From the Eppley Institute for Research in Cancer and
Allied Diseases and § Department of Pathology and
Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
68198 and the
NCI, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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Previously, it has been shown that
differentiation of embryonal carcinoma (EC) cells turns on the
expression of functional transforming growth factor type- receptors.
Here, we show that the type II receptor (T
R-II) gene is
activated at the transcriptional level when EC cells differentiate. We
show that the differentiated cells, but not the parental EC cells,
express transcripts for T
R-II. In addition, the
expression of T
R-II promoter/reporter gene constructs
are elevated dramatically when EC cells differentiate and we identify
at least two positive and two negative regulatory regions in the 5'
flanking region of the T
R-II gene. Moreover, we identify
a cAMP response element/activating transcription factor site that acts
as a positive cis-regulatory element in the T
R-II promoter, and we demonstrate that the transcription factor ATF-1 binds
to this site and strongly stimulates the expression of the T
R-II promoter/reporter gene constructs when ATF-1 is
overexpressed in EC-derived differentiated cells. Equally important, we
identify a negative regulatory element in a 53-base pair region that
had previously been shown to inhibit strongly the expression of
T
R-II promoter/reporter gene constructs. Specifically,
we demonstrate that this region, which contains an inverted CCAAT box
motif, binds the transcription factor complex NF-Y (also referred to as
CBF) in vitro. Furthermore, expression of a
dominant-negative NF-YA mutant protein, which prevents DNA binding by
NF-Y, enhances T
R-II promoter expression. Together,
these studies suggest that the transcription factors ATF-1 and NF-Y
play important roles in the regulation of the T
R-II
gene.
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INTRODUCTION |
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Transforming growth factor type-
(TGF-
)1 refers to a
complex family of genetically distinct polypeptides that are secreted by virtually all cells and which exert potent effects on cell proliferation, differentiation, extracellular matrix production, and
immunoregulation (1-4). Based on these activities and the defined
spatial and temporal pattern of expression of the three mammalian
isoforms of TGF-
(TGF-
1, TGF-
2, and TGF-
3) during mouse
embryogenesis, it has been argued that the TGF-
s play important roles in the generation and modification of extracellular signals that
direct critical processes during mammalian development (5-12). This is
borne out by the induction of fetal defects in embryos that are unable
to produce the different isoforms of TGF-
(13-17), in particular
TGF-
2, and in embryos that cannot produce functional TGF-
receptors due to inactivation of the T
R-II gene by gene targeting (18). Given the importance of TGF-
during development, it
is not surprising that defects in TGF-
signal transduction have also
been implicated in the pathological processes of many diseases,
including arthritis, ulcerative diseases, atherosclerosis, and
glomerulonephritis (2, 19). Equally important, cells that lose the
ability to respond to TGF-
are more likely to exhibit uncontrolled
growth and to undergo neoplastic transformation (20-27).
TGF-s primarily exert their biological effects through interactions
with three distinct high affinity TGF-
cell surface receptors
(designated types I, II, and III). All three receptors have been cloned
and characterized (28-33). Both the type I (T
R-I) and type II
(T
R-II) receptors are transmembrane serine/threonine kinases that
act in concert to mediate intracellular signaling. The type III
(T
R-III) receptor (also referred to as betaglycan) is a
transmembrane proteoglycan devoid of intrinsic signaling ability, but
which may act to present ligand to other signaling receptors (34). The
most commonly held model for the activation of the TGF-
signal
transduction cascade proposes the selective binding of TGF-
to the
type II receptor, a constitutively active kinase (35). Ligand binding
to T
R-II induces recruitment of T
R-I into a stable complex. Once
this complex is formed, T
R-II transphosphorylates T
R-I at serine
and threonine residues, resulting in signal transduction to downstream
substrates. Thus, loss of responsiveness to TGF-
could result from
changes in the expression of functional TGF-
receptors rather than
defects in the expression or activation of TGF-
ligand.
Efforts to define the roles of TGF- and their receptors have
involved the study of embryonal carcinoma (EC) cells, as they are a
model system used frequently for studying early mammalian development
(36). EC cells resemble cells of the early mouse embryo morphologically
and biochemically. Moreover, they can be induced to differentiate into
many of the cell types formed during mammalian embryogenesis (37),
making them particularly well suited for the investigation of the
signal transduction events involved in cellular differentiation.
