(Received for publication, March 5, 1997, and in revised form, June 13, 1997)
From the Cardiovascular Biology Laboratory, Harvard School of
Public Health, Boston, Massachusetts 02115 and the
§ Department of Medicine, Transforming growth factor (TGF)- Transforming growth factor- Cyclin A associates with Cdk2 in the S phase of the cell cycle and with
Cdc2 in the G2/M phase, and it is required for DNA replication in the S phase (4, 18). Cyclin A can also affect the
G1/S transition, as overexpression of cyclin A but not
cyclin D1 or E overcomes the G1/S block induced by loss of
cell adhesion (19). The human and mouse cyclin A genes have been cloned
recently and their promoters analyzed (20-24), and the CDE/CHR
consensus sequence has been implicated as a negative regulator of
cyclin A promoter activity during the cell cycle (20, 23). Although the
transcription factors that bind to the CDE/CHR site have not been
cloned, these factors bind to the site only during the G0 and the early G1 phases of the cell cycle. The
CDE/CHR-binding proteins may then repress the activity of the cyclin A
activating transcription factor (ATF) site.
The cyclin A ATF site is bound by ATF-1 and the cyclic AMP-responsive
binding protein (CREB), which function as positive regulators of the
cyclin A promoter (21, 22, 25). Also, the activity of ATF-1 and CREB is
regulated by their phosphorylation status (26). Although negative
regulation of the cyclin A promoter during progression of the cell
cycle could be mediated by the CDE/CHR site, we have shown elsewhere
that down-regulation of cyclin A promoter activity during contact
inhibition is mediated through the ATF site and that the abundance of
ATF-1 protein and mRNA is reduced (21).
We describe in this report mechanisms by which TGF- Mink lung epithelial cells (Mv1Lu, CCL-64)
were obtained from the American Type Culture Collection and cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (HyClone, Logan, UT) and antibiotics as described (21). The activity of the cyclin A promoter was very sensitive to confluence in
Mv1Lu cells, as it is in vascular endothelial cells (21, 27). Cells
were plated at a density of about 30,000 cells/cm2 on a
10-cm dish (surface area, 55 cm2) for 72 h. To keep
them from becoming confluent, the cells were trypsinized, transferred
from the 10-cm dish to a 15-cm dish, treated with TGF- Reporter constructs
containing fragments of the human cyclin A 5 Mv1Lu cells were transfected with 15 µg of luciferase construct by
the calcium phosphate method as described (28). To correct for
variability in transfection efficiency, we cotransfected 2 µg of
pCMV- For the cotransfection study, a catalytic Total RNA was prepared
from Mv1Lu cells by guanidinium isothiocyanate extraction and
centrifugation through cesium chloride (30, 31). The human cyclin A
cDNA fragment was amplified by the reverse transcription-polymerase
chain reaction as described (21). The human ATF-1 plasmid (32) and a
human CREB plasmid (33) were obtained from Drs. M. R. Green
(University of Massachusetts, Amherst, MA) and M. R. Montminy
(Harvard Medical School, Boston).
Total RNA was fractionated on 1.3% formaldehyde-agarose gels and
transferred to nitrocellulose filters. The filters were hybridized with
a randomly primed, 32P-labeled cyclin A cDNA probe. The
hybridized filters were washed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate at
40 °C and autoradiographed on x-ray film or stored on phosphor screens for 8-10 h (6). To correct for differences in RNA loading, the
filters were washed in a 50% formamide solution at 80 °C and rehybridized with a radiolabeled 18 S rRNA oligonucleotide probe (34).
