(Received for publication, August 31, 1995; and in revised form, October 31, 1995)
From the
The Waf1/Cip1 protein induces cell cycle arrest through inhibition of the activity of cyclin-dependent kinases and proliferating cell nuclear antigen. Expression of the WAF1/CIP1 gene is induced in a p53-dependent manner in response to DNA damage but can also be induced in the absence of p53 by agents such as growth factors, phorbol esters, and okadaic acid. WAF1/CIP1 expression in U937 human leukemic cells is induced by both phorbol ester, a protein kinase C activator, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Both of these agents induce the differentiation of these leukemic cells toward macrophages. We demonstrate that phorbol esters and okadaic acid stimulate transcription from the WAF1/CIP1 promoter in U937 cells. This transcription is mediated by a region of the promoter between -154 and +16, which contains two binding sites for the transcription factor Sp1. Deletion or mutation of these Sp1 sites reduces WAF1/CIP1 promoter response to phorbol ester and okadaic acid, while a reporter gene under the control of a promoter containing only multiple Sp1 binding sites and a TATA box is induced by phorbol ester and okadaic acid. The WAF1/CIP1 promoter is also highly induced by exogenous Sp1 in the Sp1-deficient Drosophila Schnieder SL 2 cell line. These results suggest that phorbol ester and okadaic acid activate transcription of the WAF1/CIP1 promoter through a complex of proteins that includes Sp1 and basal transcription factors.
Treatment of the human myeloid leukemic cell line U937 with
phorbol esters such as phorbol myristate acetate (PMA), ()an
activator of protein kinase C, leads to macrophage/monocyte-like
differentiation over a 72-h period(1, 2) . This
process involves changes in cell-substrate adherence, growth arrest in
late G
, and increased expression of monocyte
markers(3, 4) . Similarly, treatment of U937 cells
with okadaic acid, a natural product isolated from the black sponge and
a potent inhibitor of protein phosphatases 1 and 2A, also induces
differentiation of these cells(5) , cell cycle arrest, and
eventual (72-h) apoptosis(6) . Both PMA and okadaic acid induce
expression of the cyclin-dependent kinase inhibitor, WAF1/CIP1(7, 8) .
WAF1/CIP1 expression is induced by the p53 protein following irradiation of
cells(9, 10) , but p53-independent expression of WAF1/CIP1 is associated with differentiation of myocytes (11, 12) , of HL 60 leukemic cells(13) , and
of a number of other cells. WAF1/CIP1 is expressed in a number
of tissues over the course of murine development, and expression in
most tissues is not dependent on the presence of p53(14) .
p53-independent expression of the WAF1/CIP1 gene can be
induced in cultured cells by a number of agents, including, besides PMA
and okadaic acid, platelet-derived growth factor, fibroblast growth
factor, and transforming growth factor (15, 16) .
Preliminary analysis of the WAF1/CIP1 promoter suggests
that the elements mediating response to serum in fibroblasts are
located at least 1.9 kb upstream from the transcription start
site(14) , while responsiveness to tumor growth factor is
mediated by elements located somewhere in the promoter sequences 1.3 kb
upstream of the transcription start site(17) . We now report
that two sites that bind the transcription factor Sp1, located
approximately 115 and 65 base pairs upstream from the transcription
start site of the WAF1/CIP1 gene, are necessary for normal
levels of basal, PMA-induced, and okadaic acid-induced transcription.
These sites are also necessary for induction of the WAF1/CIP1 promoter by exogenous Sp1 in the Sp1-deficient Drosophila Schneider SL 2 cell line. Finally, our observation that a reporter
plasmid containing multiple Sp1 binding sites and a TATA box shows
transcriptional induction in response to PMA and okadaic acid in U937
cells suggests that the activity of Sp1 is sufficient for induced
transcription of the WAF1/CIP1 gene.
Figure 6: Exogenous Sp1 stimulates transcription of the WAF1/CIP1 promoter in the Sp1-deficient Drosophila Schneider SL 2 cell line. 4 µg of the indicated WAF1/CIP1 reporter constructs were transfected into SL 2 cells either with (+) or without(-) 100 ng of the Sp1 expression vector, pPacSp1. The numbers below the Sp1 (+) lanes give average induction in response to Sp1 calculated from four independent transfection experiments.
