(Received for publication, September 14, 1995; and in revised form, November 29, 1995)
From the
To define the cis-acting elements involved in the regulation of
the murine vitronectin (Vn) gene in inflammation, the 5`-flanking
region was isolated, fused to the luciferase reporter gene, and the
basal and interleukin 6 (IL-6)-stimulated transcriptional activity was
tested in transfection experiments using Hep3B cells. Treatment with
IL-6 induced this construct by more than 20-fold, whereas the
corresponding 5`-flanking region of the human Vn gene was not
stimulated. Transfection studies using murine Vn constructs with serial
5`-deletions revealed that two sequences were important in the IL-6
response, and specific mutations in both sequences abolished the
response. A 2-base pair mutation converted the human sequence to that
of a murine IL-6 responsive element and partially conveyed IL-6
inducibility. In contrast, transforming growth factor stimulated
the human construct and the endogenous Vn gene in human Hep3B cells in
a dose-dependent manner, whereas the murine construct was not
responsive. The transforming growth factor
responsive region was
localized to a 30-base pair fragment with little homology to the murine
sequence. These studies reveal that the structural basis for the
differential regulation of the human and murine Vn genes resides in the
differences in promoter sequence.
Vitronectin (Vn) ()is not only a member of a group of
cell adhesion molecules that mediate adhesion through a common
Arg-Gly-Asp containing sequence but also appears to regulate
proteolytic enzyme cascades, including the complement, coagulation, and
fibrinolytic systems(1, 2, 3) . Thus, Vn may
provide a unique regulatory link between cell adhesion, humoral defense
mechanisms, and cell invasion(1) .
Although Vn biosynthesis
was originally only detected in hepatocytes (4) , recent
studies provide evidence that low but significant levels are also
expressed in normal and diseased extra-hepatic
tissues(5, 6, 7) . Vitronectin biosynthesis
also appears to be regulated. Transforming growth factor
(TGF
) modulates Vn gene expression in the human hepatocarcinoma
cell line HepG2(8) , and rodent Vn genes are regulated by
lipopolysaccharide during acute systemic
inflammation(7, 9) . Endotoxin induces a number of
cytokines in vivo, including TNF
, IL-1, and
IL-6(10, 11, 12) . Interestingly, Vn gene
expression in the rat liver was induced within 1 h after
intraperitoneal injection of purified rat IL-6(9) , suggesting
that rodent Vn genes are directly responsive to IL-6. These latter
observations identify Vn as an acute phase reactant.
Little is known about the Vn promoters and their regulation. The immediate 5`-flanking region of the human Vn gene has been isolated, and the 5`-end of the mRNA was determined in primer extension experiments(13) . However, this fragment lacked a TATA box consensus sequence at the expected distance from the cap site, and the transcriptional activity of this fragment was not tested(13) . Thus, it is unclear whether this sequence contained a functional promoter.
In view of
these recent results, a detailed comparative analysis of the Vn
promoter of the human and murine genes was performed. IL-6 stimulated
the transcriptional activity of the murine but not of the human
promoter, while TGF specifically up-regulated the human but not
the murine promoter. Evidence is presented that the two promoters
contain different cis-acting elements that account for these
differences in regulation.
Figure 2:
Regulation of the endogenous Vn gene in
Hep3B cells by IL-6 and TGF. Confluent Hep3B cells were
serum-starved for 24 h and incubated with the indicated concentration
of TGF
or IL-6 (500 units/ml) for an additional 24 h. The
accumulation of Vn in the conditioned medium was determined by a
sandwich enzyme-linked immunosorbent assay (see ``Materials and
Methods''). Inset, Hep3B cells were serum-starved for 24
h and metabolically labeled with [
S]methionine
for an additional 24 h in serum-free medium (lane 1) or in
medium containing either IL-6 (500 units/ml, lane 2) or
TGF
(1 ng/ml, lane 3). The radiolabeled Vn was collected
by immunoprecipitation using monoclonal antibody 1244 raised against
human Vn and protein A-Sepharose and analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography.
In the present study, the immediate 5`-flanking region of the murine Vn gene was isolated and sequenced (not shown). The major cap sites were determined by primer extension analysis and were located at two adenosine residues 84 and 95 nucleotides upstream of the methionine initiation codon (not shown). The adenosine representing the most distal transcription start site (i.e. 95 nucleotides upstream of the initiation codon) was designated as +1. The sequence data indicated that the murine Vn gene resembles the majority of eukaryotic genes in that a ``TATA box'' (position -30 to -24) is located at the conserved distance from the cap site(21) .