Furthermore, they provide a useful tool for the identification of
mechanisms involved in carcinogenesis, as EC cells are tumorigenic
whereas their differentiated cells are largely non-tumorigenic.
Using this model system, we demonstrated that EC cells do not express
detectable cell surface receptors for TGF- until after they are
induced to differentiate (38). Equally important, the up-regulation in
the expression of TGF-
receptors by the EC-derived differentiated
cells coincides with the ability of exogenous TGF-
to inhibit their
proliferation as well as to their loss of tumorigenic potential (38,
39). In the present study, we examined the expression of the
T
R-II gene both in F9 EC cells and their differentiated counterparts. Our findings demonstrate that few, if any,
T
R-II transcripts are expressed by F9 EC cells, whereas
there is a dramatic increase in the expression of T
R-II
mRNA when F9 EC cells are induced to differentiate. This
observation is supported by the differences in expression of various
T
R-II promoter/reporter gene constructs in EC cells and
their differentiated counterparts, arguing strongly that the large
increase in the steady-state levels of T
R-II mRNA
that accompany differentiation is due, at least in part, to an increase
in the transcription of the T
R-II gene promoter. In
addition, our results identify both positive and negative regulatory
regions in the T
R-II promoter that appear to contribute
significantly to the transcriptional activity of the
T
R-II gene in both EC and EC-derived differentiated
cells. In this regard, mutation of a CRE/ATF motif (
196 to
185)
within one of the positive regulatory regions reduces substantially
T
R-II promoter activity in the EC-derived differentiated
cells. Gel mobility shift analyses demonstrates that the transcription
factor ATF-1 is able to bind to this CRE/ATF motif in vitro.
We demonstrate further that expression of ATF-1 in vivo
up-regulates T
R-II promoter activity in the
differentiated cells, most likely through the CRE/ATF motif.
Conversely, we have identified an inverted CCAAT box motif (
83 to
74) that appears to negatively influence the transcriptional activity
of the T
R-II promoter in both EC cells and their
differentiated counterparts. The T
R-II CCAAT box motif is
bound in vitro by the transcription factor complex, NF-Y
(also referred to as CBF) in both cell types. Importantly, we
demonstrate that expression of a dominant-negative NF-YA mutant
protein, which prevents DNA binding of the NF-Y transcription factor
complex, specifically enhances the expression of T
R-II
promoter/reporter gene constructs in both F9 EC cells and their
differentiated cells. Together, these studies suggest that ATF-1 and
NF-Y play important roles in the regulation of the T
R-II
gene.
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MATERIALS AND METHODS |
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Cells and Culture Conditions-- Stock cultures of mouse F9 EC, mouse PYS-2 and mouse PSA-5E cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Intergen Company, Purchase, NY) as reported previously (40, 41). Differentiation of F9 EC cells was induced by treatment with 5 µM all-trans-retinoic acid (RA, Eastman Kodak Co.). Stock cultures and all experimental cultures were maintained at 37 °C in a moist atmosphere of 95% air and 5% CO2.
RNA Isolation and Northern Blot Analysis-- Poly(A)+ RNA was isolated from nearly confluent cultures of F9 EC cells and F9-differentiated cells by the Invitrogen FastTrack 2.0 Kit (Invitrogen, San Diego, CA). F9-differentiated cells were derived from F9 EC cells treated with 5 µM RA for 5 days. For Northern blotting, 3 µg mRNA samples from each cell type were denatured in 1× MOPS running buffer (40 mM MOPS, pH 7, 10 mM sodium acetate, and 1 mM EDTA), 2.2 M formaldehyde, and 50% formamide at 65 °C for 15 min and electrophoresed in a 1% agarose gel containing 1× MOPS running buffer in 0.22 M formaldehyde. After electrophoresis, the fractionated mRNA was transferred to an MSI nylon membrane (Fisher Scientific, Pittsburgh, PA) in 10× SSPE (1.5 M NaCl, 0.1 M NaH2PO4, and 10 mM EDTA, pH 7.4). Following transfer, the membrane was baked at 80 °C for 2 h.
