(Received for publication, November 19, 1996, and in revised form, January 23, 1997)
From the Istituto di Ricerche Farmacologiche "Mario
Negri," via Eritrea, 62, 20157 Milan, Italy and the
§ Section of General Pathology and Immunology, University of
Brescia, 25123 Brescia, Italy
The "long pentraxins" are an emerging family
of genes that have conserved in their carboxy-terminal halves a
pentraxin domain homologous to the prototypical acute phase protein
pentraxins (C-reactive protein and serum amyloid P component) and
acquired novel amino-terminal domains. In this report, a genomic
fragment of 1371 nucleotides from the human "long pentraxin" gene
PTX3 is characterized as a promoter on tumor necrosis
factor- (TNF
) and interleukin (IL)-1
exposure in transfected
8387 human fibroblasts by chloramphenicol acetyltransferase and RNase
protection assays. In the same cells, the PTX3 promoter
does not respond to IL-6 stimulation. Furthermore, IL-1
and TNF
responsiveness is not seen in the Hep 3B hepatoma cell line. The
minimal promoter contains one NF-
B element which is shown to be
necessary for induction and able to bind p50 homodimers and p65
heterodimers but not c-Rel. Mutants in this site lose the ability to
bind NF-
B proteins and to respond to TNF
and IL-1
in
functional assays. Sp1- and AP-1 binding sites lying in proximity to
the NF-
B site do not seem to play a major role for cytokine
responsiveness. Finally, cotransfection experiments with expression
vectors validate that the natural promoter contains a functional
NF-
B site.
The human gene hPTX3 has been recently cloned from
interleukin-1 (IL-1b)1-stimulated
endothelial cells (1) and from tumor necrosis factor-
(TNF
)-stimulated fibroblasts (2). PTX3 belongs to the
family of pentraxins (so named because they are assembled in pentamers) that include C-reactive protein (CRP) and serum amyloid P component (SAP) from several different species (3) and which are markers of the
acute phase. Moreover, while the 3
half of PTX3 can be aligned with the full-length sequences of CRP and SAP (4) (pentraxin domain), the 5
half of the protein does not show significant homology
with other known proteins. PTX3 is indeed the first isolated member of a new group of proteins, known as "long pentraxins," which have different 5
-termini upstream from their pentraxin domains
(5-9).
While the classical pentraxins CRP and SAP are almost exclusively
produced by the liver in response to IL-6 in combination with IL-1 and
TNF, PTX3 shows a more promiscuous response in that its
expression in vitro can be induced in endothelial cells,
hepatocytes, fibroblasts, and monocytes. In all cases, the gene is
rapidly and directly induced by exposure to IL-1, TNF
, and
lipopolysaccharide (LPS), but not by IL-6, the mRNA peaking 4-6 h
after the stimulation (1, 4, 10). This induction is paralleled by
de novo transcription of the gene (10) and is transient in
that no more message is detectable after 24 h (1, 10).
The mouse homologue, mPTX3, shows a similar exon/intron organization and 82% identity at the amino acid level with hPTX3 (4). When C57BL mice were injected i.v with LPS to induce an acute phase response, mPTX3 expression was markedly induced in vivo after 4 h in several muscular organs, including the heart and the thigh (4). In situ hybridization studies showed that endothelial cells within the muscular tissues were the major responder cell type. Interestingly, in striking contrast with CRP and SAP, no mRNA for mPTX3 could be detected by Northern analysis in the liver (4).
Similar promiscuous in vivo expression has also been observed for the other "long pentraxins" in organs as diverse as the brain and the testis (6-9).
To begin to understand the molecular mechanisms underlying the regulated expression of the first cloned long pentraxin PTX3, we cloned and characterized the promoter of hPTX3.
The human fibrosarcoma 8387 (11) and the human hepatoma Hep 3B (12) cell lines were maintained in Dulbecco's modified Eagle's medium (Seromed, Biochrom KG, Berlin, Germany), supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 50 µg/ml gentamycin (Life Technologies, Inc., Paisley, Renfewshire, United Kingdom), and 20 mM L-glutamine (Seromed).
