From the Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, January 7, 2003, and in revised form, February 11, 2003
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ABSTRACT |
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We have shown previously that expression
of the murine CTP:phosphocholine cytidylyltransferase (CT) Phosphatidylcholine
(PC)1 is the most abundant
phospholipid in mammalian cellular membranes. Besides having a
structural role in membranes and lipoproteins (1-3), PC plays an
important role in signal transduction as a source of lipid second
messengers (1-3). In all nucleated cells, PC is made primarily through
the CDP-choline pathway in which the key enzyme is CTP:phosphocholine cytidylyltransferase (CT) (2-6).
Two genes that encode CT activity have been identified and
characterized. CT In the last several years, studies have shown that CT can be regulated
at both the transcriptional level and post-transcriptionally. CT
mRNA is increased after partial hepatectomy in rats (13), after
stimulation with colony-stimulating factor 1 in macrophages (24), and
during development and growth (25-28). It remains to be determined
whether these increases in the message levels are the result of CT The murine CT The importance of PC metabolism in cell proliferation has been under
intense investigation, and it has become clear that the GTP exchange
protein, Ras, might play an important role in linking these two
processes (38-44). Both PC synthesis and degradation are stimulated in
Ras-transformed C3H10T1/2 murine embryonic fibroblasts (45), NIH-3T3
fibroblasts (46), and in human keratinocyte cell line, HaCaT (47). The
oncogenic transformation in mouse cells leads to increased activity of
choline kinase and decreased activity of CT (45, 46), whereas in human
keratinocytes CT activity and choline uptake were increased, but
choline kinase activity did not change (47). Based upon those data and
our finding that CT Antibodies and Reagents--
All reagents were molecular biology
grade. The inhibitors of p38MAPK (SB202190) and MEK1
(PD98059) were from Calbiochem. The transfection reagent DOTAP was from
Avanti Polar Lipids (Birmingham, AL). Phospho-MAPK antibody sampler
containing phospho-p42/44MAPK, phospho-p38MAPK,
and phospho-SAPK/JNK-specific rabbit polyclonal antibodies was from New
England BioLabs, Mississauga, Canada. A murine monoclonal phosphotyrosine-specific antibody, PY20, was from BD Biosciences, Canada. Cell culture media and reagents were from Invitrogen.
Plasmid Constructs--
5'-Deletion luciferase reporter
constructs encoding the murine CT Cell Culture and MAP Kinase Inhibition--
Murine embryo
fibroblasts (C3H10T1/2) and the Ha-Ras-transformed clone
ras11A were kindly provided by Dr. C. Kent (University of
Michigan) (45). They were grown at 37 °C in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin under a humidified 5% CO2 atmosphere at 37 °C. The ras11A cell line
is neomycin-resistant and was grown in the presence of 400 µg/ml
G418. During experiments, G418 was not added to the medium, as
recommended (45). The MAP kinase inhibitors PD98059 and SB202190 were
dissolved in dimethyl sulfoxide. An aliquot of each inhibitor solution
was added to the medium, and the final concentration of the vehicle in
the medium was adjusted to 0.1% (v/v). The control medium contained the same amount of the vehicle.
Transient Transfections--
Fibroblasts were transfected using
a DOTAP liposomal method (30). Control and Ha-Ras-transformed
fibroblasts were plated at a density of 2 × 106
cells/60-mm dish and transfected the next day with 2.5 µg of specific
CT CT Enzymatic Activity--
CT activity in total cell
homogenates, cytosol, and microsomes was assayed in the presence of
PC-oleate vesicles by monitoring the conversion of
phospho[3H]choline to CDP-[3H]choline as
described previously (36). Briefly, cells were collected in a
homogenization buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM NaF,
100 µM Na3V04, and 100 µg/ml
each leupeptin and aprotinin), sonicated for 25 s at 4 °C, and
the lysate was either stored at Immunobloting Analysis of CT
Immunoblotting was performed by incubation of the membranes with either
anti-M (1:2,000), anti-CT Activation States of p42/44MAPK and
p38MAPK--
Nuclear extracts were prepared at described
(30, 52). Equal aliquots of nuclear proteins or cell lysates (20-50
µg) from control and transformed cells were separated by
electrophoresis on a 10% SDS-polyacrylamide gel, then transferred to
polyvinylidene difluoride membranes. To investigate the activation
state of different MAP kinases in response to Ha-Ras transformation, we
employed specific antibodies directed against the phosphorylated forms of p42/44MAPK and p38MAPK. The immunoblotting
procedure was as described above for CT.
Immunoprecipitation and Immunoblot Analysis of Sp3-related
Proteins--
Nuclear proteins were prepared as described (30, 52).
Cells grown in 100-mm dishes were scraped into 1 ml of an
immunoprecipitation buffer (phosphate-buffered saline (PBS) containing
50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mM
dithiothreitol). The cell debris was removed by centrifugation at
10,000 × g for 10 min at 4 °C. Total proteins (500 µg) or nuclear proteins (100 µg) from control and
Ha-Ras-transformed cells were incubated with 2 µg of Sp3-specific
polyclonal antibody (H-225, 200 µg/ml; sc-13018, Santa Cruz
Biotechnology) for 1 h at 4 °C. Subsequently, 20 µg of
protein A-agarose was added, and the samples were mixed gently
overnight at 4 °C. The immunoprecipitates were collected by
centrifugation at 1,000 × g for 5 min at 4 °C, and the pellet was washed four times with PBS. Finally, the pellet was
resuspended in 40 µl of electrophoresis sample buffer and boiled for
2-3 min. Immunoprecipitated proteins were resolved on a 12%
SDS-polyacrylamide gel. For immunoblotting, proteins were transferred
to a polyvinylidene difluoride membrane that was blocked with PBS
containing 6% powdered milk, 0.5% Tween 20, then probed with anti-Sp3
antibody at a dilution of 1:500 for 1 h at room temperature. After
three washes with PBS containing 0.5% Tween 20, blots were incubated
with horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution
1:2,000) for 1 h, washed with PBS-Tween, and proteins were
visualized using enhanced chemiluminescence. Phosphorylated Sp3 was
detected using an anti-phosphotyrosine antibody (PY-20 at 1:1,000
dilution) by reprobing the same blot using a protocol similar to that
described for CT. In some experiments, Sp3-related proteins and
tyrosine phosphorylation were measured directly by immunoblotting
(31).