To establish the transcriptional activity of the
5`-flanking region of the murine Vn gene, a 575-base pair fragment
(-528/47) was amplified by PCR and cloned upstream of the
luciferase reporter gene. The sequence of the murine 5`-flanking region
also was aligned with that of the human sequence, and the corresponding
human DNA fragment (1079/1652; numbering according to (13) )
was PCR amplified from Hep3B genomic DNA, cloned directly in front of
the luciferase reporter gene, and sequenced (compare Fig. 6).
The transcriptional activities of the resulting constructs were
compared to the parental reporter gene construct (p19LUC; (16) ) after transfection into human Hep3B hepatoma cells. Cell
lysates were prepared 48 h after transfection and analyzed for
luciferase activity. The -galactosidase gene, driven by the
cytomegalovirus promoter, was cotransfected with the luciferase
reporter gene constructs to correct for differences in DNA uptake. The
promoterless reporter gene plasmid (p19LUC) produced barely detectable
luciferase activity, whereas both Vn promoter constructs exhibited
promoter activity approximately 300-fold above background in Hep3B
cells (not shown). These results indicate that the immediate
5`-flanking regions of both genes contain a functional, active
promoter. The transcriptional activity of these Vn reporter gene
constructs agrees with the observation that the endogenous human Vn
gene is expressed by this cell line(19) .
Figure 6:
Schematic alignment of the murine and
human Vn promoters and 5`-flanking regions. The solid boxes in
the upper panel (murine Vn) represent the two IL-6 responsive
elements, whereas the solid box in the lower panel (human Vn) represents the TGF responsive element. The arrows indicate the direction of transcriptional initiation.
The 5` and 3` boundaries of the full-length promoter constructs are
indicated. The numbering of the murine construct is based on the
experimentally determined transcriptional start sites, and the
adenosine representing the most distal transcription start site was
designated as +1. The numbering of the human construct is based on
the published sequence(13) .
Experiments were
performed to characterize the effects of a number of cytokines and
growth factors on the transcriptional activity of the Vn genes. 1 day
after transfection, the cells were incubated for 24 h in the presence
of the indicated agents, and cell lysates were prepared and analyzed
for luciferase activity. The results were compared to those for the
Rous sarcoma virus long terminal repeat promoter (Table 2). The
transcriptional activity of the murine Vn promoter was strongly
up-regulated by IL-6, supporting the notion that the murine Vn gene is
regulated as an acute phase reactant (Table 2). Both the basal
and the IL-6-induced transcriptional activities of the murine Vn
promoter were reduced by IL-1 (Table 2) and TNF (not shown),
and this down-regulation was dose-dependent with respect to the
concentration of IL-1 and TNF
employed (not shown). Surprisingly,
the transcriptional activity of the human Vn promoter was not
stimulated by IL-6 (Table 2). In contrast, the human Vn promoter
was up-regulated by TGF
, whereas this growth factor was
ineffective on the murine Vn promoter (Table 2). Also, IL-1 (Table 2) and TNF
(not shown) did not suppress the
stimulatory effect of TGF
on the human Vn promoter. The results
for the Vn promoters were compared to that for the human type 1
plasminogen activator inhibitor gene, a typical TGF
-responsive and
acute phase gene(22) . A DNA fragment of the human type 1
plasminogen activator inhibitor gene (-800/75) was strongly
stimulated (150-fold) by TGF
(1 ng/ml) as previously reported,
whereas the response to IL-6 (500 units/ml; 2-fold induction) and a
combination of IL-6 and dexamethasone (500 units/ml IL-6,
10
M dexamethasone; 6-fold induction) was
rather limited in comparison to the murine Vn gene.
The differential
regulation of the Vn promoters by IL-6 and TGF was compared in
dose-response experiments (Fig. 1). The transcriptional activity
of the murine Vn promoter was up-regulated by IL-6 in a dose-dependent
manner, with half-maximal stimulation at approximately 250 units/ml (Fig. 1A). In contrast, IL-6 has only a minimal effect
on the human Vn promoter (Fig. 1A) or the Rous sarcoma
virus promoter (not shown). In contrast, TGF
stimulated the
transcriptional activity of the human Vn promoter in a dose-dependent
manner, with half-maximal stimulation at approximately 0.3 ng/ml (Fig. 1B). The murine Vn promoter was not stimulated by
TGF
concentrations up to 10 ng/ml (Fig. 1B).
Figure 1:
Regulation of the Vn promoters by IL-6
and TGF. Constructs containing either the murine Vn
(-528/47, open circles) or the human Vn (1079/1652, closed circles) promoters were transfected into Hep3B cells.