Plasmid ConstructionA 1.37-kilobase
EcoRI-PvuII genomic fragment from P2 phage
(1), which spans nucleotides
1317 to +54 relative to the
transcription start site, was blunted and subcloned into the
XhoI-digested and blunted pBL CAT 3 vector (13) (giving
1317-CAT). Following PstI-XbaI digestion, the
latter plasmid was used as a substrate to generate a set of deletion
clones with ExoIII (Stratagene, La Jolla, CA) digestion,
mung (Stratagene) blunting, and subsequent religation. Deletion clones
covering the whole 5
-flanking region of the hPTX3 gene were
sequenced by the Sanger dideoxy method (14). Three of them,
387-CAT,
180-CAT, and
74-CAT, were subsequently used in functional
assays.
The plasmid carrying a mutation in the 96 NF-
B site (
180 m
NF-
B-CAT) was produced by polymerase chain reaction (PCR) technique. The plasmid
180-CAT was used as a substrate for amplification with
four synthetic oligonucleotides (Duotech srl, Milan, Italy): oligonucleotide I (5
-CTATTACGCCAGCTGGCG-3
) was designed on a pUC-derived sequence from the pBL CAT 3 vector in sense orientation; oligonucleotide IV (5
-CAACGGTGGTATATCCAGTG-3
) was designed on the CAT
sequence in antisense orientation. Oligonucleotide II (5
-TGCATCTGAATTTGGTGGGGGAGG-3
) was designed in
antisense orientation on hPTX3 promoter from nucleotide
115 to nucleotide
97 with an additional 11 nucleotides at the 5
end; 6 of them (underlined) give rise to a XbaI
restriction site. Oligonucleotide III
(5
-TGCCCGTTACCGCAGTGCCACC-3
) was designed in sense orientation on the hPTX3 promoter from nucleotides
88 to
69; an additional tail of 9 nucleotides includes
(underlined) a XbaI site. Two PCR reactions were
performed with oligonucleotides I/II and III/IV, respectively; the
fragments obtained were separated on 5% native polyacrylamide gel,
eluted, and cut with HindIII/XbaI and
XbaI/XhoI, respectively. Finally, the digested
fragments were inserted into a
HindIII/XhoI-digested pBL CAT 3 vector. The
resulting construct (
180 m NF-
B-CAT) carries a substitution of 8 bases at the NF-
B site (ATTCTAGA instead of GGGAACTC).
To obtain the mutant plasmid 180 m Sp1-CAT, we used an
oligonucleotide carrying two mutations (underlined) in the
123 Sp1 site (oligonucleotide V
5
-CTCTCCCACCCACCCCTCCCCCACCAAAT-3
, spanning from
nucleotide
131 to nucleotide
101). This mutagenic primer, together
with the flanking oligonucleotides I and IV and with the
SmaI linearized wild-type
180-CAT plasmid as a substrate were used to produce a DNA fragment carrying the desired mutations according to the PCR mutagenesis technique as described (15). Similarly, to obtain the
180 m AP-1-CAT mutant plasmid, we used a
mutagenic primer (oligonucleotide VI
5
-CCACCAGCATTACTTCATCCCCATTC-3
spanning from
nucleotide
74 to nucleotide
46) carrying three substitutions
(underlined) in the
65 AP-1 site. The double mutant
180
m NF-
B/Sp1-CAT was obtained using oligonucleotides I, IV, and V and
the SmaI linearized
180 m NF-
B-CAT plasmid as a
substrate for PCR reaction. The mutated fragments obtained were
purified by polyacrylamide gel electrophoresis, eluted, digested with
HindIII/XhoI, and cloned into a pBL CAT3 vector.
All the PCR reactions above described were carried out using Pfu DNA
polymerase (Stratagene).
