(Received for publication, December 19, 1995; and in revised form, February 27, 1996)
From the
The 92-kDa type IV collagenase (92-kDa gelatinase B also
referred to as MMP-9), which plays a critical role in extracellular
matrix degradation, is regulated by growth factors that mediate their
effects through the ras proto-oncogene. The current study was
undertaken to determine the transcriptional requirements for the
induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector
encoding an activated Ha-ras increased 92-kDa gelatinolytic
activity and stimulated (over 10-fold) the activity of a CAT reporter
driven by 670 nucleotides of 5` flanking sequence of the 92-kDa
gelatinase B gene. Transient assays using a CAT reporter driven by 5`
deleted fragments of the 92-kDa gelatinase B promoter indicated that a
region spanning -634 to -531 was required for optimal
induction of the promoter. The individual deletion, or mutation, of a
PEA3/ets(-540) motif, AP-1 sites(-533, -79),
a NF-B(-600) consensus sequence, and a GT box(-52)
substantially reduced the activation of the promoter by ras.
An expression vector encoding the PEA3 transcription factor caused a
3-fold stimulation of the wild type but not the
PEA3/ets-deleted 92-kDa gelatinase B promoter. Co-expression
of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B
promoter-driven CAT reporter. The signaling pathway mediating the
induction of 92-kDa gelatinase B promoter activity by ras was
examined. The expression of a phosphatase (CL100) which inactivates
multiple mitogen-activated protein kinase members abrogated the
stimulation of 92-kDa gelatinase B promoter activity by ras.
However, the expression of a kinase-deficient mitogen-activated protein
kinase kinase 1 (MEK1) did not prevent the activation of the 92-kDa
gelatinase B promoter by ras and a constitutively activated
c-raf expression vector was insufficient for 92-kDa gelatinase
B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B
promoter by ras requires multiple elements including closely
spaced PEA3/ets and AP-1 sites and is MEK1-independent.
The 92-kDa type IV matrix metalloproteinase (92-kDa gelatinase B
also known as MMP-9) plays a major role in cell migration in both
physiological and pathological processes (1, 2, 3) by facilitating the destruction of
the type IV collagen-containing basement membrane which separates the
epithelial and stromal compartments(4) . The 92-kDa type IV
collagenase is secreted as a proenzyme (5) and subsequently
activated by multiple enzymes, including cathepsin G, trypsin,
stromelysin 1(6) , and 72-kDa gelatinase A (7) by the
removal of 73 amino acids from the amino terminus of the protease. The
active enzyme, which is capable of digesting native type I, III, IV,
and V collagens at nondenaturing temperatures(4, 6) ,
consists of five domains: the amino-terminal and zinc-binding domains
shared by all members of the metalloproteinase family, a
collagen-binding fibronectin-like domain, a carboxyl-terminal
hemopexin-like domain, and a unique 54-amino acid proline-rich domain
homologous to the 2 chain of type V collagen (5) .
The
92-kDa gelatinase B is encoded by a 7.7-kilobase pair gene, which spans
13 exons(8) . Transcription of the 92-kDa gelatinase B gene,
which yields a 2.5-kilobase mRNA(8, 9) , is regulated
by 670 bp ()of regulatory sequence, which includes binding
sites for AP-1, NF-
B, AP-2, and Sp1(9) . Mutation, or
deletion, of the NF-
B, AP-1, and Sp1 motifs located 600, 79, and
558 nucleotides upstream of the transcriptional start site,
respectively, reduced or abolished the ability of tumor necrosis
factor-
to stimulate the 92-kDa gelatinase B promoter in OST
osteosarcoma and HepG2 hepatoma cells(9) . On the other hand,
activation of the 92-kDa gelatinase B promoter by v-src in HT
1080 fibrosarcoma cells was attributed to binding sites for
AP-1(-79) and a Sp1-binding GT box at -52(10) .
Several lines of evidence suggest an important role for ras in the regulation of 92-kDa gelatinase B expression. First, the
amount of this collagenase is increased by growth factors including
transforming growth factor- and epidermal growth factor (11) , which via their transmembrane receptors, signal through
the Ras GTP-binding protein(12) . Second, and equally
important, the ras oncogene, itself, is a potent stimulus for
92-kDa gelatinase B secretion in rat embryo cells(13) .
