(Received for publication, July 17, 1995; and in revised form, October 25, 1995)
From the
Angiogenesis, the formation of new blood vessels, is induced by various growth factors and cytokines that act either directly or indirectly. Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and therefore has a central role in physiological events of angiogenesis. Interleukin-6 (IL-6) expression on the other hand is elevated in tissues that undergo active angiogenesis but does not induce proliferation of endothelial cells. We demonstrate using Northern analysis that treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA. The level of induction is comparable to the documented induction of VEGF mRNA by hypoxia or cobalt chloride, an activator of hypoxia-induced genes. In addition, it is demonstrated by transient transfection assays that the effect of IL-6 is mediated not only by DNA elements at the promoter region but also through specific motif(s) located in the 5`-untranslated region (5`-UTR) of VEGF mRNA. Our results imply that IL-6 may induce angiogenesis indirectly by inducing VEGF expression. It is also shown that the 5`-UTR is important for the expression of VEGF. The 5`-UTR of VEGF is exceptionally long (1038 base pairs) and very rich in G + C. This suggests that secondary structures in the 5`-UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms.
Angiogenesis, the formation of new blood vessels, is a multistep
process in which many growth factors and cytokines have an essential
role. Two types of angiogenesis promoting agents were characterized: 1)
those that indirectly stimulate angiogenesis like tumor necrosis factor
(TNF-
) (
)and transforming growth factor
(TGF-
). 2) Those that act as direct angiogens such as acidic and
basic fibroblast growth factor (aFGF and bFGF, respectively) or
vascular endothelial growth factor
(VEGF)(1, 2, 3, 4, 5, 6) .
VEGF is a very potent angiogenic agent that acts as specific mitogen for vascular endothelial cells through specific cell surface receptors. VEGF is encoded by a single gene, however, 4 isoforms of 206, 189, 165, and 121 amino acids long are produced as a result of alternative splicing(7, 8) . The 165-amino acid long isoform is the most abundant. However, no differences in the biological activities of these isoforms have been reported although differences in their receptor binding abilities and their capability to interact with the ECM were demonstrated(9, 10) .
The promoter region
of VEGF has been cloned, sequenced, and found to contain numerous
putative binding sites for various transcription factors such as AP1,
AP2, and SP1(11) . Interestingly, VEGF contains a very long
(1038 bp) 5`-UTR that was also noted in a related cytokine,
Platelet-derived growth factor (PDGF), and found to contain all the DNA
binding motifs described above. This 5`-UTR is characterized by a high
G + C content upstream to the translation initiation site which is
also characteristic of 5`-UTRs corresponding to PDGF, placenta growth
factor and TGF-(12, 13, 14) .
VEGF expression was reported in normal tissues like lung, kidney, adrenal gland, liver, stomach, heart, and peritoneal macrophages and during normal physiological conditions such as cyclical corpus luteum formation, wound healing, and embryo development. Conversely, the expression of VEGF was also reported during abnormal physiological conditions such as tumor angiogenesis(2, 15, 16, 17, 18, 19) .
The expression of VEGF promotes the formation of new blood vessels
in a regulated mechanism that is sensitive to hypoxia and is not
completely characterized. Recently, it was demonstrated that
physiological conditions such as hypoxia (or treatment with cobalt,
that mimics hypoxia) can induce VEGF
expression(20, 21) . In addition, induced expression
of VEGF was also noted in cells treated with interleukin-1
(IL-1
), TGF-
, PDGF-B, and
12-O-tetradecanoylphorbol-13-acetate(22, 23, 24, 25) .
Yet, the molecular mechanism governing the expression of VEGF are not
characterized and it is likely that other cytokines might promote the
expression of VEGF as well.
In this work, we have studied the effect of various cytokines on the expression of VEGF. We show that VEGF mRNA is induced by IL-6. This induction is also demonstrated by transient transfection assays in which the promoter region of VEGF including its exceptional 5`-UTR were connected to the reporter gene chloramphenicol acetyltransferase (CAT). We show that DNA element(s) located upstream to the transcription initiation site mediate in part the response to the IL-6. In addition, we demonstrate that the induction of VEGF is also mediated through specific DNA element(s) located at the 5`-UTR of the gene. The data suggests that the 5`-UTR elements cooperate synergistically with DNA elements located upstream to the transcription initiation site. It is also shown that 5`UTR of VEGF has an important role in the expression of VEGF. Finally, we propose that IL-6 should be considered as an indirect inducer of angiogenesis that exerts its activity through the induction of VEGF.
