(Received for publication, November 6, 1996)
From INSERM U 55, Institut Fédératif de
Recherche du Centre Hospitalo-Universitaire Saint-Antoine,
Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine,
75571, Paris Cedex 12, France and the ¶ Program in Molecular
Medicine, Department of Biochemistry and Molecular Biology, University
of Massachusetts Medical School and Howard Hughes Medical Institute,
Worcester, Massachusetts 01605
Transforming growth factor (TGF-
) is a
multifunctional factor that induces a wide variety of cellular
processes which affect growth and differentiation. TGF-
exerts its
effects through a heteromeric complex between two transmembrane
serine/threonine kinase receptors, the type I and type II receptors.
However, the intracellular signaling pathways through which TGF-
receptors act to generate cellular responses remain largely undefined.
Here, we report that TGF-
initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways including Rho-like GTPases, mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun abolishes TGF-
-mediated signaling. Therefore, the SAPK/JNK activation
contributes to TGF-
signaling.
Members of the Ras superfamily of small GTPases play essential
roles in the regulation of diverse cellular functions such as growth
control, differentiation, vesicular transport, motility, and
cytoskeletal organization (1-3). The Rho family of GTPases, which
includes Rho, Rac, and CDC42, have been implicated in distinct dynamic
processes involving the actin cytoskeleton: the formation of filopodia
and lamellipodia by CDC42 and Rac, respectively, and the assembly of
focal adhesions and stress fibers by Rho (3, 4). In addition to their
effects on the actin cytoskeleton, Rho, Rac, and CDC42 also have a role
in regulating cell proliferation, transcription, and cell
transformation (5-10). Recently, downstream mediators linking Rho-like
GTPase activation to nuclear events were identified (5-8). Rac1 and
CDC42H were shown to play a critical role in activation of members of
the mitogen-activated protein kinase
(MAPK)1 group, the stress-activated protein
kinases (SAPKs also known as c-Jun N-terminal kinases (JNKs)), in
response to growth factors, such as tumor necrosis factor- or
epidermal growth factor (6-8). The SAPK pathway involves sequential
activation of MAPK kinase kinase (MEKK1), MAPK kinase 4 (MKK4),
SAPK/JNK, and c-Jun (11-15).
Transforming growth factors (TGFs-
) belong to a family of
multifunctional cytokines that regulate cell proliferation,
differentiation, motility, and extracellular matrix formation (16-19).
TGF-
signals by simultaneously contacting two transmembrane
serine/threonine kinases known as the type I and type II receptors
(19-21). The type II receptor can directly bind TGF-
, but is
incapable of mediating responses in the absence of a type I receptor
(20, 21). Bound TGF-
is recognized by type I receptor, which is then
phosphorylated by the receptor II kinase, thereby allowing propagation
of the signal to downstream components (20, 21). To date, the
postreceptor mechanisms of action of TGF-
and the TGF-
-related
cytokines remains unresolved. Recently, TAK1, a potential component of
both TGF-
and bone morphogenetic protein 4 (BMP4) signaling
pathways, has been described as a member of the MAPKK kinase (MAPKKK)
family (22). Thus, a MAPK cascade might be involved in signaling by
TGF-
and TGF-
-related cytokines. In the current study, we provide
strong evidence for the involvement of the JNK/SAPK pathway in
TGF-
-mediated signaling. Furthermore, we demonstrate that Rho-like
GTPase function is critical for the activation of gene expression by
TGF-
.
pcDNA3CDC42H(QL), pcDNA3RhoA(QL),
pcDNA3Rac1 (QL), pCEV29CDC42H(N17), pCEV29RhoA(N19),
pCEV29Rac1(N17), and pcDNA3-HA-JNK1 were gifts from S. Gutkind. The
p3TP-Lux reporter construct (a gift from Dr. Joan Massagué)
contains three consecutive
12-O-tetradecanoylphorbol-13-acetate response elements, the
plasminogen activator inhibitor (PAI-1) promoter, and a luciferase
reporter gene. pXFTR1R4(KR) and pXFT
RII(KR)15 were kindly
provided by Dr. Rick Derynck. Expression plasmid for the
dominant-negative mutant of Jun pCMVTAM67 was a gift from Dr. Michael
Birrer. GST-Jun, the kinase-inactive MEKK1 mutant (pCMV5 MEKK1
(K432A)), and the dominant-interfering pcDNA3-Flag-MKK4 (Ala) and
pcDNA3-Flag-JNK1 (Ala183 and Phe185)
mutants have been described previously (23-25). GST-Jun-(1-79) was
expressed in Escherichia coli as described (13).