However, while the role of ras in the regulation of this
collagenase is clear, the events downstream of ras that lead
to induction of 92-kDa gelatinase B synthesis have not been elucidated. ras can stimulate multiple signaling
pathways(14, 15, 16, 17) . One of
these involves the sequential activation of the serine-threonine kinase
c-raf, mitogen-activated protein kinase kinase (MEK1), and the
extracellular signal-regulated kinases
(ERKs)(18, 19, 20, 21) . The ERKs,
in turn, increase the synthesis and/or activity of several
transcription factors including c-fos(22) and ets(23, 24) family members. Alternatively, ras can modulate gene expression through other signaling pathways,
which can be distinguished from each other on the basis of their
utilization of the jun amino-terminal kinase
(JNK)(25, 26, 27) , a fos-related
kinase(15) , or phosphatidylinositol-3-OH kinase (17) .
Accordingly, the purpose of the current study was twofold: first, to
identify the cis acting elements in the 92-kDa gelatinase B
promoter that mediate the inductive effects of ras on the
expression of this gene, and second, to examine the signaling events
connecting ras with the nuclear regulators of 92-kDa
gelatinase B expression.
Figure 1:
c-Ha-ras expression in OVCAR-3 cells increases 92-kDa gelatinolytic
activity and 92-kDa gelatinase B promoter activity. Panel A,
OVCAR-3 cell were transiently transfected at 50% confluence with
varying amounts of a c-Ha-ras expression vector
(H-ras) or the empty vector control (pSV2 neo). After 6 h, the
cells were rinsed and the medium changed. The cells were replenished
with serum-free medium 24 h later and cultured for an additional 48 h.
Conditioned medium was collected, and cells were enumerated.
Conditioned medium, normalized to cell number, was electrophoresed in a
7.5% SDS-polyacrylamide gel containing 0.1% gelatin. The gel was
incubated at room temperature for 2 h in the presence of 2.5% Triton
X-100 and subsequently at 37 °C overnight in a buffer containing 10
mM CaCl, 0.15 M NaCl, and 50 mM Tris (pH 7.5). The gel was then stained for protein with 0.25%
Coomassie. Panel B, OVCAR-3 cell were transiently transfected,
as for panel A with 6 µg of a CAT reporter driven by the
wild-type (670 bp) 92-kDa gelatinase B promoter (MMP-9 CAT), 4 µg
of a
-galactosidase-expressing vector in the presence or absence
of varying amounts of a c-Ha-ras expression vector
(H-ras) or the empty vector (pSV2 neo). Cells were cultured
for an additional 48 h and lysed by freeze-thawing. Cell extracts
(equal protein) were incubated with
[
C]chloramphenicol for 8 h. The mixture was
extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The data are typical of three separate
experiments.
Using identical transfection conditions, OVCAR-3 cells were co-transfected with a CAT reporter driven by the wild type 92-kDa gelatinase B promoter (MMP-9 CAT) and the ras expression vector or the empty vector (pSV2 neo). The cells were harvested 48 h later and assayed for CAT activity. 92-kDa gelatinase B promoter activity was activated 13-fold with 4 µg of the ras expression vector over that achieved with the empty expression vector (pSV2 neo), while 1 µg of the oncogene caused a 6-fold induction of promoter activity (Fig. 1B). These data suggested that the increased synthesis of 92-kDa gelatinase B brought about by the activated ras is likely a reflection of a trans-activation of the 92-kDa gelatinase B promoter.
Figure 2:
Activation of the 92-kDa gelatinase B
promoter by c-Ha-ras requires a region between -634 and
-531 in the 5` flanking sequence. OVCAR-3 cells were transiently
transfected, as described in the legend to Fig. 1B using 4 µg of the c-Ha-ras expression vector or an
equimolar amount of the empty expression vector (pSV2 neo), 6 µg of
a CAT reporter driven by the indicated 5` deleted fragment of the
92-kDa gelatinase B (MMP-9 CAT) promoter, and a
-galactosidase-expressing vector. The cells were harvested and
extracts (equal protein) were incubated with
[
C]chloramphenicol for 8 h. The mixture was
extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The experiment was carried out three
times.