Filters were prehybridized for 3 h at 42 °C in a solution consisting of 5× SSPE, 5× Denhardt's solution, 50% deionized formamide, 1% SDS, and 100 µg/ml denatured salmon testis DNA. Hybridization was performed in the same buffer with 2 × 106 cpm/ml of 32P-labeled probe for 16-18 h at 42 °C. The 32P-labeled probe was obtained by SacI digestion of the human TExpression Plasmids--
TR-II
promoter/chloramphenicol acetyltransferase (CAT) reporter
gene expression plasmids were generated and amplified by polymerase
chain reaction using genomic DNA containing the 5'-untranslated region
of the human T
R-II gene as a template and cloned into pGEM4-SVOCAT (42). The constructs were named
pT
RII-n, where n is the distance in
nucleotides from the transcription initiation site identified by
Humphries et al. (43) and Bae et al. (42). The
expression plasmids pECEATF-1 and
pECEATF-2 were provided by Dr. Michael O'Reilly
(University of Rochester, Rochester, NY). These plasmids contained the
human ATF-1 and ATF-2 cDNAs (44) inserted
into the expression plasmid pECE (45). The eukaryotic expression
plasmid pNFYA29 was obtained with permission of Dr. R. Mantovani from Dr. Peter Edwards (UCLA School of Medicine). This
plasmid uses an SV40 promoter to drive the expression of the
NFYA29 mutant protein (46). The normalization plasmid,
pCMV-
(CLONTECH) contains the
-galactosidase reporter gene under the control of the CMV
immediate early promoter (47). The plasmid pDOL-CMV-CAT
contains the CAT reporter gene under the control of the
CMV immediate early promoter (48) and was used as a positive control to monitor the general transcriptional activity of F9 EC and
F9-differentiated cells. All plasmids were verified by sequencing, and
purified by Qiagen tip-500 columns.
Transient Transfection Assay--
F9 EC cells, F9-differentiated
cells (day 3), PYS-2 cells, and PSA-5E cells were transfected by the
calcium phosphate precipitation method as described previously (49).
Typically, 20 µg of each TR-II promoter/CAT
plasmid was co-transfected with 2 µg of the internal standard,
pCMV-
. After an overnight incubation with the
DNA-CaPO4 precipitate, the cells were washed twice with
serum-free medium and refed with Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. CAT activities were
determined 48 h after transfection by the method of Seed and Sheen
(50) and normalized to
-galactosidase activity by the method of
Rosenthal (51) to adjust for differences in transfection efficiency
(52). In some experiments, 10 µg of pT
RII-n constructs
were co-transfected with optimal concentrations of the expression
plasmids containing human ATF-1 or ATF-2
cDNAs or the dominant negative mutant, NFYA29.
Preparation of Nuclear Extracts and Gel Mobility Shift Assay-- Nuclear extracts from F9 EC and F9-differentiated (day 5) cells were prepared as described previously (53, 54) with slight modifications of the original method of Dignam et al. (55). Nuclear extracts were prepared in the presence of the following protease inhibitors: pepstatin A, antipain, chymostatin, and leupeptin (all at 1 µg/ml), PMSF (1 mM), soybean trypsin inhibitor (20 µg/ml), benzamidine (2.5 mM), and aprotinin (2.5 kallikrein-inactivating units/ml). Nuclear extracts also contained protein phosphatase inhibitors: (NH4)2MoO4 (1 mM) and NaF (5 mM). Protein concentrations were determined using the Pierce Micro BCA protein assay reagent (Pierce).
Gel mobility shift assays were based on the method of Fried and Crothers (56) as described previously (53). Reaction mixtures were incubated for 20 min at room temperature with 12 µg of F9 EC cell and F9-differentiated cell nuclear extracts. Each reaction mixture contained 1 µg of poly(dI)-poly(dC) (Amersham Pharmacia Biotech) as nonspecific competitor DNA. The double-stranded oligodeoxynucleotide (dsODN) probes containing the wild type or mutant sequences of the human T ![]() |
RESULTS |
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Expression of TR-II mRNA in F9 EC and F9-differentiated
Cells--
T
R-II expression is normally associated with ligand
binding and growth responsiveness of cells to TGF-
(21-23, 58). To identify possible mechanisms for the significant increase in TGF-
ligand binding and growth responsiveness that is observed when EC cells
are induced to differentiate (38), we initially examined mRNA
expression of the T
R-II gene in F9 EC cells and their
differentiated counterparts. Northern blot analysis of
poly(A)+ RNA from undifferentiated F9 EC cells detected
little or no transcript of approximately 5.2 kilobases that corresponds
to the size predicted for the T
R-II gene (Fig.
