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INTRODUCTION |
Tissue transglutaminase
(TGase)1 is a member of a
family of enzymes that catalyzes a calcium-dependent
transamidation reaction that results in the covalent linkage of donor
glutamine residues of one protein to acceptor primary amino groups of
another protein or polyamine (1, 2). This modification of proteins by
TGase has been implicated in regulating numerous physiological
processes including endocytosis (3), axonal growth (4), cell
differentiation (5), and cell survival (6-8). However, the most
frequently reported cellular outcome associated with aberrant TGase
transamidation activity has been the induction of apoptosis (8-12),
suggesting that the extent of TGase enzymatic activity may be crucial
for determining the exact cellular effects induced by the TGase.
In addition to transamidation activity, TGase also undergoes a GTP
binding/GTP hydrolysis cycle, a feature that distinguishes it from the
other transglutaminases (2). GTP-bound TGase participates in signaling
pathways that link cell surface receptors to intracellular effectors.
The most studied example of this involved the up-regulation of
phospholipase C activity by the
1-adrenergic receptor
(13, 14), where it was shown that the
1-adrenergic
receptor stimulated the GTP binding activity of TGase. The GTP-bound
TGase species, in turn, was shown to stimulate phosphoinositide lipid
hydrolysis by phospholipase C. The ability of TGase to transduce
signals adds another dimension to its functionality and may help
explain how the same protein can be linked to several, sometimes
opposing, cellular responses. This point is exemplified by the recent
finding that a transamidation-defective form of TGase protected cells from heat shock-induced apoptosis (8), indicating that the GTP
binding/GTPase activity of TGase likely mediated the survival advantage.
Despite advances in the characterization of TGase functions and their
impact on cellular processes, the events that lead to a completely
activated TGase species (a molecule having transamidation activity and
capable of binding GTP) in vivo are not completely understood. One aspect of TGase regulation involves altering the expression levels of the protein. In several cell types, the TGase protein is expressed at low levels (4, 7, 15), and increases in TGase
expression and activation can be induced following chronic stimulation
with growth factors (16), chemotherapeutic agents (17), cytokines (18),
and retinoids (19). Retinoic acid (RA) is the most consistent inducer
of TGase expression and activation, and a mechanism by which RA
up-regulates TGase expression has been elucidated. RA binds to retinoic
acid receptors (RARs) and stimulates their association with specific
DNA motifs located in the promoter region of the TGase gene, resulting
in transcription (2). Other than modifying gene transcription, RARs
also activate signaling molecules. The signaling potentials of STAT3,
PI3K, and ERK have been shown to increase following RA treatment
(20-22), suggesting that some RA-mediated effects may require the
combined activation of certain signaling proteins and the
transcriptional capability of the RARs.
For some time our laboratory has been interested in understanding the
regulation and function of TGase in cells. Contrary to findings linking
TGase to apoptosis (8-12), we have shown that RA-induced TGase
expression provided a protective effect from N-(4-hydroxyphenyl)retinamide (HPR)-mediated apoptosis in
HL-60 and NIH3T3 cells (7). Moreover, RA stimulation resulted in the
activation of the anti-apoptotic molecule PI3K, which was necessary for
the induction of TGase expression and GTP binding activity (23). Given
that the regulation of TGase expression is linked to PI3K activation
and that growth factors frequently activate PI3K, we considered whether
co-treatment of NIH3T3 cells with growth factors and RA would
potentiate the induction of TGase expression, as well as the level of
transamidation or GTP binding activity, compared with RA treatment
alone. Epidermal growth factor (EGF) was chosen as the co-stimulus
because it is not only an effective activator of PI3K but it also
up-regulates TGase activity in hepatocytes (24). However, we found that
rather than enhancing the induction rate or the expression levels of
TGase, EGF suppressed the ability of RA to stimulate TGase expression.
Activation of the Ras-ERK pathway was sufficient to impart this
inhibitory effect of EGF, since cells with continuous Ras signaling
also limited RA-induced TGase expression, and blocking ERK activation
in these cells restored the ability of RA to up-regulate TGase
expression. These findings prompted us to question how EGF-regulated
processes would be affected by exogenous TGase expression. Cells
expressing TGase displayed a growth and survival advantage over control
cells, and EGF-mediated proliferation and resistance to serum
deprivation-induced apoptosis was not compromised by the overexpression
of TGase. These findings demonstrate that EGF inhibits RA-induced TGase expression in NIH3T3 cells by activating the Ras-ERK pathway, whereas
limiting TGase expression does not appear to be critical for EGF to
elicit its effects. When coupled with our earlier findings linking the
activation of PI3K to the induction of TGase expression, we begin to
appreciate the complex interplay of signaling pathways that regulate
TGase in cells. Moreover, these results are consistent with the idea
that EGF-coupled signaling does not counteract the up-regulation of
TGase as a means of reversing a TGase-mediated growth inhibitory or
apoptotic activity as TGase exhibits mitogenic and survival activities
rather than serving as a suppressor of cell growth. Thus, the effects
of EGF receptor signaling on TGase expression are more likely the
outcome of EGF-directed effects on the broad profile of RA-stimulated
gene expression, in order to repress the anti-mitogenic effects of
other RA-induced gene products.