The hepatoma cells HepG2 were maintained in RPMI
containing 10% heat-inactivated fetal calf serum (FCS), MDCK cells in
RPMI containing 5% FCS, and CHO cells in RPMI containing 10% FCS. For gene expression analysis, cells were plated to semiconfluency and
24 h later transfected with expression vectors by the
LipofectAMINETM method (Life Technologies, Inc.). Cells
were subsequently incubated in the presence or absence of human
TGF-1 (2 ng/ml) for 12 h. Extracts were then prepared and
assayed for luciferase activity using the luciferase assay system
described by the manufacturer (Promega). Light emission was measured
during the initial 30 s of the reaction using a luminometer. The
luciferase activities were normalized on the basis of CAT expression
from pCAT-control vector (Promega) and protein content.
For assaying JNK activity, cells were
lysed at 4 °C in lysis buffer containing 25 mM HEPES (pH
7.5), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% Triton
X-100, 0.5% sodium deoxycholate, 20 mM
-glycerophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and
20 µg/ml leupeptin, and lysates were clarified by centrifugation. Endogenous JNK was immunoprecipitated with polyclonal anti-JNK antibody
(Santa Cruz). Immune complexes were collected by binding to protein
G-Sepharose, washed extensively in lysis buffer, and resuspended in 30 µl of kinase reaction mixture containing 12.5 mM MOPS (pH
7.5), 12.5 mM
-glycerophosphate, 7.5 mM
MgCl2, 0.5 mM EGTA, 0.5 mM sodium
fluoride, 0.5 mM sodium vanadate, 2 µg of GST-Jun, 20 µM unlabeled ATP, and 5 µCi of
[
-32P]ATP. After incubation at 30 °C for 20 min,
kinase reaction products were analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography. The HA epitope-tagged JNK1
activity was also assayed by immunocomplex kinase assays after
immunoprecipitation with the HA monoclonal antibody 12CA5.
Proteins were separated on 10% SDS-polyacrylamide gels and electroblotted onto HybondTM membranes (Amersham). After blocking, the membranes were probed with anti-JNK polyclonal antibody (Santa Cruz) or anti-HA (12CA5) as described elsewhere (26). The immunoblot was then developed using enhanced chemiluminescence detection according to the manufacturer's protocol (Amersham).
We explored the possibility that the Rho family of GTPases are
potential downstream effectors of TGF- receptors because these proteins are involved in signaling to the nucleus leading to
transcriptional activation (5-8). Members of this family function as
binary switches by cycling between the active GTP-bound state and the
inactive GDP-bound state. These GTPases can be activated through
substitution of glutamine by a leucine residue in a position analogous
to that of codon 61 of Ras. Such a mutation has been shown to inhibit the GTPase activity of most of these proteins (8, 27). To investigate
whether Rho-like GTPases are intermediates in a TGF-
-initiated signaling pathway leading to transcriptional activation, we tested the
ability of constitutively activated mutants of RhoA, Rac1, and CDC42Hs
to signal transcriptional responses that are typical of TGF-
. A
TGF-
reporter construct (p3TP-Lux) containing a luciferase gene
controlled by a TGF-
-inducible promoter was used to monitor TGF-
-induced changes in gene expression in HepG2 cells (20, 21, 26).
Transient transfection of p3TP-Lux into HepG2 cells resulted in a
strong induction of luciferase activity in response to TGF-
1 (Fig.
1A). Cotransfection of expression plasmids
encoding the constitutively activated small GTPases RhoA-QL, Rac1-QL,
or CDC42H-QL stimulated by themselves luciferase activity with an efficiency approaching that of TGF-
stimulation in the case of Rac1-QL (Fig. 1A). Addition of TGF-
potentiated the
responses of the reporter gene to activated Rho-related GTPases,
although to a variable extent. Whereas CDC42H-QL did not enhance
luciferase activity and activated RhoA caused only a very modest
increase, overexpression of the activated form of Rac1 led to
superinduction of luciferase activity (Fig. 1A).
Cotransfection of increasing amounts of Rac1-QL expression plasmids
potentiated TGF-
-induced reporter activity in a
concentration-dependent manner (Fig. 1B). In a
control experiment, expression of the dominant-negative forms of
TGF-
types I (T
RI) and II (T
RII) receptors abolishes both control and TGF-
-induced transcriptional activation, indicating that
this effect is specific to TGF-
(Fig. 1A).
To provide further evidence for the involvement of Rho-like GTPases in
TGF- signaling, we examined the effect of dominant-negative mutants
of RhoA, Rac1, and CDC42H on transcriptional activation by TGF-
.