8387 human fibrosarcoma (11)
and Hep 3B (12) human hepatoma cells were grown to confluence,
collected after trypsinization, and cultured at a density of 8 × 105 (8387 cells) or 1 × 106 (Hep 3B) in
100-mm dishes 24 h before the transfection. Cells were transfected
by the calcium phosphate precipitation method (16) with 15 µg of CAT
reporter plasmid together with 2 µg of pSV- plasmid carrying the
-galactosidase gene under the control of the SV40 promoter (Promega
Corp., Madison, WI). Cells were left in contact with DNA for 12 (8387 cells) or 16 (Hep 3B) h. The medium was then replaced with fresh
medium. Cells were left to recover for 5 h and then stimulated
with human recombinant TNF
(500 U/ml, BASF/Knoll, Ludwigshafen am
Rhein, Germany) or with human recombinant IL-1
(100 ng/ml,
Dompé, L'Aquila, Italy) for 24 h. Human recombinant IL-6
(Immunex Corp., Seattle, WA) was used at 50 units of Cess/ml.
The cotransfection assays were performed by transfecting 8387 cells
with 15 µg of CAT containing constructs together with 0.5 µg of
pRSPA expression vector containing the cDNAs coding for NF-B p50
and p65 driven by Rous sarcoma virus promoter (17) (a kind gift of Dr.
Gary J. Nabel), either alone or in combination in a 1:1 ratio. After
transfection the cells were stimulated with TNF
(500 units/ml) or
left untreated.
Cells were harvested and lysed by three cycles of freezing and thawing
in 250 mM Tris buffer, pH 7.6. The transfection
efficiencies were measured by -galactosidase activity determination
in the same amount of cell lysate (usually 40 µg of proteins),
measured by an enzymatic assay with chlorophenol
red-
-D-galactopyranoside (Boehringer Mannheim GmbH,
Mannheim, Germany) as a substrate. Protein concentrations were
determined with the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA).
CAT reactions were carried out at 37 °C for 3 h in an 80-µl
reaction mixture containing 40 µg of cellular extracts, 0.5 µCi of
[14C]chloramphenicol (Amersham International, Little
Chalfont, UK), and 5 mM acetyl-CoA (Boehringer Mannheim).
The products were separated on a thin layer chromatography sheet, and
the percentage of conversion to the acetylated form of chloramphenicol
was quantified by scintillation counting. CAT activity values were
normalized to the -galactosidase activity.
A 382-base pair (bp)
HindIII-EcoRI fragment from the construct
74-CAT, comprising the nucleotides spanning from
74 to +54 (PvuII site) of the hPTX3 promoter, and a portion
of the CAT gene (up to the EcoRI site) were subcloned into
the pGEM-4 plasmid (Promega). The plasmid was linearized with
HindIII, and a 32P-labeled RNA probe was
generated with SP6 RNA polymerase and [32P]UTP according
to the manufacturer's instructions.
Six plates per plasmid were transfected with the same precipitation
mixture, and then three were treated with TNF (500 units/ml) for
4 h and three were left untreated. At the end of the incubation time, the cells were extracted in guanidinium isothiocyanate, and the
RNA was purified as described previously (18).
7.5 µg of total RNA or yeast tRNA control were hybridized with 2 × 105 cpm CAT riboprobe in 86% deionized formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES, pH
6.7. The hybridization was carried out for 18 h at 55 °C. The
hybridization mix was then diluted 10 times in a buffer containing 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.3 M NaCl, 0.5 unit/ml RNase A (Ambion, Austin, TX), and 100 units/ml RNase T1 (Ambion). Incubation was carried out for 45 min at
37 °C. In the same hybridization mixture, 5 × 105
cpm of a -actin riboprobe transcribed from the plasmid
pTRI
ACT-human (Ambion) by SP6 RNA polymerase were added.
After inactivation of the enzymes, the hybridization products were extracted in phenol-chloroform, precipitated, and loaded onto an urea/polyacrylamide 6% gel.