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EXPERIMENTAL PROCEDURES |
Materials--
LY294002 and PD98095 were obtained from
Calbiochem, and EGF was from Invitrogen. RA, HPR, platelet-derived
growth factor (PDGF), and monodansyl cadaverine were purchased from
Sigma. The TGase antibody was obtained from Neomarkers; the actin
antibody was from Sigma, and the phospho-ERK and ERK antibodies were
from New England Biolabs. [
-32P]GTP was purchased from
PerkinElmer Life Sciences, and all additional material was obtained
from Fisher unless stated otherwise.
Cell Culture--
NIH3T3 cells were grown in Dulbecco's
modified Eagle's medium containing 10% calf serum and 100 units/ml
penicillin, and MCF-7 cells were grown in RPMI 1640 medium containing
10% fetal bovine serum and 100 units/ml penicillin. The cell lines
were maintained in a humidified atmosphere with 5% CO2 at
37 °C. For the various treatments described, the cells were grown to
near confluence in medium containing 10% serum, and then medium
containing 1% serum with 5 µM RA (unless indicated
otherwise), or 100 ng/ml EGF, ± 10 µM LY294002 or ± 10 µM PD98095, were added for the times indicated
under "Results." Cells were rinsed with phosphate-buffered saline
(PBS) and then lysed with cell lysis buffer (10 mM
Na2HPO4, 150 mM NaCl, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% NaF, 1 mM NaVO4, 25 mM
-glycerophosphoric acid, 100 µg/ml phenylmethanesulfonyl fluoride,
and 1 µg/ml each aprotinin and leupeptin, pH 7.35). The lysates
were clarified by centrifugation at 12,000 × g for 10 min at 4 °C. Protein concentrations were determined using the
Bio-Rad DC protein assay.
Western Blot Analysis--
Total cell lysates from each sample
were combined with Laemmli sample buffer, boiled, and subjected to
SDS-PAGE. The proteins were transferred to nitrocellulose filters and
blocked with TBST (20 mM Tris, 137 mM NaCl, pH
7.4, and 0.02% Tween 20) containing 5% nonfat dry milk. The filters
were incubated with the various primary antibodies diluted in TBST
overnight at 4 °C and then washed 3 times with TBST. To detect the
primary antibodies, anti-mouse or -rabbit conjugated to horseradish
peroxidase (Amersham Biosciences), diluted 1:5000 in TBST, was
incubated with the filters for 1 h, followed by 3 washes with
TBST. The protein bands were visualized on x-ray film after exposing
the filters to chemiluminescence reagent (ECL, Amersham
Biosciences).
Photoaffinity Labeling of TGase--
Photoaffinity labeling of
TGase was performed by incubating whole cell lysates with 5 µCi of
[
-32P]GTP in 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 20% (w/v)
glycerol, 100 mM NaCl, and 500 µM AMP-PNP for
10 min at room temperature. The samples were placed in an ice bath and irradiated with UV light (254 nm) for 15 min, mixed with 5× Laemmli sample buffer, and boiled. SDS-PAGE was performed, followed by transfer
to nitrocellulose filters and exposure on x-ray film.
Nuclear Condensation or Blebbing Assay--
Cells were seeded in
6-well dishes and grown in complete medium for 2 days. The cells were
then incubated in medium containing 1.5% serum ± 5 µM RA (unless indicated otherwise) and ± 100 ng/ml EGF for 2 days. The cultures were then incubated with serum-free medium
for an additional day and were fixed and stained with
4,6-diamidino-2-phenylindole (2 µg/ml) for viewing by fluorescence
microscopy. Apoptotic cells were identified by condensed nuclei and/or blebbing.
Cell Growth Assays--
Cells were seeded at 1 × 105 cells/well and grown in medium containing 1.5%
serum ± 5 µM RA and ± 100 ng/ml EGF. Every 2 days the medium was changed. At 0, 2, 4, and 6 days of treatment, the cells were collected and counted.