1)(30). However, the intensity of this transcript increased substantially (>15-fold) after F9 EC cells were
induced to differentiate with RA (Fig. 1). Thus, it appears that there
is a strong correlation between the expression of T
R-II mRNA in F9 EC cells and F9-differentiated cells and the ability of
each cell type to bind and respond to TGF-
.
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Transcriptional Regulation of the TR-II Gene--
Due to the
large increase in the steady-state levels of T
R-II
mRNA when F9 EC cells are induced to differentiate, we examined the
transcriptional activity of the T
R-II gene promoter in F9 EC cells and their differentiated counterparts. These studies employed
chimeric gene constructs in which various amounts of the 5'-flanking
region of the human T
R-II gene were inserted upstream of
the CAT reporter gene in the plasmid pGEM-SVOCAT
(42). Transient transfection of HepG2 cells with these constructs had previously identified several distinct regulatory regions in the T
R-II promoter including: two positive regulatory regions
(
219 to
172 and +1 to +35), two negative regulatory regions (
1240 to
504 and
100 to
67), and the core promoter region (
47 to
1)(42). In the current study, the T
R-II
promoter-CAT constructs, pT
RII-1883/+50,
pT
RII-274/+50, and pT
RII-137/+50 were
transiently transfected into F9 EC cells. (These constructs contain a
common 3' end located at +50 in relationship to the primary
T
R-II transcription start site (42, 43), and increasing
amounts of the T
R-II 5'-flanking sequence ranging from
nucleotide
1883 to
137.) Consistent with the virtual absence of
T
R-II transcripts in EC cells (Fig. 1), virtually no CAT
activity was detected over background (Fig. 2). This result is unlikely to be
explained by low transfection efficiency, as both the normalizing
plasmid, pCMV-
, and positive control plasmid,
pDOL-CMV-CAT, are expressed strongly (data not shown). In
stark contrast to our observations in F9 EC cells, all of the
constructs expressed substantially greater levels of CAT activity in
the F9-differentiated cells (Fig. 2). Equally important, the 9-fold
increase in transcriptional activity of pT
RII-274/+50
when compared with pT
RII-137/+50 suggests the presence of
a strong positive regulatory element(s) located in the region between
137 and
274 of the T
R-II gene promoter. Furthermore,
the 2-fold decrease in transcription of the
pT
RII-1883/+50 construct relative to
pT
RII-274/+50 points to a weak negative regulatory
element(s) located in the region between
274 and
1883 (Fig. 2).
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Identification of a Cis-regulatory Element That Elevates TR-II
Promoter Activity in F9-differentiated Cells--
The large difference
between the level of CAT activity expressed in the differentiated cells
by constructs pT
RII-274/+50 and
pT
RII-137/+50 suggests the presence of a positive
regulatory element(s) located in the region between
274 and
137.
Studies by Bae et al. (42) identified a putative CRE/ATF
site located between
196 and
190 that contributed significantly to
the basal transcriptional activity of the T
R-II gene in
HepG2 cells. This observation along with our previous findings
demonstrating that a CRE/ATF element located in the TGF-
2
gene promoter was essential for its expression in EC-differentiated
cells (59) as well as other cell types (60, 61), led us to examine the
effect of the putative CRE/ATF site on T
R-II promoter
expression in F9-differentiated cells. For these studies, we utilized a
set of shorter constructs that eliminated a second positive regulatory
region (+1 to +35) identified by Bae et al. (42) in HepG2
cells. Similar to our observations with the pT
RII-274/+50
and pT
RII-137/+50 constructs, there was about a 9-fold
difference in the expression of pT
RII-219/+2 when
compared with pT
RII-137/+2 (compare Figs. 2 and
3), suggesting that in F9-differentiated
cells the function of the region between
219 and
137 is not
dependent on the putative downstream positive regulatory
element(s).
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Analysis of the Binding of Nuclear Proteins from F9 EC Cells and
Their Differentiated Cells to the CRE/ATF Site in the TR-II
Promoter--
Previous studies in HepG2 cells (42), as well as studies
in F9-differentiated cells (Fig. 3), demonstrate the importance of the
CRE/ATF site for expression of the T
R-II gene. However, the factor(s) that binds to this site has not been identified. This led
us to initially examine the in vitro binding of nuclear proteins to the CRE/ATF site. Gel mobility shift analysis of
radiolabeled dsODNs containing the T
R-II CRE/ATF motif
with nuclear extracts prepared from F9 EC and F9-differentiated cells
resulted in the formation of a single prominent DNA-protein complex
that migrated with similar mobility in each extract (Fig.
