(Received for publication, November 21, 1994; and in revised form, January 20, 1995)
From the
Transforming growth factor- (TGF-
) is a potent growth
inhibitor of a variety of epithelial cell types. The primary signaling
mechanism involved in mediating this and other cellular effects of
TGF-
is still unknown. We report here that both TGF-
and TGF-
resulted in a rapid activation of
mitogen-activated protein kinase (MAPK) p44
,
occurring within 5-10 min of growth factor addition. This effect
occurred in exponentially proliferating cultures of intestinal
epithelial (IEC) 4-1 cells under conditions in which DNA synthesis was
inhibited by 95% to 98%. Furthermore, TGF-
induced a
sustained activation of p44
under these
conditions, lasting for at least 90 min after initial growth factor
treatment. Another TGF-
-sensitive epithelial cell line (CCL 64)
displayed a similar rapid increase in p44
activity when treated with TGF-
. In
contrast, in IEC 4-6 cells that are resistant to TGF-
effects on
growth and DNA synthesis, TGF-
treatment did not
result in an activation of p44
. In contrast to
the results in proliferating cultures, treatment of quiescent cultures
of IEC 4-1 cells with TGF-
resulted in no significant
change in either DNA synthesis or p44
activity
within 15 min of TGF-
addition. In contrast, addition of the
growth-stimulatory combination of factors (epidermal growth factor
+ insulin + transferrin = EIT) to quiescent and
proliferating IEC 4-1 cells stimulated DNA synthesis and resulted in a
sustained activation of p44
. Together, our
results suggest an association between activation of p44
and both TGF-
-mediated growth inhibition and
EIT-mediated growth stimulation. This suggests that the specificity for
the cellular effects of growth factors may not occur at the level of
MAPK activation per se, but rather at downstream events that
include phosphorylation of distinct transcriptional complexes and
activation of a select assortment of genes. With regard to TGF-
specifically, we have proposed a model to explain how activation of
p44
may be associated with a growth-inhibitory
response.
Transforming growth factor- (TGF-
) (
)regulates a multitude of biological functions in a variety
of cell types and is a potent growth inhibitor of epithelial
cells(1, 2, 3) . The TGF-
family
includes 3 mammalian isoforms (referred to as TGF-
,
TGF-
, and TGF-
) that elicit cellular
responses by interacting with specific membrane-bound proteins.
Although several TGF-
-binding proteins have currently been
described, the type I and type II receptors are thought to be the
primary signal transducing receptors in most cell types(4) . A
number of species of the type I and II receptor classes have been
cloned and have been shown to contain a serine/threonine kinase
domain(5, 6, 7, 8, 9, 10, 11) .
Recently, it was reported that in a yeast genetic screen immunophilin
FKBP-12 specifically bound to the type I receptor(12) . In
mammalian systems, however, no coupling components or endogenous
substrates for the kinase activity have been reported. Moreover, the
exact mechanism(s) for signaling from the receptors have not been
elucidated.
Evidence has indicated that TGF- may signal
exclusively through a receptor heterocomplex of type I and type II
receptors, in which the cytoplasmic kinase domain of both receptors are
essential for signal
initiation(13, 14, 15, 16, 17, 18) .
In contrast, additional findings suggest that TGF-
may mediate
some cellular responses by signaling through either the type I receptor
or the type II receptor, indicating that two distinct
receptor-associated signaling pathways may control a separate
assortment of TGF-
responses(19, 20) . Additional
studies have reported the existence of several TGF-
receptor
complexes consisting of different receptor
subtypes(8, 10, 11, 21, 22) .
For example, TGF-
receptor type II has been shown to complex with
TGF-
receptor type III and with different type I receptors, some
of which also bind activin, a member of the TGF-
superfamily(8, 10, 11, 21, 22) .
Thus, TGF-
may regulate its diverse actions by specifically
engaging different receptor subtypes into functional multimeric
complexes.