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RESULTS |
TGase Protects NIH3T3 Cells from Serum Deprivation-induced
Apoptosis--
Exposure of cells to RA limits cell growth or induces a
cell death response in several cell types suggesting that retinoids may
be efficacious in the treatment of certain forms of cancer. More recent
work has shown that exposure of GL-15 glioma cells to 1 µM RA promoted cell proliferation (20), and findings from our laboratory demonstrated that RA affords protective effects to HL-60
and NIH3T3 cells from the apoptotic agent
N-(4-hydroxyphenyl)retinamide (HPR) (7). We decided to test
whether RA treatment could also protect NIH3T3 cells from serum
deprivation-mediated cell death. Mouse fibroblast cells were chosen for
this study because it was determined previously that exposure of NIH3T3
cells to RA caused the up-regulation of TGase expression, as well as
stimulated the GTP binding and transamidation activities of TGase, and
that the RA-induced TGase activity limited the effectiveness of HPR at inducing apoptosis (7). Consistent with these findings, Fig. 1A shows that NIH3T3 cells
treated with 5 µM RA for 2 days resulted in an increase
in TGase protein levels (Fig. 1A,
TGase) and
yielded a corresponding stimulation of GTP binding activity (Fig.
1A, [
-32P]GTP
binding) as indicated by the incorporation of
[
-32P]GTP. The extent of TGase expression and GTP
binding activity stimulated at significantly higher levels of RA (50 µM) was comparable with that obtained with 5 µM RA, indicating that the latter level was sufficient to
yield a maximum TGase response.

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Fig. 1.
RA-induced TGase expression protects cells
from serum deprivation-mediated apoptosis. A, cells
were exposed to media containing low serum and different concentrations
of RA for 2 days and then were lysed. The cell extracts were used to
determine TGase GTP binding activities using an affinity labeling assay
with radioactive GTP as outlined under "Experimental Procedures."
Western blot analysis using TGase and actin antibodies was performed to
assess the expression levels of each of these proteins. B,
NIH3T3 cells expressing only vector (vector) or WT TGase
(HA-tagged WT TGase) were maintained in low serum ± RA for 2 days
and then the cells were deprived of serum. A day later the cells were
collected and scored for apoptosis as described under "Experimental
Procedures." The inset depicts the expression levels of
the RA-induced and exogenously expressed TGase in the clones prior to
serum starvation.
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We then examined whether RA protected these cells from serum
deprivation-induced apoptosis. The fibroblasts were stimulated with or
without RA for 2 days and then were stressed by serum starvation, and
the resulting apoptotic rates were compared. Cells that were not
treated with RA were susceptible to apoptosis, as nearly 70% of the
cells displayed nuclear condensation or blebbing, a characteristic
unique to cells undergoing apoptosis (Fig. 1B, vector). To
confirm that these cells were apoptotic, a second assay that detects
the activation of caspases was also performed. Total cell lysates from
cells that were or were not deprived of serum were subjected to Western
blot analysis with an antibody that recognizes the cleaved (activated)
form of caspase 3. Consistent with cells undergoing programmed cell
death, cleaved caspase 3 was readily detected in the serum-deprived
cells but not in the actively growing cells (data not shown). Treatment
with 5 µM RA resulted in a dramatic reduction in the rate
of cell death compared with control cells (Fig. 1B, vector),
demonstrating that RA can provide a protective effect from serum
deprivation-induced apoptosis as well as HPR-mediated apoptosis (7).
The increase in TGase expression caused by 5 µM RA (Fig.
1B, inset, vector) can account for the pro-survival effect
of RA, because simply overexpressing TGase in NIH3T3 cells (Fig.
1B, inset, WT TGase) is sufficient to inhibit apoptosis
resulting from serum deprivation, and does so as effectively as
treating cells with 5 µM RA (Fig. 1B, WT TGase).
TGase has long been considered an apoptotic or cell differentiation
factor (5, 8-12), yet the data presented here and in our previous work
(6, 7) have implicated TGase as a survival factor. We questioned
whether the anti-apoptotic effect of TGase was unique to NIH3T3 cells
or whether TGase might provide a protective effect to other cell
lineages. To address this possibility, an analysis of TGase expression
and GTP binding activity in various cell lines was performed. We
reasoned that if TGase activation were detrimental to cell
proliferation then it would not be detected in any actively growing
cells. However, if TGase was linked to a survival response, then its
activity could be enhanced in some cell types. The data presented in
Fig. 2 shows that treatment with RA
increased TGase expression and GTP binding activity in all of the cell
lines assayed, indicating that various cell types express functional
RARs and can induce TGase expression and activation similar to NIH3T3
cells. The monkey kidney cell line COS7, the breast tumor cell line
MDAMB231, and the brain tumor cell line U87 all displayed substantial
levels of TGase expression even prior to RA treatment. Cervical
carcinoma (HeLa) cells displayed detectable TGase expression in the
absence of RA treatment; however, incorporation of
[
-32P]GTP into TGase only occurred after treatment
with RA (25). In contrast, the TGase expressed in MDAMB231 and U87
cells was able to incorporate [
-32P]GTP in the absence
of RA stimulation (Fig. 2). Thus different cell lines display varying
degrees of basal TGase expression and GTP binding activity, arguing
against a general role for chronic TGase GTP binding activity in
limiting cell proliferation and/or promoting apoptosis.

