(Received for publication, August 28, 1995; and in revised form, October 16, 1995)
From the
Transforming growth factor 1 (TGF-
1) is a
multifunctional cytokine that positively or negatively regulates the
proliferation of various types of cells. In this study we have examined
whether or not the activation of the mitogen-activated protein (MAP)
kinases is involved in the transduction of cell growth modulation
signals of TGF-
1, as MAP kinase activity is known to be closely
associated with cell cycle progression. Although TGF-
1 stimulated
the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to
detectably stimulate the tyrosine phosphorylation and activation of the
41- and 43-kDa MAP kinases at any time point up to the reinitiation of
DNA replication. TGF-
1 also failed to stimulate the expression of
the c-fos gene. Furthermore, TGF-
1 synergistically
enhanced the mitogenic action of epidermal growth factor (EGF) without
affecting EGF-induced MAP kinase activation in these fibroblasts, and
it inhibited the EGF-stimulated proliferation of mouse keratinocytes
(PAM212) without inhibiting EGF-induced MAP kinase activation. Thus,
the ability of TGF-
1 to modulate cell proliferation is apparently
not associated with the activation of MAP kinases. In this respect,
TGF-
1 is clearly distinct from the majority, if not all, of
peptide growth factors, such as platelet-derived growth factor and EGF,
whose ability to modulate cell proliferation is closely associated with
the activation of MAP kinases. These results also suggest that the
activation of MAP kinases is not an absolute requirement for growth
factor-stimulated mitogenesis.
Transforming growth factor- (TGF-
) (
)is the
prototype of a large family of cytokines that regulate a wide variety
of cellular processes including cell proliferation, cell
differentiation, cell motility, cell organization, and extracellular
matrix production (reviewed in (1, 2, 3, 4) ). The TGF-
family
includes three mammalian isoforms, TGF-
1, TGF-
2, and
TGF-
3, which have similar biochemical and biological
characteristics. The effects of TGF-
on cell growth control are
complicated and vary dramatically depending on the target cell type,
the cell density, and the presence of other growth factors in the
culture medium. Although these biological functions of TGF-
have
been intensively studied over the past decade, the biochemical
mechanisms that underlie these complex effects are largely unknown.
TGF- generates diverse cellular responses by interacting with
specific membrane-bound proteins. Affinity labeling with radioiodinated
TGF-
has identified a number of different sizes of receptors and
binding proteins; these include type I (M
=
53,000), type II (M
= 75,000), type III (M
= 280,000), type IV (M
= 60,000), type V (M
=
400,000), and type VI (M
= 180,000)
receptors, as well as several other membrane binding proteins of M
= 40,000, 60,000, and 140,000 (reviewed
in (5, 6, 7) ). Among these receptors and
membrane binding proteins, the most widely distributed are the type I,
II, and III receptors. The way these different receptors contribute to
the multiple functions of TGF-
is unclear. However, recent genetic
and biochemical evidence suggests that the type I and type II
receptors, both of which have transmembrane serine/threonine kinase
structures, are essential for eliciting the many effects of
TGF-
(8, 9, 10, 11) , while the
type III receptor (betaglycan) is involved in the presentation of the
ligand to the signaling receptors(12, 13) .
TGF- has emerged as a positive or negative regulator of cell
proliferation; it stimulates the growth of certain mesenchyme-derived
cells but acts as a potent growth inhibitor of many other cell types
such as epithelial and endothelial
cells(1, 2, 3, 4) . All these
effects of TGF-
have been suggested to be mediated, as described
above, by the activation of a heteromeric receptor complex that
consists of the type I and type II
receptors(8, 9, 10, 11) . Thus, in
order to gain further insight into these dual effects of TGF-
on
cell growth regulation, it appears critical to determine the exact
molecular signaling mechanism from the receptors to the nucleus. Up
until now, the majority of reports pertaining to TGF-
-regulated
cell growth control have focused on the identification of the nuclear
components that are regulated by this factor in inducing its
growth-inhibitory effects. For example, in epithelial cells, TGF-
decreases c-myc, p34
, cdk4,
and B-myb expression(14, 15, 16, 17, 18, 19) ,
decreases the phosphorylation of the retinoblastoma protein and
p34
(20, 21) , prevents cdk2 activation(22) , and regulates several G
cyclins and cell cycle-associated cyclin-cdk inhibitors(23, 24, 25, 26) . In
contrast to these nuclear effects of TGF-
, very little is known
about how it regulates cytoplasmic signaling components in cells where
TGF-
is neither growth-stimulatory nor growth-inhibitory.
