(Received for publication, July 5, 1995)
From the
Human TNF-stimulated gene 14 (TSG-14) encodes a secreted 42-kDa
glycoprotein that shows significant homology to proteins of the
pentraxin family, which includes the acute phase reactants C-reactive
protein and serum amyloid P component. Levels of TSG-14 protein (also
termed PTX-3) become elevated in the serum of mice and humans after
injection with bacterial lipopolysaccharide, but in contrast to
conventional acute phase proteins, the bulk of TSG-14 synthesis in the
intact organism occurs outside the liver. In the present study we
cloned and partially sequenced murine genomic TSG-14 DNA. Analysis of
the coding region predicts a high degree of amino acid sequence
homology between murine and human TSG-14 (88 and 75% identity in the
first and second exons, respectively). The promoter of the TSG-14 gene
lacks consensus sequences for either a TATA box or CCAAT box. Primer
extension analysis and S1 nuclease protection assay revealed one major
transcription start site, situated within a consensus sequence for an
initiator element. Sequence analysis of a 1.4-kilobase pair
fragment of the 5`-flanking region of the TSG-14 gene revealed the
presence of numerous potential enhancer binding elements, including six
NF-IL6-like sites, four AP-1, one AP-2, one NF-
B, two Sp1, two
interferon-
-activated sites (GAS), one Hox-1.3, and five binding
sites for Ets family members. Transfection of BALB/c 3T3 cells with
promoter DNA fragments linked to the luciferase reporter gene revealed
that the 5`-flanking region of the TSG-14 gene comprises elements that
can mediate a basal level of transcription and inducibility by TNF.
TNF-stimulated gene 14 (TSG-14) ()was originally
isolated by differential hybridization of a cDNA library from
TNF-treated normal human FS-4 foreskin fibroblasts(1) . TSG-14
cDNA encodes a secreted glycoprotein of 42 kDa(2) . An
essentially identical cDNA termed PTX-3 was cloned by Breviario et
al.(3) from IL-1-stimulated human vascular endothelial
cells. In its C-terminal half TSG-14 protein shows significant sequence
homology (20-30%) with members of the pentraxin family of
proteins(2, 3) . The pentraxin proteins exhibit a
characteristic discoid arrangement of five noncovalently bound subunits
and share an eight-residue amino acid homology domain (``pentraxin
signature''). The pentraxin family comprises two major acute phase
proteins, the serum amyloid P component (SAP) and C-reactive protein
(CRP) with a molecular mass of approximately 25 kDa (4, 5) and four larger proteins with molecular mass
around 40-50 kDa:
TSG-14/PTX3(1, 2, 3, 6) , Xenopus laevis protein (XL-PXN1)(7) , an acrosomal
protein (apexin) from guinea pig sperm(8) , and a neuronal
pentraxin protein(9) . A remarkable feature of the smaller
pentraxins is their high degree of conservation throughout evolution
since they have been described in species as diverse as man, mouse,
guinea pig, rat, rabbit, marine teleosts, and Limulus
polyphemus, an
invertebrate(10, 11, 12, 13) .
While apexin, XL-PXN1, and neuronal pentraxin have only recently been identified, SAP and CRP were described about 50 years ago and have been extensively studied. SAP and CRP are acute phase proteins whose serum concentrations increase dramatically in response to trauma or inflammation(4, 5) . Human and murine CRP and SAP genomic DNAs have a similar organization and show significant sequence homology(14, 15, 16, 17) . However, CRP and SAP show some species specific differences in their regulation. CRP is highly inducible in humans but not in mice during inflammation, whereas SAP is the major acute phase protein in mice, but its concentration stays relatively constant in human serum(4) . In order to elucidate the activation of these genes, their promoters have been analyzed structurally and functionally. Several studies have identified sequences in the 5`-flanking regions of the CRP and SAP genes that can mediate their inducibility by the inflammatory cytokines IL-1, TNF, and IL-6. Human and murine CRP genes were shown to contain in their 5`-flanking regions a TATA box along with binding sites for transcription factors of the C/EBP (NF-IL6) family that, along with the so-called acute phase response factor/Stat3, are thought to mediate cytokine inducibility(16, 18, 19, 20, 21, 22, 23) . The presence of specific binding sites for hepatocyte nuclear factor 1 in the CRP and SAP promoters was implicated in the hepatocyte-specific expression of these genes(19, 22) .