The filters were scanned, and radioactivity was measured on a
PhosphorImager running the ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Nuclear extracts from Mv1Lu cells
were prepared as described (21). Protein concentrations in nuclear
extracts were measured by the Bio-Rad protein assay system, which is
based on the Bradford method (35). Double-stranded oligonucleotide
probes synthesized according to the sequence of the human cyclin A ATF
site (bp To examine the phosphorylation of CREB and
AFT-1, we separated 30 µg of nuclear extract (prepared from Mv1Lu
cells treated with or without TGF- To identify sequences that
mediate down-regulation of cyclin A promoter activity by TGF-
To determine the effect of TGF- To identify proteins that bind to the ATF-site, we performed gel
mobility shift analysis with antibodies. Before addition of the probe,
Mv1Lu cell extracts were incubated with or without antibody to ATF-1,
CREB, or ATF-2 (Fig. 3). The faint X
complex visible in Fig. 2C disappeared in Fig. 3, even in
nuclear extracts preincubated without an antibody. Preincubation of
nuclear extracts with antibody specific to ATF-1 or CREB, but not
ATF-2, markedly decreased the density of the DNA·protein complexes
(Fig. 3). In addition, the anti-CREB antibody produced a supershifted
DNA·protein·antibody complex (indicated by an
asterisk). These data identify ATF-1 and CREB as the major
binding proteins for the cyclin A ATF site in Mv1Lu cells, as we have
observed previously in vascular endothelial cells (21).
At
24 h, TGF-
If TGF-
In contrast with PKA, protein Ser/Thr phosphatases type 1 (PP1) and 2A
(PP2A) dephosphorylate CREB (39-41). Inhibitors of CREB/ATF-1 phosphatases should therefore increase phosphorylation of CREB/ATF-1. We first examined the effect of okadaic acid (42), an inhibitor of PP1
and PP2A, on TGF-
Although it has been shown that a functional TGF- Phosphorylation of ATF-1 and CREB is crucial to their ability to
trans-activate target genes (26). Phosphorylation of CREB at
serine 133 is required for it to bind to CBP (CREB-binding protein) and
activate transcription of its target genes (46-48), and
phosphorylation at serine 63 is important to the activity of ATF-1 (36,
37, 49). PKA and Ca2+, calmodulin-dependent
protein kinases have been shown to phosphorylate ATF-1 and CREB (36,
37, 49). Recently RSK-2 (a member of the pp90rsk family and a
downstream kinase of the p21ras signaling pathways) has also
been shown to function as a CREB kinase (50). It appears that multiple
signal pathways converge at the point of CREB/ATF-1 phosphorylation to
regulate the activity of these transcription factors. Also, the Ser/Thr
phosphatases PP1 and PP2A have been shown to dephosphorylate CREB and
decrease its activity (39-41). Thus, the total activity of ATF-1 and
CREB depends on their phosphorylation state, which in turn reflects a
balance between the kinases and phosphatases (26).
Although we did not examine the effect of TGF- The suppression of the inhibitory effect of TGF- Our data indicate that TGF- We extend our gratitude to Edgar Haber for
his continued enthusiasm and support of our work. We thank Dr. Haber
for reviewing the manuscript, Bonna Ith for technical assistance, and
Thomas McVarish for editorial assistance.
Pulmonary Divisions,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 prevents
cell cycle progression by inhibiting several regulators, including
cyclin A. To study the mechanisms by which TGF-
1 down-regulates
cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A promoter into mink lung epithelial cells in the absence and presence of TGF-
1. The TGF-
1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolished down-regulation. Surprisingly, although TGF-
1 treatment for 24 h markedly decreased cyclin A promoter activity, it did not
decrease the abundance of the ATF-binding proteins ATF-1 and cyclic
AMP-responsive binding protein (CREB). However, we detected 90 and 78%
reductions (by Western analysis) in phosphorylated CREB and ATF-1,
respectively, in mink lung epithelial cells treated with TGF-
1.
TGF-
1-induced down-regulation of cyclin A promoter activity was
reversed by okadaic acid (a phosphatase inhibitor) and by
cotransfection with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A). These data indicate that TGF-
1
may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.
