contains
critical regulatory elements necessary to direct tissue-specific
CD14 expression in monocytes and hepatocytes (9, 41). Within this fragment, a 0.7-kb enhancer located 
6 kb 5'
of the CD14 transcription initiation sites appears to be
essential for CD14 expression in hepatocytes, while
additional upstream elements are required to direct transcription in
monocytes (41).
Our present data show that a common SNP in the proximal CD14
promoter results in increased transcriptional activity. A reporter
vector containing CD14/-159T was more active than the
C allelic variant in transient transfection assays using
CD14-expressing monocytic cells. This increase in activity was
paralleled by a decreased affinity of the interactions between the Sp1,
2, and 3 proteins and the polymorphic GC box in the CD14
promoter. All Sp family members contain a highly conserved DNA binding
domain and bind the same consensus sequence, but show promoter
context-related differences in their transactivating properties
(35). It is generally believed that the function of Sp
protein-dependent promoters is regulated by the relative ratio between
activating and repressing members of the Sp family. In this scenario,
the -159/CT polymorphism increases transcription by
lowering the affinity of the CD14 regulatory region for Sp3,
a factor known to inhibit the activity of a number of promoters
(40, 43, 44). This line of reasoning is strongly supported
by the comparison of the transcriptional activities of the C
and T allele in two cell lines that exhibit opposite
patterns of Sp1/Sp2 and Sp3 expression. Indeed, transcription of the
T allele was increased in the monocytic cell line Mono Mac 6
in which Sp3 expression is low. By contrast, in Sp3-rich hepatocytic
HepG2 cells, the CD14/-159CT transcriptional difference
was lost. Thus, the affinity of the promoter GC box and the ratio
between activating and repressing Sp family members play a complex but
critical role in regulating the transcription of the two allelic
variants of the CD14 promoter.
In our population studies, levels of serum sCD14 were higher in the
CD14/-159T homozygotes (18), and as described
in this study, the CD14/-159T reporter construct was
transcriptionally more active than the C allelic variant in
monocytic cells but not in hepatocytes. Yet sCD14 is known to be
secreted by both monocytes (8) and hepatocytes (9, 10). Thus, it is possible that genetic variation in the
CD14 proximal promoter may affect its ability to engage in
long-range interactions with liver-specific, promoter-distal elements
such as the enhancer situated 6 kb upstream of the CD14
transcription start site (41). These elements are not
included in the reporter constructs analyzed in this study.
More and more reports are becoming available that describe novel polymorphisms in disease-related genes, and associations between these SNPs and specific phenotypes. However, few mechanistic studies have so far addressed the functional impact that SNPs in regulatory regions have on the control of gene expression (45, 46, 47, 48). A common theme of these studies, as well as in ours, is that, unlike conventional vectors containing artificial mutations, most reporter constructs that model naturally occurring polymorphic variants exhibit relatively modest differences in transcriptional activity when compared with their wild-type counterpart in transient transfection assays. However puzzling, this paradigm is consistent with the basic notion that these instances of genetic variation may be insufficient to cause disease when taken individually. However, they may induce significant modulation of gene expression when they become involved in gene-by-gene and/or gene-by-environment interactions. Thus, complex phenotypes and/or polygenic diseases would not be expected to emerge as all-or-none events, but rather to result from a combination of small quantitative effects. This may be especially true of innate immunity, the genes for which are highly conserved across species and therefore are not likely to tolerate dramatic shifts in their functional patterns. Therefore, we envision a scenario in which a constellation of variations in critical genes fine-tunes innate and adaptive immune responses, and the way they interface with the environment, resulting in a wide spectrum of related phenotypes.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tricia D. LeVan, Arizona Respiratory Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, AZ 85724. E-mail address: Tricia.LeVan{at}resp-sci.arizona.edu
3 Abbreviations used in this paper: mCD14, membrane CD14; sCD14, soluble CD14; RLA, relative luciferase activity; SNP, single nucleotide polymorphism; C/EBP, CCAAT/enhancer-binding protein.
Received for publication November 9, 2000. Accepted for publication September 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation 99:3218.25-dihydroxyvitamin D3. J. Immunol. 153:3276.[Abstract]
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F. D. Martinez Gene-Environment Interactions in Asthma: With Apologies to William of Ockham Proceedings of the ATS, January 1, 2007; 4(1): 26 - 31. [Abstract] [Full Text] [PDF]
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L. Cameron, R. B. Webster, J. M. Strempel, P. Kiesler, M. Kabesch, H. Ramachandran, L. Yu, D. A. Stern, P. E. Graves, I. C. Lohman, et al. Th2 Cell-Selective Enhancement of Human IL13 Transcription by IL13-1112C>T, a Polymorphism Associated with Allergic Inflammation J. Immunol., December 15, 2006; 177(12): 8633 - 8642. [Abstract] [Full Text] [PDF]
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 R. C. Barber, L.-Y. E. Chang, B. D. Arnoldo, G. F. Purdue, J. L. Hunt, J. W. Horton, and C. C. Aragaki Innate Immunity SNPs are Associated with Risk for Severe Sepsis after Burn Injury Clin. Med. Res., December 1, 2006; 4(4): 250 - 255. [Abstract] [Full Text] [PDF]
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 M. Kabesch A Glitch in the Switch?: Of Endotoxin, CD14, and Allergy. Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 365 - 366. [Full Text] [PDF]
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A. Simpson, S. L. John, F. Jury, R. Niven, A. Woodcock, W. E. R. Ollier, and A. Custovic Endotoxin Exposure, CD14, and Allergic Disease: An Interaction between Genes and the Environment Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 386 - 392. [Abstract] [Full Text] [PDF]
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 S. P. Wiertsema, S.-K. Khoo, G. Baynam, R. H. Veenhoven, I. A. Laing, G. A. Zielhuis, G. T. Rijkers, J. Goldblatt, P. N. LeSouef, and E. A. M. Sanders Association of CD14 Promoter Polymorphism with Otitis Media and Pneumococcal Vaccine Responses. Clin. Vaccine Immunol., August 1, 2006; 13(8): 892 - 897. [Abstract] [Full Text] [PDF]
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, n = 3), and Mono Mac 6 cells
(n = 4). C, Reporter analysis of
CD14 promoter variants. Mono Mac 6 and HepG2
cells were transiently transfected with -159C/luc,
-159T/luc, or pGL3-Basic (20 µg). Luciferase activity was
normalized for transfection efficiency using a control plasmid pRL-CMV
(25 ng) and protein concentration. Results are reported as fold
increase in the RLA of the CD14 reporter constructs compared
with the empty control vector pGL3-Basic. The figure compares the
mean ± SEM of results obtained in 14 experiments with Mono Mac 6
cells (MM6) and three experiments with HepG2 cells.