Dominant-negative mutants were generated through substitution of
threonine at position 17 to asparagine. The analogous mutation in
p21ras increases its affinity for GDP. This results in
sequestration of guanine nucleotide exchange factors making them
unavailable for activation of endogenous p21ras and thereby
blocking downstream signaling events (28). By analogy to the activity
of N17Ras, the mutants of CDC42H, Rac1, and RhoA have similarly been
shown to function as dominant-negative molecules (6, 8).
As shown in Fig. 1C, TGF--induced transcription of the
reporter gene was effectively inhibited by N17Rac1 expression. However, induction of transcription by TGF-
was also inhibited by expression of either N19RhoA or N17CDC42H (Fig. 1C). Hence, functional
RhoA and CDC42H also appear to be required for TGF-
-mediated
signaling, even though activated RhoA and CDC42H are not sufficient for
activation (Fig. 1A). This situation is remarkably similar
to that of the Ras-mediated signaling pathways. For example, activated
Ras is not sufficient to activate serum response factor-linked
signaling, yet expression of the dominant-interfering Ras derivative
N17Ras inhibits activation of the serum response factor-linked pathway by extracellular stimuli that act through G protein-coupled receptors (5). Additional evidence that RhoA and CDC42H are components in the
TGF-
signaling pathway is provided by our findings that overexpression of N19RhoA and N17CDC42H together with N17Rac1 abolishes
completely transcriptional activation in response to TGF-
, whereas
overexpression of N17Rac1 alone had a less inhibitory effect on
reporter gene activity (Fig. 1C).
Together, these results provide strong evidence that RhoA, Rac, and
CDC42H play an important role in TGF- receptor signaling. However,
our data differ significantly from that recently published by Mucsi
et al. (29). They demonstrated that expression of the dominant-negative mutant of Rac, but not dominant-negative mutants of
CDC42H and Rho, inhibited transcriptional activation by TGF-
in
NIH3T3 cells. The apparent discrepancy between these findings and ours
might be due to cell type differences or could reflect the possibility
that receptor activation generates another signal that synergizes with
RhoA and CDC42H to activate gene expression. In this context, recent
studies have shown that RhoA, Rac1, and CDC42H signal to the nucleus in
a cell type-specific manner (30). Furthermore, we have previously shown
that TGF-
induces rapidly (2 min) the activity of a 78-kDa
serine/threonine kinase p78 in HepG2 cells for which TGF-
can act as
a growth-inhibitory factor (26). In contrast, there was no apparent
induction of p78 activity when the assay was done with NIH3T3 cells, a
cell line that failed to undergo growth arrest in response to TGF-
.
Identification of the mechanisms by which Rho, Rac, and CDC42H pathways
link TGF-
-receptor activation to nuclear events would help clarify this issue.
Recently, Rac and CDC42H have been shown to activate the JNK signaling
pathway leading to c-Jun transcriptional activation (6, 8). To
determine whether SAPK/JNKs are activated in response to TGF-, we
tested SAPK/JNK activity in an immune-complex kinase assay using
GST-Jun-(1-79) as substrate (6, 8). Fig. 2A
shows that exposure of HepG2 to TGF-
caused a marked and persistent increase in SAPK/JNK activity, with maximal activation at 12 h. The effect of TGF-
on SAPK/JNK activity was
dose-dependent (Fig. 2B). In comparison with the
immediate and transient JNK activation induced by other stimuli such as
tumor necrosis factor-
(5 min) and anisomycin (6, 8, and data not
shown), we did not detect a significant increase of GST-Jun
phosphorylation before 1 h. However, the time course of SAPK/JNK
activation by TGF-
is similar to that induced by radiation (15, 31).
Furthermore, using HepG2 cells transiently transfected with the
hemagglutinin (HA)-tagged SAPK/JNK1 (p46) and SAPK/JNK2 (p54), the two
forms recognized by the antibody used to detect the endogenous
SAPK/JNKs (Fig. 2A), we found that TGF-
increased both
SAPK/JNK1 and SAPK/JNK2 activities (data not shown). Identical results
were obtained with the MDCK and CHO cell lines, suggesting similarities
in stimulus-response coupling mechanisms of TGF-
receptors (Fig.
2C). In contrast, recent studies using NIH3T3 cells have
shown that TGF-
stimulation did not significantly induce the
activation of SAPK/JNK at any time up to 3 h, although the
addition of TGF-
to these cells led to the increase in MAPK activity
under these experimental conditions (29). This may simply represent a
difference in the delay of JNK induction by TGF-
in NIH3T3 cells.
Consistent with this hypothesis, JNK activation did not occur until 4 to 6 h after exposure of MDCK cells to TGF-
(data not shown).
