(Received for publication, August 3, 1995; and in revised form, September 26, 1995)
From the
The transforming growth factor s (TGF-
s) are a group
of multifunctional growth factors that inhibit cell cycle progression
in many cell types. The TGF-
-induced cell cycle arrest has been
partially attributed to the regulatory effects of TGF-
on both the
levels and activities of the G
cyclins and their
cyclin-dependent kinase partners. The ability of TGF-
to inhibit
the activity of these kinase complexes derives in part from its
regulatory effects on the cyclin-dependent kinase inhibitors,
p21/WAF1/Cip1, p27
, and p15. Upon treatment of cells
with TGF-
, these three inhibitors bind to and block the activities
of specific cyclin-cyclin-dependent kinase complexes to cause cell
cycle arrest. Little is known, however, on the mechanism through which
TGF-
activates these cyclin-dependent kinase inhibitors. In the
case of p21, TGF-
treatment leads to an increase in p21 mRNA. This
increase in p21 mRNA is partly due to transcriptional activation of the
p21 promoter by TGF-
. To further define the signaling pathways
through which TGF-
induces p21, we have performed a detailed
functional analysis on the p21 promoter. Through both deletion and
mutation analysis of the p21 promoter, we have defined a 10-base pair
sequence that is required for the activation of the p21 promoter by
TGF-
. In addition, this sequence is sufficient to drive
TGF-
-mediated transcription from a previously nonresponsive
promoter. Preliminary gel shift assays demonstrate that this TGF-
responsive element binds specifically to several proteins in
vitro. Two of these proteins are the transcription factors Sp-1
and Sp-3. These studies represent the initial steps toward defining the
signaling pathways involved in TGF-
-mediated transcriptional
activation of p21.
The transforming growth factor s (TGF-
s), (
)a group of protein hormones that regulate many cellular
functions, inhibit cell proliferation by causing growth arrest in the
G
phase of the cell
cycle(1, 2, 3, 4) . Progression
through G
is dependent on the sequential formation,
activation, and subsequent inactivation of the G
cyclin-cyclin-dependent kinase complexes, primarily cyclin
D-cyclin-dependent kinase 4 and cyclin E-cyclin-dependent kinase 2
complexes(5, 6) . The TGF-
-induced G
cell cycle arrest has been attributed to the regulatory effects
of TGF-
on both the levels and activities of these G
cyclins and cyclin-dependent
kinases(7, 8, 9) . The inhibition of G
cyclin-cyclin-dependent kinase complex activity by TGF-
is
mediated in part through several members of a recently described family
of low molecular weight cyclin dependent kinase inhibitors. These
cyclin-dependent kinase inhibitors, which include p21/WAF1/Cip1,
p27
, p57
, p18, p16, and p15, physically
associate with their target cyclins, cyclin-dependent kinases, or
cyclin-cyclin-dependent kinase complexes to inhibit their activities
(reviewed in (10, 11, 12, 13) ).
TGF-
regulates the activities of three of these cyclin-dependent
kinase inhibitor family members: p27
, p15, and p21
(reviewed in Refs. 10 and 11).
p21 was first cloned and
characterized as an important effector that acts to block cyclin
E-cyclin-dependent kinase 2 complex kinase activity in p53-mediated
cell cycle arrest induced by DNA damage (14, 15, 16, 17, 18, 19, 20, 21) .
p21 has subsequently been shown to be induced by TGF- (22, 23, 24) and has been implicated as an
effector of the TGF-
growth inhibitory signaling
pathway(22) . The increase in p21 seen on TGF-
treatment
is mediated, in part, through a transcriptional activation of the p21
promoter by TGF-
via a p53-independent mechanism(22) .
Although great strides have been made in understanding the
mechanisms through which TGF- inhibits the G
cyclincyclin-dependent kinase complexes, the signaling pathways between
the TGF-
receptor complex and these downstream targets remain
unknown. The finding that TGF-
transcriptionally activates the p21
promoter opens an opportunity to study these signaling pathways. We
describe here a detailed functional analysis of the p21 promoter as a
first step in defining the pathway through which TGF-
activates
p21 expression and causes cell cycle arrest.