Electromobility Shift Assays--
Nuclear extracts from Ha-Ras
and control fibroblasts were prepared 24 h after serum stimulation
as described (30, 52). The double stranded CT RNA Preparation and Reverse Transcriptase-mediated PCR of
CT Statistical Analysis of Data--
Calculations of the average
values, standard deviations, and the comparison of means by Student's
t test were performed by Microsoft Excel (Microsoft Inc.).
Densitometric analyses of the CT Expression of Ha-Ras Increases Active
p44/42MAPK--
It is well documented that
deregulated cell proliferation is a consequence of activated Ras
signaling through the MAP kinase cascade, which is a critical component
of the proliferative response (39-41). Activated Ras signals directly
through Raf kinase with the subsequent activation of MEK1/2 kinases and
results in the phosphorylation and activation of
p44/42MAPK. Typically, the
Raf/MEK1/2/p42/44MAPK pathway is strongly stimulated by
growth factors and mitogenic stimuli, whereas in contrast, two other
signaling pathways, mediated by p38MAPK and
p46/54JNK, are activated primarily by cellular stresses
that include heat, UV radiation, and hypoxia (42-44, 53) and usually
are antiproliferative and apoptotic (54-56). We, therefore, determined
whether p44/42MAPK was activated to a greater extent in
fibroblasts constitutively expressing oncogenic Ha-Ras. The
phosphorylation of p44/42MAPK was analyzed by antibodies
that specifically detected phosphorylated, and therefore active, forms
of p42/44MAPK and p38MAPK. Using the
anti-phospho-p42/44MAPK antibody we observed bands
corresponding to p42/p44MAPK in the nuclear proteins from
both control and Ha-Ras-transformed cells (Fig.
1). The nuclear proteins from transformed
cells contained significantly more phosphorylated
p42/44MAPK, whereas p38MAPK was only weakly
phosphorylated in Ha-Ras-transformed cells relative to control cells
(Fig. 1).
Ha-Ras Transformation Decreases Total CT Enzymatic Activity but
Increases CT
We next performed immunoblotting analyses of the different CT isoforms
(Fig. 2A). Densitometric
analysis of the immunoreactive bands showed that CT
Because cells constitutively expressing Ha-Ras contained more active
nuclear p42/44MAPK activity compared with the control cells
(Fig. 1), we investigated whether or not the MEK1/2 inhibitor PD98059
altered the amount of CT CT CT Sp3 Binding to the CT Ha-Ras Transformation Increases Sp3 Protein Expression and Modifies
Sp3 Proteins Post-translationally--
We next investigated whether
the increase in Sp3 binding to the promoter (Fig. 4B) was
caused by an increase in the levels of Sp3 protein and/or a consequence
of differences in post-translational modifications of Sp3. The
existence of multiple Sp3 proteins has been shown to be caused by the
presence of an internal translation initiation start site, which
results in a truncated Sp3 that has opposing activity to the
full-length Sp3 protein (50, 60). We therefore immunoprecipitated
proteins from cell lysates and nuclear extracts from transformed and
control cells using a Sp3-specific antibody. Fig.
5 (lanes 3 and 4)
clearly demonstrates that in nuclear extracts from Ha-Ras-transformed
cells the amounts of both the full-length Sp3
(a1, 116 kDa, a2, 97 kDa) and truncated Sp3 (b1, 70 kDa and
b2, 66 kDa) are increased compared with control
cells. The doublets typically observed for each isoform,
a1/a2 and
b1/b2, likely result from
differences in mobility of post-translationally modified Sp3 proteins,
primarily from differences in phosphorylation. In the nucleus of
Ha-Ras-transformed cells 2.1-fold more of the slowest migrating form of
Sp3, a1, is present than in control nuclei. Moreover, the a2 form is 1.4-1.9-fold more
abundant in Ha-Ras-transformed cells than in control cells. However,
the a3 form of Sp3 (~90 kDa), which represents
yet another modified full-length Sp3 protein, was more dominant in
control cells than in Ha-Ras-transformed cells (2.2.-fold more in
lysates and 2.1-fold more in the nuclear extract). No differences in
the amounts of the truncated Sp3 b1 and
b2 forms between control and Ha-Ras-transformed
cells were observed in the whole cell lysates. However, in nuclear
extracts the amount of the b1 form was increased
by 50%, and the b2 form was increased by 30%
in Ha-Ras cells relative to control cells.
Next, we inspected the murine Sp3 (GenBank XP_130306) for putative
phosphorylation sites by using the NetPhos 2.0 algorithm (61). The
computer analysis did not reveal any conserved tyrosine phosphorylation
sites in the Sp3 protein. There are, however, two nonconsensus tyrosine
kinase sites at positions 87 (LQGNYIQSP) and 347 (CGKVYGKTS) which
prompted our further analysis, shown in Fig. 5 (lanes 1 and
2). The immunoprecipitated Sp3 proteins were reprobed with
an anti-phosphotyrosine antibody and compared with the Sp3 immunoblots.