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Fig. 2.
Loss of regulation of TGase GTP binding
activity in certain cell lines. Cell lines were grown for 2 days
in complete media and then placed in low serum. The following day,
cells were exposed to low serum ± 5 µM RA for 2 days and then lysed. The cell extracts were used to determine the
expression level of TGase by Western blot analysis and GTP binding
activity by photoaffinity labeling as outlined under "Experimental
Procedures."
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EGF Stimulation of NIH3T3 Cells Limits the RA-induced Expression
and Activation of TGase--
RA treatment has been shown to result in
the up-regulation of a growing number of intracellular signaling
proteins including ERK (22), STAT3 (20), and PI3K (21), and it was
demonstrated recently (23) that PI3K activity was essential for
RA-induced TGase expression. Each of these signaling molecules is
frequently used by growth factors, such as EGF, to regulate a variety
of cellular processes (26), raising the possibility that TGase expression and GTP binding activity may be positively regulated by
growth factor stimulation in NIH3T3 cells. As shown in Fig. 3A, treatment of NIH3T3 cells
for 3 days with EGF does not lead to an increase in TGase expression
nor does EGF stimulate the GTP binding activity of TGase. Because
RA-mediated TGase expression requires PI3K, together with the fact that
EGF directly activates PI3K, we questioned whether the co-stimulation
of cells with RA and EGF might synergize to elicit a more rapid
induction of TGase expression or increase the overall level of TGase
protein compared with RA stimulation alone. However, instead of
increasing either of these parameters, EGF significantly diminished,
but did not completely block, the ability of RA to augment the
expression and GTP binding activity of the TGase (Fig. 3A).
The EGF-mediated inhibition of retinoid-induced TGase expression was
not dependent on the concentration of RA used to stimulate these cells,
as exposure with up to 75 µM RA was not able to overcome
the inhibitory effects of 30 min of EGF pretreatment (Fig.
3B). It is worth noting, however, that if TGase expression
was initially up-regulated by RA, followed by stimulation of the cells
with EGF for 3 additional days, the growth factor was ineffective at
decreasing TGase expression or GTP binding activity (Fig.
3C), despite being capable of fully activating ERK (Fig.
3D). Taken together, these findings indicate that although
RA and EGF activate common signaling proteins, there must be some
degree of signaling specificity since only RA stimulates TGase
expression. More importantly, EGF-mediated signaling inhibits retinoid-induced TGase expression in NIH3T3 cells only when the cells
are pretreated with EGF or when EGF and RA are added together, whereas
EGF appears to be ineffective at down-regulating TGase expression and
GTP activity when added subsequent to RA treatment.

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Fig. 3.
EGF inhibits the ability of RA to induce
TGase expression and GTP binding activity. Cells were grown to
near confluence and then were placed in low serum. The following day,
cells were treated singly with 5 µM RA or 100 ng/ml EGF
or co-stimulated with 5 µM RA and 100 ng/ml EGF for 2 days and then lysed (A), pretreated with 100 ng/ml EGF for
30 min before being exposed to various doses of RA for 2 days and then
lysed (B), pretreated with 5 µM RA or 100 ng/ml EGF for 2 days before being exposed to medium ± 5 µM RA and ± 100 ng/ml EGF for 3 additional days
before being lysed (C), or pretreated with 5 µM RA for 2 days before being stimulated with 100 ng/ml
EGF for the times indicated and then lysed (D). The cell
extracts were used to determine the expression level of TGase by
Western blot analysis and GTP binding activity by photoaffinity
labeling as outlined under "Experimental Procedures." The
activation state of ERK was measured via Western blot analysis with a
phospho-specific ERK antibody (P-ERK).