The cells were treated with the indicated concentration of IL-6 (panel A) or TGF
(panel B) for 24 h and assayed
for luciferase activity (see ``Materials and Methods''). The
-fold induction is plotted on the y axis (linear scale)
against the concentration of cytokine or growth factor on the x axis (linear scale).
These results raised the possibility that either the human and
murine endogenous Vn genes are differentially regulated or important
regulatory elements (e.g. more distal DNA fragments, intron or
exon sequences) that were not contained within the reporter gene
constructs are required for the regulation. To discriminate between
these two possibilities, the regulation of the endogenous human Vn gene
was studied in human hepatoma cells. TGF stimulated the
accumulation of Vn in the conditioned media of Hep3B cells in a
dose-dependent manner, whereas IL-6 (500 units/ml) had no effect (Fig. 2). The same was true using IL-6 concentrations up to
5,000 units/ml (not shown). Similar results were obtained when
radiolabeled Vn was immunoprecipitated using a monoclonal antibody
specific for human Vn (Fig. 2, inset). Quantification
of the radioactivity of the Vn containing bands revealed that IL-6
reduced Vn biosynthesis by approximately 25%, whereas TGF
stimulated the accumulation of Vn in the conditioned medium
approximately 3-fold (not shown). In contrast, we were unable to
immunoprecipitate radiolabeled Vn from the extracellular matrix (not
shown), indicating that the majority of the synthesized Vn accumulated
in the conditioned medium. The metabolic activity of the
TGF
-stimulated wells was determined by trichloroacetic acid
precipitation of radiolabeled proteins and was not significantly (i.e. less than 30% variation) different from that of
untreated or IL-6-treated cells (not shown). It should be noted that
TGF
, but not IL-6, stimulated the accumulation of Vn in the
conditioned medium of human HepG2 cells approximately 2-fold,
consistent with previous reports(8) . These results clearly
indicate that the endogenous human Vn gene and the human promoter
construct studied are regulated in a similar fashion.
Experiments were performed to specifically localize the region(s) in the Vn promoters that mediate inducibility in Hep3B cells. In the first set of experiments, Hep3B cells were transfected with a series of overlapping 5`-deletion constructs of the murine Vn promoter and then incubated for 24 h in the presence of 500 units/ml IL-6. Cell lysates were prepared and analyzed for increased luciferase activity (Fig. 3). Initial studies implicated two areas in the regulation by IL-6 (Fig. 3, panel A), a distal element located between -428 and -328, and a proximal element between -100 and -91. The latter sequence contains a C/EBP consensus sequence (see also Fig. 7, top panel). To more precisely define the distal IL-6 responsive sequence, several progressive 5`-deletions were made through this region (Fig. 3, panels B and C). When sequences between -403 and -383 were deleted, the level of IL-6 induction fell from 16-fold to 4-fold. The remaining level of induction was still constant in deletions to -353 (Fig. 3, panel B). To further define this distal IL-6-responsive element, the sequence between -403 and -383 was scanned by deleting 3-4 bases at a time (Fig. 3, panel C). Constructs containing 5`-deletions to -399 retained full IL-6 inducibility, whereas further deletion to -393 progressively reduced the -fold induction to 6-fold (Fig. 3, panel C). These results suggest that the distal IL-6 regulatory sequence is located between -399 and -393. To confirm these results in the context of the intact promoter (-528/47), both regulatory sequences were scrambled by site-directed mutagenesis. IL-6 dose-response experiments revealed that the IL-6 inducibility of the murine Vn promoter was drastically reduced (Fig. 4). These results indicate that at least two different sequences, located between -399/-393 and -100/-92, are involved in the regulation of the murine Vn gene by IL-6.
Figure 3: Deletional analysis of IL-6 responsive elements as shown by stimulation of the murine Vn promoter by IL-6. Panel A, a series of progressive 5`-deletions were prepared between -528 and -91. Panel B, A 5`-deletion series between -428 and -353 was prepared. Panel C, a more detailed analysis of the active region (-403 to -393) is shown. The resulting constructs were transfected into Hep3B cells and assayed for luciferase activity following treatment with 500 units/ml IL-6 (see ``Materials and Methods''). The 5` boundaries are indicated to the left of each construct. The -fold increase in luciferase activity following IL-6 treatment is presented to the right of each construct. The solid box represents the ``TATA'' box at position -24. The arrow indicates the location and direction of transcriptional initiation at position 1.
