From the Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The activity of the transcription factor NF- Gene expression is regulated by the interplay of different
transcription factors which bind to specific DNA recognition motifs and
cooperate with the basal machinery to initiate transcription. During
the last few years, an emerging body of evidence is revealing the
importance of crossed interactions between members of distinct families
of transcription factors to form higher complexes, enabling the
accurate regulation of this process. The serum response element (SRE)1 is a specific DNA
sequence which is found in the promoter of several immediate-early
genes (2). The prototypic c-fos SRE binds a ternary complex
composed of a homodimer of p67SRF (serum response factor)
and a third subunit, p62TCF (ternary complex factor), which
belongs to the Ets family of accessory proteins. These TCF factors have
the ability to bind a purine-rich motif 5' to the SRF-binding site,
known as the Ets recognition domain, only when SRF is bound to DNA, and
include Elk-1, SAP-1, and SAP-2/ERP/NET proteins. The formation of this SRE binding-ternary complex requires the conserved B-box motif of the
Ets subunits and sequences located in the core domain of the SRF
protein (coreSRF), which is a region that is also
responsible of its DNA binding and dimerization capabilities
(2-8).
The SRE has shown to be necessary and sufficient for the rapid
induction of the c-fos proto-oncogen in response to
different external stimuli such as serum, growth factors, and phorbol
esters (2, 9). Furthermore, this DNA motif is a point of convergence of
different signal transduction cascades activated by an extensive range
of agonists. The regulation of the activity of the SRE is mediated by
two different signaling pathways. The first mechanism is elicited by
the multiple phosphorylation of TCFs within their C-terminal
transactivation domain. Such phosphorylation can be triggered by
distinct families of mitogen-activated protein kinases. Actually,
whereas the classical Ras-Raf-MEK-ERK cascade is responsible of the
phosphorylation of TCFs proteins after growth factors or phorbol esters
stimulation (2, 10-14), JNK/SAPK and p38 have also been shown to
phosphorylate TCFs in response to certain cytokines and environmental
stress conditions (15-17).
The second signaling pathway which controls an efficient
transcriptional activation through the SRE site is mediated by the SRF.
Hill et al. (18) showed that this TCF-independent regulation is modulated by members of the Rho family of small GTPases, RhoA, Rac1,
and Cdc42Hs. Indeed, stimulation of the transcriptional activity of the
SRF induced by serum, lysophosphatidic acid (LPA), and
AlF4- (an activator of heterotrimeric G proteins) is
signaled by RhoA in NIH 3T3 cells, since the expression of the C3
component of the Clostridium botulinum toxin efficiently
blocks this effect. Fromm et al. (19) have also found that
the G The precise mechanism by which Rho proteins can modulate the
transcriptional activation through the c-fos SRE is ill
defined. It has been proposed that this regulation may be targeted by a second, unknown accessory protein, distinct from TCFs, which could interact with the DNA-bound p67SRF (3, 18, 23, 24). As
other results suggest that the SRF contains different sequences which
can inhibit its own transactivation capability (25), it is possible
that such interaction could relieve the transactivation domain from
this inhibitory effect. And, moreover, this putative recognition factor
should be a critical target for Rho family-mediated signal transduction pathways.