Electrophoretic Mobility Shift AssayNuclear extracts were
prepared from 8387 cells that were stimulated with TNF (500 units/ml) for 3 h or left untreated as described (19). The
oligonucleotides utilized (Duotech) correspond to the NF-
B site at
position
96 and its flanking sequences both in a wild type
(5
-AATTCAGGTTACC-3
) and a mutated form (5
-AATTCAGGTTACC-3
). 10 µg of nuclear
proteins were incubated with 50 pg of 32P-labeled
oligonucleotides and 1 µg of poly(dI·dC) (Pharmacia Biotech,
Uppsala, Sweden) in 15 µl of binding reaction buffer (40 mM Tris (pH 7.5), 120 mM KCl, 8% Ficoll, 4 mM EDTA, 1 mM dithiothreitol, and 10%
glycerol) for 20 min at room temperature. A 1000-fold molar excess of
cold oligonucleotide was used for competition assays. Competition
assays were performed using oligonucleotides carrying wild-type or
mutant NF-
B sites and with an oligonucleotide carrying a functional
NF-
B site from human immunodeficiency virus type I (HIV-I) long
terminal repeat (LTR) (5
-GATCCAGAGGAGAGGC-3
) (20) which was also used for a standard binding reaction as a positive
control. The resulting complexes were separated from the free probe by
electrophoresis in a 5% native polyacrylamide gel in 0.5%
Tris-buffered EDTA. In supershift analysis we used serum 1141 raised
against an amino-terminal peptide of human p50 (21), serum 1226 raised
against a carboxy-terminal peptide of human p65 (21), and serum 1136 raised against a carboxy-terminal peptide of human c-Rel (21); after 20 min of preincubation on ice, a standard binding assay was
performed.
To
analyze the promoter region of the hPTX3 gene, genomic DNA
sequences upstream the cDNA-encoding sequence were studied. An
EcoRI-PvuII fragment, which spans nucleotides
1317 to +54 (and does not include the ATG) (Fig.
1A), was subcloned for further investigation
in the pBL CAT 3 expression vector (13) and identified as
1317-CAT
(Fig. 1B). A series of deletion mutants obtained by
ExoIII/mung directional deletions was selected as shown in Fig. 1B.
The sequence of the 5-flanking region of the hPTX3 gene
(Fig. 1A) revealed features of an eukaryotic promoter, such
as the presence of a number of potential binding sites for
transcription factors. We have identified one NF-IL 6, two NF-
B, one
AP-1, two Pu.1, three PEA 3, one Ets-1, and two Sp1 consensus sequences (Fig. 1A). No obvious TATA or CAAT consensus box was found.
The previously identified transcription start site (1), however, corresponds to a pyrimidine-rich 7-nucleotide consensus (22) sequence
which has been reported to act in TATA-less promoters and is underlined
in Fig. 1A.
While this article was in preparation, the sequence of the
mPTX3 became available (23), and the alignment shows an
overall 50.1% conservation, with the last 380 nucleotides showing a
66% conservation (Fig. 1A). Furthermore, two of the
reported potential binding sites, the NF-B and the AP-1 sites at
positions
96 and
65, respectively, are maintained at
approximately the same positions in both sequences (Fig.
1A).
The deletion mutants
schematically shown in Fig. 1B were used for expression
studies in 8387 human fibrosarcoma cells and in Hep 3B human hepatoma
cells. After calcium phosphate-mediated transfection of the cells,
cultures were left untreated or were treated with TNF or IL-1
for
24 h. Data from at least four separate experiments are shown in
Fig. 2. The
1317-CAT construct shows a 5.3-fold basal
activity with respect to the empty vector, and a comparable level is
observed also with the
387 construct, implying that the 1000 intervening nucleotides do not contribute significantly to this basal
activity. In contrast, the
180 construct has a 2-fold higher basal
level, while further deletion up to
74 abolishes almost completely
the activity.
TNF exposure (gray bars) results in a 2.5-fold induction
with the
1317,
387, and
180 CAT constructs, but it is completely inactive on the
74 construct. IL-1
exposure (hatched
bars) induced a quite similar effect in the conditions tested. On
the contrary, IL-6 assayed on 8387 cells transfected with the
1317
construct was completely inactive and did not modify the responsiveness to TNF
(data not shown). In the same experimental setting, an artificial CAT reporter construct containing four tandem NF-
B sites
from IL-6 promoter (24) cloned into a pBL CAT 2 vector (13) gave a mean
fold induction of 4.5 ± 0.35 times on TNF
induction compared
with the untreated cells (data not shown).
To validate these results with a different approach, we analyzed the
8387-transfected cells by RNase protection. As shown in Fig.