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EGF is a well established mitogenic and anti-apoptotic factor (26),
whereas RA has classically been considered to be a cell differentiation
factor (27). Because of the opposing functions attributed to EGF and
RA, it became important to determine how these factors, alone and in
combination, affect the apoptotic and cell growth rates of NIH3T3
cells. As expected, EGF caused a 50% reduction in the rate of cell
death induced by serum starvation (Fig.
4A) and stimulated a 2.5-fold
increase in cell number following 6 days of growth factor treatment
(Fig. 4B). Exposure to 5 µM RA protected the
cells from serum deprivation-mediated apoptosis to an extent similar to
that observed with EGF (Fig. 4A). Combining both EGF and RA
did not significantly reduce the apoptotic rate any further than
treating with either factor alone. This was to be expected since the
protective effects of RA from stress-induced apoptosis appear to occur
via the up-regulation of TGase activity (Fig. 1B), and EGF
suppresses most of the RA-induced TGase expression and thereby
eliminates the anti-apoptotic effect of RA. As a result, the extent of
resistance from serum deprivation-induced apoptosis caused
by co-stimulation with RA and EGF is similar to that obtained when
treating with EGF alone. Interestingly, exposure of cells to 5 µM RA, which strongly inhibits the proliferation of the
breast carcinoma cell line MCF-7 (Fig. 4C) and
differentiates the leukemia cell line HL-60 (28), did not block the
basal or EGF-mediated growth of NIH3T3 cells (Fig. 4B).
Moreover, both the untreated and EGF-stimulated cells appeared to grow
slightly better in the presence of RA. These findings suggest that 5 µM RA, which is capable of eliciting an anti-apoptotic
effect in mouse fibroblasts (Fig. 1B), also does not inhibit
the growth of these cells, regardless of whether or not RA is able to
up-regulate TGase expression and activity.

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Fig. 4.
RA does not compromise EGF-mediated cell
growth and protection from apoptosis. A, NIH3T3 cells
were exposed to low serum ± RA and ± EGF for 2 days and
then were serum-starved. The following day, the cells were scored for
programmed cell death as described under "Experimental Procedures."
The assay was performed 3 times, and the average percentage of cell
death is shown. B, NIH3T3 cells were seeded at 1 × 105 cells/well and grown in low serum medium ± 5 µM RA and ± 100 ng/ml EGF for the times indicated
and then counted. C, MCF-7 cells were seeded at 1 × 105 cells/well and grown in low serum ± 5 µM RA and counted at the times indicated.
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Ras Activity Is Sufficient to Suppress RA-induced TGase
Expression--
To explore further the relationship between
retinoid-induced TGase expression and the negative effects that EGF
treatment has on this process, we set out to define the signaling
cascade(s) used by EGF to inhibit RA-induced TGase expression. We first
examined whether the suppressive effects of EGF on the RA-mediated
expression and GTP binding activity of TGase were specific for EGF or
if other mitogenic agents could also inhibit RA-induced TGase in these
cells. For these studies, 20% calf serum was used as a general mitogenic factor since serum is essential for long term culturing of
cells, and the addition of serum to serum-deprived cells stimulates the
activities of several protein kinases (29). In addition, PDGF, a
natural ligand for NIH3T3 cells, was also tested for its ability to
block RA-mediated TGase up-regulation. Fig.
5A shows again that
pretreatment of cells with EGF inhibited the ability of RA to induce
TGase expression and GTP binding activity by about 90%. Cells
maintained in medium containing 20% calf serum or PDGF together with
RA for 2 days also resulted in a nearly complete inhibition of
RA-mediated TGase expression.

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Fig. 5.
Dominant-active Ras, but not Cdc42, can mimic
the ability of EGF to inhibit RA-induced TGase expression.
A, cells maintained in low serum for 1 day were pretreated
with 100 ng/ml EGF, 20% calf serum (20% CS), or 50 ng/ml
PDGF for 1 h and then exposed to 5 µM RA for 2 days
and lysed. B, cells expressing only vector
(vector), a dominant-active form of Ras (Ras
G12V), and a dominant-active form of Cdc42 (Cdc42 F28L)
were exposed to low serum ± 5 µM RA for 2 days and
then lysed. The whole cell extracts from each assay were used to
determine the expression levels of TGase and ERK by Western blot
analysis and GTP binding activity by photoaffinity labeling as outlined
under "Experimental Procedures."