Figure 3:
Stimulation of 92-kDa gelatinase B
promoter activity by c-Ha-ras requires binding sites for AP-1,
PEA3/ets, NF-B, and Sp1, as well as a GT box. OVCAR-3
cells were transiently transfected, as described in the legend to Fig. 1B using 4 µg of the c-Ha-ras expression vector (H-ras) or an equimolar amount of the
empty vector (pSV2 neo), 6 µg of a CAT reporter driven by the
wild-type (wt), point-mutated (mt), or deleted (del) fragments of the
92-kDa gelatinase B promoter (MMP-9 CAT) and a
-galactosidase-expressing vector. Construct names indicate the
binding site that was mutated or deleted and its position relative to
the transcription start site. All mutants and deletions are in context
of 670 bp of 5` flanking sequence. Cell extracts (equal protein) were
incubated with [
C]chloramphenicol for 8 h. The
mixture was extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The data are representative of three
or more separate experiments.
Additionally, a number of other transcription factor
binding sites along the full length of the promoter were also required
for its stimulation by ras. Thus, mutation of an Sp1 motif at
-558 (-558 Sp1 mt), and an AP-1 binding site at -79
(-79 AP-1 mt) previously, shown to be required for the tumor
necrosis factor-- or phorbol ester-dependent stimulation (9) of 92-kDa gelatinase B, all substantially reduced the
ability of ras to stimulate the 92-kDa gelatinase B promoter (Fig. 3B). Also, the 92-kDa gelatinase B promoter
mutated at an NF-
B site at -600 (-600 NF-
B mt)
was stimulated far less (Fig. 3A) than the wild type
promoter. Mutation of an Sp1-binding GT box (-52 GT mt) located
52 nucleotides upstream of the transcriptional start site and which is
required for 92-kDa gelatinase B promoter stimulation by
v-src(10) , practically abolished the induction by ras (Fig. 3B). The observation that 90 bp of
5` flanking sequence was insufficient for ras stimulation (Fig. 2), combined with the finding that mutation of the AP-1
site at -79 or the GT box at -52 abrogated ras stimulation, suggests that these motifs are required for but, by
themselves, insufficient to mediate the ras induction of
92-kDa gelatinase B. An NF-
B-like motif (39) located at
-615 did not appear to be required for optimal 92-kDa gelatinase
B promoter activity since mutation of this site (-615 NF-
B
mt) did not diminish the stimulation by ras (Fig. 3A). We also found that deletion of a
sequence spanning -175 to -146 (175-146 del), which
was shown to mediate the induction of the osteopontin gene by ras(40) , did not impair the stimulation of 92-kDa
gelatinase B promoter activity by the oncogene (Fig. 3B).
Figure 4:
Stimulation of 92-kDa gelatinase B
promoter activity by the co-expression of a PEA3-encoding vector. Panel A, OVCAR-3 cells were transiently transfected, as
described in the legend to Fig. 1B using 6 µg of a
CAT reporter driven by either the wild-type 92-kDa gelatinase B
promoter (MMP-9 wt) or the same promoter with a deletion of the
PEA3/ets site at -540 (-540 PEA3 del) with
expression vectors encoding PEA3 (PEA3 pcDNA3) or ras (H-ras) or the empty expression vectors (pcDNA3 and pSV2
neo, respectively). The expression plasmids were equimolar with each
other and one-fourth of the molar equivalent of the CAT reporter. Panel B, OVCAR-3 cells were transiently transfected, as
described for panel A but using 3 µg of a CAT reporter
driven by either a promoter containing three PEA3 binding sites (TORU
CAT), or the same vector lacking the PEA3 binding sites (pBL CAT). The
amounts of the expression constructs were identical to that used in panel A. For both panels A and B, the cells
were harvested and assayed for -galactosidase activity. Cell
extracts, corrected for differences in transfection efficiency, were
incubated with [
C]chloramphenicol for 8 h. The
mixture was extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The experiment was carried out three
and two times for panels A and B,
respectively.