3, lane 1, the undigested riboprobe
corresponds to the predicted size of 383 nucleotides. In the
transfected 8387 cells (lanes 4-13), the protected band is
309 bp in all cases, thus showing the use of the same transcriptional
start site in the artificial constructs as in the wild-type gene
(1).
While no precise measurement can be made for the baseline values of the
different constructs among them due to possible variation in
transfection efficiency, for each experimental group the untreated and
treated cells can be compared. TNF indeed increases the
transcription of the three responsive constructs by 3-6-fold (as
determined by densitometric scanning) (lanes 4-9), whereas
construct
74 shows no activity in both unstimulated and stimulated
cells (lanes 12 and 13). These data have been
reproduced in three separate experiments.
To study the cellular specificity of the TNF/IL-1
responsiveness,
we transfected the same constructs in the human hepatoma cell line Hep
3B; although PTX3 mRNA is inducible in these cells by
TNF
and IL-1
exposure (1), we could not observe, either in
unstimulated or in TNF
-stimulated cells, a significant CAT activity
with respect to the empty vector-transfected cells (data not shown)
despite a good transfection efficiency. On the other hand, the
artificial CAT reporter construct containing four tandem NF-
B sites
(see above) showed, under the same experimental conditions, a full
responsiveness to TNF
(>15-fold; data not shown). All these data
together imply that further genomic elements or posttranslational modifications may crucially contribute to the hepatic transcription of
PTX3 detectable in vitro.
The sharp
difference in the activity between 180 and
74, the presence of an
NF-
B element at position
96 (Fig. 1A), and the known
effect of NF-
B in TNF
- and IL-1
-mediated responses prompted us
to analyze the involvement of NF-
B in PTX3 regulation by
electrophoretic mobility shift assay. An oligonucleotide corresponding to the sequence from position
103 to
81 of hPTX3 was utilized. A
low level of binding activity was detectable in untreated 8387 cells as
two separate bands (Fig. 4A, lane 1), and
they were clearly increased after a 3-h TNF
stimulation (lane
2). The specificity of the binding activity is documented by the
complete competition with the cold specific oligonucleotides
(lane 3) and with an oligonucleotide containing a canonical
NF-
B site from HIV-1 LTR (lane 5) (20).
We also generated a mutant hPTX3 NF-B oligonucleotide which did not
contain a NF-
B-binding site. This mutated oligonucleotide was unable
to compete for the binding of the NF-
B proteins to the wild-type
sequence (lane 4) and, on the other hand, when used as a probe, did not
show binding activity in either untreated (lane 6) and
TNF
-treated (lane 7) 8387 cells.
Supershifting with antibodies clearly indicated that the two bands correspond to the p50/p65 heterodimer and to the p50/p50 homodimer, respectively (Fig. 4B, lanes 3 and 4). Furthermore, c-Rel is not present in this complex (lane 5) as demonstrated by the lack of supershifting, similar to what is observed with an irrelevant antibody (lane 6).
We further compared under the same experimental conditions the binding
activity of 8387 nuclear extracts on an oligonucleotide containing the
PTX3 NF-B site and on an oligonucleotide containing a
canonical NF-
B-binding site from HIV-1 LTR, which has been previously reported to give rise to only one retarded complex (20). As
shown in Fig. 4C, while the binding on the PTX3
oligonucleotide gave rise to two retarded complexes, corresponding to
p50/p50 and p50/p65 homo- and heterodimers, only the upper band was
present in the binding to the HIV-1 LTR NF-
B site.
To more directly assess the functional relevance of the
NF-B site, we mutagenized this site in the
180 construct, (
180 m
NF-
B-CAT). The mutated sequence is identical to the degenerated oligonucleotide that we had used in the gel retardation experiments (Fig. 4A, lanes 6 and 7). When the
180 m NF-
B-CAT construct was
analyzed by CAT analysis and RNase protection, it was evident that
despite a detectable level of basal activity, cytokines exposure did
not lead to any significant induction of its transcription (Fig. 2,
180 m NF-
B-CAT, and Fig. 3, lanes 10 and 11), thus implying an
NF-
B mediated induction of the transcriptional activity of the hPTX3
promoter by TNF
and IL-1
.