Differences in phosphorylation of Sp3 proteins between control and
Ha-Ras-transformed cells are evident in both whole cell lysates and
nuclear extracts. In addition, an unknown protein of molecular mass
>150 kDa was immunoprecipitated which was more highly phosphorylated
in the Ha-Ras cells. Furthermore, Fig. 5 (lanes 1 and
2) shows less phosphorylation of Sp3
a1 and more phosphorylation of Sp3
a3 in nuclear extracts from the transformed
cells than from the control cells. Phosphorylation of Sp3
a2 and Sp3
b1/b2 was not observed. The results in Fig. 5 indicate that the full-length Sp3
a3 and an unknown protein that
coimmunoprecipitates with Sp3, are more phosphorylated in the
Ha-Ras-transformed cells, but other Sp3 forms are less phosphorylated
compared with control cells. Furthermore, the amounts of full-length
Sp3 (a1 and a2) and
truncated Sp3 (b1 and b2)
are augmented by Ha-Ras transformation resulting in stronger binding of
these Sp3 species to the CT
Based upon our previous results (30, 31), which demonstrated that Sp1
also plays an important role in the regulation of expression of
Ctpct, we next determined whether the amount of Sp1 was
increased by increased expression of Ha-Ras. We found, however, that
the amount of Sp1 protein was unchanged (data not shown), consistent
with the data in Fig. 4B that show no difference in binding
of Sp1 to the promoter probe between control and Ha-Ras-transformed cells.
Transient Expression of Sp3 Increases the Amount of Both CT We have found that in C3H10T1/2 fibroblasts Ha-Ras activates the
expression of Sp3 via the p42/44 MAP kinase pathway, thereby increasing
the amounts of CT Mechanistic Studies on Activation of the CT
Previously we established that the CT
The stronger binding of Sp3 to the CT
Given the overwhelming evidence for the role of Sp1 in growth
regulation and tumor development (62, 63), it is surprising that Sp3 is
solely implicated in the regulation of CT Ha-Ras Activation of CT
Our data show that in Ha-Ras-transformed fibroblasts, the levels of
CT
CT phosphorylation has been studied extensively. It has been clearly
established that soluble, inactive forms of CT are more highly
phosphorylated than are membrane-bound, active forms (79, 80). CT
activity also varies inversely with CT phosphorylation during the
cell-cycle (81), and the translocation of CT between membranes and
cytosol is influenced by phosphorylation/dephosphorylation (4, 79, 80).
CT dephosphorylation is secondary to its association with membranes,
suggesting that CT interactions with lipids could be a more significant
factor for enzymatic activity than phosphorylation (82, 83). Insulin
and epidermal growth factor stimulate the phosphorylation of CT in HeLa
cells in vivo, and p42/44MAPK is capable of
phosphorylating purified CT
CT
Alteration in lipid composition can also post-translationally regulate
CT
Whether the decreased CT activity in Ha-Ras-transformed cells is linked
to direct post-translational modifications of both CT isoforms,
differences in the type, content and/or location of regulatory lipids,
or other mechanisms, would be difficult to resolve.
gene is
regulated during cell proliferation (Golfman, L. S., Bakovic, M.,
and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692). We have now characterized the role of Ha-Ras in the
transcriptional regulation of the CT
gene. The expression of CT
and CT
2 proteins and mRNAs was stimulated in C3H10T1/2 murine
fibroblasts expressing oncogenic Ha-Ras. Incubation of cells with the
specific inhibitor (PD98059) of p42/44MAPK decreased the
expression of both CT isoforms. Transfection of fibroblasts with CT
promoter-luciferase constructs resulted in an ~2-fold enhanced
luciferase expression in Ha-Ras-transformed, compared with
nontransformed, fibroblasts. Electromobility shift assays indicated
enhanced binding of the Sp3 transcription factor to the CT
promoter
in Ha-Ras-transformed cells. Expression of several forms of Sp3 was
increased in nuclear extracts of Ha-Ras-transformed fibroblasts
compared with nontransformed cells. Tyrosine phosphorylation of one Sp3
form was decreased, whereas phosphorylation of two other forms of Sp3
was increased in nuclear extracts of Ha-Ras-transformed cells. When
control fibroblasts were transfected with a Sp3-expressing plasmid, an
enhanced expression of CT
and CT
was observed. However, the
expression of CT
or CT
was not increased in Ha-Ras-transformed cells transfected with a Sp3 plasmid presumably because expression was
already maximally enhanced. The results suggest that Sp3 is a
downstream effector of a Ras/p42/44MAPK signaling pathway
which increases CT
gene transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is expressed in many cells and tissues (7-13) and
has a predicted structure that contains catalytic, phosphorylation, and
lipid binding domains as well as a nuclear localization sequence (8,
14-21). Recently, CT
has been identified in human tissues and
appears to exist as two splice variants, CT
1 and CT
2, differing at their C termini (22, 23). Like CT
, CT
1/2 contains catalytic and lipid binding domains. However, CT
1 lacks the phosphorylation domain, and both CT
1 and CT
2 lack the nuclear localization
sequence (23).
and/or CT
1/2 mRNA stability, an increase in gene transcription,
or a combination of both.
gene (Ctpct) was cloned and characterized
by Tang and co-workers (29). The Ctpct promoter contains
several putative elements for binding transcription factors, including Ap1, an overlapping site for nuclear factor-
B, E2F, and Elk1, one
sterol response element, as well as three elements for Sp-related factors (30). We have shown that Sp1, Sp2, and Sp3 bind competitively to three GC-rich elements and that relative promoter activity depends
upon the abundance of these factors (31). We further established that
transcription enhancer factor-4 can bind to an upstream regulatory
element (
103/
82) and enhance the CT
gene expression through its
interactions with the basal transcriptional machinery (32). In
agreement with the finding that lipoprotein deficiency induces the
expression of CT
mRNA and protein in alveolar type II epithelial
cells (33), and our observation that the CT
promoter contains a
putative sterol response element (30), studies by Kast et
al. (34) indicate a role for cholesterol/sterol response
element-binding protein and the functionality of the sterol
response element in the regulation of CT
gene expression in Chinese
hamster ovary cells and THP-1 cells. On the other hand, Lagace et
al. (35) have shown that cholesterol/sterol response element-binding protein can stimulate PC biosynthesis without altering
transcription of the CT
gene. Recently we discovered that increased
transcription of the CT
gene occurs during the S phase of the cell
cycle in murine fibroblasts (36). However, this type of regulation was
not evident in type II lung epithelial cells (37), further suggesting a
cell type-specific regulation of the CT
gene.