Figure 5:
Stimulation of the 92-kDa gelatinase B
promoter by c-Ha-ras is attenuated by the expression of a
transactivation domain-lacking c-jun mutant. Panel A,
OVCAR-3 cells were transiently transfected, as described in the legend
to Fig. 1B, using 6 µg of a CAT reporter driven by
670 bp of 5` flanking sequence of the 92-kDa gelatinase B gene (MMP-9
CAT), a -galactosidase-expressing vector, and, where indicated,
expression vectors encoding c-Ha-ras (H-ras) and a
transactivation domain-lacking c-jun protein (TAM-67) (or an
equimolar amount of the empty vector-CMV vector). Panel B,
OVCAR-3 cells were transiently transfected, as described in the legend
to Fig. 1B using 3 µg of a reporter plasmid (pBL
CAT) fused to three tandem AP-1 repeats (3X AP1 pBLCAT), a
-galactosidase-expressing vector, and, where indicated, 4 µg
of the c-Ha-ras expression vector (H-ras), or an
equimolar amount of pSV2 neo. For panels A and B, the
cells were harvested, equal amounts of extracted protein incubated with
[
C]chloramphenicol for 8 (panel A) or
1.5 h (panel B). The mixture was extracted with ethyl acetate
and subjected to thin layer chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The data are typical of two different
experiments.
Figure 6:
Abrogation of the
c-Ha-ras-dependent stimulation of 92-kDa gelatinase B promoter
activity by co-expression of a MAPK-inactivating phosphatase. OVCAR-3
cells were transiently transfected as described in the legend to Fig. 1B using 6 µg of a CAT reporter driven by the
92-kDa gelatinase B promoter (MMP-9 CAT), a
-galactosidase-expressing vector, and, where indicated, 4 µg
of the c-Ha-ras expression vector (Ha-ras), an
equimolar amount of pSV2 neo, with or without either an expression
vector encoding CL100 (CL100 pSG5) or the empty vector (pSG5). Cell
extracts (equal amounts of protein) were incubated with
[
C]chloramphenicol for 8 h. The mixture was
extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. The experiment was carried out
twice.
Figure 7:
Expression of a catalytically inactive MEK
attenuates the ability of c-Ha-ras to stimulate a promoter
driven by the urokinase but not by the 92-kDa gelatinase B promoter.
OVCAR-3 cells were transiently transfected, as described in the legend
to Fig. 1B using 6-8 µg of a CAT reporter
driven by the wild type 92-kDa gelatinase B (MMP-9 CAT, panel A) or urokinase (u-PA CAT) (62) promoters (panel B), a
-galactosidase-expressing vector, and, where indicated, 4 µg
of the c-Ha-ras expression vector (Ha-ras), (or an
equimolar amount of pSV2 neo), or an expression vector encoding either
the wild-type MEK1 (MEK1) or a kinase-inactive MEK1 sequence (K97M).
Cell extracts, corrected for differences in transfection efficiency (as
determined by
-galactosidase activity), were incubated with
[
C]chloramphenicol for 8 h. The mixture was
extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. Mock, no DNA added. The
experiment was carried out on two separate
occasions.
To further address the role of MEK1 in the regulation of 92-kDa gelatinase B promoter activity by ras, we determined if the expression of a constitutively activated c-raf (BXB) (18) could induce the 92-kDa type IV collagenase promoter since c-raf lies directly upstream of MEK1(19, 21) . The BXB expression construct encodes an in-frame deletion of amino acids 26-302 in the regulatory region of c-raf(18) . Expression of the constitutively activated serine-threonine kinase did not stimulate 92-kDa gelatinase B promoter activity (Fig. 8A) over that achieved with the empty expression vector (pMNC). However, the constitutively activated serine-threonine kinase caused over a 4-fold stimulation of a CAT reporter driven by the urokinase-type plasminogen activator promoter (Fig. 8B), indicating that the lack of effect on 92-kDa gelatinase B promoter activity was not a consequence of an ineffective expression of this construct in OVCAR-3 cells. Thus, the expression of a constitutively activated c-raf is an insufficient stimulus for 92-kDa gelatinase B expression in these cells.