The majority of reports pertaining to the
growth-inhibitory effect of TGF- have focused on defining
components that are regulated by this factor within the nucleus. For
example, in epithelial cells, TGF-
decreases c-myc,
p34
, cdk4, and B-myb expression(23, 24, 25, 26, 27, 28, 29, 30) ,
decreases the phosphorylation of the retinoblastoma protein and
p34
(31, 32, 33) ,
increases the expression of c-jun and
c-fos(34, 35, 36) , stimulates the
phosphorylation of cAMP-responsive element binding
protein(37) , prevents cdk2 activation(38) ,
and regulates several G1 cyclins and cell cycle-associated
cyclin-cdk inhibitors(39, 40, 41, 42) . In
contrast to these nuclear effects of TGF-
, we have reported the
first direct evidence for a rapid activation of a cytoplasmic signaling
component for TGF-
in epithelial cells. That is, we have shown
that TGF-
and TGF-
resulted in a
rapid activation of p21
in TGF-
-sensitive
IEC 4-1 cells, but not in TGF-
-resistant IEC 4-6
cells(43) . This finding was unexpected, since activation of
Ras was thought to be associated with mitogenic responsiveness or
stimulation of cellular growth(44) . However, multiple
transducing signals are thought to converge downstream of Ras activity
at the mitogen-activated protein kinases (MAPKs)(45) .
Therefore, it is possible that other signaling molecules, functioning
either in parallel with or downstream from Ras in the TGF-
pathway, may alter or reverse the stimulatory signals transmitted by
p21
. Thus, it was of interest to examine the
effect of TGF-
on MAPK activity.
MAPKs, also known as
extracellular signal regulated kinases (ERKs) constitute a family of
serine/threonine kinases ranging in molecular mass from 42 kDa to 97
kDa. Two MAPK proteins, p42 and
p44
, require phosphorylation on both tyrosine
and threonine residues for full enzymatic
activation(46, 47) . Additionally, they are activated
in association with stimulation of cell growth by a range of different
agents, including insulin(48, 49) ,
thrombin(50) , epidermal growth factor(51) , and
several hematopoietic growth factors(52, 53) .
Moreover, nerve growth factor has been shown to activate these MAPK
forms in association with the induction of differentiation in PC12
cells(49, 54) . Recently, several Ras-independent
pathways have been shown to induce MAPK activity as well(55) .
Once activated, p42
and p44
phosphorylate a variety of substrates found within the
cytoplasm and the nucleus(45) . Recent studies have
demonstrated that p42
and p44
can be translocated to the nucleus when they are
persistently activated (56, 57) . With transient
induction, however, MAPK activity is localized in the
cytoplasm(56, 57) . Hence, p42
and p44
are thought to serve as
intermediaries connecting the two subcellular compartments.
In this
report, we utilized the same two clonal intestinal epithelial (IEC)
cell lines which were used to demonstrate an association between
activation of Ras and growth inhibition by TGF-(43) . We
have previously described the isolation of these TGF-
-sensitive
and -resistant cell clones(58) . Moreover, hypersensitivity of
the IEC 4-1 cells to TGF-
, relative to the parental cells from
which they were derived, was explained by a 5- to 10-fold increase in
both total receptor numbers per cell and TGF-
binding to the type
I and type II signaling receptors(58) . Here, we demonstrate
that TGF-
activates p44
in epithelial
cells for which TGF-
is growth-inhibitory. In contrast, TGF-
did not activate this kinase in proliferating and quiescent cultures of
epithelial cells that were resistant to the growth-inhibitory effects
of this polypeptide.
Figure 1:
Effect of TGF- on
[
H]thymidine incorporation and
p44
activity in proliferating cultures of IEC
4-1 cells. A, exponentially proliferating IEC 4-1 cells were
treated with or without TGF-
(10 ng/ml) for 24 h in
serum-free medium, after which time [
H]thymidine
incorporation was determined, as described under ``Experimental
Procedures.'' Results are expressed as x ± S.D. (n = 3). B, near-confluent IEC 4-1 cells were
treated for the indicated times with TGF-
(10 ng/ml).
Cell lysates were incubated with either an antibody specific for
p44
(SC-93) or nonimmune rabbit IgG
(nonspecific control). In vitro phosphorylation of MBP by
p44
was then analyzed as described under
``Experimental Procedures.'' Phosphorylated MBP was
visualized by autoradiography. C, equal loading was determined
by incubation of transferred proteins with p44
antibody (SC-93), followed by ECL detection. h.c. IgG = heavy chain IgG. D, plot of betagen scan
results, x ± S.D. (n = 4 or 5);
statistical significance was determined for all times except the 90-min
point, which is represented as the mean of two experiments. *, p < 0.01.