Treatment of U937 cells with either PMA or okadaic acid induces accumulation of WAF1/CIP1 mRNA (Fig. 1). Treatment with 100 nM PMA results in maximum levels of RNA by 2-4 h, while cells treated with 100 nM okadaic acid do not accumulate maximal levels of WAF1/CIP1 mRNA until 8 h (Fig. 1). This difference in the rate of induction between these agents is based on the concentration of activator employed, since higher concentrations of okadaic acid, 500 nM, result in increases in the level of WAF1/CIP1 mRNA levels at earlier time points (Fig. 1).
Figure 1: Accumulation of WAF1 mRNA in response to treatment of U937 cells with PMA or okadaic acid. U937 cells were treated with 100 nM PMA, 100 nM okadaic acid, or 500 nM okadaic acid as indicated above the lanes. At the indicated times, cells were collected and used to prepare RNA for Northern blot analysis. 20 µg of total RNA was loaded in each lane, and filters were first probed with radiolabeled WAF1/CIP1 cDNA and then stripped and reprobed with tubulin cDNA as a loading control.
To determine whether PMA and okadaic
acid stimulate transcription from the WAF1/CIP1 promoter,
contructs containing varying lengths of the WAF1/CIP1 promoter
in front of a CAT reporter gene were transfected into U937 cells. Each
set of transfected cells was split into equal aliquots, which were
treated with PMA or okadaic acid or left untreated as controls. The
smallest construct, containing 170 base pairs of promoter sequence
(from 154 bases upstream of the transcription start site at +1 to
+16), was fully inducible by both PMA and okadaic acid (Fig. 2). When compared with the -2320/+16 construct (Fig. 2), the -154/+16 construct was more strongly
induced by PMA (19.5- versus 7.3-fold) and okadaic acid (16.1- versus 9.8-fold), suggesting that upstream elements that
repress transcription may be found between -2320 and -154.
A series of intermediate constructs displayed a gradual increase in
response as promoter sequence was deleted from -2320 to
-154, but all constructs were induced by PMA and okadaic acid. ()The two p53 binding sites identified at positions
-2.3 kb and -1.4 kb do not play any role in the induction
by these two agents since they are deleted in the smaller constructs
without any affect on transcription. Further analysis of promoter
sequences downstream from -154 revealed that a deletion of
promoter sequences between -122 and -61 eliminated both
basal and inducible promoter activity (see Fig. 3). When the
-122/-61 deletion is introduced into a construct containing
2.3 kb of upstream WAF1/CIP1 promoter sequence, induction of
transcription in response to PMA or okadaic acid is lost (Fig. 2), demonstrating that these sequences are important for
inducible transcription and cannot be replaced by upstream elements.
These elements are also important for induction in response to p53.
Although U937 cells contain no wild-type p53, cotransfection of a wild
type p53 expression vector induces transcription from the undeleted
2.3-kb WAF1/CIP1 promoter (Fig. 2). The
-122/-61 deletion, however, abolishes induction in response
to p53. Similar results were obtained when the two promoter constructs
were compared in the GM glioma cell line, which contains a wild type
p53 gene under the control of a steroid-inducible promoter (data not
shown). These results indicate that WAF1/CIP1 promoter
elements within the -122/-61 region are necessary for both
p53-dependent and p53-independent induction.
Figure 2:
Promoter sequences between -122 and
+16 mediate phorbol ester and okadaic acid induction of the WAF1/CIP1 promoter. A, diagrammatic representation of
CAT constructs containing between 2.3 kilobases and 170 bases of WAF1/CIP1 promoter sequence. All constructs shown have a 3`
terminus at +16, where +1 is the transcription start site. B, constructs were electroporated into U937 cells; one-third
were treated with 200 nM PMA, one-third were treated with 100
nM okadaic acid, and one-third were untreated controls, as
indicated below the panels. 24 h later CAT activity
was assayed; for each construct, one assay is shown and average
induction in response to PMA or OK calculated from three independent
transfection experiments is listed below each lane.
All transfections included cytomegalovirus/-galactosidase plasmid;
cell extracts were assayed for galactosidase activity to ensure equal
transfection efficiency (galactosidase assays not shown). C,
U937 cells were cotransfected with the indicated WAF1/CIP1 promoter constructs and vectors expressing either wild-type p53 (WT) or mutant p53 (Mut). CAT activity was assayed 24
h after transfection.