Mitogen-activated protein (MAP) kinases, also known as extracellular
signal-regulated kinases (ERKs), are representative cytoplasmic
signaling components. They are activated in many cell types by diverse
extracellular stimuli that elicit a wide array of physiological
responses such as cell division, differentiation, and secretion. MAP
kinases are serine/threonine kinases that phosphorylate a variety of
regulatory proteins, which include other protein kinases
(p90, MAP kinase-activated protein kinase 2,
etc.), cytoplasmic phospholipase A
, cytoskeletal proteins
(microtubule-associated proteins), and transcription factors (c-Jun,
c-Myc, p62
, etc.) (reviewed in (27, 28, 29, 30, 31, 32) ).
Thus, MAP kinases are thought to function as key intermediaries in the
intracellular signal transduction networks. The most widely studied
members of the MAP kinase family are the 43- and 41-kDa MAP kinases
(ERK1 and ERK2, respectively); these are activated by phosphorylation
on both threonine and tyrosine residues (33, 34) by a
dual specificity kinase, MAP kinase/ERK kinase
(MEK)(35, 36) . MEK activity is in turn regulated by
serine phosphorylation catalyzed by MEK activators; the major MEK
activator is a serine/threonine kinase,
Raf-1(37, 38, 39) . Furthermore, it has
recently been reported that the activated Ras-mediated translocation of
Raf-1 to the plasma membrane is one of the necessary events for Raf-1
to be activated following
phosphorylation(40, 41, 42) . However, the
identity of the kinase(s) that phosphorylate Raf-1 is poorly understood
at present. Interesting in this respect, a rapid activation of Ras by
TGF-
has been reported in intestinal epithelial (IEC 4-1)
cells, where TGF-
was found to be growth-inhibitory(43) .
In this report, we have examined whether or not the effects of
TGF- in modulating cell proliferation are associated with the
activation of 41- and 43-kDa MAP kinases. Our results demonstrate that
(i) TGF-
1 alone can stimulate the growth of Swiss 3T3 and Balb 3T3
cells without MAP kinase activation, (ii) TGF-
1 synergistically
enhances the mitogenic action of EGF in these fibroblasts without
affecting EGF-induced MAP kinase activation, and (iii) TGF-
1
inhibits the EGF-stimulated proliferation of mouse keratinocytes
without inhibiting EGF-induced MAP kinase activation.
Figure 2:
Mitogen-induced activation of 41- and
43-kDa MAP kinases in Balb 3T3 and Swiss 3T3 cells. Growth-arrested
Balb 3T3 or Swiss 3T3 cells were treated with 20 ng/ml of PDGF-BB, 20
ng/ml of PDGF-AA, or 2.5 ng/ml of TGF-1 for the indicated periods
of time. Cells were then lysed, and cell lysates (20 µg protein)
were resolved by SDS-PAGE, blotted, and probed with anti-MAP kinase
antibody or anti-phosphotyrosine antibody, followed by ECL detection. Closed arrowheads indicate positions of the phosphorylated
(activated) forms of 41- and 43-kDa MAP kinases (pp41, pp43), while open arrowheads indicate positions of the unphosphorylated
form of these MAP kinases (p41, p43). MAP kinase assay was performed by
incubating cell lysates (10 µg of protein) with anti-MAP kinase
antibody, followed by the kinase reaction and resolution on SDS-PAGE;
phosphorylated MBP was detected by autoradiography (
P-MBP). Data shown are representative of three to five
separate experiments that gave essentially the same
results.
Figure 3:
Kinetics of MAP kinase activation by
PDGF-BB and TGF-1 in Balb 3T3 cells. Growth-arrested Balb 3T3
cells were treated with 20 ng/ml of PDGF-BB or 2.5 ng/ml of TGF-
1
for the indicated periods of time. MAP kinase assay was performed by
incubating cell lysates (10 µg of protein) with anti-MAP kinase
antibody followed by the kinase reaction; radioactivity incorporated
into MBP was determined as described under ``Experimental
Procedures.'' Inset shows the prolonged kinetics of MAP
kinase activation. Each value represents the mean ± S.E. of
triplicate determinations of a representative experiment. Similar
results were obtained in five independent
experiments.