promoter activity and CT
mRNA increased
after growth stimulation by serum (36), we investigated the role of Ha-Ras in the regulation of the CT
gene. We demonstrate that the
Ras/p42/44MAPK signaling pathway plays a role in the
regulation of expression of both CT
and CT
which is at least
partially mediated by the transcription factor Sp3.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter, LUC.C5 (
2068/+38),
LUC.C7 (
1268/+38), LUC.C8 (
201/+38) and LUC.D1 (
90/+38), LUC.D2
(
130/+38), and LUC.D3 (
52/+38), and the vector enabling expression
of
-galactosidase, pBK
Gal, have been described previously (30).
The Sp1 expression plasmid pPacSp1 and the pPacO-control vector were
gifts from Dr. R. Tjian (48). The Sp3 expression plasmid, pPacSp3, was
from Dr. J. Noti (49), and human Jun and Rb expression plasmids, pCMV-Jun, pCMV-hRb, respectively, and control vector pCMVO were from
Dr. J. M. Horowitz (50).
-luciferase reporter plasmid with or without the indicated amounts
of Sp1 and Sp3 expression plasmids (pPacSp1 or pPacSp3) or control
vector (pPacO). Transfected cells were grown overnight in normal
medium, then growth was arrested in a low serum medium (0.5% fetal
calf serum) for 2 additional days. The arrested cells were stimulated
to grow by the addition of 10% serum and 24 h later collected for
further analysis. The pBK
Gal vector, encoding
-galactosidase, was
cotransfected as an internal control to measure differences in
transfection efficiency. Luciferase and
-galactosidase activities
were measured using a luciferase and
-galactosidase assay system
(Promega). The amount of cellular protein was measured by the Bio-Rad method.
70 °C or added immediately (25 µg of protein) to a CT-activity assay buffer (57.8 mM
Tris-HCl, pH 7.5, 40 mM NaCl, 1.78 mM EDTA, 8.9 mM magnesium acetate) containing 1.5 mM [3H]phosphocholine, 3 mM CTP, and 0.2 mM PC-oleate (1:1) vesicles in a final volume of 100 µl.
The reaction was incubated 15 min at 37 °C and stopped by boiling
for 2 min. The supernatant was collected by centrifugation at 500 × g for 5 min and an aliquot spotted on a Silica Gel G60
thin-layer plate. The plate was developed in a solvent mixture of
methanol, 0.6% NaCl, and saturated ammonia (10:10:0.9, v/v) and
CDP-[3H]choline quantified by liquid scintillation counting.
and CT
--
Cell lysates from
control and Ha-Ras-transformed cells (20-50 µg of protein) were
prepared as described previously (36) and separated on 10% denaturing
polyacrylamide gels. The proteins were transferred onto polyvinylidene
difluoride membranes (Bio-Rad) at ~150 mA and 4 °C for 1 h.
After checking for protein loading with Ponceau S dye, the membranes
were incubated overnight with 5% skim milk in TTBS (20 mM
Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20). The
membranes were washed in TTBS and incubated at 25 °C for 2 h
with polyclonal antibodies raised against CT (anti-M), CT
, CT
1/2,
and/or CT
2. The CT (anti-M) antibody was a rabbit polyclonal
antibody directed against a peptide corresponding to amino acids
256-288 (M domain) of rat liver CT
from Dr. R. Cornell (51). The
anti-CT
rabbit polyclonal antibody, corresponding to the first 17 amino acids of human CT
, the rabbit anti-human CT
1/
2 (B2
epitope) antibody, corresponding to amino acids 5-22 of CT
1/2, and
the rabbit anti-human CT
2 antibody (B3 epitope), corresponding to
amino acids 347-365 of CT
2, were all gifts from Dr. S. Jackowski
(22, 23).
(1:1,000), anti-CT
1/2 (1:1,000) or
CT
2 (1:5,000) as the primary antibody. The membranes were washed
five or six times for 15 min each with TTBS, then incubated with goat
anti-rabbit antiserum (1:5,000 dilution; horseradish peroxidase-conjugated (Roche Molecular Biochemicals) at room
temperature for 1 h. The membranes were washed five or six times
with TTBS, developed with enhanced chemiluminescence reagent (Pierce),
and exposed to XAR-5 film (Kodak). To reprobe the blots, the membranes were stripped in 100 mM mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8, at 50 °C for 30 min and subjected
to the above immunoblotting procedure.
promoter probes D1
(
98/+38) and D2 (
130/+38) containing Sp binding sites were prepared
from LUC.D1 and LUC.D2 reporter vectors by restriction digestion and
purification (30). The promoter fragments were 3'-end labeled with
[32P]dCTP and Klenow polymerase and the binding of Sp1
and Sp3 analyzed after separation of DNA-protein complexes on 5%
nondenaturing polyacrylamide gels followed by autoradiography (30,
31).