Figure 4:
The induced CAT activity in response to
treatments with IL-6 and cobalt is mediated by DNA motifs located at
the promoter region and at the 5`-UTR of VEGF. A, the promoter
region of VEGF including the 5`-UTR (3.4-kb segment) was connected
to CAT reporter gene, construct p3.4CAT. In addition, three internal
deletions were generated in the 5`-UTR of the p3.4CAT construct that
resulted in the formation of p3.4CAT
NrX (a deletion of 83 bp),
p3.4CAT
Nhe (a deletion of 695 bp), and p3.4CAT
NX (a
deletion of 989 bp). All 4 constructs are illustrated. Each construct
was transfected into L8 cells that were treated with IL-6 or cobalt
chloride (Co) or not treated(-) and CAT assays were
performed as described under ``Materials and Methods.'' B, a deletion of 1571 bp at the 5` end of all p3.4CAT
constructs was generated resulting in p1.8CAT constructs illustrated in
the figure. The various constructs were transfected into L8 cells as
described in panel A. In each set of experiments (panels A or B) the CAT activity was calculated in relative units
and the results are shown next to the corresponding CAT spots.
Abbreviations in the figure represent restriction sites (RI), EcoRI;, N, NarI; Nhe, NheI; X, XhoI. Arrow, transcription
start site; numbers, relative distance from the translation
start site. Statistical significance was determined by the t test with a probability value (p) <
0.1.
Figure 1:
Effect of cytokines and cobalt chloride
on VEGF mRNA expression in A431 cells. A431 cells were treated with the
various cytokines at indicated times or cobalt chloride for 6 h (for
details see ``Materials and Methods'') and total RNA was
prepared. Northern blot analysis was performed using P-labeled DNA fragments corresponding to VEGF (upper
panels) and interferon regulatory factor-1 (middle
panel). Lower panel shows the amounts of 28 S
RNA.
It was demonstrated by Keshet and co-workers (32) that IL-6 is expressed during the angiogenesis that accompanies folliculogenesis of the maternal decidua and during early postimplantation development. Therefore, we have focused on the effect of IL-6 on VEGF expression. Since A431 cells are not very suitable for transfection, the ability of IL-6 to induce VEGF mRNA was tested in two rat-derived cell lines, L8 cells (skeletal muscle myoblasts) and C6 cells (Glioma cells) in which VEGF is hypoxia-inducible(20) . The results shown in Fig. 2clearly demonstrate that VEGF mRNA expression in the two cell lines was augmented by both IL-6 and cobalt. Thus, the effect of IL-6 on VEGF expression is not limited to A431 cells.
Figure 2: Effect of IL-6 and cobalt chloride on VEGF mRNA expression in L8 and C6 cells. C6 (black bars) and L8 (gray bars) cells were treated with IL-6 (80 ng/ml) for 24 h or cobalt chloride (550 µM) for 6 h. Cells were harvested, total RNA was prepared, and Northern blot analysis was performed using VEGF probe as described in the legend to Fig. 1. Relative intensities of bands corresponding to VEGF mRNA (presented in relative units) were quantitated and plotted following PhosphorImager analysis.
Figure 3: IL-6 enhances CAT activity of reporter plasmid driven by VEGF genomic region. L8 cells were transfected with 5 µg of p3.4CAT plasmid (see illustration under Fig. 4A) and 24 h later the cells were washed and treated with either IL-6 at the indicated concentrations (ng/ml), or cobalt chloride (550 µM) or not treated (for details see ``Materials and Methods''). The actual CAT assay spots corresponding to the faster migrating form of monoacetylated chloramphenicol are placed under the relevant bar. Statistical significance was determined by the t test with a probability value (p) < 0.05.
We first wanted to test the effect of the
5`-UTR on the promoter region. For that purpose, we have compared the
CAT activity of the full-length construct, p3.4CAT, with that of the
other 3.4-kb constructs containing deletions in that region. The
results of these transfection experiments are shown in Fig. 4A. As expected, both IL-6 and cobalt promoted CAT
activity of the full-length promoter that also contains 5`-UTR
(construct p3.4CAT) as compared to CAT activity of untreated cells.
However, in cells transfected with the construct p3.4CATNX, in
which most of the 5`-UTR was deleted (except for 49 bp downstream to
transcription start site), no significant change in CAT activity in
response to IL-6 or cobalt was detected. Interestingly, this construct
(p3.4CAT
NX) still contains ``a classical promoter
region'' which is missing the 5`-UTR. However, the CAT activity
derived by this construct in untreated cells was reduced by 50% when
compared to that of cells transfected with the full-length construct.
We have also studied the effect of shorter internal deletions in the
5`-UTR on the ability to promote CAT activity in response to IL-6 and
cobalt. Deletion of only 83 bp in the 5`-UTR near the translation start
site (p3.4CAT
NrX) resulted in a significant reduction in CAT
activity of the untreated transfected cells (at least by 30%) and in a
weak response to IL-6, although the response to cobalt was still
sustained. A reporter construct containing an internal deletion of 695
bp out of the 1038 bp of the 5`-UTR (p3.4CAT
Nhe) was also tested.