4). The protein(s) in each complex
appears to bind specifically to the CRE/ATF site of the probe, as a
25-fold excess of both unlabeled wild-type probe and unlabeled probe
(M1) mutated slightly upstream (
203 to
200) of the CRE/ATF site
competed effectively for the formation of DNA-protein complex, whereas
a 25-fold excess of unlabeled probe (M2) mutated within (
195 to
192) the CRE/ATF site competed only very weakly (Fig. 4). Moreover, a
25-fold excess of unlabeled probe containing the essential CRE/ATF
element (
74 and
67) from the human TGF-
2 gene
promoter (53, 60) also competed effectively for the formation of the
DNA-protein complex binding to the T
R-II CRE/ATF (Fig.
4). Similarly, the unlabeled wild-type T
R-II probe and
not the CRE/ATF mutant counterpart (M2) was able to compete effectively
for the factors that bind to the CRE/ATF motif of the
TGF-
2 gene (data not shown).
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ATF-1 Is Present in the DNA-Protein Complexes Formed between the
TR-II CRE/ATF Site and Nuclear Extracts from F9 EC and
F9-differentiated Cells--
To identify the transcription factor(s)
present in the DNA-protein complexes formed between the
T
R-II CRE/ATF site and nuclear extracts prepared from F9
EC and F9-differentiated cells, we used a battery of antibodies that
individually recognize transcription factors that bind to CRE/ATF
motifs, including antibodies that recognize ATF-1, ATF-2, c-Jun, CREB,
and CREM. Each of these antibodies were incubated with nuclear extracts
prepared from F9 EC and F9-differentiated cells and analyzed by gel
mobility shift assay with the T
R-II CRE/ATF specific
probe. Only ATF-1, or a closely related transcription factor, appears
to be present in the DNA-protein complexes formed with the F9 EC (Fig.
5) and F9-differentiated (Fig.
6) cell nuclear extracts, as determined
by both the change in migration and intensity of the prominent
DNA-protein complex. However, neither of the heterodimeric partners of
ATF-1 identified to date, CREB and CREM, were detected in the
DNA-protein complex, suggesting that ATF-1 is binding either as a
homodimer or as a heterodimer with an as yet unidentified
transcription factor.
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The Transcription Factor ATF-1 Up-regulates the TR-II Promoter
in Vivo--
Despite the finding that ATF-1 binds to the
T
R-II CRE/ATF site in vitro, it was possible
that other members of the CREB/ATF family of transcription factors are
responsible for the basal transcriptional activity mediated through the
CRE/ATF site in vivo. Therefore, to examine the ability of
ATF-1 to influence the expression of the T
R-II gene, we
employed eukaryotic expression vectors in transient transfection assays
that express either ATF-1 (pECEATF-1) or ATF-2
(pECEATF-2) proteins under the control of the
SV40 promoter. When F9-differentiated cells were
co-transfected with pECEATF-1 and the
pT
RII-219/+2 promoter/reporter construct, T
R-II promoter activity was up-regulated by over 40-fold
compared with the expression of the pT
RII-219/+2
construct co-transfected with pSV
(an SV40
promoter vector without ATF-1 or ATF-2) (Fig. 7). In contrast, co-transfection of the
pECEATF-2 plasmid with pT
RII-219/+2
resulted in only a modest (4-fold) induction of T
R-II
promoter activity (Fig. 7). The increase in pT
RII-219/+2 expression by ATF-1 does not appear to be due to general effects on
cellular transcription, as overexpression of ATF-1 had only a minor
effect (<3-fold induction) on two other T
R-II promoter constructs (pT
RII-137/+2 and pT
RII-47/+2)
that do not contain the CRE/ATF site (Fig.
8). Similarly, overexpression of ATF-1 did not have a significant effect on the CMV promoter (Fig.
8). Together, our findings argue that ATF-1 contributes to the
expression of the T
R-II gene in EC-differentiated
cells.