Figure 7: Introduction of IL-6 responsiveness to the human Vn promoter. Two adjacent single-point mutations (position 1491 C to T, 1492 A to G) were introduced into the human Vn promoter by site-directed mutagenesis (see ``Materials and Methods''). The resulting construct was transfected into Hep3B cells and then assayed for luciferase activity following treatment with the indicated concentration of IL-6 (filled squares). The -fold induction was compared to the wild-type mouse (open circles) and wild-type human Vn promoters (closed circles), respectively. The -fold induction is plotted on the y axis (logarithmic scale) against the concentration of IL-6 on the x axis (linear scale).
Figure 4: Mutational analysis of the IL-6 responsive elements in the murine Vn promoter. The regions encompassing the proximal (-100/-92) and distal (-399/-391) IL-6 responsive elements were scrambled by site-directed mutagenesis in the intact Vn promoter (-528/47) and assayed for luciferase activity following treatment with the indicated concentration of IL-6. The -fold induction is plotted on the y axis (linear scale) against the concentration of IL-6 on the x axis (linear scale). Closed circles, wild-type murine Vn promoter; open circles, mutations in both the proximal and distal IL-6 responsive elements.
In the second set of experiments, a series of
5`-overlapping deletion constructs of the human Vn promoter were
transfected into Hep3B cells and then incubated for 24 h with 1 ng/ml
TGF. These initial studies implicated the region between 1179 and
1279 in the regulation by TGF
(Fig. 5, panel A).
The responsive sequence was further defined by several progressive
5`-deletions. An initial 2-fold loss in inducibility was observed by
deletion to 1190, and further deletion to 1212 abolished the majority
of the TGF
response (Fig. 5, panel B). Inspection
of the sequence revealed no evidence for any reported TGF
responsive elements. The results are summarized in Fig. 6.
Figure 5:
Deletional analysis of the Vn promoter and
5`-flanking region as shown by stimulation of the human Vn promoter by
TGF. Panel A, a series of progressive 5`-deletions were
prepared between 1079 and 1379. Panel B, a more detailed
analysis of the region between 1179 and 1245 is depicted. The resulting
constructs were transfected into Hep3B cells and then assayed for
luciferase activity following treatment with 1 ng/ml TGF
(see
``Materials and Methods''). The 5` boundaries are indicated
to the left of each construct. The -fold increase in
luciferase activity following TGF
treatment is presented to the right of each construct. The arrow at position 1
indicates the location and direction of transcription
initiation.
Alignment of the murine Vn promoter sequence (-528/47) with that of the corresponding human gene (1079/1652) revealed that the overall sequence identity is 65%. Notably, the upstream IL-6 responsive sequence was also present in the human Vn promoter, whereas the downstream sequence contained two mismatches in the center of the recognition sequence (Fig. 7, top panel). Site-directed mutagenesis was employed to convert the human downstream sequence into the murine sequence. The resulting reporter gene construct was transiently transfected into Hep3B cells, and the luciferase activity was compared to the wild-type human and murine promoters after treatment with IL-6 (Fig. 7). The mutant human Vn promoter was stimulated by IL-6 in a dose-dependent manner, indicating the importance of the downstream IL-6 responsive element in a heterologous promoter.
Although the human and murine Vn proteins are functionally
indistinguishable(4) , we provide evidence that the two
5`-flanking regions fused to a reporter gene are regulated quite
differently. Specifically, IL-6 stimulated the murine construct in a
dose-dependent manner but was ineffective when the human construct was
studied. In contrast, the human but not the murine construct was
strongly stimulated by TGF. A number of observations support the
notion that the endogenous genes and the reporter gene constructs are
regulated in a similar fashion. First, TGF
stimulated the
accumulation of Vn in the conditioned medium of Hep3B cells in a
dose-dependent manner (Fig. 2), consistent with recent reports
on HepG2 cells, another hepatocarcinoma cell line(8) . It
should be noted that in the latter study, the steady-state level of Vn
mRNA increased upon stimulation with TGF
(8) , indicating
that the stimulation of Vn biosynthesis by TGF
is, at least in
part, on the transcriptional level. This observation is consistent with
preliminary results that TGF
but not IL-6 increases the
steady-state level of Vn mRNA in Hep3B cells approximately 2-fold (not
shown). In addition, IL-6 failed to elicit stimulation of the
endogenous Vn gene in Hep3B cells (Fig. 2), consistent with the
lack of effect on the reporter gene construct (Fig. 1). Thus,
the endogenous human Vn and the human Vn promoter reporter gene
construct appear to be regulated in a similar fashion. Second, the
endogenous murine and rat Vn genes are stimulated in acute inflammation in vivo(7, 9) and purified IL-6-induced rat
hepatic Vn gene expression within 1 h after injection, suggesting a
direct effect on the transcriptional activity of rodent Vn
genes(9) . In agreement with these observations, the murine Vn
promoter fused to the luciferase reporter gene construct was strongly
induced by IL-6 ( Table 2and Fig. 1). These results
indicate that with respect to IL-6 induction, the murine reporter gene
construct and the endogenous gene are regulated in a similar fashion.