--
Total RNA from control and Ha-Ras-transformed cells grown
in the presence or absence of MAP kinase inhibitors was extracted using
TRIAZOL reagent (Invitrogen) (36). The RNA was reverse transcribed with
a first strand cDNA synthesis kit (Superscript II, Invitrogen)
according to the manufacturer's protocol. The reverse-transcribed
mRNA (0.5-3 µg) was amplified using PCR primed with forward
(5'-ATGCACAGTGTTCAGCCAA-3') and reverse (5'-GGGCTTACTAAAGTCAACTTCAA-3') primers corresponding to the CT
gene. The signal from the CT
mRNA transcript was normalized to the signal obtained from
glycerol-3-phosphate dehydrogenase as a control using the primer pair
5'-TCCACCACCTGTTGCTGTA-3' (forward) and 5'-ACCACAGTCCATGCCATCAC-3'
(reverse) or with the signal from cyclophilin using the primers
5'-TCTTCTTGCTGGTCTTGCCATTCC-3' (forward) and
5'-TCCAAAGACAGCAGAAAACTT-3' (reverse). 30 cycles of PCR amplification
at 95 °C for 1 min, 45° for 1 min, and 72 °C for 2 min produced
~200-bp fragments for CT
and ~250-bp fragments for
glyceraldehyde-3-phosphate dehydrogenase; 33 cycles at 94 °C for 1 min, 60 °C for 2 min, 72 °C for 2 min produced ~300-bp fragments for cyclophilin.
and/or CT
protein mass and
mRNA expression were performed by Scion Image acquisition and
analysis software (Scion Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (42K):
[in a new window]
Fig. 1.
Nuclear p42/p44MAPK, but not
p38MAPK, is activated in Ha-Ras-transformed C3H10T1/2
fibroblasts. Control and Ras-transformed fibroblasts were grown in
10% serum-containing medium. Cell lysates were harvested 24 h
after serum treatment and nuclear extracts prepared. The
phosphorylation of p42/44MAPK and p38MAPK was
determined by immunoblotting. Each lane contains 50 µg of
nuclear protein. Immunoblots were probed with
anti-phospho-p42/p44MAPK antibody and
anti-phospho-p38MAPK antibody. The immunoblots were also
stained with Ponceau S to verify equal loading of protein in each
lane (not shown). Results were similar in two independent
experiments.
and CT
2 Protein Mass in a
p42/44MAPK-dependent Manner--
It
was of interest to know whether or not CT activity was affected by the
Ras-signaling pathway. Table I shows
total CT enzymatic activities in control and Ha-Ras-transformed cells
stimulated by serum in the presence and absence of specific inhibitors
of p42/p44MAPK and p38MAPK. The flavone
compound PD98059 is a specific inhibitor of MEK1/2 kinase and has been
used extensively for investigating the physiological function of
p42/p44MAPK (57). The pyridylimidazole compound SB202190 is
a p38MAPK inhibitor (58, 59). Neither compound inhibits
other known related kinases (57-59). The results shown in Table I
reveal that, similar to published data (45), in Ha-Ras-transformed
cells CT activity was 65% (p < 0.05) less than in the
control cells. An equivalent decrease was also observed in the cytosol
and microsomes of Ha-Ras-transformed cells compared with control cells
(data not shown). Paradoxically, in neither control cells nor
Ha-Ras-transformed cells was the CT activity modified by kinase
inhibitors, suggesting a complex regulation of CT activity by Ras
signaling (Table I).
Ha-Ras transformation inhibits whereas MEK1 (PD98059) and
p38MAPK (SB202190) inhibitors do not alter total CT
(CT + CT
) enzymatic activity
protein levels
increased 1.8 ± 0.2-fold in Ha-Ras-expressing cells relative to
control cells. With the CT
2-specific antibody (Fig. 2B)
Ha-Ras-transformed cells contained significantly higher amounts (1.5- ± 0.1-fold) of immunoreactive CT
2 protein than did the control
cells (CT
1 was not detectable by the CT
1/2 antibody).
View larger version (38K):
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Fig. 2.
The amount of CT and
CT
2 protein is increased by
Ha-Ras/MEK1/2/p42/44MAPK signal transduction pathway.
A, effect of Ha-Ras transformation (upper panel)
and MEK1/2 inhibition by PD98059 (lower panel) on the amount
of CT
protein. B, effect of Ha-Ras transformation
(upper panel) and MEK1/2 inhibition by PD98059 (lower
panel) on the amount of CT
2 protein. In the upper
panels, quiescent cells were grown in 10% serum for 24 h
without PD98059. In the lower panels of both A
and B quiescent cells were grown in the presence of 10%
serum for 24 h with the indicated concentrations of PD98059.
Results are representative of four independent experiments.
and CT
2 proteins (Fig. 2). Because
MEK1/2 phosphorylates and activates p42/p44MAPK, the
inhibitor would be expected to inhibit the phosphorylation of
p42/p44MAPK and their downstream targets. The addition of
the 20-150 µM MEK1/2 inhibitor to the cells reduced the
amount of CT
(Fig. 2A) and CT
2 (Fig. 2B) in
a dose-dependent manner. The magnitude of inhibition by 150 µM PD98059 in control cells was 37 and 50% for CT
and CT
2, respectively, and in Ha-Ras-transformed cells was 61 and 87%
for CT
and CT
2, respectively.
mRNA Is Increased in Ha-Ras-transformed Cells in a
p42/44MAPK-dependent
Manner--
Based upon the above results and our previous findings
that serum growth factors stimulate the expression of the CT
gene at
the transcriptional level (36), we next investigated whether CT
mRNA was increased by overexpression of Ha-Ras. Fig.
3A shows that CT
mRNA
is increased in Ha-Ras-transformed cells in the absence of serum
(2.5-2.8-fold) as well as after serum stimulation (4.0-6.5-fold).
Fig. 3B demonstrates that 150 µM
p42/44MAPK inhibitor (PD98059) reduces CT
mRNA
expression by 70% in both control and Ha-Ras cells. The
p38MAPK inhibitor SB202190 did not decrease CT
mRNA
expression in control and Ha-Ras cells. Together, these results suggest
that the increase in CT
protein mass shown in Fig. 2 is caused by an
increased transcription of the CT
gene in Ha-Ras-transformed cells.
This increase is p42/44MAPK-dependent and is
increased further by serum.
View larger version (21K):
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Fig. 3.