Our group has recently demonstrated that the Rho family of small
GTPases can efficiently induce the transcriptional activity of the
nuclear factor- Different interactions of the NF- In the present study, we have investigated the implications of
RhoA-dependent activation of NF- Cell Culture and Transfections--
Simian COS-7 fibroblast-like
cells were cultured in Dulbeccos's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum and 1 mM
glutamine. Murine NIH 3T3 fibroblasts were grown in DMEM with 10%
newborn calf serum. For transient expression assays, cells were
transfected in 60-mm dishes by the calcium phosphate method as
described (44). The amount of plasmidic DNA was kept constant at 9-10
µg/plate with the corresponding empty vector. The total amount of DNA
was kept at 30 µg/100-mm plate with calf thymus DNA (Boehringer
Mannheim). After the precipitate was removed, cells were incubated in
DMEM, 0.5% fetal calf serum for the next 24 h. Cells were
stimulated during the last 5 h of culture where indicated and
harvested for the different assays. Transfection efficiency was
normalized by co-transfection of the plasmid pCMV- Plasmids--
pCDNAIIIB plasmid (Invitrogen) and derived
expression vectors encoding for constitutively activated RhoA (QL),
Rac1 (QL), and Cdc42Hs (QL) proteins have been described previously
(45). ( Gene Expression Analysis--
Analysis of the NF-
Measurement of the activation of the chromosomal SRE- Western Blot Assays--
For protein expression assays, cells
were transfected with the corresponding plasmids and incubated in DMEM,
0.5% fetal calf serum for the next 24 h. The lysis was performed
in buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate, 10 mM Na4P2O7, 50 mM sodium fluoride, 20 µg/ml leupeptin, 20 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The total amount of protein was determined with a commercial kit based on
the Bradford method (Bio-Rad). Lysates were obtained in 1 × SDS
Laemmli buffer. Thirty micrograms of total protein were analyzed by
SDS-electrophoresis on 10% polyacrylamide gels (SDS-PAGE). After
transferring to nitrocellulose, the blots were incubated with the
corresponding rabbit antiserum (p50, RelA(p65), C/EBP Rho Proteins Activate Transcription through
Artificious regulation of the SRE-driven transcription due to the
characteristic episomal replication of a transfection system based on
COS-7 cells could affect the results. In order to eliminate this
possibility we also investigated whether SRE-dependent
transcription could be stimulated by Rho proteins in cells stably
transfected with a plasmid carrying the SRE-binding site in a
non-episomal vector. NIH 3T3 cells were stably transfected with the
pSV2Neo vector along with the SRE- NF-
For that purpose, an inhibitor of the NF-
We also explored whether the activation of the 4 × SRE-CAT
reporter by specific Rho-related exchange factors was affected by the
expression of I
Next, COS-7 cells were co-transfected with the corresponding cDNAs
of the constitutively activated Vav or Ost genes, along with the
pRcCMV-I Expression of the I
Since transactivation of the 4 × SRE-CAT induced by RhoA seemed
to be dependent on the NF- RelA and p50 Subunits Cooperate with RhoA in the Transactivation
through the SRE-binding Site--
The mechanism by which NF-
COS-7 cells were co-transfected with a suboptimal dose of the
expression vector corresponding to RhoA QL and increasing
concentrations of the cDNA of the RelA or p50 protein, along with
the 4 × SRE-CAT plasmid. Expression of the RelA subunit along
with the constitutively activated RhoA GTPase cooperated in the
transactivation of the SRE-dependent reporter (Fig.
4C). Franzoso et al. (42) have previously shown
that RelA but not p50 synergizes with SRF in the transactivation of the
interleukin 2R- NF-
Transcriptional activity assays were carried out co-transfecting the
expression vector encoding for the constitutively activated form of
RhoA along with the cDNAs of the RelA and the C/EBP Inhibition of the RhoA-dependent Activation of the
SRE-binding Site by a Specific Inhibitor of the C/EBP Family of
Transcription Factors--
The C/EBP
COS-7 cells were co-transfected with the 4 × SRE-CAT reporter and
the expression vector corresponding to the constitutively activated
RhoA, along with the cDNA for LIP (Fig.
