(Received for publication, August 16, 1995; and in revised form, September 18, 1995)
From the
Transforming growth factor (TGF-
) causes growth
arrest in the G
phase in many cell types. One probable
pathway for this growth inhibition is through the TGF-
-mediated
up-regulation of the cyclin-dependent kinase (CDK) inhibitor
p15
, which specifically inhibits the enzymatic
activities of CDK4 and CDK6. An active cyclin D-CDK4/6 complex is
required for pRb phosphorylation to allow the cell cycle to progress
from G
to S phase. To study the molecular mechanism of the
p15
induction by TGF-
, we isolated a
780-base pair promoter sequence of the human p15 gene and
inserted this fragment upstream of a luciferase reporter gene. When
this construct was transiently transfected into HaCaT cells, luciferase
activity was induced more than 10-fold upon TGF-
treatment,
indicating that the induction of p15
expression by TGF-
is partly exerted at the
transcription level. Promoter deletion analysis revealed that the
sequence from -110 to -40 relative to the transcription
start site is capable of conferring the 10-fold induction by TGF-
.
Within this region there are three Sp1 consensus sites. Mutation of one
of these sites, GGGGCGGAG, substantially reduced both the induction by
TGF-
and the basal promoter activity, whereas mutations in the
other two Sp1 sites and the spacer sequences had little effect. In
addition, gel mobility shift assay indicates that the transcription
factors Sp1 and Sp3 bind to this Sp1 site. Taken together, these data
suggest that a specific Sp1 consensus site is involved in the mediation
of TGF-
induction as well as the basal promoter activity of the p15 gene and that Sp1 and Sp3 transcription factors might be
involved in this regulation.
Transforming growth factor s (TGF-
s) (
)represent a large family of cytokines with diverse
activities in the regulation of cell growth, differentiation, and
morphogenesis(1, 2, 3) . TGF-
causes
growth inhibition of most epithelial, endothelial, fibroblast,
neuronal, lymphoid, and hematopoietic cell types(3) . TGF-
treatment induces growth arrest in the G
phase of the cell
cycle, and this effect has been attributed largely to an inhibition of
phosphorylation of the retinoblastoma susceptibility gene product, pRb (4) . Progression through the G
phase of the cell
cycle requires phosphorylation of pRb by G
cyclin-dependent
kinase (CDK) complexes, particularly the cyclin D-CDK4 and cyclin
D-CDK6 complexes(5) . Phosphorylation of pRb releases
transcription factors, including members of the E2F transcription
factor family, required for the G
to S phase transition of
the cell cycle(6) .
Two distinct families of CDK inhibitors,
represented by p16 and p21, have been identified recently and shown to
be capable of binding to and inhibiting the activities of various CDK
enzymes (for a recent review, see (5) ). The p16 family of CDK inhibitors specifically interacts with two
closely related CDK proteins, CDK4 and CDK6, both of which have been
strongly implicated as the physiological pRb kinases. One member of
this family, p15
, was specifically
up-regulated by TGF-
in human keratinocyte HaCaT
cells(7) . The steady-state level of p15
mRNA was induced 30-fold upon TGF-
treatment,
implicating p15
as a primary effector of the
TGF-
-mediated cell cycle arrest(7) . Previously it was
shown that treatment of HaCaT cells with TGF-
caused rapid
transcriptional induction of the p21 gene (also known as cip1/ WAF1/sdi1) through a p53-independent
pathway, suggesting that p21 is also involved in mediating the cell
cycle arrest caused by TGF-
(8) .
The signaling pathways
downstream of the TGF- receptor complex that lead to the
inhibition of cell cycle progression are still poorly understood. Since
both CDK inhibitors, p15 and p21, are up-regulated by TGF-
treatment, they could be coordinately regulated through a similar
mechanism. In this study, we attempt to elucidate the mechanism through
which TGF-
specifically up-regulates the expression of
p15
by a detailed analysis of the
p15
promoter sequences.
Figure 3:
Scanning mutation analysis of the p15
promoter. A, scanning mutation constructs, p15P113-LS1 through
p15P113-LS6, are shown with the mutated sequences. Sequences from
-113 to +5 of the wild type promoter, p15P113, are also
shown and the three Sp1 consensus sites underlined. The
transcription initiation site is indicated by an arrow. Fold
induction by TGF- treatment as measured in panel B is
shown for each construct. B, the scanning mutants were assayed
for luciferase activities as described in the legend of Fig. 1.
Figure 1:
Deletion analysis of the p15 promoter.
Luciferase (Luc) constructs with progressive deletions of the
p15 promoter sequences are shown. The restriction sites used in the
construction of these deletion constructs are indicated. The initiator (Inr) sequence is indicated as an open box, and the
transcription initiation site is indicated by an arrow. The
deletion constructs were transiently transfected into HaCaT cells and
the RLU measured after cells were treated with or without 100 pM human TGF-1. Each bar represents the mean RLU of two
duplicate experiments under the same assay condition. The fold
inductions by TGF-
treatment are shown to the left of bar
graph.