Figure 3: Regions containing Sp1 consensus binding sites mediate promoter response to PMA and okadaic acid. A diagram of CAT contructs with WAF1/CIP1 promoter sequence deleted between -154 and -61 is shown in the upper part of the figure. The lower part shows CAT assays from U937 cells transfected with the WAF1/CIP1 promoter construct indicated above each panel and then split into three aliquots and treated with 200 nM PMA, treated with 100 nM OK, or left untreated(-) as indicated beneath the panels. The basal transcription and -fold activation numbers shown below are the averages of three independent transfection experiments. All experiments included one transfection with the -154/+16 WAF1/CIP1 promoter construct as a standard for basal activity. -Fold activation for each construct was calculated based on the basal activity of that particular construct.
The WAF1/CIP1 promoter sequence between -122 and -61 does not
contain consensus binding sites for factors such as AP 1, Egr-1, or
NF-B, which are known to activate other genes in response to
phorbol esters or okadaic acid(22, 23) . However, the
region does contain several consensus binding sites for the
transcription factor Sp1(7) . DNase I footprint analysis of
this region indicated that at least two Sp1 consensus binding sites,
centered around -115 and -67, fell within protected
regions.
PMA treatment of cells did not cause any
noticeable change in the footprints, suggesting that the binding of
factors to this region of the WAF1/CIP1 promoter is not
enhanced by PMA. To verify that these potential Sp1 binding sites were
important for induction of transcription in response to PMA and okadaic
acid, a series of CAT contructs were generated with deletions or
mutations of the Sp1 consensus sequences found in the
-122/-61 region of the WAF1/CIP1 promoter
(diagrammed in Fig. 3and Fig. 4). These constructs were
transfected into U937 cells and analyzed for basal activity and for
transcriptional response to PMA and okadaic acid ( Fig. 3and Fig. 4). As mentioned above, deletion of the
-122/-61 sequence, which includes several Sp1 consensus
binding sites, markedly decreased both basal and induced transcription.
All transcription experiments included one transfection using the
-154/+16 promoter construct; the basal transcription of this
construct was set at 1.0, and basal transcription of all other
constructs was normalized to this value. For each construct, percentage
conversion of chloramphenicol to the acetylated form in PMA or okadaic
acid-treated cells was divided by percentage conversion in untreated
cells to obtain values for induced transcription.
Figure 4: A consensus Sp1 binding site between bases -117 and -112 is necessary for WAF1/CIP1 promoter activation in response to PMA or okadaic acid. Mutated PCR primers were used to introduce base pair changes to the wild type WAF1/CIP1 promoter sequence, which are indicated by underlining. Consensus Sp1 binding sites are shown above the sequence. U937 cells were transfected as described in Fig. 4; the numbers represent an average of three independent transfections, and were calculated as in Fig. 4.
Deletion of sequences between -131 and -117, immediately 5` to the upstream Sp1 consensus binding site, decreased PMA induction by a small amount and decreased okadaic acid induction approximately 50%. A further deletion of sequences from -117 to -100, which eliminates the Sp1 binding sites entirely, markedly decreased basal transcription and induction in response to PMA and okadaic acid. In comparison, deletion of the downstream region between -81 and -62 knocked out okadaic acid response while having little effect on PMA response. PMA induction appears to require only the upstream element, while okadaic acid induction requires both the upstream and downstream elements.
To more precisely examine the role of the upstream Sp1 consensus site in mediating WAF1/CIP1 transcription, mutations were introduced into the -131/+16 promoter construct (Fig. 4). Mutation of three bases in the first Sp1 site decreased both basal and induced transcription by PMA and okadaic acid; the reduction in activity was approximately the same as that observed when the region was deleted. This result confirms that the Sp1 binding site is necessary for induction. Mutation of bases outside the Sp1 consensus sequence had little effect on promoter activity (Fig. 4).