The amount of CT
mRNA is altered by serum, Ha-Ras transformation, and
inhibition of p42/44MAPK. A,
effect of serum and Ha-Ras transformation on CT
mRNA abundance
in fibroblasts. Control and transformed cells were incubated without
serum for 48 h. At this time, either total RNA was isolated, or
the serum-starved cells were incubated for an additional 24 h with
10% serum. mRNA abundance was estimated by reverse
transcriptase-based PCR. Total mRNA was reverse transcribed using
primers specific for CT
and glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) (Control), and PCR was performed within
the linear range of concentrations of mRNA and number of PCR
cycles. CT
mRNA expression was normalized to
glyceraldehyde-3-phosphate dehydrogenase mRNA, and the abundance of
CT
mRNA was calculated relative to that in control cells grown
without serum. B, effect of the MEK1 inhibitor PD98059 and
the p38MAPK inhibitor SB202190 on CT
mRNA abundance
in control and Ha-Ras-transformed fibroblasts. The experiment was
performed as in A except that cyclophilin served as control
for the reverse transcriptase-based PCR. mRNA abundance is given
relative to that of control cells grown without inhibitors.
Promoter Activity Is Increased in Cells Overexpressing
Ha-Ras--
To examine further the transcriptional regulation of the
CT
gene, we investigated CT
promoter activity in transiently
transfected cells using a series of truncated promoter-luciferase
reporters (Fig. 4A). We have
shown previously that C3H10T1/2 fibroblasts support the activation of
CT
promoter-luciferase constructs (30, 31). Fig. 4A shows
that control and Ha-Ras-transformed cells both have the ability to
direct luciferase gene expression, as expected (31). Interestingly, the
promoter activity of each of the promoter-reporter constructs tested,
including the basal promoter construct
52/+38 (LUC.D3), is
persistently 1.5-3-fold higher in the Ha-Ras cells than in control
cells, suggesting that the basal promoter region is sufficient to drive
the increased luciferase expression in the cells constitutively
expressing Ha-Ras.
View larger version (21K):
[in a new window]
Fig. 4.
Promoter activation of the
CT gene by Ha-Ras transformation and binding
of transcription factor Sp3. A, CT
promoter-luciferase reporter activity in control and Ha-Ras-transformed
fibroblasts. A series of truncation mutants of the CT
promoter
linked to a luciferase reporter, spanning
201 bp to +38 bp of the
promoter region (2.5 µg), and the SV-40 promoter-luciferase control
vector (SV-40; 2.5 µg) were transiently cotransfected with the
pSV
-galactosidase expression vector (2.5 µg). Luciferase activity
was normalized to
-galactosidase activity and is relative to that of
the SV-40 control. Cells were serum deprived for 3 days prior to the
transfection, then stimulated with serum for 24 h, and luciferase
assays were performed. Results are the means ± S.D. of three
independent experiments, each performed in triplicate. B,
Ha-Ras transformation increases the binding of Sp3, but not Sp1, to the
CT
promoter. Electrophoretic mobility shift assays were performed
using control and Ha-Ras-transformed nuclear fractions and were
incubated with 32P-labeled oligonucleotide consisting of
the region
90/+38 bp of the CT
proximal promoter and basal
Sp-cis-acting elements. The complexes were resolved by
electrophoresis on a nondenaturing 5% polyacrylamide gel. The bands
corresponding to the complexes for Sp3 (I and II) and Sp1 are
indicated. This experiment was repeated twice with similar
results.
Promoter Is Increased in
Ha-Ras-transformed Cells--
The basal promoter region of the CT
gene contains complex, overlapping binding sites for nuclear
factor-
B, Elk1, E2F, and Sp1. Previously, we investigated the role
of the Sp site located at position
39/
9 (30, 31). The role of Sp1,
Sp2, and Sp3 in binding to this site was established by mutation and
overexpression analysis in insect and mammalian cells, including
C3H10T1/2 murine fibroblasts (30, 31). We have demonstrated previously
that Sp1 and Sp3 specifically and competitively bind to this promoter region as well as to two other regions of the CT
promoter at
88/
50 and
148/
128. We therefore performed
electromobility shift assays with nuclear proteins from control and
Ha-Ras-transformed cells to determine whether the increase in CT
promoter activity in the Ha-Ras cells was caused by changes in protein
binding to the promoter. Fig. 4B shows that the protein
profile from control and Ha-Ras cells is the same, suggesting that
stimulation by Ha-Ras transformation of CT
promoter activity is not
the result of the binding of a new transcription factor. We
demonstrated previously by supershift analysis in C3H10T1/2 cells using
antibodies specific for the Sp1 and Sp3 proteins that these proteins
bind the promoter probe
90/+38 (30, 31). From Fig. 4B, it
can be seen that there is a stronger binding of the Sp3-related nuclear
proteins in the transformed, compared with the control, cells. A
similar binding profile was observed when a different
promoter probe,
130/+38, which contained Sp binding sites, was used
(data not shown). From these results and from our previously published
data (30, 31), we conclude that the increase in CT
promoter activity in the Ha-Ras-transformed cells shown in Fig. 4A is, at
least in part, a consequence of an increased binding of the
transcription factor Sp3.
View larger version (59K):
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Fig. 5.
Differential expression and phosphorylation
of full-length Sp3 species and truncated Sp3 in control and
Ha-Ras-transformed fibroblasts. Control and Ha-Ras cell lysates
(500 µg; upper panel) and nuclear proteins (100 µg;
lower panel) were immunoprecipitated with anti-Sp3 antibody.
Phosphorylation of Sp3-related proteins (lanes 1 and
2) was detected by anti-phosphotyrosine antibodies, and Sp3
proteins (lanes 3 and 4) were identified by using
anti-Sp3 antibodies. The full-length Sp3 proteins, a1,
a2, and a3, and truncated Sp3 proteins,
b1 and b2, are described under "Results." An
unknown protein that coprecipitates with Sp3 proteins is also indicated
(*). The ~50 kDa bands in lanes 3 and 4 represent IgG. Molecular size markers are indicated on the left
side.
promoter (Fig. 4B).
and
CT
2 Protein--
Mutation and transfection analyses (31) revealed
that Sp3 is functionally equivalent to Sp1 and can act as an
independent transcriptional activator of the CT
promoter. We have
also demonstrated that overexpression of Sp3 in mammalian cells
(including C3H10T1/2 murine embryo fibroblasts) stimulates CT
promoter-luciferase reporter activity (31). Thus, to determine whether
Sp3 increases the amount of CT proteins we analyzed the expression of
CT proteins after transfection of control and Ha-Ras fibroblasts with
various amounts (0-12 µg) of the Sp3-expression plasmid, pPacSp3.