A 780-bp genomic DNA fragment, which contains sequences
upstream of the previously reported 5`-ends of the p15 cDNA(7, 11) , was cloned from a human genomic
library. The 5`-end of the p15 mRNA was mapped to the adenosine in the
sequence CCCCACTCT as shown in Fig. 3A by S1 nuclease
protection assay (data not shown). Thus, the cloned 780-bp DNA fragment
largely contains the p15 promoter sequence. The sequence around the
initiation site matches the initiator sequence as defined by Smale and
Baltimore(15) . No apparent TATA sequence was found around the
-25 to -30 region. Therefore, the p15 promoter may be
defined as a TATA-less/initiator promoter. The p15 promoter sequences
are highly GC-rich (70% G + C from -200 to -1).
To
determine its inducibility by TGF-, the 750-bp p15 promoter
sequence was inserted upstream of a luciferase reporter gene in the
vector pGL2-basic (Fig. 1). When the resultant construct,
p15P751-luc, was transiently transfected into HaCaT cells, a
10-15-fold induction of luciferase activity was routinely
observed upon TGF-
treatment as measured by RLU (Fig. 1).
Thus, the p15 promoter is capable of being induced by TGF-
, and
the transcription activation is at least partly responsible for the
accumulation of p15 mRNA upon TGF-
treatment(7) .
To
identify specific promoter elements that confer the TGF-
induction, a series of 5` processive promoter deletion constructs were
generated (Fig. 1). These deletion constructs were transiently
transfected into HaCaT cells and assayed for luciferase activities in
the absence or presence of TGF-
. Fig. 1shows that
deletions up to the position of -110 relative to the initiation
site did not change the fold of induction by TGF-
although the
overall promoter activities in the presence of TGF-
dropped about
3-fold. Deleting sequences from -110 to -30, however,
abolished TGF-
induction (Fig. 1), indicating that a
TGF-
-responsive element is located in this region.
There are
three potential Sp1 binding sites within the -110 to -30
sequences upstream of the transcription initiation site of the p15 gene. Previously, a GC-rich sequence (GCCTCC) capable of binding
to Sp1 and Sp3 was shown to be responsible for the induction of p21 gene by TGF- treatment(30) . Interestingly, this
sequence is identical to the first Sp1 consensus site in the -110
to -30 fragment of the p15 promoter. To test the possibility that
this sequence, or the other two Sp1 consensus sites, may be involved in
the mediation of p15 promoter induction by TGF-
, we generated more
5` promoter deletion constructs to delete either one (p15P97-luc), two
(p15P69-luc), or all three Sp1 consensus sites (p15P47-luc) and assayed
for their luciferase activities in the absence or presence of TGF-
(Fig. 2). Fig. 2shows that deletion of the first Sp1
consensus site had little effect on either the induction fold by
TGF-
or the basal promoter activity of the p15 luciferase
construct, whereas deletion up to the second Sp1 consensus site reduced
TGF-
induction dramatically from 19-fold of the wild type promoter
to 2.8-fold. Deletion of all three Sp1 consensus sites reduced
TGF-
induction only slightly further to 1.5-fold near the
background level. These data suggest that the second Sp1 consensus site
is the most critical sequence for either the induction by TGF-
or
the basal promoter activity of the human p15 promoter.
Figure 2:
Deletion analysis of the Sp1 consensus
sites on the p15 promoter. p15P113-luc, which contains three Sp1
consensus sites and is fully inducible by TGF-, is shown.
Luciferase (Luc) constructs with one (p15P97), two (p15P69),
and three (p15P47) Sp1 consensus sites deleted are also shown. These
constructs were transiently transfected into HaCaT cells and the
luciferase activity assayed as described in the legend of Fig. 1. Fold induction by TGF-
is indicated. Inr,
initiator.
To confirm
the importance of the second Sp1 consensus site in conferring the
transcription inducibility of the p15 promoter by TGF-, a series
of scanning mutation constructs were made in the promoter context of
p15P113, which contains 113 base pairs upstream of the transcription
initiation site and is fully capable of being induced by TGF-
(Fig. 3A). The scanning constructs were transiently
transfected into HaCaT cells and assayed for their luciferase
activities in the absence or presence of TGF-
1. With the exception
of p15P113-LS3, all mutants did not change significantly the fold of
induction upon TGF-
treatment or the basal transcription activity (Fig. 3B). Mutant p15P113-LS3, which contains a
mutation in the second Sp1 site within the -110 to -30
region, decreased the fold of induction from 12-fold of the wild type
promoter to 4-fold (Fig. 3B). In addition, the basal
promoter activity of p15P113-LS3 was also reduced significantly,
approaching the background level (Fig. 3B). Together
with the promoter deletion analysis, these data suggest that a specific
Sp1 consensus site is important for the mediation of TGF-
induction of the p15 gene as well as its basal promoter
activity.