Figure 1:
Deletion analysis of
the p21 promoter. Full-length and deletion p21 promoter reporter
constructs were transfected into HaCaT, and TGF--induced
luciferase activity was measured in relative light units (RLU). Fold induction was calculated by comparing the
luciferase activity of cells treated with TGF-
and untreated
controls. In each experiment, duplicate transfections were performed.
These results are from a representative experiment. The underlined sequence was found to be essential for TGF-
-mediated
activation of the promoter constructs. A nearly consensus p53 binding
site is indicated at the 5` end of the full-length promoter
construct.
Mutagenesis of p21P 93-S was performed using the Muta-Gene M13 In Vitro mutagenesis kit (Bio-Rad). The oligonucleotides used in the mutagenesis reactions are as follows: p21P 93-S mut#1, CCTCAAGGAGGCGGGGTTTCTAGATATCGAATTCCTG; p21P 93-S mut#2, CCCGGGCCCGCCTCAGTTCTAGATAACCCGCGCTC; p21P 93-S mut#3, CCGCCCGCCCGGATTTCTAGAGAGGAGGCGGG; p21P 93-S mut#4, CCTGATATACAACCGTATTCTAGAAGCCCGCCTCAAGGCGGG; p21P 93-S mut#5, CAGCGCGGCCCTGACGCGTCTAGACCCCGCCCGG; p21P 93-S mut#6, GCTGGCGCAGCTCAGCATCTAGACAATATACAACCGCC; p21P 93-S mut#2.2, CCCGCCTCAAGGATCCGGGACCCGC; p21P 93-S mut#2.3, CCCGCCTCAAGCCGGCGGGACCCGCGCTC. The presence of the desired mutations was determined by sequence analysis.
The DNA construct P1634, which
contains a consensus TATA box and initiator sequence, was a gift from
Dr. S. Smale(26) . The consensus TATA and INR sequences
contained on a BglII-HindIII fragment were
subcloned into pGL2-basic to create pGL2 T+I. pGL2 T+1 1
T
RE, 2
T
RE, and 4
T
RE were
created by first synthesizing complementary oligonucleotides
representing the sequences in the p21 promoter between bases -71
and -86. These oligonucleotides were annealed and cloned into the EcoRV site of P1634. The BglII-HindIII
fragment of the resulting construct was subcloned into pGL2-basic. The
number and orientation of the inserts was determined by sequence
analysis.
Binding reactions were performed at 4 °C. 3 µl
of nuclear extract (3-6 µg of protein) was incubated with 1
µg of poly(dI-dC) in 11 µl of hypotonic lysis buffer to give a
final salt concentration of 100 mM NaCl. Nonlabeled specific
competitors were then added, and after a 5-min incubation, 40,000 cpm
of radiolabeled probe was added. Binding reactions were resolved on a
0.5 TBE, 5.5% acrylamide gel at 200 volts for 2 h at 4 °C.
The gels were subsequently dried, and autoradiography was performed.
For Sp-1 and Sp-3 supershift assays, 2 µl of Sp-1 or Sp-3 specific
polyclonal antibody (27) was added to the binding reaction
prior to the addition of radiolabeled probe.
Using two
convenient SmaI sites in this region, the deletion construct
p21Psma was created, which contains the p21 promoter sequences from
base -111 through the transcriptional initiation site (15) . This construct was activated by TGF- to a level
similar to that of the full-length p21P, further defining the TGF-
responsive element to a 50-base pair region between -62 and
-111. In addition to decreasing TGF-
-mediated activation of
transcription, removal of this 50 base pair region also decreases basal
promoter activity. When this 50-base pair region was removed from the
full-length p21 promoter, the TGF-
-induced luciferase activity of
the resulting construct, p21PSma
2, was significantly less than
that of the wild type promoter (Fig. 1). These results suggest
that the 2.3-kilobase pair 5` end of the p21 promoter proximal to the SmaI site at -111 is not required nor sufficient for
induction of the promoter by TGF-
. Finally, the construct p21P
93-S, which contains the promoter sequences between -62 and
-93, was activated by TGF-
to a level similar to that of the
wild type promoter (Fig. 2). Thus, the TGF-
responsive
element is harbored in a 32-base pair region between -93 and
-62.