The fact that
IL-1 and TNF, two major mediators of inflammation, can induce
transcriptional activation of the TSG-14 gene, as demonstrated by
nuclear run-on analysis(2) , and that TSG-14 is structurally
related to two major acute phase proteins suggested that TSG-14 plays a
role in the inflammatory response. Indeed, the appearance of TSG-14
protein in the serum was induced in mice after LPS
injection(6) . In addition, the concentration of TSG-14 was
increased in the serum of human volunteers injected with LPS and in the
sera of patients with bacterial septicemia (6) . ()In both the human volunteers and in mice, immunoblot
analysis of serum samples showed no detectable TSG-14 protein before
LPS stimulation, followed by the appearance of a prominent 42-kDa
TSG-14 band, with peak levels detected at 4-6 h after injection
with LPS. However, when attempts were made to identify the cellular
source of TSG-14 in LPS-injected mice, no TSG-14 mRNA was detected in
the liver and several other organs (spleen, kidney, and
lung)(6) . Alles et al.(24) recently
demonstrated that TSG-14/PTX-3 is abundantly synthesized in cells of
the human monocyte-macrophage lineage. Thus, TSG-14 is different from
CRP, SAP, and other classical acute phase proteins produced by
hepatocytes in that it is produced mainly by fibroblasts, endothelial
cells, and monocytes.
Although human TSG-14/PTX-3 genomic DNA has
been isolated and shown to contain three exons(3) , no
information on the promoter region was available. In order to
investigate TSG-14 gene regulation and its tissue-specific expression,
we decided to clone the murine TSG-14 genomic DNA and to analyze its
5`-flanking region. In this study, we describe the cloning and
sequencing of approximately 1.4 kb of the TSG-14 promoter, of the first
exon, the first intron, the second exon, and part of the second intron.
With the aid of a murine cell line that expresses TSG-14 after
treatment with IL-1 or TNF, we determined the transcriptional start
site of TSG-14 mRNA. Initial information obtained by transient
transfection analysis of the TSG-14 promoter suggests a complex
mechanism of regulation of gene expression. An interesting feature of
the murine TSG-14 promoter is the presence of six potential NF-IL6
elements along with potential AP-1 and NF-B binding sites. The
absence of elements associated with the hepatocyte-specific expression
of CRP and SAP is consistent with the lack of a significant level of
TSG-14 gene expression in the mouse liver.
Figure 1: Organization and nucleotide sequence of the murine TSG-14 gene. A, partial restriction map of the 2708-bp BamHI fragment spanning the 5`-flanking region, exon 1, intron 1, exon 2, and part of intron 2. The presence of conserved residues GT and AG marking intron/exon boundaries is indicated. B, nucleotide sequence of the 2708-bp fragment. The transcription start site (as determined in subsequent experiments) is designated +1. The coding sequences for exon 1 and exon 2 are boxed. Consensus sequences for known transcription factors are boxed, with abbreviations for the corresponding transcription factors shown above the sequences (see also Table 1).
Figure 2: Alignment of the predicted murine and human TSG-14 amino acid sequences encoded by exons 1 and 2. Conserved residues are boxed. Conservative substitutions are indicated by asterisks placed above the residue in the murine sequence. The human TSG-14 sequence is from (2) .
In the
promoter region of TSG-14, we were unable to find consensus sequences
for either a TATA-box or a CCAAT-box. However, we have determined the
presence of several consensus binding sequences for known transcription
factors. Among these sequences, we have identified six NF-IL6-like
recognition sequences, four AP-1, one AP-2, one NF-B, two Sp1, two
GAS, one Hox-1.3 site, and five consensus sequences for the binding of
different members of the Ets family of transcription factors (Fig. 1B, Table 1). Another intriguing element
found in the TSG-14 promoter is an alternating CA residues stretch of
44 located between nucleotides -645 and -601. A similar
sequence has been found in several other genes such as the 5`-flanking
region of the iNOS gene (26) and the Scya5 gene (27) .