Figure 1:
Mitogen-induced DNA
synthesis reinitiation in Balb 3T3 and Swiss 3T3 cells. Growth-arrested
Balb 3T3 or Swiss 3T3 cells were exposed to varying concentrations or
2.5 ng/ml of TGF-1, 20 ng/ml of PDGF-AA, 20 ng/ml of PDGF-BB, or
20 ng/ml of EGF. The rate of DNA synthesis was measured 12 h after
exposure of the cells to mitogens by adding BrdU to the cultures
followed by incubation for 16 h. BrdU-labeled nuclei were determined in
a total of more than 800 nuclei using 2 coverslips/experimental
condition. Data shown are representative of four separate experiments
that gave essentially the same results.
PDGF-AA, PDGF-BB, and EGF rapidly activated both of
the 41- and 43-kDa MAP kinases in growth-arrested Balb 3T3 and Swiss
3T3 cells. Activation of these MAP kinases was detected by the
appearance of their active forms, which show reduced mobility in
SDS-PAGE due to phosphorylation of specific threonine and tyrosine
residues(33, 34) , by analyzing their tyrosine
phosphorylation, and by a direct in vitro kinase assay of MAP
kinase immunoprecipitates using MBP as the substrate. These analyses
always gave essentially the same time course profile of MAP kinase
activation. As shown in Fig. 2and Fig. 3, the activation
of the MAP kinases in PDGF-AA/-BB-stimulated Balb 3T3 cells reached a
maximal level within 10 min of the addition of PDGF-AA/-BB and then
declined gradually. However, considerable activation (20% of
maximum) could still be detected 3 h after stimulation. In contrast,
TGF-
1 stimulation of quiescent Balb 3T3 and Swiss 3T3 cells did
not significantly induce the tyrosine phosphorylation or the activation
of 41- and 43-kDa MAP kinases at any time point up to 16 h, although
the addition of TGF-
1 to these cells led to the reinitiation of
DNA synthesis after a lag time of
12 h (data not shown).
Figure 4:
Mitogen-induced expression of c-fos gene in Balb 3T3 and Swiss 3T3 cells. Growth-arrested Balb 3T3
cells were treated with of 2.5 ng/ml of TGF-1, 20 ng/ml of
PDGF-AA, or 1 ng/ml of EGF for the indicated periods of time, while
growth-arrested Swiss 3T3 cells were treated with 2.5 ng/ml of
TGF-
1 or 20 ng/ml of PDGF-BB. Twenty µg of total RNA was
separated by formaldehyde/agarose gel electrophoresis, blotted, and
hybridized with
P-labeled v-fos cDNA probe. The
amounts of 28 and 18 S ribosomal RNA are shown as internal standards.
The values for the percentage of labeled nuclei in each
mitogen-stimulated Balb 3T3 cells were 0.3% (unstimulated), 15.5%
(TGF-
1), 17.6% (PDGF-AA), or 8.8% (EGF), which were determined by
using sister cultures corresponding to those shown in the figure. Similar results were obtained in three independent
experiments.
Figure 5:
Effect of combinations of growth factors
on DNA synthesis reinitiation and MAP kinase activation in Balb 3T3
cells. Growth-arrested Balb 3T3 cells were treated with 0.25 ng/ml of
TGF-1, 1 ng/ml of EGF, or 2.5 ng/ml of PDGF-BB, separately or in
combination (TGF-
1/EGF (A); TGF-
1/PDGF-BB (B); EGF/PDGF-BB (C)). The rate of DNA synthesis was
measured as described in the Fig. 1legend. Dotted
lines/arrowheads indicate values for sum of the
percentage of BrdU-labeled nuclei in the cells stimulated with these
growth factors separately (right panel). MAP kinase activity
was determined after stimulating cells with growth factors for the
indicated periods of time as described in the Fig. 3legend,
using sister cultures corresponding to those used for the measurement
of DNA synthesis. Each value represents the mean ± S.E. of
duplicate determinations of a representative experiment (left
panel). Similar results were obtained in three independent
experiments.
Thus, although TGF-1 influenced the mitogenic
potential of EGF and PDGF-AA/-BB in Balb 3T3 and Swiss 3T3 cells quite
differently, it did not affect significantly the degree of MAP kinase
activation induced by all of these growth factors at any time point
analyzed (Fig. 5, A and B). In sharp contrast,
the degree of MAP kinase activation, especially at the sustained phase
(30
60 min following stimulation), was markedly enhanced in those
fibroblasts treated simultaneously with EGF and PDGF-AA/-BB compared
with fibroblasts treated separately with these mitogens. Fig. 5C shows the typical results of such an analysis
for Balb 3T3 cells stimulated with a combination of EGF and PDGF-BB.