To test for binding of Sp1 (or other factors) at these sites, double-stranded oligonucleotides containing WAF1/CIP1 promoter sequence from -128 to -99 or from -86 to -57 were used for gel mobility shift experiments with nuclear extracts from U937 cells (Fig. 5). Both oligonucleotides bind to a set of three proteins or protein complexes, which closely resemble the set of proteins previously observed to bind to both Sp1 sites and retinoblastoma control elements(24, 25) . These proteins are usually designated 1A, 1B, and 2, (see Fig. 5); 1A has been identified as the Sp1 gene product based on interactions with anti-Sp1 antibodies(25) , and the other bands are postulated to be Sp1-related proteins(26) . As shown in Fig. 5, preincubation of U937 nuclear extract with anti-Sp1 antibodies (21) disrupts binding of the 1A protein to both WAF1/CIP1 promoter oligonucleotides. All three proteins (1A, 1B, and 2) can also be competed off the WAF1/CIP1 -128/-99 promoter oligonucleotide with excess unlabeled -128/-99 oligonucleotide or an oligonucleotide containing the SV 40 Sp1 binding site (Promega) but not by an oligonucleotide that does not contain an Sp1 consensus sequence (see ``Materials and Methods'' for sequence), suggesting that the proteins that bind to both footprinted regions are Sp1 or Sp1-related factors.
Figure 5: Sp1 and related proteins bind to the WAF1/CIP1 promoter at the sites protected from DNase I digestion. Gel mobility shift experiments were performed using either radiolabeled -128/-99 oligonucleotide or radiolabeled -86/-57 oligonucleotide, containing the sequences from the WAF1/CIP1 promoter protected from DNase I digestion and necessary for PMA/okadaic acid induction, as shown in Fig. 3and Fig. 4. Oligonucleotides were incubated with U937 cell nuclear extract and preimmune serum or anti-Sp1 serum (first four lanes on the left). Nuclear extracts were also incubated with radiolabeled -128/-99 WAF1/CIP1 promoter oligonucleotide and with cold competitor oligonucleotides (right). The competitor oligonucleotides used are indicated above the lanes: the -128/-99 oligonucleotide itself, a WAF1/CIP1 promoter oligonucleotide containing no Sp1 sites, and an oligonucleotide containing the SV 40 Sp1 binding site. The set of proteins that bind to the Sp1 consensus sites are commonly designated 1A, 1B, and 2, as indicated on the left.
To verify that Sp1 activates transcription of the WAF1/CIP1 promoter, WAF1/CIP1 contructs were transfected into the Sp1-deficient Drosophila Schneider SL 2 cell line either in the presence or in the absence of the Sp1 expression vector pPacSp1 (27) . In the Sp1-deficient Drosophila cells, WAF1/CIP1 constructs containing sequence from -117 to +16 are highly induced in response to exogenous Sp1 expression (Fig. 6). Deletion of either the upstream or downstream Sp1 binding sites individually has a partial effect on the level of expression in response to Sp1, but deletion of both sites results in a much greater reduction in promoter activity induced by cotransfecting the Sp1 expression vector (Fig. 6). This result is similar to the pattern observed in U937 cells for response to PMA or okadaic acid, although deletion of individual sites can have a more severe effect on response to PMA or okadaic acid in the U937 cells.
Sp1 has been shown to interact with the TATA box binding protein (TBP)-associated protein TAF110 and, in collaboration with another TBP-associated protein, TAF250, activate transcription(28, 29) . If Sp1 and the complex of TBP proteins are sufficient for induction of transcription in response to PMA or okadaic acid, it is predicted that transcription of a reporter plasmid containing only multiple Sp1 binding sites and a TATA box would be stimulated by PMA or okadaic acid treatment of U937 cells. To evaluate this possibility the plasmid pGAGC6, containing six Sp1 binding sites and an adenovirus TATA box, was transfected into U937 cells, and the cells were treated with PMA or okadaic acid. Both PMA and okadaic acid induced transcription from this vector, while control vector not containing Sp1 sites was not induced (Fig. 7).
Figure 7: Transcription of a reporter plasmid containing six Sp1 binding sites is induced by PMA and okadaic acid in U937 cells. Plasmid pGAGC6, containing six Sp1 binding sites and an adenovirus TATA box upstream of a luciferase reporter gene, was transfected into U937 cells. Cells were treated with PMA or okadaic acid as in Fig. 2; cell extracts were then assayed for luciferase activity. -Fold induction is an average calculated from at least four independent transfection experiments. Vector without Sp1 sites (pGAM) was used as a control.