It has been suggested that such ``TG element'' sequence may
potentially have a Z-DNA conformation and serve as an enhancer element (28) .
Figure 3: Analysis of TSG-14 mRNA expression in different murine cell lines. Cells were treated for 4 h with TNF (50 ng/ml) or IL-1 (2 ng/ml) or left untreated. RNA was isolated, separated, and immobilized on nylon membranes as described under ``Experimental Procedures.'' The blot was first hybridized with a NcoI/HindIII fragment containing the second exon of the murine TSG-14 gene (TSG-14) and then stripped and rehybridized with a probe for pHe7 cDNA hybridizing with a housekeeping mRNA (pHe7). A, lanes 1 and 2, mouse embryonic fibroblasts; lanes 3 and 4, BALB/c 3T3 cells; lanes 5 and 6, J774 cells; lanes 7 and 8, 63 cells; lanes 9 and 10, J744E cells; lanes 1, 3, 5, 7, and 9, untreated cells; lanes 2, 4, 6, 8, and 10, TNF-treated cells. B, lane 1, mouse embryonic fibroblasts; lane 2, BALB/c 3T3 cells; lane 3, J774 cells; lane 4, 63 cells; lane 5, J744E cells. All samples in panel B are from IL-1-treated cells.
Figure 4:
Determination of the transcription start
site of the TSG-14 gene by S1 nuclease mapping and primer extension. A, S1 nuclease mapping. Poly(A) mRNA isolated
from untreated BALB/c 3T3 cells (CTRL) or from cells treated
with TNF for 4 h (TNF) was annealed to a 90-mer
oligonucleotide spanning nucleotides -14 to +76 of the
TSG-14 gene. The lane marked PROBE was loaded with the
labeled 90-mer oligonucleotide alone. The pGEM plasmid digested by AluI was used in the lane labeled MARKERS. The arrow points to the band obtained by S1 mapping. B,
primer extension. Poly(A)
mRNA from control (CTRL) or TNF-treated BALB/c 3T3 cells (TNF) was
hybridized to a 21-mer oligonucleotide corresponding to positions
+122 to +143. Sequencing with this oligonucleotide was used
for calibration. The start site of transcription is marked by an arrow, and the corresponding nucleotide is indicated by an asterisk.
Figure 5:
Transcriptional activity of the TSG-14
promoter and its deletion constructs linked to the luciferase reporter
gene upon transfection into BALB/c 3T3 cells. A, the deletion
constructs shown were used for transfection of BALB/c 3T3 cells. After
transfection cells were either left untreated or treated with TNF (100
ng/ml) for 24 h. The relative luciferase activity was determined by
normalization to the -galactosidase activity. Fold induction represents the ratio of relative luciferase activity in
TNF-treated over control cells. Each value is the mean and standard
deviation from six independent experiments. p values were
determined by the t test for paired samples. B,
actual results representative of the experiments summarized in panel A. Means and standard deviations of three independent
experiments are shown. Base-line relative luciferase activity (i.e. value obtained after transfection of cells with
``empty'' luciferase vector) was approximately 3000 and did
not increase after TNF treatment.
Human TSG-14 cDNA encodes a 42-kDa glycoprotein with
significant sequence homology to the acute proteins CRP and SAP and
other members of the pentraxin family(2) . The fact that TSG-14
is inducible by the proinflammatory cytokines TNF and IL-1, together
with the demonstration that TSG-14 protein levels are elevated in the
serum of mice and humans after LPS injection(6) , strongly
suggests that TSG-14 plays a role in inflammation. To date no
information regarding TSG-14 gene regulation by TNF and IL-1 has been
available. Therefore, we cloned and sequenced a 2.7-kb fragment of the
murine genomic TSG-14 DNA including 1.4 kb of its 5`-flanking
region. A comparison of the intron-exon structure of the isolated
portion of the murine gene with the earlier published organization of
the human TSG-14/PTX-3 gene (3) revealed a close similarity. In
addition, a high degree of homology was found between the coding
sequences of the murine DNA and the earlier determined human TSG-14
cDNA sequence(1, 2, 3) . At the predicted
amino acid level there is 88% identity (93% homology, if conservative
substitutions are included) and 75% identity (81% homology) between
sequences encoded by murine and human exons 1 and 2, respectively.