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Identification of a Cis-regulatory Element Located between 83 and
74 of the T
R-II Promoter--
Previous studies by Bae et
al. (42) identified a regulatory region located between
137 and
47 in the T
R-II promoter that had a strong negative
effect on the basal transcriptional activity of the T
R-II
gene in HepG2 cells. However, the location of the negative regulatory
element and, more important, the transcription factor that binds to
this site were not identified. Utilizing a set of T
R-II
promoter/CAT deletion constructs that ranged from nucleotide
219 to
47, and each ending at +2, we determined that the region
located between
100 and
47 also suppresses T
R-II promoter activity in F9-differentiated cells (data not shown, also see
Fig. 10). Sequence analysis of this region identified a 10-base pair
putative cis-regulatory element (TGATTGGCAG) located between
83 and
74 that contains an inverted CCAAT box motif. Moreover, expression of
T
R-II promoter/reporter constructs with this site
mutagenized is elevated when transfected into HepG2 cells.2 Interestingly, this
sequence is identical to the core sequence (
789 to
780) of a
negative regulatory element identified in the human CYP1A1
gene using HepG2 cells (62, 63). Moreover, this same sequence has been
demonstrated to be an essential cis-regulatory element of the
fibroblast growth factor-4 (FGF-4) gene in F9 EC cells
(64-66). In both the CYP1A1 gene and the FGF-4
gene, it was determined that the transcription factor complex NF-Y was
able to bind in vitro to the core CCAAT box motif.
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The Transcription Factor Complex NF-Y Is Present in the DNA-Protein
Complexes Formed between the TR-II Putative CCAAT Box Motif and
Nuclear Extracts from F9 EC and F9-differentiated Cells--
Previous
studies using gel mobility supershift analysis determined that the
transcription factor complex, NF-Y is one of the transcription factors
that binds to the TGATTGGCAG sequence in both the FGF-4 gene
(64-66) and human CYP1A1 gene (63). The ability of
oligonucleotides containing the FGF-4 CCAAT box and the
CYP1A1 NRE to compete effectively for the binding of
proteins to the T
R-II CCAAT box motif raised the
possibility that NF-Y may also bind to the T
R-II CCAAT
box motif. As NF-Y is reported to be a trimeric complex containing
NF-YA, NF-YB, and NF-YC (also known as CBF-B, CBF-A, and CBF-C,
respectively), we used an antibody that specifically recognizes the
NF-YA subunit to characterize the DNA-protein complexes that form
between the T
R-II CCAAT box motif and nuclear proteins
from both F9 EC and F9-differentiated cells. In both cell types, only
the slower of the two closely migrating distinct DNA-protein complexes
was supershifted by the addition of the NF-YA antibody, whereas the
nonspecific IgG antibody had no effect on the mobility of any of the
DNA-protein complexes (Fig. 9, A and B). In
addition, the NF-YA antibody also appears to recognize the less
distinct slowest migrating DNA-protein complex formed with nuclear
extract from F9 EC cells (Fig. 9A), suggesting that NF-Y may
be interacting with another factor(s). Thus, it appears that the NF-Y
transcription factor complex binds the T
R-II CCAAT box
motif in vitro and differentiation does not overtly affect
the ability of NF-Y to bind to this site. In contrast to the slower
migrating DNA-protein complex, the faster migrating complex observed in
both F9 EC cells and their differentiated counterparts was not
supershifted by the NF-YA antibody (Fig. 9, A and
B). Hence, the factor(s) in this DNA-protein complex appears
to be distinct from NF-Y and thus far it has not been identified.
NF-Y Influences TR-II Expression in Vivo--
The results of
our transient expression studies, combined with our in vitro
binding analyses to the T
R-II CCAAT box motif, implies a
role for NF-Y in T
R-II transcription. However, additional studies are required to address the question of whether NF-Y affects T
R-II expression in vivo. To this end,
Mantovani et al. (46) described a mutant NF-YA protein,
NFYA29, which contains mutations in three amino acids of the DNA
binding domain of NF-YA. The NFYA29 protein continues to bind to the YB
subunit of NF-Y, but not to DNA, thereby functioning as a dominant
negative repressor of NF-Y-mediated effects on transcription (46).
Results from studies co-expressing this dominant-negative NF-YA
demonstrated an in vivo role of NF-Y in the
sterol-dependent expression of the farnesyl diphosphate (FPP) synthase gene, the 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) synthase gene (67), and the FGF-4
gene (66).