The levels of both CT
(1.2-fold and 1.4-fold) and CT
2 (1.15-fold and 1.5-fold) proteins increased in experiments with 3 and 6 µg of
the pPacSp3 plasmid, respectively, for control cells only (Fig. 6). No
increase in CT
and CT
2 proteins was apparent in Ha-Ras cells
after transfection with 3 and 6 µg of pPacSp3 because CT expression was already increased. At higher plasmid
concentration, 12 µg of pPacSp3, the CT
protein decreased to the
"basal" levels (transfections with empty plasmid pPacO), and in
case of CT
2 the detectable protein declined below the basal
level (0.7-fold and 0.5-fold in the Ha Ras and control cells,
respectively). These results show for the first time that expression of
Sp3 increases the level of CT
and CT
2 proteins (Fig.
6).
View larger version (28K):
[in a new window]
Fig. 6.
The amount of CT
protein is increased by transfection of cDNA encoding
Sp3. Sp3 proteins were expressed in control and Ha-Ras-transformed
cells by transient transfection of 3, 6, and 12 µg of pPacSp3 or 12 µg of pPacO control vector. The total amount of DNA was kept constant
at 12 µg by the addition of the appropriate amounts of empty control
vector. After 48 h, cells were harvested and 50 µg of proteins
from cell lysates was loaded onto 7% SDS-polyacrylamide gels. The
expression of CT
and CT
2 was assessed by immunoblotting. Two
independent experiments were performed with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and CT
2 mRNA and protein. Our data indicate
that when Sp3 binds to one or more sites on the CT
promoter, transcription of the CT
gene is stimulated. Because the CT
promoter has not yet been characterized, it is not possible to
determine whether Sp3 directly or indirectly governs the increased
amount of CT
2 in the Ha-Ras-transformed cells.
Gene--
The focus
of the current study was to elucidate the mechanism by which the
expression of CT
mRNA and protein is increased in
Ha-Ras-transformed fibroblasts. Previously, we established that growth
stimulation by serum increased the expression of CT
mRNA during
the cell cycle (36). The results presented here demonstrate that the
p42/44MAPK signaling pathway is responsible for the
serum-induced increase in CT
mRNA and protein that is further
magnified by constitutive activation of p42/44MAPK in
transformed cells. In accordance with those results, when signaling via
p42/44MAPK was inhibited, or when serum was eliminated from
the growth medium, the expression of CT
mRNA and protein was
decreased. To identify the regulatory cis-acting elements
responsible for the Ras/p42/44MAPK regulation of the CT
gene, we used luciferase-reporter mutants spanning the promoter region
from
201 bp to +38 bp. We demonstrated that the promoter
activity for all constructs was persistently higher in transformed cells.
basal promoter is completely
inactive if transcription is not supported by Sp1 or Sp3 nuclear
factors, as in insect cells naturally lacking those factors (31).
Transient expression of Sp1 or Sp3 initiates CT
basal transcription
in insect cells, suggesting that the interactions of Sp1 and Sp3 with
the basal promoter and general transcription factors are critical for
the CT
transcriptional activation (31). The basal CT
promoter Sp
element is located in the vicinity of the transcription initiation
site, at position
22/
15 bp, and could bind Sp1 and Sp3 together
with other nuclear proteins, possibly including the basal transcription
factors (30, 31). We have now established that Sp3 from transformed
cells binds stronger to the CT
promoter than Sp3 from untransformed
cells and that Sp1 binds equally in both cells (Fig. 4B),
suggesting that Sp3, not Sp1, could be solely responsible for the
up-regulation of CT
gene in transformed cells.
promoter in transformed cells
is probably a combination of increased mass and modified phosphorylation of several Sp3 protein species. Sp3 exists in two main
isoforms (50) of which we showed that the full-length Sp3 a
(stimulatory form) is primarily overexpressed and post-translationally
modified by Ha-Ras/p42/44MAPK. The truncated form Sp3
b (repressive form) was increased modestly by the Ha-Ras
transformation and was not affected by tyrosine phosphorylation (Fig.
5). At lower Sp3 cDNA concentrations the protein expression of both
CT
and CT
2 increased in control cells, whereas in transformed
cells, overexpression of Sp3 did not alter the CT
and CT
2 levels
because they were already elevated. However, at higher Sp3 cDNA
concentrations, the levels of CT
and CT
2 were decreased below or
returned to the basal level even in transformed cells, suggesting that
Sp3 also acted as a transcriptional repressor. A dual property of Sp3
has been manifested in previous studies for the regulation of other
genes (62, 63) including our own work with the Sp3 regulation of CT
promoter in insect cells (31). That Sp3 is a multiple regulator is also
supported by the findings that at higher concentrations, Sp3 is
normally inhibitory by a DNA binding-independent mechanism that
involves competition for components of the basal transcriptional
machinery (62) or titration of the promoter-specific transcription
factors (64).
during oncogenic
transformation and that neither the Sp1 binding nor abundance appears
to be important for CT
gene expression. Sp1 is a well known target
for Ras/p42/44MAPK regulation by phosphorylation, which
typically does not involve changes in the Sp1 protein mass (65-68).