(TGF-
)1 inhibits
proliferation of most normal cell types in culture and in
vivo (1, 2). Because TGF-
arrests progression of the cell cycle
in mink lung epithelial (Mv1Lu) cells when added both early and late in
the G1 period (3), the inhibitory effect of TGF-
could
be mediated by more than one mechanism. The progress of the cell cycle
is regulated by the sequential expression of cyclins, followed by the
activation of their associated cyclin-dependent kinases
(Cdks) (4). Inhibitors of the Cdks block cell cycle progression (5-8). The many cell cycle regulators that TGF-
has been shown to affect include Cdk2, Cdk4, p15INK4b, p21Waf1/Cip1,
p27Kip1, and cyclin A (9-17).
1 down-regulates
cyclin A promoter activity in mink lung epithelial cells. A
TGF-
1-induced down-regulation of cyclin A promoter activity was
mediated through the ATF site. Twenty-four hours of TGF-
1 treatment
decreased cyclin A mRNA and promoter activity but not the abundance
of the ATF-binding proteins ATF-1 and CREB. Using antibodies specific
for phosphorylated ATF-1 and CREB, we detected a significant reduction
in phosphorylated ATF-1 and CREB only in TGF-
1-treated cells. The
TGF-
1-induced down-regulation of cyclin A promoter activity was
reversed by cotransfection with expression plasmids harboring
cAMP-dependent protein kinase (PKA) or simian virus 40 small tumor antigen (Sm-t).
Cell Culture
1, and
harvested at various times thereafter.
-flanking region were
inserted into the promoterless luciferase reporter plasmid pGL2-Basic
(Promega, Madison, WI) as described (21). Cyclin A plasmids contained a
3500-bp SmaI-SmaI restriction fragment
(~bp
3200 to +245), a 761-bp SacI-SmaI
fragment (bp
516 to +245), and fragments of 471 bp (
266/+205), 338 bp (
133/+205), and 132 bp (
133/
2). The ATF consensus sequence
(TGACGTCA) in the plasmids
266/+205 and
133/+205 was mutated to
TGCCCCCA by polymerase chain reaction to generate the plasmids
mut 266/+205 and mut
133/+205, as described elsewhere (21).
GAL (containing the potent cytomegalovirus enhancer and
promoter driving the
-galactosidase gene) in all experiments. The
luciferase assay and
-galactosidase assay were performed as
described (21), and the ratio of luciferase activity to
-galactosidase activity in each sample was used as a measure
of normalized luciferase activity. Each construct was transfected at
least two times, and each transfection was done in quadruplicate. Data
for each construct are presented as the mean ± S.E.
-subunit of PKA, obtained
from Dr. R. A. Maurer (Oregon Health Sciences University, Portland, OR) (29) was cloned into pcDNA3 (Invitrogen, San Diego, CA). As a negative control, we cloned an inactivated form of the PKA
catalytic
-subunit (mut-PKA) into the same vector. The expression plasmids pCEP4 Sm-t and pCB6-I-1, encoding full-length Sm-t and a
constitutively active form of I-1, respectively, were provided by Drs.
A. Alberts (Imperial Cancer Research Fund, London, United Kingdom) and
S. Shenolikar (Duke University, Durham, NC).
84 to
63, 5
TGAATGACGTCAAGGCCGCGAG 3
) were radiolabeled
as described (34). A typical binding reaction mixture contained DNA
probe at 20,000 cpm, 1 µg of poly(dI-dC)·poly(dI-dC), 25 mM HEPES (pH 7.9), 40 mM KCl, 3 mM
MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, and 3 µg of nuclear extract in a final
volume of 25 µl. The reaction mixture was incubated at room
temperature for 20 min and analyzed by 5% native polyacrylamide gel
electrophoresis in 0.25 × TBE buffer (22 mM Tris
base, 22 mM boric acid, and 0.5 mM EDTA). To
characterize specific DNA-binding proteins, we incubated nuclear
extracts with various antibodies for 12 h at 4 °C before addition of the probe. The polyclonal antibodies were raised against human ATF-1 and ATF-2 (Upstate Biotechnology, Inc., Lake Placid, NY)
and CREB (New England Biolabs, Beverly, MA).