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DISCUSSION |
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TGF- ligand binding and cellular responsiveness to TGF-
increase dramatically when EC cells are induced to differentiate, which
suggests strongly that TGF-
receptors are differentially expressed
by EC cells and their EC-derived differentiated counterparts (38). In
this report, we examined the expression of the T
R-II gene
in F9 EC and F9-differentiated cells, as T
R-II expression is normally associated with ligand binding and growth responsiveness of
cells to TGF-
(21-23, 58). Our studies demonstrate that the expression of T
R-II is closely associated with the induction of
differentiation in F9 EC cells. Few, if any, transcripts for the
T
R-II gene are detected in F9 EC cells, whereas there is a dramatic increase in the mRNA steady-state levels of this gene in
F9-differentiated cells. Equally important, the high levels of
expression of T
R-II promoter/CAT reporter chimeric gene
constructs in F9-differentiated cells, but not in their
undifferentiated parental cells, argues strongly that the increase in
the steady state levels of T
R-II mRNA results, at
least in part, from increased transcription of the T
R-II
gene. Furthermore, this study identifies two different transcription
factors, ATF-1 and NF-Y, that bind in vitro and in
vivo to a positive regulatory element and negative regulatory
element, respectively.
The up-regulation of the TR-II gene during
differentiation coincides with the ability of TGF-
to both bind and
inhibit the growth of the EC-derived differentiated cells as well as to
their loss of tumorigenic potential. Therefore, EC cells provide a
powerful model system for studying the mechanisms that control
T
R-II expression during normal development as well as
during the processes of malignant transformation. In regard to the
latter, several lines of evidence suggest that T
R-II may
act as a tumor suppressor gene. A number of tumors, including small
cell lung carcinoma (26), thyroid tumors (27), and prostate
adenocarcinoma (25) show a loss of or reduced expression of functional
TGF-
type II receptors. Additionally, transfection of wild-type
T
R-II constructs into hepatoma cells and MCF-7 breast
carcinoma cells lacking functional type II receptors restores
sensitivity to TGF-
and suppresses their tumorigenic phenotype (21,
22). Moreover, gross structural mutations of both T
R-II
alleles has been observed in 71-90% of colorectal tumors and cell
lines and in 71% of gastric cancer cell lines with microsatellite
instability (23, 24, 68). However, in a number of instances in which
cells fail to express the T
R-II gene at the RNA or
protein level, no apparent structural mutations within the coding
region of the gene were observed. This raises the possibility that
defects in the promoter region of the T
R-II gene and/or
in the mechanisms regulating the transcription of the
T
R-II gene may play important roles in the aberrant
expression of T
R-II in certain neoplasms. In this regard,
the low levels of T
R-II mRNA expressed by A431 tumor
cells is thought to be a result of a point mutation located in the 5'
flanking region of the T
R-II promoter (69).
To understand further the mechanism(s) that control the regulation and
expression of the TR-II promoter during differentiation, our studies also examined the expression of T
R-II
promoter/reporter gene constructs in the differentiated cells derived
from various EC cell lines to identify putative DNA regulatory elements
that are required for normal T
R-II promoter expression.
The data presented demonstrates the existence of at least four distinct
regulatory regions in the T
R-II promoter including, two
positive regulatory regions located between
274/
100 and
47/+2,
and two negative regulatory regions located between
1883/
274 and
100/
47. In addition, we determined that a putative CRE/ATF site
located at
196 to
190 exerts a significant positive influence on
T
R-II promoter activity in the differentiated cells,
whereas a putative inverted CCAAT box motif located at
83 to
74
exerts a significant negative influence on T
R-II promoter
activity in both F9 EC cells and their differentiated counterparts.
In regard to the CRE/ATF motif, mutation of this site reduces
transcription of wild-type TR-II promoter/reporter
constructs in the differentiated cells by approximately 50%, which is
similar to what was observed in HepG2 cells (42). Gel mobility shift analysis further demonstrated that protein complexes containing the
transcription factor ATF-1 specifically recognize and bind the
T
R-II CRE/ATF site. Overexpression of ATF-1 in
F9-differentiated cells resulted in a dramatic increase in
T
R-II promoter activity most likely through the CRE/ATF
site, suggesting that ATF-1 not only interacts with this site in
vitro but also in vivo. Interestingly, the expression
of the TGF-
2 gene in EC cells and their differentiated counterparts is also regulated by mechanisms involving the binding of
ATF-1 to an essential CRE/ATF element (7, 53, 59, 60).