Even as a target for p42/44MAPK, we report that Sp1 is not
a significant regulator of CT
gene expression after oncogenic
transformation and that Sp3 is predominantly regulating CT
by means
of the Ras/p42/44MAPK signaling pathway. To our knowledge
this is the first time that Sp3, independently from Sp1, appears to
regulate gene expression by means of the Ras/p42/44MAPK
signaling pathway. Interestingly, ERK2 (p42MAPK) but not
ERK1 (p44MAPK) could also be preferentially stimulated by
Sp3, not Sp1 (69), which taken together with our findings suggest the
existence of a positive feedback mechanism for the observed selective
up-regulation of Sp3 protein in transformed cells. The most recent
evidence from gene disruption of Sp3 (70) suggests that Sp3 has a
distinct regulatory function from Sp1 and other members of the broader Sp1/Krüppel-like family of transcription factors (62). Sp3 is
essential for postnatal survival, and Sp3-deficient embryos are
growth-retarded and die at birth of respiratory failure (70). The
observed breathing defect remains obscure, and surfactant protein
expression was not found to be different from that in the Sp3 wild type
embryos (70). However, given that CT
plays a central regulatory role
in the production of the lung phospholipid surfactant, PC, it may be
that the deletion of the Sp3 gene would diminish CT
expression and,
thus, PC production causing lung failure. It is highly likely that Sp3
deletion will also abolish the expression of CT
; however, the
precise mechanisms for how Sp3 regulates the expression of CT
will
remain unknown until the promoter of the murine CT
gene is isolated
and fully characterized.
Expression--
Expression of
constitutively active Ras induces cellular proliferation and usually is
transforming. The mechanism by which Ras mediates these effects is
believed to be through a direct interaction with downstream effectors
resulting in a persistent activation of MAP kinase signaling pathways
(39-41). It is well established that one characteristic of transformed
cells is perturbation of lipid synthetic and degradative pathways
(71-73), suggesting an active role of lipids in oncogenic
transformation. Expression of oncogenic Ras correlates with increased
levels of phosphocholine and phosphoethanolamine (45, 73-75),
diacylglycerols (76), inositol phosphates (77), and arachidonic acid
(78). The stimulated degradation of phospholipids is accompanied by an
increased biosynthesis (45, 73-75), demonstrating an accelerated
turnover of lipids in transformed cells. Increased amounts of
phosphocholine and phosphoethanolamine have been implicated in tumor
development (78), during which choline/ethanolamine kinases are
typically up-regulated, whereas enzymes involved in the subsequent
metabolism of phosphocholine and phosphoethanolamine (i.e.
CTP:phosphocholine- and phosphoethanolamine cytidylyltransferases) are
either up-regulated (47) or down-regulated (45, 46) depending on the
cell type.
and CT
2 proteins and mRNAs are increased relative to
control cells. However, we were surprised that the increased amounts of
CT
and CT
2 proteins in the Ha-Ras-transformed cells did not
correlate with an increase in CT activity. On the contrary, CT activity
was significantly lower in transformed cells than in control cells even
though more CT protein was present. Other studies have also reported
that CT activity is lower in Ha-Ras-transformed cells than in control
cells (45). Moreover, CT activity was not affected by the specific
inhibitor of p42/44MAPK (PD98059) in transformed cells
(that had constitutively active p42/44MAPK) or in control
cells in which p42/44MAPK was activated by serum. The
inhibitor PD98059, however, decreased the protein mass of both CT
and CT
2 as would be expected if their expression were regulated by
p42/44MAPK. Thus, Ha-Ras/p42/44MAPK signaling
simultaneously increases the amount of CT protein yet decreases CT activity.
(65). Thus, phosphorylation of CT might
be enhanced in the Ha-Ras-transformed cells because of constitutive
activation of p42/44MAPK.
has been discovered only recently (22, 23), and its
post-translational regulation by phosphorylation has not been reported.
However, CT
2 phosphorylation domains are highly homologous to the
phosphorylation domains of CT
and possibly could be targeted by
similar mechanisms. Which of the numerous Ser/Thr phosphorylation sites
of CT
and CT
are selective targets for Ras/p42/44MAPK
signaling is presently unknown.
(and possibly CT
) activity and might contribute to the
modification of CT activity in Ha-Ras-transformed cells. Ample evidence
suggests that the cellular lipid content is changed after oncogenic
transformation (45-47, 71-73, 76). For example, recent data show that
increased cell proliferation and activation of the
p42/44MAPK signaling cascade alter the expression and/or
activity of several genes of phospholipid metabolism, including
phospholipase C (76), phospholipase A2 (71), the
level and/or phosphorylation of lipid transport proteins, apoA-I (85),
apoC-III (86), low density lipoprotein receptor (87), and the activity
of lipid-related transcription factors sterol response element-binding
protein (88) and peroxisomal proliferator activated receptor-
(84) which regulate many genes involved in lipid metabolism,
perhaps including the CT
gene (34).
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ACKNOWLEDGEMENTS |
---|
We thank Sandra Ungarian for excellent technical assistance and Prof. Jean Vance for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Canadian Institutes of Health Research.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.
Present address: Dept. of Human Biology and Nutritional Sciences,
University of Guelph, Animal Science/Nutrition Bldg., Rm. 346, Guelph,
Ontario NIG2W1, Canada.
§ These authors contributed equally to this work.
¶ Postdoctoral fellow of the Alberta Heritage Foundation for Medical Research. Present address: Human Cancer Genetics, The Ohio State University, 420 West 12th Ave., Columbus, OH 43210.
Medical scientist of the Alberta Heritage Foundation for
Medical Research and Canada Research Chair in Molecular and Cell Biology of Lipids. To whom correspondence should be addressed: Dept. of
Biochemistry, University of Alberta, 328 Heritage Medical Research
Centre, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-8286; Fax: 780-492-3383; E-mail: dennis.vance@ualberta.ca.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300162200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PC, phosphatidylcholine; CMV, cytomegalovirus; CT, CTP:phosphocholine cytidylyltransferase; LUC, luciferase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PBS, phosphate-buffered saline; Rb, retinoblastoma; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase.
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REFERENCES |
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