We attempted to confirm these results using a number of established
murine hepatoma cell lines. Unfortunately, none of the cells studied
expressed Vn. Information regarding the regulation of the endogenous
murine Vn gene by TGF
is not available. However, the
identification of a TGF
responsive sequence in the human Vn
promoter construct and the lack of the corresponding sequence in the
murine Vn promoter (see below) would predict that the endogenous murine
Vn gene is not stimulated by TGF
.
Acute phase genes fall into
two categories. Type 1 acute phase genes require IL-1 and IL-6 for
maximal stimulation, whereas induction of type 2 acute phase genes
requires only IL-6(23) . The observation that maximal
stimulation of the murine Vn gene is achieved by IL-6 alone (Table 2), whereas a combination of IL-1 and IL-6 (Table 2)
or TNF and IL-6 (not shown) actually leads to a down-regulation of
the IL-6 induction, suggests that Vn is a type II acute phase protein.
The molecular basis for the differential regulation of the human and murine Vn genes was elucidated. The studies summarized in Fig. 3and Fig. 4demonstrate that the response of the murine Vn promoter to IL-6 is contained within two separate regions. This conclusion is based on both 5`-deletional analysis of these regions and scrambling of the respective sequences by site-directed mutagenesis in the context of the whole promoter. The proximal sequence at -100 to -92 is identical to the IL-6 responsive transcription control element present in several acute phase genes, including fibrinogen and C-reactive protein (consensus sequence (T/A)T(C/G)TGGGA(A/T), (24) ). Thus, it is likely that NF IL-6 binds to this sequence, leading to an up-regulation of Vn gene expression. The second sequence (-399/-391) shares only 3 bases with this consensus sequence, raising the possibility that transcription factor(s) distinct from NF IL-6 may bind to this site. Clearly, additional studies are required to identify the nature of the transcription factor(s) that interact with each site. The importance of the proximal sequence was confirmed with a heterologous promoter (i.e. the human Vn promoter) that lacked IL-6 responsiveness.
The TGF responsive sequence was localized to a 33-base pair
stretch between 1179 and 1212, and this sequence appears to be
unrelated to previously described TGF
responsive elements. The
human and murine 5`-untranslated sequences were aligned, and while the
overall identity was 65%, the sequence between 1179 and 1212 was only
45% conserved, and no continuous stretch with more than 3-base pair
matches was detected. This comparison thus strongly suggests that the
TGF
responsive element is not contained within the murine Vn
promoter.
The physiological significance of the regulation of the Vn genes remains to be elucidated. The homeostatic response of the body to trauma or infection consists of an orderly and orchestrated series of events that results in the halting of the process of injury, the protection of the rest of the organism against further injury, and the initiation of repair processes aimed at returning the body to normal function. One of the striking changes that occurs during these events is the dramatic alteration in the plasma concentration of a series of proteins, collectively called acute phase proteins (reviewed in (24, 25, 26, 27) ). Many of the acute phase proteins act as antiproteinases, opsonins, or blood-clotting and wound-healing factors, which may protect against generalized and local tissue destruction associated with inflammation(28) . Vn shares several functional similarities with classical acute phase proteins, including regulatory functions in the complement, blood coagulation, and fibrinolytic systems (reviewed in (1) and (2) ). The physiological significance of the up-regulation of rodent Vns should probably be viewed in the context of these functions.
Interestingly, the human Vn gene appears
to be regulated quite differently. Species differences with respect to
acute phase regulation have been reported for a number of acute phase
proteins, the most prevalent example being C reactive protein, the
classical human, but not murine acute phase protein(24) .
However, we are unaware of any example that the corresponding mammalian
genes are either regulated by a cytokine (i.e. IL-6) or growth
factor (i.e. TGF). The physiological significance of the
regulation of human Vn by TGF
remains to be elucidated. Recent
reports indicate that the Vn gene is expressed at significant levels at
extrahepatic sites(7) . This observation raises the possibility
that the up-regulation of the Vn gene by TGF
could be of
importance for stimulating Vn biosynthesis in localized areas of
inflammation and tissue damage.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X72091[GenBank].