Figure 6:
Effect of combination of TGF-1 and
EGF on DNA synthesis reinitiation and MAP kinase activation in PAM212
cells. Growth-arrested PAM212 cells were exposed to 2.5 ng/ml of
TGF-
1 (T) or 20 ng/ml of EGF (E), separately or
in combination (E/T). A, the rate of DNA
synthesis was measured 12 h after exposure of the cells to growth
factors by adding BrdU to the cultures followed by incubation for 10 h.
The percentage of labeled nuclei were determined in
400
nuclei/coverslip, and each value represents the mean ± S.E. of
determinations on three coverslips/experimental condition of a
representative experiment. B, cell lysates (10 µg protein)
of PAM212 cells treated with growth factors for the indicated periods
of time were resolved by SDS-PAGE, blotted, and probed with anti-MAP
kinase antibody (Anti-MAPK) or anti-phosphotyrosine antibody (Anti-pTyr), followed by ECL detection. Closed arrowheads indicate positions of the phosphorylated (activated) forms of 41-
and 43-kDa MAP kinases (pp41, pp43), while open arrowheads indicate positions of the unphosphorylated form of these MAP
kinases (p41, p43). C, MAP kinase assay was performed by
incubating cell lysates (10 µg of protein) with anti-MAP kinase
antibody followed by the kinase reaction; radioactivity incorporated
into MBP was determined as described under ``Experimental
Procedures.'' Each value represents the mean ± S.E. of
duplicate determinations of a representative experiment, using sister
cultures corresponding to those used to measure DNA synthesis (A). Similar results were obtained in two independent
experiments.
EGF rapidly and markedly induced tyrosine
phosphorylation and activation of the 41- and 43-kDa MAP kinases in PAM
212 cells as it did in fibroblasts (Fig. 6, B and C). TGF-1 alone did not induce MAP kinase activation in
PAM 212 cells at any time point analyzed. Also, TGF-
1 did not
affect EGF-induced MAP kinase activation in these epithelial cells when
the two growth factors were added simultaneously, although under these
conditions EGF's capacity to stimulate cell proliferation was
markedly reduced.
We have examined whether or not the MAP kinase cascade is
involved in the signaling pathway of TGF- in eliciting its effects
to modulate cell proliferation. The MAP kinase cascade is the major
cytoplasmic kinase pathway activated commonly by numerous mitogenic
stimuli that interact with a diversity of structurally distinct
receptors(27, 28, 29, 30, 31, 32) ,
and it has recently been shown that the activation of the cascade is
necessary for the proliferation of
fibroblasts(51, 52) . For the analysis, we focused on
the activation of the 41- and 43-kDa MAP kinases, as these kinases
stand at a key position in the cascade and play a role in integrating
multiple mitogenic signaling pathways that involve Ras, Raf-1, Mos, MEK
kinase-1, protein kinase C, and even certain heterotrimeric G
proteins(27, 28, 29, 30, 31, 32) .
We demonstrate in this report that TGF-1 fails to detectably
activate the 41- and 43-kDa MAP kinases in Swiss 3T3 and Balb 3T3 cells
where TGF-
1 is clearly mitogenic. TGF-
1 also failed to induce
c-fos gene expression in these cells (Fig. 4). This
response is mediated by the serum-response element, which is bound in a
ternary complex containing the transcription factors p67
and p62
(53) , and phosphorylation of the
p62
by MAP kinases results in enhanced ternary complex
formation with consequent induction of c-fos expression(49, 50) . In some of our experiments,
induction of a minimum level of the c-fos gene expression was
observed in Swiss/Balb 3T3 cells treated with TGF-
1 for 30 min but
only on overexposed autoradiograms (more than a 10 times longer
exposure than that shown in Fig. 4). More significantly, under
our experimental conditions, TGF-
1 stimulated DNA synthesis
reinitiation in Balb 3T3 cells to an approximately equal or even higher
level than did PDGF-AA or EGF. Thus, it seems unlikely that such an
extremely low level of c-fos gene expression could have any
consequences for the cell or represent a significant response to the
extracellular stimuli. Furthermore, TGF-
1 had neither a
synergistic nor an antagonistic effect on the activation of the 41- and
43-kDa MAP kinases when used in combination with other growth factors,
although TGF-
1 apparently synergized (in fibroblasts) or
antagonized (in epithelial cells) the mitogenic potential of several
growth factors such as EGF (Fig. 5A and Fig. 6).