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The above finding implies that signal transduction pathways commonly
activated by growth-promoting factors may be responsible for the
inhibition of retinoid-mediated expression of TGase. The small
GTP-binding proteins Ras and Cdc42 are two signaling molecules that are
often activated by mitogens (30-33) and aberrant activation of either
small G-protein in fibroblasts leads to cellular transformation (34, 35). NIH3T3 cells stably overexpressing dominant-active forms
of Ras (Ras(G12V)) and Cdc42 (Cdc42(F28L)) are available in our
laboratory (Fig. 5B), and each of these cell lines has been
extensively characterized and shown to display properties related to
transformation including growth in low serum, resistance to apoptosis,
and anchorage-independent growth. We have used these cell lines to
determine whether activation of Ras or Cdc42 mimics the ability of EGF
to inhibit RA-induced TGase expression. Control cells, as well as cells
expressing Ras(G12V) and Cdc42(F28L), were placed in low serum with or
without 5 µM RA for 3 days, and the cells were then
analyzed for TGase expression and GTP binding capability. Cells
expressing Cdc42(F28L) showed an induction of TGase expression and GTP
binding activity by RA that was similar to that observed in control
cells (Fig. 5B). In contrast, the expression level and GTP
binding activity of TGase induced by RA treatment of
Ras(G12V)-expressing cells was significantly reduced compared with
control cells or cells expressing Cdc42(F28L). These findings indicate
that the ability of EGF to activate Ras, but not Cdc42, was sufficient
for the EGF-mediated inhibition of RA-induced TGase expression.
Inhibition of ERK Activity Restores the Ability of RA to
Up-regulate TGase Expression in Ras-transformed Cells--
Ras can
regulate the activities of multiple signaling molecules, and ERK is
perhaps the most well studied (26). The activation of ERK by Ras
proceeds through the sequential activation of several intermediate
protein kinases that have been collectively referred to as the Ras-ERK
pathway. The signal transducing end point of the Ras-ERK cascade occurs
when ERK becomes phosphorylated by its immediate activator, MEK, and
translocates to the nucleus where it modulates the activity of
transcription factors to promote cell growth (36). Given that
Ras(G12V), but not Cdc42(F28L), chronically activates ERK (37, 38),
together with the finding that Ras(G12V)-expressing cells show a loss
in the ability of RA to induce TGase expression, raises the possibility
that Ras utilizes ERK to regulate negatively the retinoid-induced
expression of TGase. To determine whether this is the case, we
inhibited ERK activation in cells expressing dominant-active Ras using
a specific chemical inhibitor that blocks the activation of MEK. As
shown in Fig. 6A, incubation
of cells expressing dominant-active Ras with the MEK inhibitor,
PD98095, by itself did not result in increased TGase expression.
However, when cells incubated with PD98095 were exposed to RA for 2 days, increases in TGase expression and GTP binding activity were
observed. Parallel assays conducted on control cells and
Cdc42(F28L)-expressing cells showed that PD98095 had no effect on the
ability of RA to induce TGase expression or GTP binding activity (Fig.
6A). The fact that PD98095 treatment enhanced RA-induced
TGase expression only in cells expressing Ras(G12V) implies that the
MEK inhibitor was specific for down-regulating Ras-activated ERK and
was not having a more general inhibitory effect. The level of
RA-mediated TGase expression and GTP binding activity observed in the
Ras(G12V)-expressing cells, under conditions where ERK activity was
inhibited, was comparable with the level of TGase expression and
activation induced by RA in control cells (Fig. 6B). This
suggests that of the several signaling proteins regulated by Ras,
activation of ERK is sufficient for the Ras-mediated inhibition of
RA-induced TGase expression.

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Fig. 6.
Inhibiting ERK activity in cells expressing
dominant-active Ras restored the ability of RA to up-regulate TGase
expression and GTP binding activity. A, vector only
(vector), dominant-active Ras (Ras G12V), and
dominant-active Cdc42 (Cdc42 F28L) expressing cells were
exposed to low serum medium containing 10 µM LY294002
(LY) or 10 µM PD98095 (PD) ± 5 µM RA for 2 days and then lysed. B, cells
expressing dominant-active Ras (Ras G12V) were exposed to medium
containing 10 µM LY294002 (LY) and 10 µM PD98095 (PD) singly or combined together
with (or without) 5 µM RA for 2 days and then lysed. The
cell extracts from each assay were used to determine TGase GTP binding
activities using an affinity labeling assay with radioactive GTP as
outlined under "Experimental Procedures." Western blot analysis
using a TGase antibody was utilized to assess the expression levels of
this protein.