In order to determine
whether MAPK activity would be altered by TGF-, we examined the
time-dependent effects of TGF-
on in vitro p44
activity. IEC 4-1 cells, plated as for Fig. 1A, but treated with TGF-
(10
ng/ml) for various times, were lysed and immunoprecipitated with either
nonimmune rabbit IgG (nonspecific control) or with an antibody specific
for p44
. Immunoprecipitates were then incubated with
MBP, and in vitro phosphorylation of MBP was examined by
SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 1B). As Fig. 1B illustrates for
the 0- and 5-min time points, MBP phosphorylation was not observed in
the samples immunoprecipitated with nonimmune rabbit IgG. This was the
case for all time points (data not shown). Equal loading was determined
by immunoblotting transferred proteins with a
p44
-specific antibody (Fig. 1C). As Fig. 1C depicts, p44
was equally
immunoprecipitated and equally loaded at all time points examined.
Further, as expected, the nonimmune rabbit IgG immunoprecipitation
control did not contain the p44
protein; however, the
IgG heavy chain is visible in these lanes (Fig. 1C, Rab
IgG lanes, 0 and 5 min). Relative levels of MBP phosphorylation were
determined by betagen scanning. The mean MBP phosphorylation levels
from 4 or 5 experiments are plotted in Fig. 1D, and the
level of statistical significance at individual time points is
provided. The results indicate that treatment of exponentially
proliferating cultures of IEC 4-1 cells with TGF-
(10
ng/ml) resulted in a subtle, but sustained, activation of p44
(Fig. 1, B and D). As Fig. 1D depicts, p44
activity was increased by 1.6-fold
within 5 min of TGF-
addition and then continued to
increase as a function of incubation time. By 30 min, p44
activity had reached maximal levels of 1.9-fold above basal
activity. This activity slightly decreased by 60 min, but was still
sustained at levels 1.5-fold above basal activity for at least 90 min (Fig. 1, B and D). Thus, TGF-
produced a sustained activation of p44
under
conditions that resulted in a 98% growth inhibition.
Figure 2:
Effect of TGF- on
[
H]thymidine incorporation and
p44
activity in proliferating cultures of IEC
4-1 and CCL 64 cells. A, exponentially proliferating IEC 4-1
cells were incubated with or without TGF-
(10 ng/ml)
for 24 h prior to determination of [
H]thymidine
incorporation, as in Fig. 1. Results are presented as x ± S.D. (n = 3). B, exponentially
proliferating CCL 64 and IEC 4-1 cells were treated with
TGF-
(10 ng/ml) for 10 min. In vitro phosphorylation of MBP by p44
was
determined as for Fig. 1. Equal loading was verified by
Coomassie staining of the gels used for autoradiography. C,
plot of betagen scan results are from two separate experiments,
expressed as x ± range.
Additionally, we wished to determine whether
p44 activation by TGF-
occurred in epithelial cell
types other than IEC 4-1 cells. Thus, we chose to examine the effects
of TGF-
on the mink lung epithelial cell line CCL 64.
TGF-
has previously been shown to reversibly inhibit
the DNA synthesis of CCL 64 cells by more than 90% at a concentration
of 2.5 ng/ml(60) . Under the conditions we utilized, a 97%
inhibition in DNA synthesis was observed with TGF-
(10
ng/ml) treatment (data not shown). Moreover, a 10-min treatment with
TGF-
(10 ng/ml) induced p44
activity by
approximately 2.0-fold in the CCL 64 cells (Fig. 2, B and C). Thus, the stimulation of MAPK by TGF-
is not
limited to intestinal epithelial cells and can be detected in lung
epithelial cells as well.
Figure 3:
Effect of TGF- on
[
H]thymidine incorporation and
p44
activity in proliferating cultures of IEC
4-6 cells. A, exponentially proliferating IEC 4-6 cells were
incubated for 24 h with or without TGF-
(10 ng/ml).
Thymidine incorporation was determined as for Fig. 1. Results
are expressed as x ± S.D. (n = 3). B, near-confluent IEC cells were treated for the indicated
times with TGF-
(10 ng/ml). In vitro phosphorylation of MBP by p44
was then
determined as for Fig. 1. Equal loading was determined as for Fig. 2. C, plot of betagen scan results for IEC 4-1
cells is expressed as x ± S.D. (n = 5);
IEC 4-6 results plotted are from two separate experiments, expressed as x ± range.