When introduced into the cells, the level of CAT activity as compared
to the full-length construct was also reduced by 25% but no significant
response to either cobalt or IL-6 was noted. These results imply that
the 5`-UTR potentiates the expression of the VEGF promoter and that
response elements to either IL-6 or cobalt may be located within that
region.
Fig. 4B shows the results of transient
transfection experiments using the 1.8-kb group of CAT constructs which
are similar to the 3.4-kb constructs except for a deletion of 1751 bp
at the 5` end of the available DNA sequence of the promoter region of
VEGF (see illustrations in Fig. 4B). In general, the
results of the 1.8-kb group resemble that of the 3.4-kb group except
for the fact that the basal CAT activity and responses to IL-6 or
cobalt were reduced by 25-50%. The 1.8CAT construct promoted
response to both IL-6 and cobalt. However, in comparison to the 3.4CAT
construct the extent of increased CAT activity was only
1.4-1.5-fold and not 3-3.5-fold as for the 3.4CAT
construct. Similarly, deletion of 83 bp (p1.8CATNrX) resulted in
a modest response to cobalt treatment and a weak response to IL-6. The
remaining constructs, p1.8CAT
Nhe and p1.8CAT
NX, promoted
CAT activities in untreated transfected cells that were 4-fold lower as
compared to that of cells transfected with the full-length 3.4-kb
construct. No significant response to IL-6 was observed with these
constructs and the response to cobalt was residual. These results
indicate that the 1751-bp deleted segment contains DNA motifs that work
cooperatively with DNA or RNA elements located at the 5`-UTR and
confirm the fact that this region is essential for expression of VEGF.
Formation of new blood vessels, angiogenesis, is mediated
through the activation of parental vessels by specific inducers that
either act directly or indirectly. VEGF is a direct angiogenic agent
and its role in promoting angiogenesis during normal and abnormal
physiological conditions is well documented(1) . This suggests
that its expression is subjected to complex regulatory mechanisms.
Several stimulators of VEGF expression have been reported including
hypoxia and cobalt chloride (20, 21, 31, 33) , and various
cytokines and growth factors such as IL-1, TGF-
, and
PDGF-B(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) .
We have studied the effect of IL-6 on VEGF expression. IL-6 is a multi-functional cytokine that is produced by many cells and has pleiotropic effects. IL-6 acts on a wide range of tissues and cell lines and promotes cell growth and differentiation on the one hand and growth arrest on the other hand(36, 37, 38) . Previously, it was demonstrated that the in vivo expression of IL-6 accompanies vascularization in the reproductive tissues(32) . Moreover, IL-6 expression has been noted during wound healing and tumor growth(38, 39) . It was suggested that IL-6 may induce angiogenesis and may also induce motility of cells such as endothelial cells(32, 38, 40) . We provide evidence indicating that IL-6 may promote angiogenesis through the induction of VEGF expression. This induced expression is mediated by specific DNA motifs located on the putative promoter region of VEGF as well as by specific elements located in the 5`-UTR. IL-6 exerts its biological effects through association with specific cell surface receptors resulting in the activation of specific transcription factors that interact with two types of cis-acting DNA control elements mediating IL-6 response. Type I elements have a consensus sequence agTgNNGYAA which serves as binding sites for NF-IL-6, and type II IL-6 response elements have a consensus sequence CTGGGA that binds IL-6-RE binding protein(41, 42, 43) . Interestingly, two type I response elements are located at DNA sequences flanking the translation initiation site of VEGF. The first is located between positions -796 and -804 (GTGCTGGAA) within the 5`-UTR and the second is located between positions -2313 and -2320 (TGAGGGAA) upstream to the transcription initiation site. Our results provide evidence that both elements might be functional. When DNA sequences between positions -1829 and -3400 were deleted, the response to IL-6 was decreased by 2-fold in comparison to the response of the full-length promoter plus 5`-UTR. This suggests that part of the IL-6-REs are confined to the deleted region (Fig. 4). In addition, internal deletions within the 5`-UTR abrogated the effect of IL-6, indicating that a possible functional IL-6-RE is also contained within that region. These findings indicate that the putative IL-6-REs at the promoter region of VEGF and its 5`-UTR are not only functional but also operate in a cooperative manner. Our results, however, do not enable us to determine whether motifs in the 5`-UTR regulate the expression of VEGF at the transcriptional or post-transcriptional level.
Recently, it was
reported that IL-6-like VEGF is induced in response to
hypoxia(44) . Thus, our findings together with these recent
results suggest that induction of IL-6 by hypoxia may promote the
expression of VEGF that eventually leads to angiogenesis. The
involvement of IL-6 in angiogenesis is apparently not limited to the
hypoxia conditions since it is also produced during wound healing (39) and ovulation(32) . Therefore, IL-6 should be
considered as an indirect angiogenic factor. Interestingly,
-macroglobulin is an acute-phase protein that is also
induced by IL-6(42, 43) . It was reported that VEGF is
inactivated by binding to
-macroglobulin(45) .