The association of WAF1/CIP1 expression with differentiation in many types of cultured cells suggests that p53-independent induction of WAF1/CIP1 may have a role in cell differentiation in vivo; the pattern of WAF1/CIP1 expression during mouse embryogenesis also correlates with terminal differentiation of skeletal muscle, cartilage skin, and nasal epithelium(12) . Identification of the WAF1/CIP1 promoter elements that mediate p53-independent induction of transcription should help to identify the signal transduction pathways that stimulate expression of WAF1/CIP1 during cell differentiation.
Since WAF1/CIP1 expression is stimulated
by a number of agents, including platelet-derived growth factor,
fibroblast growth factor, interleukin 2, tumor growth factor ,
phorbol esters, okadaic acid, retinoic acid, and vitamin
D
(15, 30) , it is possible that a number
of elements in the WAF1/CIP1 promoter function together to
precisely regulate the level of WAF1/CIP1 expression. Such
multiple signals might be necessary to generate sufficient WAF1/CIP1 expression for growth inhibition, since there is
some evidence that the ratio of WAF1/CIP1 protein to target molecules,
such as cyclin-dependent kinases, determines its effect(31) .
This idea is supported by the fact that the WAF1/CIP1 promoter
element responsible for induction in response to serum in fibroblasts
appears to be located between -1,817 and -4699 bases
upstream of the transcription start site(14) , while induction
in response to tumor growth factor
in SW480 cells is mediated by
elements between -61 base pairs and -1.1 kb
upstream(17) . These results suggest that there are at least
two p53-independent pathways for induction of WAF1/CIP1 transcription.
We now report that induction of the WAF1/CIP1 promoter in U937 leukemic cells by PMA and okadaic acid involves Sp1 binding sites located approximately 115 and 80 bases upstream of the transcription start site; loss of these sites results in lack of response to PMA and okadaic acid as well as loss of WAF1/CIP1 promoter induction in response to exogenous Sp1 in the Sp1-deficient Drosophila Schneider SL2 cell line. The upstream(-115) Sp1 site is vital for full response of the promoter to PMA in U937 cells, but all potential Sp1 binding sites between -122 and -61 must be deleted to abolish induction by exogenous Sp1 in the SL2 cells. The requirements for utilization of an Sp1 binding site in U937 cells may be more stringent due to the presence of Sp1-related proteins (26) or Sp1-inhibitory proteins (25) that are not present in the Drosophila SL2 cells. The fact that the reporter plasmid pGAGC6 (containing multiple Sp1 binding sites upstream of a TATA box) is induced by PMA and okadaic acid in U937 cells suggests that Sp1 may be sufficient for increased transcription of the WAF1/CIP1 promoter in response to PMA or okadaic acid treatment.
The Sp1 transcription factor is found in glycosylated and phosphorylated forms, but little is known about how these modifications affect function(32) . Interactions between Sp1 and the retinoblastoma protein have also been reported; a 20-kDa inhibitor of Sp1 (Sp1-I) was identified that also bound to Rb, and it was proposed that Rb binds and inactivates Sp1-I, leading to transcriptional activation by Sp1(24) . A 74-kDa protein that binds to the transactivation domain of Sp1 and inhibits Sp1-mediated transactivation has also been identified(33) . These reports suggest that there are multiple inhibitors of Sp1, which could inhibit interaction of Sp1 with the transcription factor IID complex(28) . It is also possible that proteins of the transcription factor IID complex are modified in response to PMA, or that the composition of the transcription factor IID complex is altered by gain or loss of TBP-associated factors (TAFs). Recent studies have shown that some TAFs may serve as coactivators to mediate transcriptional regulation(34) .
Signal transduction
pathways that activate or inactivate such Sp1 inhibitors may serve to
regulate Sp1 transcriptional activation and may therefore regulate WAF1/CIP1 and other genes involved in cell differentiation. A
number of signal transduction pathways composed of kinase cascades have
been described in the literature(35) . However, a variety of
signals that activate either the mitogen-activated protein kinase
pathway, the stress-activated protein kinase pathway, or the p38 kinase
pathway fail to induce WAF1/CIP1 transcription in U937 cells.
These signals include UV irradiation, activated mitogen-activated
protein kinase kinase, and osmotic shock. This suggests
that the signals that induce WAF1/CIP1 transcription via Sp1
may be part of an as yet unidentified pathway.