All these findings show no apparent association between MAP kinase
activation and the ability of TGF-1 to modulate cell
proliferation. The cytoplasmic signaling pathway of TGF-
1 appears
to be totally independent of the MAP kinase cascade. Thus, TGF-
1
is clearly distinct from the majority, if not all, of other peptide
growth factors such as EGF, PDGF, and fibroblast growth factor whose
ability to modulate cell proliferation is closely associated with the
activation of MAP kinases(54) . In this respect,
interleukin-4(55) , activin A (another member of the TGF-
superfamily)(56) , and thyrotropin (57, 58) have been reported to induce cellular
proliferation without activation of MAP kinases, although thyrotropin
has also been shown to induce the activation of MAP kinases in human
thyroid cells(59) .
Although it has previously been proposed
that TGF- promotes the growth of cells via the induced expression
of PDGF-AA(60, 61, 62) , recent studies have
demonstrated that TGF-
can stimulate cell proliferation by a
PDGF-independent mechanism(63) . Our results showing that
TGF-
1-treatment of cells did not induce MAP kinase activation and
c-fos gene expression at any time point analyzed (Fig. 2Fig. 3Fig. 4) suggest that the action of
TGF-
to stimulate proliferation of Swiss 3T3 and Balb 3T3 cells is
unrelated to that of PDGF-AA/-BB, because these mitogens clearly
induced the activation of MAP kinases and c-fos gene
expression soon after binding to their receptors. The mitogenic
potential of TGF-
1 was not inhibited by the depletion of protein
kinase C in Balb 3T3 and Swiss 3T3 cells, which were pretreated with
200 ng/ml of phorbol 12-myristate 13-acetate for 24 h (data not shown),
would also support the above conclusion; the capacity of PDGF-AA/-BB to
reinitiate DNA synthesis was reduced to
50% in such phorbol
12-myristate 13-acetate-pretreated cells compared with the untreated
native cells(45) . However, we cannot exclude the possibility
that TGF-
1 induces the production of a novel growth factor, which
acts as a direct mitogen in fibroblasts through a MAP kinase-/protein
kinase C-independent mechanism.
As far as we know, TGF-1 does
not induce the activation of other recently identified cytoplasmic
signaling pathways such as the JAK/STAT pathway or the JNK(SAPK)
pathway in Swiss 3T3 cells. (
)Activation of the former has
been commonly observed in cells stimulated with a variety of cytokines
(interferons, interleukins, erythropoitin, etc.) and also with EGF and
PDGF (reviewed in (64) ), while the later pathway has been
reported to be activated by treatment of cells with UV radiation,
proinflammatory cytokines, and environmental stress (reviewed in Refs.
31, 32, and 65). Thus, the signaling pathway of TGF-
1, beginning
with the cell surface receptors that have intrinsic serine/threonine
kinase activity, seems quite unique and apparently independent of those
well-characterized cytoplasmic kinase cascades. The precise cytoplasmic
signaling pathway for TGF-
1 through which it elicits its complex
effects on the regulation of cellular growth remains to be elucidated.
Recently, TGF- has been shown to induce the rapid activation of
p21
(43) , and of a MAP kinase
(p44
) (66) in proliferating cultures but not in
quiescent cultures of intestinal epithelial (IEC 4-1) cells for
which TGF-
is growth inhibitory. However, activation of the
p44
was
2-fold at most, and appearance of the
active form of p44
, which has reduced mobility in
SDS-PAGE due to the phosphorylation of specific threonine and tyrosine
residues was not clearly observed in the TGF-
-treated IEC
4-1 cells. It seems likely that only a marginal part of the
cellular pp44
was activated. Such a small level of MAP
kinase activation could play a role only if it exceeds the threshold
level in each cell.
In conclusion, we have demonstrated in this
report that TGF-1 is able to modulate cellular proliferation by a
signaling pathway that is totally independent of the MAP kinase
cascade. Our results also suggest that the activation of MAP kinases is
not an absolute requirement for the transition of cells from the
arrested state (G
) through G
into S phase.