To identify protein factors interacting with the second
Sp1 site, we used DNA sequences from -68 to -82 on the p15
promoter covering the second Sp1 site in the gel mobility shift assay.
As shown in Fig. 4, lane 2, three distinctive protein
complexes (I, II, and III) were observed when this probe was incubated
with nuclear extract prepared from HaCaT cells. All three complexes are
specific to the probe since they were readily competed by an excess of
cold homologous competitor (Fig. 4, lanes 9 and 10) but were resistant to the competition of nonspecific
oligonucleotides (Fig. 4, lanes 7 and 8).
Complex I was abolished when antibody against the human Sp1
transcription factor was included in the binding reaction and therefore
represents the complex formed between Sp1 and the second Sp1 site on
the p15 promoter (Fig. 4, lane 4). Similarly, complexes
II and III represent the complexes formed between Sp3 and the second
Sp1 site on the p15 promoter since both complexes were abolished when
the binding reaction includes antibody against the transcription factor
Sp3 (Fig. 4, lane 6). Together with the promoter
mutation analysis, these data show that the second Sp1 site within the
-110 to -30 region on the p15 promoter, which is capable of
conferring the TGF- inducibility of the p15 promoter, binds to the
transcription factors Sp1 and Sp3.
Figure 4:
Factors binding to the second Sp1 site on
the p15 promoter. Complementary oligonucleotides covering the second
Sp1 site on the p15 promoter were labeled with
[-
P]ATP and used in the gel mobility shift
assay. Three protein-DNA complexes (I, II, and III), as indicated by arrows, were observed when the probe was incubated with 1
µg of nuclear protein (lane 2). In lanes 3 and 4, preimmune sera or antibody against human Sp1 were included
in the binding reaction, respectively. In lanes 5 and 6, preimmune sera or antibody against human Sp3 were included
in the binding reaction. In lanes 7 and 8, a 50- or
250-fold excess of nonspecific oligonucleotides was included in the
binding reaction. In lanes 9 and 10, a 50- or
250-fold excess of the homologous oligonucleotides was included in the
binding reaction. Lane 1 is probe
alone.
Sp1 consensus sites are present
in numerous gene promoters including many housekeeping genes and
cellular proto-oncogenes(16, 17, 18) . The
well characterized transcription factor Sp1 binds to its cognate
binding site and activates transcription presumably through its
interaction with the TBP-associated factor 110 (TAF110) (19) . Recent studies suggest that Sp1 and a member of the Sp1
family, Sp3, are involved in the regulation of many growth-related
cellular genes, including c-fos, c-myc, TGF-
1,
and TGF-
3 genes, through a cis-acting element termed the pRb
control element(16, 20, 21, 22) . A
model has been proposed to postulate that the functional interaction
between pRb and Sp1 in vivo results in the
``superactivation'' of Sp1-mediated
transcription(16) . Here we show that a specific Sp1 consensus
site is involved in the mediation of TGF-
induction of the p15 gene and that the Sp1 and Sp3 proteins could bind to this specific
Sp1 site. The same site is also responsible for the basal promoter
activity. Evidence is accumulating that basal transcription machinery
is also capable of being regulated in cells, presumably through the
action of TBP-associated factors (TAFs). For example, p53 transcription
activation is mediated by TAF
40 and TAF
60 (23) and the presence of TAF
150 and
TAF
250 stabilized the preinitiation complex assembled on
an initiator-containing promoter while it destabilized the complex on
an initiator-less promoter(24) . A temperature-sensitive
mutation in TAF
250 has been shown to cause cell cycle
arrest(25, 26) . It is possible that some TAFs could
relay the cues received from growth signals and affect the
transcription preinitiation complex assembled on certain growth-related
genes.
A GC-rich sequence in the p21 promoter capable of binding to
the Sp1 and Sp3 proteins was shown to be responsible for the induction
of the p21 gene by TGF- in HaCaT cells(30) .
Interestingly, this GC-rich sequence, termed T
RE for
TGF-
-responsive element, was capable of conferring TGF-
inducibility when inserted into an exogenous promoter(30) .
Based on these studies, it is conceivable that the same GC-box binding
factor(s) may be involved in the coordinate regulation of both p15 and p21 genes. Several proteins capable of binding to
those GC-rich sequences including the Sp1 consensus sites have been
identified. Some of these factors have extensive homologies with Sp1
and thus belong to the Sp1 transcription factor family, such as Sp2,
Sp3 and
Sp3(16) , whereas others are entirely different
from Sp1, such as ETF and GC-box binding protein
(GCF)(27, 28) . Notably, Sp3 and GC-box binding
protein have been shown to bind to the GC-boxes, including Sp1
consensus sites, and repress transcription from certain
genes(27, 29) . More experiments are needed to
determine if Sp1, its family members, or other novel GC-box binding
proteins are involved in the regulation of p15 and p21 genes by TGF-
. It is possible that the subtle sequence
variations and the optimal spacing between binding sites for various
transcription factors could alter the balance between the positive and
negative transcription regulators and consequently exert a different
mode of transcription regulation.