1 for 24 h) on a 10%
SDS-polyacrylamide gel and immunoblotted the extract with
phospho-CREB-specific (Ser-133) antibody according to the protocol
provided by the manufacturer (PhosphoPlus CREB (Ser-133) antibody kit,
New England Biolabs). This antibody specifically recognizes the
phosphorylated forms of CREB and ATF-1. The same blot was then
incubated with stripping buffer (62.5 mM Tris-HCl (pH 6.8),
2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at
50 °C and reblotted with anti-ATF-1 (C41-5.1) antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), which does not cross-react with other
members of the ATF/CREB family, and then with anti-CREB (C-21) antibody
(Santa Cruz Biotechnology), which is specific for CREB1.
The ATF Site Mediates Down-regulation of Cyclin A Promoter Activity
in TGF-1-treated Mv1Lu Cells
1, we
transfected a series of luciferase reporter gene plasmids containing
various lengths of the human cyclin A 5
flanking sequence into Mv1Lu
cells treated with or without TGF-
1 for 48 h. The ratio of
luciferase activity in TGF-
1-treated cells versus
untreated cells was calculated for each plasmid. Although TGF-
1 had
no effect on the luciferase activity of pGL2-Control (driven by the
potent SV40 enhancer and promoter), it decreased luciferase activity by
80% in all but two plasmids containing the cyclin A promoter (Fig.
1). In these two plasmids (mut
266/+205 and mut
133/+205), the ATF sequence had been mutated. These data indicate that the ATF site mediates down-regulation of cyclin A
promoter activity by TGF-
1 in Mv1Lu cells. We also determined the
time course of the TGF-
1-induced down-regulation of cyclin A
promoter activity. TGF-
1 decreased luciferase activity by 25% at
12 h and by 70% at 24 h (Fig.
2A). Considering the half-life of the luciferase protein (~6 h), inhibition of cyclin A promoter activity must have occurred before 12 h. This possibility was further supported by the finding that TGF-
1 markedly decreased cyclin A mRNA abundance at 12 h (Fig. 2B).
Fig. 1.
ATF site mediates down-regulation of the
cyclin A promoter by TGF-1. A, plasmids containing
various lengths of the 5
-flanking sequence of the cyclin A gene and a
luciferase reporter (Luc) gene are depicted. The
transcription start site and cis-acting elements are
indicated by an arrow and boxes, respectively
(21, 24). Mutation of the ATF site is denoted by an X.
B, each plasmid (15 µg) was transfected into Mv1Lu cells
by the calcium phosphate method (21, 51). Sixteen to twenty hours after
transfection, Mv1Lu cells were trypsinized, transferred from a 10-cm
plate to a 15-cm plate to avoid contact inhibition (which
down-regulates cyclin A promoter activity), treated with or without 50 pM TGF-
1 for an additional 36 h, and harvested as
described (34). For each construct, the plasmid pCMV-
GAL was
cotransfected to correct for differences in transfection efficiency,
and relative luciferase units were obtained by dividing luciferase
activity by
-galactosidase activity. Shown for each plasmid is the
ratio (percent) of relative luciferase activity in TGF-
1-treated
cells to that in untreated cells (mean ± S.E.).
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
TGF-1 treatment for 24 h decreases
cyclin A promoter activity and the abundance of cyclin A mRNA but
not that of ATF-binding proteins. A, Mv1Lu cells were
transfected with cyclin A promoter plasmid
266/+205 as described for
Fig. 1 and then treated with or without TGF-
1 (50 pM)
for the indicated times. Cells were harvested and luciferase activity
was measured as in Fig. 1. B, Mv1Lu cells were treated with
or without TGF-
1 for the indicated times and then harvested for
Northern analysis with cyclin A and 18 S probes (to correct for
differences in loading) as described (21). C, gel mobility
shift assays were performed with a double-stranded, 32P-labeled, 22-bp oligonucleotide containing the ATF site
of the cyclin A promoter. Nuclear extracts were prepared from Mv1Lu
cells treated with or without TGF-
1 for the indicated times.