Figure 4:
Effect of TGF- on
[
H]thymidine incorporation and
p44
activity in quiescent cultures of IEC 4-1
cells. A, quiescent IEC 4-1 cells were incubated for 24 h with
or without TGF-
(10 ng/ml). Thymidine incorporation
was determined as for Fig. 1. Results are expressed as x ± S.D. (n = 3). B, quiescent cells
were treated for the indicated times with TGF-
(10
ng/ml). In vitro phosphorylation of MBP by p44
was determined as for Fig. 1. Equal loading was
determined as for Fig. 2. C, plot of betagen scan
results shown in B. Results are representative of two
experiments.
Figure 5:
The effect of the growth- stimulatory
combination of factors (E + I + T = EIT) on
[H]thymidine incorporation and
p44
activity in quiescent and proliferating
cultures of IEC 4-1 cells. A, quiescent IEC 4-1 cells were
incubated for 21 h with or without EIT, after which time
[
H]thymidine incorporation levels were determined
as for Fig. 1. Results are presented as x ± S.D. (n = 3). B, quiescent IEC 4-1 cells were
treated with EIT for the indicated times, and p44
activity was determined as for Fig. 1. C,
plot of betagen scan results shown in B. D, proliferating
cultures of IEC 4-1 cells were incubated with the growth-stimulatory
combination EIT for 24 h. Thymidine incorporation was determined as for Fig. 1. Results are expressed as x ± S.D. (n = 3). E, near-confluent IEC 4-1 cells were treated
with EIT for the indicated times. In vitro phosphorylation of
MBP by p44
was then determined as for Fig. 1. F, plot of betagen scan results shown in E. Equal loading was determined by immunoblotting, as in Fig. 1. Results are representative of two
experiments.
Additionally, we wished to investigate
whether these growth factors would modulate p44 in
proliferating cultures of IEC 4-1 cells, as we have observed for
TGF-
. Fig. 5D indicates that treatment with EIT
for 24 h stimulated a 3.0-fold increase in DNA synthesis in
proliferating cultures of IEC 4-1 cells. Furthermore, a 3.1-fold
induction of p44
was detected within 5 min of growth
factor addition, which then decreased slightly to levels 2.8-fold above
basal activity for at least 20 min (Fig. 5, E and F). All lanes were equally loaded, as determined by incubating
transferred proteins with p44
antibody (SC-93), followed
by ECL detection (data not shown). Thus, this growth-stimulatory
combination of factors (EIT) stimulated DNA synthesis and activated
p44
in both proliferating and quiescent IEC 4-1 cells.
In this report, we have demonstrated that both
TGF- and TGF-
result in a rapid
activation of p44
, beginning as early as 5 min after
addition of the growth factor to proliferating cultures of epithelial
cells. In untransformed intestinal epithelial cells, maximal activation
of p44
by TGF-
occurred at 30 min, at which time
kinase activity was stimulated by 1.9-fold. Moreover, this effect
occurred under conditions in which the growth of the cells was
inhibited by at least 95-98%. This report is the first
description of an activation of this form of MAPK by a growth
inhibitor. Although a previous report described a slight activation of
a 57-kDa Erk-like protein by TGF-
in colon carcinoma
cells(61) , only a transient activation was observed. In the
untransformed epithelial cells that we have examined, TGF-
affected a sustained activation of p44
, lasting
for at least 90 min.
Recent evidence has indicated that the level
and duration of activation of MAPK are important factors in determining
which specific cellular effects will be
elicited(56, 57) . For example, a persistent
activation of MAPKs was coupled to the mitogenic potential of Chinese
hamster lung (CCL 39) fibroblast cells, whereas non-mitogens, such as
phorbol esters and -thrombin-receptor synthetic peptides, only
induced a transient activation of MAPK(56) . Conversely, EGF, a
growth stimulator of PC12 pheochromocytoma cells, resulted in a
transient activation of MAPK in these cells, whereas the induction of
differentiation by nerve growth factor in PC-12 cells was associated
with a sustained activation of MAPK(57) . Hence, the duration
of MAPK activation is not definitively correlated with a specific
cellular function, but it does appear to be regulated in a cell-type
and stimulus-specific manner. In our studies, the kinetics for
TGF-
activation of p44
closely resembled the
sustained activation displayed by the differentiation factor nerve
growth factor in PC-12 cells(57) .