Figure 2:
Mutation analysis of the p21 promoter.
Mutants #1-#6 are identical to the wild type p21P 93-S sequence
with the exception of the sequences shown for each mutant construct.
p21P 93-S mut#2.2 and mut#2.3 are identical to the wild type p21P 93-S
with the exception of the underlined bases. These constructs
were transfected into HaCaT and TGF--induced luciferase activity
was measured. Luciferase activity was normalized to protein
concentration. Fold induction was calculated by comparing the
luciferase activity of cells treated with TGF-
and untreated
controls. Transfection were done in duplicate in each experiment, and
these results are those of a representative experiment. The region of
the p21 promoter found to be necessary for induction by TGF-
has
been underlined and labeled T
RE. Several
putative Sp-1 sites are also indicated.
To more
precisely map this specific region of the p21 promoter, two additional
mutant constructs were created. p21P 93-S mut#2.2 has a mutation of
bases -78 and -79 from CC to AG, and p21P 93-S mut#2.3 has
a mutation of bases -76 and -77 from CT to GG (Fig. 2). These two mutant constructs have a significantly
reduced ability to be activated by TGF- (Fig. 2). Not only
do these mutations provide insight into the bases that are essential in
binding the factors driving TGF-
-mediated transcription, but they
serve as a useful control in the electrophoretic mobility shift assays
(EMSAs) studies described below.
Figure 3:
Control of a consensus TATA + INR
sequence by concatenated TREs. A, constructs containing a
luciferase reporter gene driven by either a consensus TATA box and
initiator sequence or a TATA box and INR sequence with the addition of
one, two, or four copies of the p21 promoter sequence between bases
-71 and -86 were created. B, these constructs were
transfected into HaCaT, and TGF-
-induced luciferase activity was
measured and normalized to protein concentration. The errors bars represent the average deviation between two separate transfections
in a single experiment.
Figure 4:
EMSA of TRE binding proteins. A, nuclear extract from TGF-
-treated or untreated HaCaT
was incubated with an end-labeled T
RE DNA probe corresponding to
regions -71 to -86 of the wild type p21 promoter sequence.
In each panel an increasing amount of unlabeled specific competitor was
used corresponding to the sequences between -71 and -86 of
the wild type p21 promoter, p21P93-S mut#2.2, or p21P93-S mut#2.3. B, DNA probes corresponding to the bases -71 through
-86 of the wild type p21 promoter, p21P93-S mut#2.2, and p21P93-S
mut#2.3 were end-labeled and used in an EMSA with both
TGF-
-treated and untreated HaCaT nuclear extract. Each EMSA was
performed in the presence and the absence of a 100-fold excess of
unlabeled DNA probe as a specific competitor. C, EMSA was
performed using the wild type T
RE probe as in A with the
inclusion of polyclonal antibodies to either Sp-1, Sp-3, or preimmune
serum.
To investigate the mechanism through which TGF- induces
the expression of p21, we have performed a detailed functional analysis
of the p21 promoter. Through both deletion and mutation analysis, we
have defined the sequences required for TGF-
-activated
transcription and demonstrated that the sequences could confer
TGF-
inducibility to a minimal basal promoter. We have termed this
sequence a T
RE, TGF-
responsive element.
EMSA with the
wild type and mutant TRE sequences and nuclear extracts from HaCaT
revealed three specific bands of retarded mobility. The presence of
these three retarded bands is correlated functionally with the ability
of the T
RE to drive TGF-
-mediated transcription. No change in
the pattern or intensity of the retarded bands was observed when
nuclear extract from TGF-
-treated HaCaT was used. Thus, TGF-
is not activating transcription by increasing the binding of these
proteins to the T
RE sequence. Consistent with this observation,
the induction of p21 mRNA by TGF-
was not blocked by pretreatment
of cells with cyclohexamide, a protein synthesis inhibitor (data not
shown). Therefore, the factors responsible for the induction of p21 by
TGF-
are likely to be pre-existing. These factors may become
phosphorylated or interact with proteins that are modified or
phosphorylated upon TGF-
treatment to activate transcription.