The activation of SAPK/JNK indicated by our observations agrees with
recent studies showing that TGF-
activates a novel MAPKKK, known as
TAK1, that may be involved in signal transduction by members of the
TGF-
superfamily. Activated TAK1 phosphorylates and promotes
activation of MKK4 (also termed SEK1 or JNKK), the kinase that controls
activation of SAPK/JNK (22). Whether TAK1 is downstream of the TGF-
receptor in the biochemical route to JNK warrants further
investigation.
To test the hypothesis that SAPK/JNK activation may contribute to the
induction of gene expression by TGF-, we examined the ability of the
dominant-interfering mutants of MEKK1 and MKK4 to block SAPK/JNK
activation and inhibit transcriptional responses following exposure of
cells to TGF-
. Expression of either MEKK1(K432A) or MKK4(Ala)
inhibited activation of co-transfected HA-tagged SAPK/JNK1 in response
to TGF-
, indicating that TGF-
signaling was specifically blocked
in the transfected cells (Fig. 3A).
Interestingly, the dominant-negative MEKK1 and MKK4 mutants had similar
effects on TGF-
-mediated transcriptional activation (Fig.
3B), which is consistent with the hypothesis that activation
of MEKK1-MKK4-SAPK/JNK signaling pathway plays a central role in
mediating these transcriptional processes. Identical results were
obtained with the MDCK cells indicating that the inhibitory effect of
dominant-negative mutants of MEKK1 and MKK4 on transcriptional
activation by TGF-
occurs in multiple cell types (Fig.
3B). Expression of MEKK1(K432A) or MKK4(Ala) is also
sufficient to block the superinduction of the gene reporter activity by
TGF-
in cells coexpressing the constitutively activated forms of
RhoA, Rac1, and CDC42H (Fig. 3C). Taken together, these
results support a model in which the SAPK/JNK cascade participates in a
signaling pathway activated by TGF-
receptors through Rho family
GTPases.
To further demonstrate a role of the SAPK/JNK cascade on
transcriptional activation by TGF-, we made the use of
dominant-interfering mutants of SAPK/JNK1 and c-Jun. c-JunTAM67 acts as
a dominant-interfering mutant because a deletion in the N-terminal
transactivation domain of c-Jun that includes the binding site for
SAPK/JNK1 (31, 32). As expected, expression of both dominant-negative
mutants of SAPK/JNK1 (JNK1(Ala183 and Phe185))
and c-Jun (TAM67) inhibited the activation of the reporter gene by
TGF-
(Fig. 4A). In a dose-response
experiment, low levels (0.01 µg) of TAM67 construct were sufficient
to inhibit TGF-
-induced gene expression significantly, and high
levels (1 µg) completely blocked activation (data not shown). In
contrast, overexpression of TAM67 had no effect on TGF-
-mediated
HA-JNK activation (Fig. 4B). From these results, it is
evident that inhibition of transcriptional responses to TGF-
occurs
at a level downstream of SAPK/JNK and that c-Jun plays an important
role in TGF-
signaling. In support of this interpretation are data
showing that TGF-
may up-regulate the expression of c-Jun product
(33). Moreover, SAPK/JNKs are thought to be responsible for
phosphorylating the transactivating domain of c-Jun protein in
vivo, and, in turn, phosphorylated c-Jun homodimers have potent
AP-1 activity and can control the expression of a number of genes,
including c-jun itself (34). We conclude that SAPK/JNK
signaling pathways contribute to the intracellular relay of
transcriptional signals originating from the TGF-
receptors.
Our present studies provide the first demonstration of the involvement
of Rho and CDC42H in TGF--mediated signaling pathways and indicate a
critical role of the SAPK/JNK cascade in delivering a signal to the
nucleus leading to transcription activation. This signaling pathway may
account for part of the genetic response of cells to TGF-
. In
addition, this pathway may contribute to, or cooperate with, the
activation of the MAD transcription factor by the TGF-
-related
factor BMP-2 (19, 35-37). Our findings raise questions concerning the
role of Rho-like GTPases and the SAPK/JNK signaling pathway in the
biological actions of TGF-
(16-19, 26, 38). Substantial evidence
has been accumulated demonstrating that Rho-like GTPases and the
SAPK/JNK cascades play essential roles in the regulation of multiple
physiological processes, including cell growth control, cell death,
cell motility, embryonic morphogenesis, and regulation of the
cytoskeleton (3, 5-8, 25, 39, 40). The identification of Rho family
GTPases and the SAPK/JNK cascade as essential components in the TGF-
signaling pathway provide new insight into the mechanism by which
TGF-
mediates its biological actions.