In both of the reports
discussed above, the prolonged phase of MAPK activation was linked to
translocation of the enzyme into the nucleus (56, 57) . Thus, the prolonged activation of
p44 by TGF-
(sustained for at least 90 min)
suggests that TGF-
may result in translocation of this form of
MAPK to the nucleus. Although we have not yet examined TGF-
effects on subcellular localization of p44
, the kinetic
profile for the activation of this kinase suggests that this
TGF-
-mediated cellular effect may provide an important link
between the cytoplasmic and nuclear events associated with TGF-
responses. Furthermore, our results indicate that the activation of
p44
by TGF-
may be associated with the
growth-inhibitory responsiveness of the IEC 4-1 cells to TGF-
.
That is, while this effect was observed in IEC 4-1 cells that are
growth-inhibited by TGF-
, the activation of p44
by
TGF-
was not observed in IEC 4-6 cells that are insensitive to the
growth-inhibitory effects of TGF-
.
Our experiments involving
TGF- addition to quiescent cells, in the absence of other
exogenous growth factors or serum, also support an association between
inhibition of DNA synthesis by TGF-
and activation of
p44
. That is, addition of TGF-
to IEC 4-1 cells,
made quiescent by growing the cells on basal medium alone for 9 days
(nutrient and growth factor deprivation), resulted in no change in DNA
synthesis and no activation of p44
within 24 h and 15
min after TGF-
addition, respectively. It was not surprising that
[
H]thymidine incorporation into DNA did not
change under the conditions of these experiments (see Fig. 4A), since treatment with TGF-
would not
result in entry of the cells into the S phase of the cell cycle.
However, it was of interest that p44
was not activated
within 15 min of TGF-
addition to quiescent IEC 4-1 cells, as was
observed after TGF-
addition to proliferating cultures. A previous
report (62) indicated that TGF-
inhibited the activation
of p44
(for periods up to 30 min) in quiescent CCL 64
mink lung epithelial cells, released from TGF-
-induced G
growth arrest by addition of serum and EGF. In these experiments,
EGF and serum were added in the absence and presence of TGF-
to
cells that had been arrested in late G
by growth of the
cells on TGF-
for 24 h prior to subsequent additions. In contrast,
our results demonstrate direct effects of TGF-
alone (i.e. in the absence of serum or other growth factors) on
p44
, a potential signaling component for mediating
growth inhibition by TGF-
.
As discussed earlier, the kinetics
for TGF- activation of p44
closely resemble those
obtained in PC-12 cells, following addition of the differentiation
factor nerve growth factor (57) . Further, TGF-
has been
shown to induce a more differentiated phenotype in some cell types,
including untransformed IEC and human colon carcinoma cells, under
certain circumstances(63, 64, 65) . Thus, it
is possible that the activation of p44
by TGF-
may
be involved in regulating cellular differentiation, rather than cell
growth. Evidence against this possibility includes the fact that
TGF-
activated p44
in CCL 64 cells, which do not
appear to undergo morphological differentiation in response to
TGF-
. Further, although we have not examined markers of
differentiation in the IEC 4-1 cells, the induction of
intestinal-specific enzymes is not always associated with TGF-
responsiveness in IEC-6 cells(63, 66) .
Although
the data in Fig. 1Fig. 2Fig. 3Fig. 4indicate an
association between TGF- activation of p44
and
growth inhibition, other results in Fig. 5, A-F,
indicate that the growth factor combination EIT can also activate
p44
in IEC 4-1 cells. However, the kinetics for MAPK
activation by EIT in both proliferating and quiescent IEC 4-1 cells
were different from those for TGF-
. That is, activation of
p44
was maximal by 5 min after EIT addition, whereas
maximal levels of activation were not attained until 30 min after
TGF-
addition. Moreover, EIT stimulated p44
activity to levels 3-4-fold above baseline values, whereas
TGF-
induced kinase activity by only 2-fold (approximately) in IEC
4-1 cells. Thus, the temporal regulation and threshold levels of MAPK
activity may specify some growth factor responses. However, MAPK
activation was sustained in association with both TGF-
-mediated
growth inhibition and EIT-mediated growth stimulation. Thus, it appears
that the specificity for the cellular effects of growth factors may
largely be determined, not at the level of MAPK activation per
se, but by subsequent events (i.e. the formation of
various complexes between MAPKs and transcription factors, the
phosphorylation and activation of distinct transcription factors, and
the temporal activation of select classes of genes).