To validate the hypothesis that NF-B is the key responsive element,
we also mutagenized the Sp 1 and AP-1 sites which are present in the
minimal promoter (as shown in Fig. 1b). The
180 m Sp 1-CAT was indeed
still responsive (Fig. 2), although at a lower level compared with the
wild type
180-CAT construct (1.7 mean fold induction over four
separate experiments upon TNF
induction). As expected, also the
double mutant
180 m NF-
B/Sp 1-CAT is not responsive to TNF
and
IL-1
stimulation, although it retains a basal activity. On the other
hand, the AP-1 mutant
180 m AP-1-CAT shows full cytokine
responsiveness (3.8 and 2.7 mean fold induction with TNF
and
IL-1
, respectively), but a much lower basal level of CAT expression
(Fig. 2).
To quantify the observed stimulations with respect to canonical NF-B
sites, we made use in the same experimental setting, of an artificial
CAT reporter construct containing four tandem NF-
B sites derived
from IL-6 promoter (24) and cloned in a pBL CAT 2 vector (13). This
reporter gave a mean fold induction of 4.5 ± 0.35 upon TNF
stimulation relatively to untreated cells (data not shown), therefore
of comparable entity to those observed with the PTX3 constructs.
To further
substantiate that the NF-B site is indeed the main functional
responsive element, we cotransfected 8387 cells with the
180-CAT and
with the
180 m NF-
B-CAT constructs as reporters together with p50
and p65 NF-
B expression vectors (17) either alone or in combination.
As shown in Fig. 5, top, cotransfection of p50 alone did
not modify the basal CAT activity by the construct in the wild type
configuration, as compared with cells transfected with the empty vector
pRSPA, while p65 and the combination of the two increased the basal
activity by a factor of 3.6 (p50/p65) to 3.9 (p65 alone). The addition
of TNF
was effective in all the experimental conditions tested (3, 2.6, 1.7, and 1.7 fold induction over the unstimulated cells in pRSPA,
p50, p65, and p50/p65 transfected cells respectively). On the other
hand, overexpression of p65 alone, or in combination with p50, as well
as addition of TNF
had no effect on the
180 m NF-
B-CAT
construct carrying the mutation in the
96 NF-
B site (Fig. 5,
bottom). These data are consistent with the hypothesis that the hPTX3
natural promoter contains a functional NF-
B site.
Under the same experimental conditions, the control reporter plasmid
containing four NF-B binding sites from the IL-6 promoter was
induced by p50/p65 overexpression by 5.9-fold, while no induction was
detectable against a pSV2 CAT reporter plasmid utilized as a negative
control (not containing a NF-
B element) when cotransfected with p50
and p65 in combination (data not shown).
In this report, we characterize the promoter of the human
PTX3 gene (hPTX3). A genomic fragment of 1317 bp,
located 5 to the transcriptional start site, responds to TNF
and
IL-1
stimulation in transiently transfected human 8387 fibroblasts
but not in human hepatoma Hep 3B cells, as measured by transfection and
CAT assays (more than 2-fold induction) and by RNase protection
analysis (3-6-fold induction). Deletion mutants show that the 180 bp
more proximal to the start site are sufficient for TNF
- and
IL-1
-inducible transcriptional activity. On the contrary, the last
74 bp are unresponsive. In the intervening 106 bp we show that a
classical NF-
B binding site is present and furthermore that p50/p50
homodimers and p50/p65 heterodimers can bind to this element after
incubation with nuclear extracts from 8387 fibroblasts. TNF
exposure
increases this NF-
B activity, while the minimal construct carrying
an inactivating mutation of this site loses the TNF
inducibility in
the same cells. Finally, we confirmed the hypothesis that NF-
B
proteins are functionally active on the hPTX3 promoter by
cotransfection with p50 and p65 NF-
B expression vectors. On the
contrary, Sp1 and AP-1 do not seem to play a major role for the
cytokine inducibility of the gene.