Figure 8:
Effect of expressing a constitutively
activated c-raf on 92-kDa gelatinase B promoter activity.
OVCAR-3 cells were transiently transfected, as described in the legend
to Fig. 1-B using 6 µg of a CAT reporter driven by the
wild-type 92-kDa gelatinase B (MMP-9-CAT, panel A) or
urokinase (uPA CAT; 62, panel B) promoters, a
-galactosidase-expressing vector, 4 µg of the c-Ha-ras expression vector (H-ras) (or an equimolar amount of the
pSV2 neo vector), or the indicated amount of an expression vector
encoding a constitutively active c-raf (BXB) or the empty
vector (pMNC). The indicated amount (0.5, 1, 2
) of plasmid
refers to molar equivalents with respect to the amount of CAT reporter.
Cell extracts were assayed for
-galactosidase activity and
equivalent amounts (after correcting for varying transfection
efficiencies) incubated with [
C]chloramphenicol
for 8 h. The mixture was extracted with ethyl acetate and subjected to
thin layer chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope. Mock, no DNA added. The
results shown are representative of duplicate
experiments.
92-kDa gelatinase B expression is regulated by ras(13) as well as by a number of growth factors, including
transforming growth factor- and epidermal growth factor, which
mediate their effects through this GTP-binding
protein(11, 12) . However, the cis-acting
elements within the 92-kDa gelatinase B promoter responsible for the
induction by ras as well as the signaling pathway involved
have not been elucidated. We report herein that the induction of 92-kDa
gelatinase B promoter activity by an activated ras is mediated
through multiple elements including two previously undescribed, closely
spaced PEA3/ets and AP-1 motifs located 540 and 533
nucleotides upstream of the transcriptional start site, respectively.
The involvement of the PEA3/ets binding site in mediating the induction of the 92-kDa gelatinase B promoter by ras is suggested from several observations. First, the ability of ras to stimulate the 92-kDa gelatinase B promoter was diminished by deletion of the PEA3/ets site at -540. Second, in OVCAR-3 cells an expression vector encoding PEA3 was sufficient to stimulate, albeit to a lesser degree to that of ras, the wild type 92-kDa gelatinase B promoter, but not the promoter devoid of this consensus sequence. The contention that the PEA3/ets motif is involved in mediating the induction of the 92-kDa gelatinase B promoter by ras is given further support by reports from other investigators. Thus, ras increases the synthesis of several Ets proteins (52) and mutated Ets proteins inhibit ras activation of transcription at least in NIH3T3 fibroblasts(53) . Our findings may explain the observation by Higashino et al.(54) that 92-kDa gelatinase B promoter activity is induced over 10-fold by the recently cloned human homologue of PEA3 (E1A-F)(42) . However, in that study, the authors did not determine if this was a direct effect of E1A-F or secondary to the production and/or activation of other transcription factors, a real possibility in light of a report (55) that the expression of at least one ets family member (c-ets-1) stimulates the jun and fos promoters, thereby increasing AP-1 activity.