With regard to
TGF- specifically, one possible explanation for the observed
activation of p44
by TGF-
, in association with an
inhibition of cell growth, involves an associated phosphorylation and
activation of Elk-1/p62
, ultimately leading to
autoinduction of TGF-
(see model in Fig. 6). This
hypothesis is based upon several previous reports. That is, it has been
demonstrated that autoinduction of TGF-
is mediated by
the AP-1 (Jun-Fos) complex in the TGF-
promoter, and
that both Jun and Fos components are required for transcriptional
activation(34) . TGF-
has also been shown to
autoinduce TGF-
expression in IEC-6 cells (66) and to induce both c-jun and c-fos expression in other cell types (34, 35, 36) . In addition, it has been shown
that p44
/p42
can efficiently
phosphorylate and activate the transactivation potential of
Elk-1/p62
in vitro and in
vivo(67, 68, 69) . Phosphorylation of
specific sites within the C-terminal domain of Elk-1/p62
enhances the formation of a ternary complex composed of
Elk-1/p62
, p67
, and the serum response
element in the c-fos promoter, thereby increasing c-fos transcription, and subsequently AP-1-dependent gene
transcription(67, 68, 69) . Thus, TGF-
addition to untransformed epithelial cells may result in activation and
nuclear translocation of p44
, followed by
phosphorylation and activation of Elk-1/p62
, increased
transcription of c-fos, elevated AP-1 activity of the
TGF-
promoter, and increased production of
TGF-
(see Fig. 6). In cell types that have the
potential to activate the latent, secreted
TGF-
(1, 2, 3) , the growth-inhibitory
effects of TGF-
would be amplified. In this way, the activation of
p44
by TGF-
may be indirectly associated with the
growth-inhibitory effects of TGF-
. However, additional experiments
are required to test this model.
Figure 6:
Model for association between activation
of p44 by TGF-
and growth inhibition.
TGF-
activates p44
within minutes of its
addition to proliferating cultures. This activation results in
phosphorylation of transcription factors such as Elk-1/p62
and in transcriptional activation of genes such as
c-fos. Elevated expression of c-fos induces an
increase in the TGF-
promoter AP-1 activity, thereby
inducing TGF-
production. This autoinduction of
TGF-
results in amplification of the growth-inhibitory
effect of this growth factor in cell types that can activate the
secreted TGF-
. R
and R
= TGF-
receptor type I and
receptor type II, Ras = p21
, MAPKK = mitogen-activated protein kinase kinase, SRF = p67
, Elk-1 = Elk-1/p62
, SRE =
serum response element, and Fos/Jun = heterocomplex of
c-Fos and c-Jun.
In summary, we have shown that a
growth inhibitor (TGF-) can activate a kinase known for its
involvement in growth-stimulatory pathways (p44
). The
activation began within 5 min of TGF-
addition to proliferating
cultures of epithelial cells, in the absence of serum or other growth
stimulators, and was sustained for at least 90 min. Moreover, the
activation occurred in cells that responded to TGF-
with an
inhibition of DNA synthesis and cell growth, but not in cells that did
not display these effects. We have proposed a model to account for the
association between activation of this kinase and the growth-inhibitory
responsiveness of the cells to TGF-
. The model involves
phosphorylation and activation of transcription factors (such as
Elk-1/p62
) that can ultimately increase AP-1-dependent
gene transcription, thereby leading to TGF-
autoinduction via the AP-1 sites in the TGF-
promoter. In keeping with this model, the TGF-
-sensitive IEC
4-1 cells produce and activate TGF-
, whereas the
TGF-
-resistant IEC 4-6 cells do not produce any detectable
TGF-
(58) . Thus, the IEC 4-6 cells may be
resistant to the growth-inhibitory effects of TGF-
due to their
inability to activate p44
and to amplify production of
TGF-
.