Addition of nuclear extracts from Mv1Lu cells resulted in three
retarded bands, X, Y, and Z. NS and Free indicate nonspecific band and free probe, respectively.
[View Larger Version of this Image (45K GIF file)]
1 Treatment for 24 h Has No Effect on the Abundance of
ATF-binding Proteins
1 on
ATF-binding proteins, we performed gel mobility shift analysis with a
22-bp probe encoding the ATF consensus sequence (21) and nuclear
extracts prepared from Mv1Lu cells treated with TGF-
1 for 12, 24, 36, and 48 h. Incubation of nuclear extract from untreated Mv1Lu
cells with the 22-bp probe resulted in three specific DNA·protein
complexes, X, Y, and Z (Fig. 2C), as we have observed in
nuclear extract from vascular endothelial cells (21). The three
complexes were competed away by an identical unlabeled oligonucleotide
but not by an oligonucleotide containing a mutated ATF site (data not shown). To our surprise, we found that TGF-
1 treatment for up to
24 h had no effect on the abundance of complexes X, Y, and Z. This
lack of effect is in sharp contrast to the significant reduction in
cyclin A mRNA abundance and promoter activity observed 24 h
after TGF-
1 treatment. TGF-
1 eventually decreased the abundance of all three complexes of ATF-binding proteins after 36 and 48 h
(Fig. 2C). This reduction in ATF-binding proteins may have
played an important role in the complete down-regulation of cyclin A gene transcription at 36 and 48 h (Fig. 2A).
Fig. 3.
ATF-1 and CREB are the ATF-binding proteins
in Mv1Lu cells. Gel shift analysis was performed as in Fig.
2C. Before addition of the probe, nuclear extracts were
incubated with antibody to ATF-1, CREB, or ATF-2. The
asterisk indicates supershifted band produced by the
anti-CREB antibody.
[View Larger Version of this Image (62K GIF file)]
1 Decreases Phosphorylation of CREB and ATF-1
1 decreased cyclin A mRNA levels and promoter
activity (Fig. 2). Surprisingly, it had no effect on the abundance of
ATF-binding proteins that regulate cyclin A promoter activity. To
investigate this paradox, we examined CREB and ATF-1 phosphorylation, because it has been shown that only the phosphorylated forms of CREB
and ATF-1 are active (26, 36-38). We performed Western analysis with
30 µg of nuclear extract from Mv1Lu cells treated with or without
TGF-
1 for 24 h and antibodies that recognized phosphorylated CREB, total CREB, phosphorylated ATF-1, and total ATF-1. Although TGF-
1 had little effect on the total amount of CREB and ATF-1, it
decreased the phosphorylated forms of CREB and ATF-1 by 90 and
78%, respectively (Fig. 4), as
measured on a PhosphorImager.
Fig. 4.
TGF-1 treatment for 24-h decreases
phosphorylated but not total nuclear CREB and ATF-1 protein.
Nuclear extract (30 µg) prepared from Mv1Lu cells treated with 50 pM TGF-
1 for 24 h was separated on
SDS-polyacrylamide gels and immunoblotted with an antibody specific for
phosphorylated CREB that recognizes the phosphorylated forms of CREB
(p-CREB) and ATF-1 (p-ATF-1). The same blot was
stripped and then reblotted with an anti-ATF-1 antibody, which
recognizes total ATF-1 but not other members of the ATF/CREB family,
and an antibody that recognizes total CREB.