Since IL-6 induces
-macroglobulin it may eventually
lead to the inactivation of circulating VEGF.
We also demonstrate
that other cytokines such as IFN- and TNF-
can also induce
the expression of VEGF mRNA. The involvement of IFN-
in angiogenic
processes have not been described before. However, IFN-
is
expressed during inflammation, rheumatoid arthritis, and wound healing.
Thus, it is probable that expression of IFN-
in response to these
disorders might be one of the signals that triggers the angiogenic
process through the induction of VEGF expression. Direct proof for such
regulatory mechanisms awaits future studies. On the other hand,
TNF-
has been implicated as an indirect angiogenic
factor(46) . Our data suggests as published very recently, that
it acts through the induction of the expression of the direct
angiogenic agent, VEGF(47) .
The 5`-UTR of VEGF is
exceptionally long (1038 bp) and contains several putative DNA motifs
for SP1, AP1, AP2, as originally noted by Tischer and
colleagues(11) , and also the putative IL-6-RE type I element.
This region is rich in G + C content. A similar structure was also
reported for PDGF-B. It was demonstrated that the PDGF 5`-UTR acts as
translational inhibitor and upon deletion translation efficiency
increases(13, 48) . Furthermore, during megakaryocytic
differentiation, in which PDGF-B is produced, translation repression is
removed by cis-acting elements(48) . Similarly, the 5`-UTR of
placenta growth factor, albeit shorter (less then 300 bp), is composed
of 73% G + C base pairs with a short open reading frame that has
an inhibitory effect on translation(12) . Our results also
demonstrate a possible role for the 5`-UTR in VEGF expression. However,
unlike the 5`-UTRs of PDGF and placenta growth factor that serve as
translation inhibitors, VEGF 5`-UTR enhances promoter activity as
determined by transient assays. A deletion of 83 bp located in the
vicinity of the translation start site resulted in a significant
decrease in CAT activity (30-40%), suggesting that this region
has a role in VEGF expression. Since this region is characterized by a
highly G + C content (83%) it is most probable that it has a role
in maintaining proper secondary structure of the 5`-UTR which is
crucial for proper translation. A possible role for secondary structure
was also suggested for PDGF 5`-UTR in inhibiting scanning mechanisms of
ribosomes. It was proposed that such structured 5`-UTRs might serve as
internal ribosome entry sites(48) . Since the 5`-UTR of VEGF
can theoretically form such structures, it is most probable that it is
also subjected to translational regulation. DNA motifs that confer
response to hypoxic treatment enable the binding of specific hypoxia
induced factor(21) . Similar DNA motifs were also observed for
VEGF, however, only the motif located at the 3`-UTR was functional
while the two homologous motifs at the promoter region of VEGF
(-2856 to -2847 and -1958 to -1949) were not
essential(49) . It was found that a putative new hypoxia
element was contained within a 100-bp region between locations
-1882 to -1782. Our results support this finding and
enables the margins of the hypoxia element to be further narrowed to
positions -1782 to -1829 (construct p1.8CAT). The magnitude
of increased CAT activity for the 3.4CAT construct following treatment
with cobalt and for the 1.8CAT construct was similar (2-fold),
implying that the hypoxia (cobalt) response element is indeed located
downstream to position -1829. When most of 5`-UTR was deleted,
the response to cobalt was not observed (Fig. 4). This is not in
agreement with the reported results of Minchenko et al.(49) that also used constructs lacking the 5`-UTR to which
luciferase was connected as the reporter gene. They demonstrated
enhanced luciferase activity in response to cobalt in HeLa cells, and
calculated their results as ratio between the activity of the reporter
gene in treated cells and untreated cells and not in absolute light
units. Therefore, it is likely that the extremely sensitive luciferase
assay enabled the detection of variations even at very weak promoter
activities. However, their results do not contradict ours showing that
the 5`-UTR is essential for VEGF expression. Differences in cell lines
should be ruled out since our data were also reproducible in HeLa cells
(data not shown).
In conclusion, we provide evidence showing that the role of IL-6 in angiogenesis is mediated through the induction of VEGF, a potent angiogenic agent. We show that putative DNA motifs located at the promoter region as well as in the 5`-UTR are necessary for the responsiveness to IL-6. In addition, it is demonstrated that 5`-UTR enhances the basal promoter activity of VEGF as determined by transient transfection assays. These results indicate that the secondary structure of 5`-UTR may be important for an efficient expression of VEGF and suggest that transcription and post-transcription control mechanisms are involved.