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Given our previous work showing that PI3K activity was required for
retinoid-stimulated TGase expression in NIH3T3 cells (23), we examined
whether PI3K had a similar role in cell lines that stably expressed the
dominant-active forms of Ras and Cdc42. In order to inhibit PI3K
activity, we took advantage of the chemical inhibitor LY294002. Each
cell line was incubated with LY294002 for 3 h prior to being
exposed to RA for 2 days, and then the expression levels and GTP
binding activity of TGase were compared for the different
conditions. Fig. 6A shows that incubation of mouse
fibroblasts expressing Cdc42(F28L) with LY294002 yielded a considerable
decrease in RA-induced TGase expression and GTP binding activity. Even
the already compromised ability of RA to induce the GTP binding
activity of TGase in Ras(G12V)-expressing cells was further accentuated
when PI3K activity was inhibited. Moreover, the ability of PD98095
treatment to restore RA-induced TGase expression in cells stably
expressing Ras(G12V) was completely abrogated with the addition of
LY294002 (Fig. 6B). These findings indicate that PI3K
activity is indispensable for RA to induce TGase expression and
activation in normal cells, as well as in both Cdc42- and
Ras-transformed NIH3T3 cells.
TGase Overexpression Enhances EGF-mediated Growth and Protection
from Apoptosis--
Activation of the EGF receptor typically
up-regulates the expression and activation of proteins that enhance
cell proliferation and resistance to cell death, and blocks the
expression and/or activation of proteins that limit cell growth and
stimulate apoptosis (26). Given that EGF inhibits RA-induced TGase
expression through activation of the Ras-ERK signaling cascade, it
seemed possible that increased TGase expression and activation would in
turn antagonize EGF-mediated signaling activities. To examine this
possibility, we utilized NIH3T3 cells that stably overexpressed
wild-type (WT) TGase or a transamidation-defective TGase mutant,
TGase(C277V) (Fig. 7A).
Because a role for TGase in endocytosis had been proposed previously
(3), coupled with fact that down-regulation of activated EGF receptors
occurs primarily via endocytosis, we first examined whether TGase might
influence the half-life of the activated EGF receptor. As an assay for
EGF receptor signaling activity, the magnitude and duration of ERK
activation were determined following EGF stimulation, using an antibody
that specifically recognizes the phosphorylated or activated form of
ERK. EGF-stimulation of control cells resulted in a rapid induction of
ERK activity that was maximal at 5 min and was reduced to basal levels
by 20 min (Fig. 7A, vector). The kinetics of EGF-stimulated
ERK activation in cells stably expressing WT TGase or TGase(C277V) were
unchanged from control cells (Fig. 7A, WT TGase and
TGase(C277V)). In addition, the magnitude of ERK
activity detected in cells expressing TGase was indistinguishable from
control cells suggesting that TGase does not alter the signaling
potential of the EGF receptor.

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Fig. 7.
Exogenous TGase expression does not
compromise EGF-mediated effects in NIH3T3 cells. Cells stably
expressing wild-type TGase (WT TGase), a
transamidation-deficient TGase mutant (C277V TGase), or
vector only (vector) were used in each of the following
assays. A, cells were grown to near confluence and
serum-starved for 1 day. The cultures were then stimulated with 100 ng/ml EGF for the times indicated and lysed. From the whole cell
extracts, the activation state of ERK was measured via Western blot
analysis with a phospho-specific ERK antibody (P-ERK), and
the expression level of TGase was determined by Western blot analysis
with an antibody for TGase. B, stable cell lines were
exposed to serum-free medium ± 100 ng/ml EGF for 2 days, and the
cells were then collected and scored for apoptosis as described under
"Experimental Procedures." C, cells were seeded at
1 × 105 cells/well and grown in low serum ± 100 ng/ml EGF for the times indicated and then counted.
|
|
We also examined whether EGF-mediated cell growth and/or resistance to
cell death was compromised by the expression of TGase. Consistent with
our earlier observation (Fig. 5A) that TGase serves as an
anti-apoptotic factor, TGase expression protected mouse fibroblasts
from apoptosis because of serum starvation, just as effectively as EGF
(Fig. 7B, vector and WT TGase). EGF treatment of
cells overexpressing either WT or transamidation-defective TGase
appeared to increase cell survival in an additive fashion, making these
cells more resistant to serum deprivation-mediated cell death compared
with that obtained upon EGF stimulation or TGase expression alone (Fig.
7B, vector and WT TGase). Comparing the proliferation rates in low serum for control cells and cells expressing WT TGase and the transamidation-defective TGase mutant also
revealed that overexpression of WT TGase provided a growth advantage.