Another important aspect of the work reported in this study is the
demonstration that a 53-base pair negative regulatory region located
between 100 and
47 contains an inverted CCAAT box motif that
negatively regulates T
R-II promoter activity. We
demonstrate further that the transcription factor complex, NF-Y, binds
to the T
R-II CCAAT box motif in vitro when
nuclear extracts from both F9 EC cells and their differentiated
counterparts are used in gel mobility supershift analyses. Importantly,
we also demonstrate that expression of the dominant-negative NFYA29
mutant protein in both F9 EC and F9-differentiated cells specifically
increases the expression of T
R-II promoter/reporter
constructs that contain the CCAAT box motif. In further support of
these findings, we have shown that NF-Y in nuclear extracts prepared
from HepG2 cells binds to the CCAAT box motif (data not shown) and
expression of T
R-II promoter/reporter constructs with
this site mutagenized is elevated when transfected into HepG2
cells.2 Thus, our findings argue strongly that in
vivo binding of NF-Y to the CCAAT box motif represses the activity
of the T
R-II promoter.
CCAAT box motifs are found in the promoter regions of many genes, and a
survey of over 500 unrelated promoter sequences determined that most
proximally located CCAAT boxes reflect the target sequence for NF-Y
rather than C/EBP or NF1 (70). NF-Y was originally identified as a
ubiquitously expressed protein that binds to the Y box motif,
originally defined as an inverted CCAAT box motif (CTGATTGGYY) in all
MHC class II genes that is critical for tissue specific gene
expression (71). Although the exact mechanism(s) by which NF-Y
regulates transcription is unclear, it has been demonstrated to act as
both a positive (66, 72, 73) and a negative (74) transcription factor.
Several studies suggest that NF-Y acts by stabilizing or recruiting the
binding of additional factors to adjacent promoter elements (75, 76).
Whether there are coordinate interactions between ATF-1 and NF-Y that
act to regulate the TR-II promoter in F9 EC and
F9-differentiated cells is unknown, however these two factors appear to
act in concert to regulate a number of other gene promoters, including
the human cyclin A gene and the rat hexokinase II gene (72, 77).
In conclusion, our studies demonstrate clearly that transcription of
the TR-II gene is influenced significantly by both
positive and negative cis-regulatory elements and trans-acting factors. However, the exact mechanism(s) by which differentiation turns on the
expression of the T
R-II gene remains to be determined. Given the important role of the TGF-
type II receptor during normal
development and in its aberrant expression in certain neoplasms, further study of this gene in this model system is clearly warranted. In addition, it will be interesting to determine whether
differentiation also leads to the transcriptional activation of the
genes for the type I and type III TGF-
receptors.
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ACKNOWLEDGEMENTS |
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We thank Michael O'Reilly for the gift of pECEATF-1 and pECEATF-2 expression plasmids and Steven Hinrichs for the gift of the ATF-1 monoclonal antibody. We thank Robert Mantovani for permission to use the pNFYA29 plasmid, which was generously provided by Peter Edwards.
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FOOTNOTES |
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* This work was supported in part by Grant CA 74771 from the NCI, National Institutes of Health and by core grants to the Eppley Institute from the American Cancer Society (SIG) and the NCI, National Institutes of Health (Laboratory Cancer Research Center Support Grant CA 36727).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported in part by a postdoctoral fellowship provided by NCI-supported Training Grant T32 CA09476 in Cancer Research.
** To whom all correspondence should be addressed at: Eppley Inst. for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. Tel.: 402-559-6338; Fax: 402-559-4651; E-mail: arizzino{at}unmc.edu.
The abbreviations used are:
TGF-, transforming growth factor-
; T
R-II, type II transforming growth
factor-
receptor; EC, embryonal carcinoma; FGF, fibroblast growth
factor; RA, retinoic acid; dsODN, double-stranded oligodeoxynucleotide; ATF, activating transcription factor; CRE, cAMP response element; CAT, chloramphenicol acetyltransferase; MOPS, 4-morpholinepropanesulfonic
acid.
2 D. Kim and S.-J. Kim, unpublished results.
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REFERENCES |
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