Because the TGF--responsive site in the p21 promoter contains a
nearly consensus Sp-1 binding site, we investigated whether the
retarded bands on EMSA represent the binding of the transcription
factor Sp-1 or other Sp-1 family members. Using Sp-1- and Sp-3-specific
polyclonal antibodies, we were able to show that both Sp-1 and Sp-3 are
capable of binding to the p21 T
RE sequence. This result alone does
not demonstrate that Sp-1 and its family members are involved in
TGF-
-mediated signal transduction. Other unidentified proteins,
capable of binding to this sequence, may be responsible for
TGF-
-activated transcription. An important role for Sp-1, however,
is supported by our recent finding that TGF-
activates
transcription from the promoter of the p15 gene, another
TGF-
-activated cyclin-dependent kinase inhibitor, through a
consensus Sp-1 site(36) . (
)In a previous study of
the mouse
2 collagen promoter, an Sp-1 site was also found to be a
critical component of the TGF-
-inducible element(29) .
Taken together, these studies suggest that a subset of
TGF-
-inducible genes may contain a similar TGF-
responsive
element that may be activated by TGF-
through Sp-1 or Sp-1-like
factors capable of recognizing an Sp-1 consensus site.
Although the
functionally defined TRE is capable of activating transcription by
itself, the region of the p21 promoter between bases -54 and
-63, which contains an Sp-1 binding site, also appears to play an
important role in TGF-
-activated transcription. Mutations in this
site reduce the TGF-
-mediated induction of the p21 promoter by
half, in contrast to mutations in the T
RE Sp-1 site, which
eliminates TGF-
-mediated induction. This suggests that these Sp-1
sites are functionally different. This may be due to differences in
their spacing from the transcriptional initiation site or due to subtle
base pair differences in their sequence.
Sp-1 binding sites have been described in many promoters, and consequently Sp-1 has fallen into the stereotype of being a ``house keeping'' transcription factor, whose activity is necessary solely for the basal transcription of many genes. This simple model for Sp-1 was complicated by the identification of a number of Sp-1-related proteins (28, 30, 31, 32, 33) . All of these Sp-1 family members are capable of interacting with a consensus Sp-1 site. Several family members are expressed in a cell type-specific manner(31, 32, 33) . These findings suggest that the presence of an Sp-1 binding site is not simply to ensure adequate basal transcription but may serve as a site for the interplay of several differentially expressed transcription factors.
Perhaps the most suggestive work demonstrating that Sp-1
may play an important role in regulated transcription is the
demonstration that Sp-1 is a critical factor in regulating
transcription mediated by the retinoblastoma protein, Rb. Several
groups have shown that Rb overexpression can lead to the activation of
a number of genes including c-Fos and
TGF-1(27, 34, 35) . The Rb control
elements (RCEs) in these promoters have been defined and found to
interact with Sp-1(27, 34, 35) . Although no
direct association between Rb and Sp-1 could be detected and no
specific regulation through protein association or phosphorylation has
been observed, the effect of Rb overexpression on this element is
believed to be through Sp-1. Experiments have shown that the binding of
Sp-1 alone is both necessary and sufficient for the ability of these
Rb-responsive promoters to be activated by Rb(35) .
A
potential connection may be made between the TGF--responsive
element defined here and the previously described RCE. Like the RCE,
the p21 T
RE is capable of binding Sp-1. The p21 T
RE,
therefore, may represent an RCE. By maintaining Rb in an
hypophosphorylated state, TGF-
may be exerting its effects on the
p21 promoter through Rb. Induction of p21 would in turn lead to a
further increase in the hypophosphorylated form of Rb, thus
establishing a positive feedback loop between p21 and Rb, ensuring an
effective G
cell cycle arrest. It should be noted that
although the model of positive feedback is attractive, it remains to be
determined if the T
RE in the p21 promoter can function as the
previously described RCEs.