[View Larger Version of this Image (40K GIF file)]
1-induced Down-regulation of Cyclin A Promoter
Activity by PKA, Okadaic Acid, and Sm-t
1 inhibits
cyclin A promoter activity by decreasing phosphorylation of ATF-1 and
CREB, agents that increase the phosphorylation of ATF-1 and CREB should
counteract this inhibitory effect. To test this hypothesis we
cotransfected into Mv1Lu cells 15 µg of cyclin A reporter plasmid
266/+205 and various amounts of a PKA catalytic subunit expression
plasmid. This form of PKA has been shown to phosphorylate and activate
ATF-1 and CREB (24). Cotransfection of the PKA expression plasmid
reversed TGF-
1-induced inhibition of cyclin A promoter activity in a
dose-dependent manner (Fig. 5). Twenty micrograms of PKA expression
plasmid completely suppressed down-regulation of cyclin A promoter
activity by TGF-
1. As a negative control, we also cotransfected a
mutant PKA expression plasmid that does not phosphorylate CREB. The
mutant PKA expression plasmid did not prevent TGF-
1-induced
inhibition of cyclin A promoter activity (data not shown).
Fig. 5.
Cotransfection of the PKA catalytic subunit
reverses TGF-1-induced down-regulation of cyclin A promoter activity
in a dose-dependent manner. Mv1Lu cells were
transfected (as in Fig. 1) with 15 µg of cyclin A plasmid DNA
(
266/+205) and 0-20 µg of an expression plasmid containing PKA
catalytic subunit DNA. The total quantity of DNA was kept constant (35 µg) by the addition of vector DNA. After transfection, the cells were
treated with 50 pM TGF-
1 for an additional 24 h and
extract was harvested for luciferase assays as described for Fig.
1.
[View Larger Version of this Image (13K GIF file)]
1-induced down-regulation of cyclin A promoter
activity. Although low doses of okadaic acid had no effect, 20 nM okadaic acid almost completely reversed the inhibitory
effect of TGF-
1 on cyclin A promoter activity (Fig.
6A). To determine whether 20 nM okadaic acid affected TGF-
1-induced decreases in ATF-1 and CREB phosphorylation, we treated Mv1Lu cells with or without
20 nM okadaic acid for 30 min before adding TGF-
1.
Okadaic acid prevented the down-regulation of ATF-1 and CREB induced by TGF-
1 (Fig. 6B). These data suggest that the
phosphorylation status of ATF-1 and CREB may be responsible for
TGF-
1-mediated down-regulation of cyclin A transcription. To test
further whether inhibition of PP1 or PP2A prevented TGF-
1-induced
down-regulation of cyclin A promoter activity, we cotransfected 7.5 µg of cyclin A reporter plasmid
266/+205 with 15 µg of expression
plasmid encoding a constitutively active form of I-1 or full-length
Sm-t, which inhibit PP1 and PP2A, respectively (39-41, 43, 44).
Cotransfection with the Sm-t, but not the I-1, expression plasmid
abolished the inhibitory effect of TGF-
1 (Fig.
7A). Furthermore, the Sm-t
expression plasmid reversed the inhibitory effect of TGF-
1 in a
dose-dependent manner (Fig. 7B). As little as
3.5 µg of Sm-t expression plasmid almost completely abolished
inhibition by TGF-
1.
Fig. 6.
Reversal of TGF-1-induced down-regulation
of cyclin A promoter activity and TGF-
1-induced phosphorylation of
ATF-1/CREB by okadaic acid. A, Mv1Lu cells were transfected
with 15 µg of cyclin A reporter plasmid DNA
266/+205 as in Fig. 1.
After transfection, the cells were treated with okadaic acid
(OA) at the indicated concentrations. The cells were then
treated with 50 pM TGF-
1 for 24 h, and extract was
harvested for luciferase assays as described for Fig. 1. B,
Mv1Lu cells were not treated (Control) or treated with
TGF-
1 for 24 h after they had been treated for 30 min with
vehicle (TGF-
1) or okadaic acid (OA/TGF-
1). Cell extracts were prepared, and Western analysis was performed with an
antibody specific for phosphorylated CREB that recognizes the
phosphorylated forms of CREB (p-CREB) and ATF-1
(p-ATF-1), as described for Fig. 4.