Whereas control cells or cells expressing the transamidation-defective TGase mutant displayed limited growth, cells expressing WT TGase grew
about 2.5-fold better than control cells (Fig. 7C). The
addition of EGF to each of the stable cell lines caused a parallel
increase in cell proliferation over the basal growth rate of each cell line, indicating that TGase did not negatively regulate EGF-mediated growth.
 |
DISCUSSION |
The goal of this study was to understand better how RA-induced
TGase expression is regulated in cells and, once expressed, what effect
TGase has on cellular processes. In mouse fibroblasts, RA enhances both
the expression and activation of the GTP binding and transamidation
activities of the TGase (7). It has been well documented that
RA-induced increases in TGase expression are mediated through the
up-regulation of the transcriptional activities of the RARs (2). Our
results provide some interesting new insights into the underlying
regulatory mechanisms for RA-induced TGase expression. For example,
although we find that the activity of the anti-apoptotic factor PI3K is
required for RA-induced TGase expression, EGF treatment of cells, which
activates PI3K, causes an inhibition of the RA-mediated up-regulation
of TGase. The inhibitory signal provided by EGF results from the
stimulation of the Ras-ERK cascade, probably explaining an earlier
report (39) that showed dominant-active Ras inhibited RA-mediated TGase
expression. However, it is not yet known how the activation of PI3K nor
the stimulation of the Ras-ERK cascade influences the induction of
TGase expression by RA, but we speculate that these pathways lead to
the activation of transcriptional regulators that act in tandem with
the RARs to enhance or inhibit transcription of the TGase gene. Support for this idea comes from the identification of several putative transcription factor-binding sites in the TGase promoter region (40,
41), including response elements for AP-1 as well as the nuclear factor
B. The activities of both AP-1 and nuclear factor
B have been
shown to be up-regulated by PI3K (42, 43), raising the possibility that
the requirement of PI3K activity for RA-stimulated TGase expression may
involve the ability of PI3K to activate one or both of these
transcription factors. Additionally, it is worth noting that the
ability of dominant-active Ras to abrogate RA-induced TGase expression
reflects a specific signaling effect rather than a general
characteristic of cellular transformation, as the expression of a
transforming mutant of Cdc42 (Cdc42(F28L)) in NIH3T3 cells did not
limit RA-induced TGase expression. Moreover, some human tumor cell
lines (U87 and MDAMB-231) actually exhibited constitutive TGase GTP
binding activity.
Thus, it seems unlikely that the negative regulatory effects elicited
by EGF on RA-mediated TGase expression are the outcome of a mitogenic
signaling pathway (e.g. EGF receptor-signaling) specifically
targeting TGase for down-regulation. If anything, the mitogenic and
survival activities exhibited by TGase under a variety of conditions
would be expected to complement rather than antagonize mitogenic
signaling activities. Rather, it seems more likely that the EGF
receptor-Ras-ERK pathway is directed in a more general sense at
RA-induced gene expression. It has been well established that RA
induces the expression of genes with anti-proliferative activity that
supports cell cycle arrest and cellular differentiation (4, 5, 28). The
targeting of RA-induced gene expression by EGF would probably ensure
that such anti-proliferative activities are not activated by
differentiation factors like RA. TGase, which we believe is activated
by RA so as to serve as an "insurance factor" against apoptosis,
would not be needed by EGF signaling events that provide anti-apoptotic activity and so can be shut down with the other RA-induced gene products.
Numerous studies have associated increased TGase expression and
activation in cells with the promotion of apoptosis or cell differentiation. For example, exogenous TGase expression sensitized the
neuroblastoma cell line SK-N-BE to apoptosis (9), whereas blocking
TGase expression in the promonocytic cell line U937, using antisense
technology, reduced the effectiveness of apoptotic stimuli to induce
cell death (10). Yet the results presented here argue for a role for
TGase in protecting NIH3T3 cells from serum deprivation-mediated
apoptosis and promoting cell proliferation. Interestingly, it was
recently reported (44) that thymocytes isolated from
TGase
/
mice were more susceptible to
apoptosis, indicating that the protective effect of TGase was not
limited to just mouse fibroblasts. How TGase can have opposing cellular
effects depending on the cell type is not clear but is of particular
interest to us. It may be that TGase elicits distinct cellular outcomes
in different cell lineages. The precedence for proteins having opposing
functions in different types of cells has been noted with c-Jun
N-terminal kinase (JNK) and Myc (45-48). In the case of JNK,
persistent JNK activation induced apoptosis in cells of neuronal origin
(45), whereas in gliomas continuous JNK signaling has been associated with transformation (46). Experiments are currently being conducted to
determine whether or not aberrant TGase activity promotes or inhibits
apoptosis in cells of different lineages. We are especially interested
in assessing the importance of the loss of regulatable TGase GTP
binding activity, exhibited by tumor cell lines U87 and MDAMB231,
for cell viability.