Hence, as is also true for other
pentraxins(10, 11, 12, 13) , TSG-14
is highly conserved during evolution, suggesting structural constraints
imposed by protein function.
Analysis of the 5`-flanking sequence of
the murine TSG-14 gene revealed no obvious consensus sequence for a
TATA- or a CCAAT-box. However a sequence that matches five out of seven
residues of the consensus sequence for initiator elements (29, 30) defines the transcriptional start site in the
TSG-14 promoter. Furthermore, sequence analysis revealed the presence
of several potential cis-acting elements in the 5`-flanking
region of the TSG-14 gene, including six NF-IL6-like sequences, four
AP-1 sites, one AP-2, one NF-B, two Sp1, and two GAS (Fig. 1B, Table 1). Studies on the regulation of
other TNF- or IL-1-inducible genes revealed that NF-
B, NF-IL6
(C/EBP-family), and AP-1 transcription factors (and cooperative
interactions among these factors) are often crucial in their regulation (31, 32, 33, 34, 35) . The
presence of multiple potential NF-IL6 binding sites in the TSG-14
promoter is of particular interest in view of the central role played
by NF-IL6 in the acute phase response and inflammation (36, 37, 38) . Whether the GAS elements are
likely to be functional is uncertain because the human TSG-14 gene is
not inducible by interferon-
, nor did interferons affect TSG-14
gene expression induced by other cytokines(1) . However,
variants of the GAS element serve as binding sites for various Stat
family proteins, important in the signaling from a variety of cytokine
and growth factor receptors (39) . Another potential binding
site identified in the murine TSG-14 promoter is the Hox-1.3 consensus
element. The homeobox gene-encoded Hox-1.3 protein is expressed mainly
during embryogenesis but also in several adult tissues, and its
expression in fibroblasts seems to correlate with cell
growth(40) . Several consensus binding sequences for
transcription factors of the Ets family have also been identified in
the TSG-14 promoter. Similar sequences have been found in a number of
cytokine genes, including the TNF-
promoter in which it is thought
to play a crucial role(41) . Similarly, the binding of the Pu.1
transcription factor in the IL-1 promoter appears to be important for
the cell-specific expression of IL-1(42) . Another interesting
element in the murine TSG-14 promoter is the 44-bp stretch of
alternating CA residues. Enhancing functions have been attributed to
this element (28) . Intriguingly enough, the same type of
sequence has been found in the intron of the human, but not the mouse,
CRP gene, and it has been hypothesized that this site may be involved
in the differential expression of CRP in human and mouse
cells(43) . While human and mouse CRP and SAP genes contain in
their promoter region at least one consensus sequence for the
liver-specific transcription factor hepatocyte nuclear factor 1, no
such sequence was found in the TSG-14 promoter. This result is in
agreement with the finding that, in contrast to CRP and SAP, TSG-14 is
not produced by the liver(6, 24) .
Initial analysis
of promoter function of the TSG-14 gene by transfection into BALB/c 3T3
cells of the 5`-flanking region linked to the luciferase gene showed
that the 1.4-kb DNA fragment mediates a relatively high level of
constitutive transcriptional activity along with some inducibility by
TNF. Deletion analysis suggests that removal of sequences upstream of
position -436 resulted in a somewhat diminished inducibility by
TNF (Fig. 5B). Even more pronounced than the decrease
in inducibility by TNF was a concurrent increase in constitutive
transcriptional activity, suggesting the presence of upstream negative
regulatory sequences. Analysis of other promoters showed that NF-IL6
sites can function both as positive and negative regulatory
elements(44) . It is conceivable that some of the multiple
potential NF-IL6 binding sites in the murine TSG-14 promoter will also
turn out to act as negative elements. Additional investigations by
deletion analysis and site-directed mutagenesis will be required to
define more precisely the cis-acting elements and trans-acting factors important in the induced expression of
the murine TSG-14 gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33842[GenBank].