[View Larger Version of this Image (22K GIF file)]
Fig. 7.
Sm-t reverses the inhibitory effect of
TGF-1 on the cyclin A promoter. A, Mv1Lu cells were
transfected with 7.5 µg of cyclin A reporter plasmid
266/+205 and
15 µg of pcDNA vector, Sm-t expression plasmid, or I-1 expression
plasmid. After transfection, the cells were treated with 50 pM TGF-
1 for 24 h, and the extracts were harvested.
B, Mv1Lu cells were transfected with 7 µg of cyclin A
reporter plasmid
266/+205 and 0-7 µg of Sm-t expression plasmid DNA. The quantity of total DNA was kept constant (14 µg) by the addition of vector DNA. After transfection, the cells were treated with
50 pM TGF-
1 for an additional 24 h, and extract was
harvested for luciferase assays as described for Fig. 1.
[View Larger Version of this Image (15K GIF file)]
receptor
complex (including both types I and II) is required for TGF-
-induced down-regulation of cyclin A promoter activity (45), the downstream inhibitory pathway has not been completely elucidated. In this report
we show that the ATF site mediates inhibition of the cyclin A promoter
by TGF-
1 in mink lung epithelial cells (Fig. 1). Treatment with
TGF-
1 for 24 h markedly decreased cyclin A mRNA levels but had no effect on the abundance of ATF-binding proteins (Fig. 2).
1 on individual
kinases and phosphatases, TGF-
1 treatment for 24 h resulted in
a marked reduction in phosphorylated ATF-1 and CREB but had little
effect on total ATF-1 and CREB (Fig. 4). Thus, TGF-
1 may suppress
cyclin A activity by reducing ATF-1 and CREB phosphorylation. This
conclusion is further supported by our observation that okadaic acid
and the PKA catalytic subunit, which increases phosphorylation of ATF-1
and CREB, overcame the inhibitory effect of TGF-
1 on cyclin A
promoter activity (Figs. 5 and 6).
1 on cyclin A
promoter activity by okadaic acid in Mv1Lu cells implies the presence
of an okadaic acid-sensitive CREB/ATF-1 phosphatase in this cell type.
PP1 is the principal CREB phosphatase in fibroblasts and thyroid
follicular cells (39, 40), whereas PP2A is the principal CREB
phosphatase in hepatocytes (41). This discrepancy may be due to
differences in protein expression among cell types or to differences in
protein phosphatase preparations (41). The relative abundance of these
phosphatases in Mv1Lu cells has not been estimated. Our finding that
cotransfection of Sm-t, but not I-1 (Fig. 7), abolished TGF-
1's
inhibitory effect indicates that PP2A may play a role in
TGF-
1-induced dephosphorylation of CREB/ATF-1 in Mv1Lu cells, but
certainly does not exclude a role for PP1-mediated dephosphorylation of
CREB/ATF-1 in this cell type.
1 may inhibit cyclin A promoter activity
by decreasing the phosphorylation and activity of CREB and ATF-1 but
not their abundance. These data illustrate one of the biochemical
pathways by which TGF-
inhibits cyclin A transcription and
progression of cell cycle.
*
This work was supported by a grant from the Bristol-Myers
Squibb Pharmaceutical Research Institute and by National Institutes of
Health Grants HL03274 (to N. E. S. S.), HL03194 (to
M. A. P.), and GM53249 (to M.-E. L.).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.
Contributed equally to this work.
**
To whom correspondence should be addressed: Harvard School of
Public Health, 677 Huntington Ave., Boston, MA 02115. E-mail: lee{at}cvlab.harvard.edu.
1
The abbreviations used are: TGF-,
transforming growth factor-
; ATF, activating transcription factor;
CREB, cyclic AMP-responsive binding protein; PKA, cyclin
AMP-dependent kinase; PP1, protein phosphatase 1; PP2A,
protein phosphatase 2A; I-1, inhibitor 1; Sm-t, simian virus 40 small t
antigen; bp, base pair(s).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.