These data show for the first time that a classical NF-B complex can
functionally interact with the "long pentraxin" hPTX3 promoter in human fibroblasts after exposure to TNF
. The different methods utilized indicate a 2-5-fold transcriptional induction of the
gene, which is in agreement with the observed increase in nuclear
runoff experiments on isolated monocytes (10).
PTX3 belongs structurally to the family of the classical
acute phase protein pentraxins, which include CRP and SAP, in several animal species. Both genes are characteristically induced by IL-6 in
combination with IL-1 and TNF mainly, if not exclusively, in hepatocytes (25-31). PTX3 was the first cloned member of
the newly emerging group of "long pentraxins," (5-9) because they
show a long amino-terminal domain fused to the carboxy-terminal
pentraxin domain (corresponding to most of the classical pentraxin
sequence). The significance of this genetic acquisition is far from
being understood, but all the long pentraxins do not show a
liver-restricted expression pattern and seem to be expressed in a much
wider spectrum of organs, such as the brain and the testis (6-9).
Furthermore, hPTX3 was shown to be transcribed after exposure to
IL-1, TNF
, and the bacterial product LPS (i.e. all
prototypical proinflammatory signals) but not by IL-6, in several
different cell types, including endothelial cells, fibroblasts,
hepatocytes, and monocytes (1, 10). Furthermore, PTX3
expression induced by IL-1
is not modified in endothelial cells and
hepatocytes by concomitant exposure to IL-6 (data not shown). The same
gene was cloned in TNF
-stimulated fibroblasts, named
TSG-14 (2), and demonstrated to be directly induced by TNF
(32, 33).
Inducibility by IL-1 and TNF
, but not by IL-6, may correlate well
with the demonstrated role of NF-
B (for review, see Refs. 34-36)
and, furthermore, with the presence of only one NF-IL 6 binding site
(37, 38) and with the absence of APRF elements (39-41) in the human
promoter. Both elements, in fact, have been demonstrated to be
necessary in multiple copies for IL-6 inducibility (42).
The murine gene (82% identical at the amino acid level) shows a similar exon/intron organization and is localized on a syntenic chromosomal region (4). It is induced in vitro only in peritoneal macrophages, in some fibroblasts, and in very few endothelial cell lines, but not in hepatocytes; on the other hand, it was induced in vivo in several muscular tissues after LPS i.v. injection (an acute phase experimental model), but not in the liver (4, 32). In addition, in situ hybridization studies have indicated that in the heart and in the thigh, the endothelial cells were the most abundant producer cell type (4).
The alignment between the human and the murine promoters shows a high
overall degree of conservation, including few hypothetical binding
sites for transcription factors, in particular the NF-B site which
is here demonstrated as functionally important for the hPTX3
gene. What are the structural reasons for the differences in the
expression of PTX3 between humans and mice is still unclear.
The reported lack of consensus sites for hepatic transcription factors
in the murine promoter (and in the human promoter as well) may account
in part for the absence of induction in the liver (another obvious
difference with the classical CRP and SAP genes (3), but the positive
elements required for its inducibility in the endothelial cells of the
muscular district have yet to be elucidated. On the other hand, recent
work with transgenic animals has shown, in the case of CRP, that the
precise characterization of the functional elements required for the
in vivo "acute phase" inducibility may require a complex
interaction between 5 and 3
elements (43), which was unexpected on
the basis of previous in vitro studies (27-29, 31).
We have described the functional role of the NF-B site in the
promoter of the hPTX3 gene for TNF
inducibility in
fibroblasts. We can only speculate at the moment, on the basis of the
large amount of published data, that this same site may be relevant also for LPS inducibility of the gene in fibroblasts as well as in
other cell types. NF-
B may interact with other factors as suggested
by others (44-48), particularly in view of the presence of AP-1 and
Sp1 sites in close proximity to the NF-
B site, and of the fact that
they both have been reported to interact functionally with NF-
B
complexes (44, 48). Indeed, the Sp1 mutant shows a reduced TNF
and
IL-1
inducibility, while the AP-1 mutant is fully responsive to
cytokines, although its basal level of expression is significantly
reduced. Further work will be required to directly address the possible
interplay of different transcription complexes on the hPTX3
promoter.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X97748[GenBank].