The PEA3/ets site at -540 was located just upstream of an AP-1 consensus
sequence(-533) and mutation of this site, like the PEA3/ets motif, practically abolished the stimulation of the 92-kDa
gelatinase B promoter by ras. It may very well be that the
cooperation of these two sites is necessary for the stimulation of
92-kDa gelatinase B promoter activity by ras. Indeed, the
involvement of juxtaposed PEA3/AP-1 elements in the regulation of
several inducible genes including type I collagenase, urokinase,
keratin 18, and tumor necrosis factor- (37, 56, 57, 58, 59) has
been reported by this and other laboratories. Thus, we recently
demonstrated that the stimulation of urokinase promoter activity by ras also required closely spaced intact PEA3/ets and
AP-1 motifs insofar as mutation of either of these sites abolished the
induction by the GTP-binding protein(33) . Similarly, the
inducibility of the type I collagenase gene by either phorbol ester or
fibronectin required the presence of an AP-1 sequence and a
PEA3/ets-like motif(37, 60) . However, while
the closely spaced PEA3/ets and AP-1 motifs are required for
the stimulation of the 92-kDa gelatinase B promoter by ras, it
is unlikely that they are sufficient for optimal expression of this
collagenase. Thus, the individual mutation of other motifs including an
Sp1-binding site(-558), a GT box(-52), an AP-1 binding
site(-79), as well as a NF-
B motif(-600) spread over
the length of the promoter substantially impaired the ability of ras to induce 92-kDa gelatinase B promoter activity. These
binding sites have been shown previously (9, 10) to be
required for the stimulation of the 92-kDa gelatinase B promoter by
v-src (-52 GT box, -79 AP-1) and by phorbol ester
(-79 AP-1, -558 SP1, and -600 NF-
B). Since
expression of PEA3 stimulated the 92-kDa gelatinase B promoter, then,
presumably, the basal level of transcription factors, which bind to the
aforementioned sequences, is sufficient to allow activated expression
of this metalloproteinase by this ets family member.
The signaling pathway connecting membrane-bound ras with the transcriptional control of 92-kDa gelatinase B expression in OVCAR-3 cells merits discussion. We initially hypothesized that this was accomplished via a pathway utilizing c-raf, MEK1, and MAPK family members(18, 20, 21) . Indeed, the ability of CL100, which inactivates multiple MAPK members(47, 48) , to abrogate the stimulation was in line with this contention. However, while these data suggested the involvement of a MAPK in mediating the stimulation of the 92-kDa gelatinase B promoter by ras, other experiments suggested that this was independent of MEK1 which activates the ERK1 and ERK2 group of MAPKs(19) . Thus, interfering with the function of MEK1 failed to block the activation of the 92-kDa gelatinase B promoter by ras. Moreover, the expression of a constitutively activated c-raf, which lies upstream of MEK1(20) , while inducing the urokinase promoter in these cells, did not stimulate a 92-kDa gelatinase B promoter-driven CAT reporter. Thus, ras regulates the promoter of this metalloproteinase either through an alternative effector to c-raf or via multiple effectors including the serine-threonine kinase. Indeed, it is now apparent that ras can modulate gene expression through a number of independent pathways, which can be distinguished from each other on the basis of their utilization of the jun amino-terminal kinase (JNK)(25, 26, 27) , a fos-related kinase (15) or phosphatidylinositol-3-OH kinase(17) . Thus, in a recent report, Al-Alawi et al.(51) showed that thyrotropin-induced mitogenesis, which was ras-dependent, bypassed the c-raf-dependent cytoplasmic kinase cascade leading those authors to speculate that in TSH-treated thyroid cells ras signals through effectors other than the well-studied cytoplasmic kinase cascade (c-raf, MEK, ERK). Our observation that CL100, which inactivates multiple MAPK members including the JNKs(47, 48) , prevented 92-kDa gelatinase B promoter stimulation by ras is consistent with the notion that a MAPK member, presumably other than ERK1 and ERK2, is involved.
In conclusion, we have shown that the induction of 92-kDa gelatinase B promoter activity by ras occurs through multiple transcriptional elements (see Fig. 9for diagrammatic representation of these elements), including previously undescribed, closely spaced PEA3/ets(-540) and AP-1 motifs(-533). Additionally, the ras-dependent expression of this metalloproteinase in OVCAR-3 cells is mediated through a MEK1-independent signaling pathway. Since the 92-kDa gelatinase B has been implicated in the invasiveness of different tumor types(1, 61) , these findings may be relevant to the development of therapeutic agents for invasive cancer which modulate 92-kDa gelatinase B expression by interfering with its paracrine and/or autocrine activation through the ras pathway.
Figure 9: Diagrammatic representation of the transcription factor binding sites in the 92-kDa gelatinase B promoter. Transcription factor binding sites are represented as boxed areas. The position of the 5` end of each motif is indicated relative to the transcription start site with the exception of the Sp1 site(9) , where the number refers to the 3` end of the motif.