6A). Expression of such a
specific inhibitor was able to block the transactivation of the
SRE-dependent promoter due to either LPA stimulation or RhoA overexpression. Furthermore, the RhoA-dependent
activation of the SRE binding motif inhibited by LIP was fully restored
by co-transfection of the full-length active C/EBP RhoA Affects the Levels of the C/EBP Rho proteins regulate critical biological processes such as cell
growth, transformation and metastasis, apoptosis, response to stress,
and certain aspects of development (44, 45, 74, 91-103). Recently, our
group has demonstrated that the human proteins RhoA, Rac1, and Cdc42Hs
(three prototype members of the Rho family) activate NF- Three prototype members of the Rho family, RhoA, Rac1, and Cdc42Hs,
induce the transcriptional activation of both NF- Previous reports showed that NF- From the experiments shown here we cannot conclude whether RelA and p50
can bind to p67SRF as prototypical NF- The region between residues 204 and 234 of RelA is involved in its
interaction with SRF, while the dimerization between RelA and p50 is
mediated by residues 222 to 231, all of them located in the Rel
homology domain (42, 104). Thus, interaction of RelA with the SRF is
feasible through residues 204 to 222. Furthermore, the positive effect
observed on SRE-dependent transcription when p50 is
overexpressed along with RhoA could be explained if the excess p50
subunit induced its dimerization with RelA and translocation to the
nucleus, where it would contribute to the activation of the SRF. On the
other hand, the region of p67SRF implicated in complexing
with RelA overlaps the previously described domain which is responsible
for neutralization of its own transcriptional activity, favoring the
hypothesis that NF- Nevertheless, the I We demonstrate that activation of the SRE by RhoA involves another
factor, the C/EBP The p20/C/EBPB
can be modulated by members of the Rho family of small GTPases (Perona,
R., Montaner, S., Saniger, L., Sánchez-Pérez, I., Bravo,
R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475).
Ectopic expression of RhoA, Rac1, and Cdc42Hs proteins induces the
translocation of NF-
B dimers to the nucleus, triggering the
transactivation of the NF-
B-dependent promoter from the
human immunodeficiency virus. Here, we demonstrate that activation of
NF-
B by RhoA does not exclusively promote its nuclear translocation
and binding to the specific
B sequences. NF-
B is also involved in
the regulation of the transcriptional activity of the c-fos
serum response factor (SRF), since the activation of a
SRE-dependent promoter by RhoA can be efficiently
interfered by the double mutant I
B
S32A/S36A, an inhibitor of the
NF-
B activity. We also present evidence that RelA and p50 NF-
B
subunits cooperate with the transcription factor C/EBP
in the
transactivation of the 4 × SRE-CAT reporter. Furthermore, RhoA
increases the levels of C/EBP
protein, facilitating the functional
cooperation between NF-
B, C/EBP
, and SRF proteins. These results
strengthen the pivotal importance of the Rho family of small GTPases in
signal transduction pathways which modulate gene expression and reveal
that NF-
B and C/EBP
transcription factors are accessory proteins
for the RhoA-linked regulation of the activity of the SRF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 subfamily of heterotrimeric G protein
subunits is able
to induce the SRE activity by a RhoA-dependent pathway. On
the other hand, Rac1 GTPase plays an essential role in the activation
of the c-fos SRE induced by certain agents such as the
epidermal growth factor and hydrogen peroxide (20-21). Whereas Hill
et al. (18) have described that Rac1 and Cdc42Hs GTPases can
activate the SRF in a RhoA-independent manner, other authors have found
a link between these signaling cascades (22).
B (NF-
B) (1, 26). The NF-
B complex is mainly
composed of two subunits of 50 and 65 kDa which are retained in the
cytoplasm by a third protein, I
B. These I
B inhibitory proteins
block the ability of the dimer to translocate to the nucleus and
activate gene expression (27-29). This transcription factor has shown
to play a relevant role in the control of cell growth and apoptosis,
along with different aspects of the immune and inflammatory responses
(30-41). RhoA, Rac1, and Cdc42Hs are able to trigger the
transactivation of the NF-
B dependent-HIV promoter in different cell
lines. The mechanism involved is the conventional nuclear translocation
of RelA/p50 and p50/p50 dimers, by a mechanism that involves the
phosphorylation and proteolytic degradation of the inhibitory subunit
I
B
. Moreover, this activation is independent of that induced by
H-Ras/Raf cascade (1, 26).
B dimers with other families of
transcription factors have been widely reported (reviewed in Ref. 27).
It has been described that RelA can act as an accessory protein for the
SRF and a physical association between both subunits have been
demonstrated in vitro (42). The formation of this complex
seems to be mediated through the Rel homology domain of RelA and the
DNA-binding domain of SRF, which has shown to exert a negative effect
on its transactivation ability. Furthermore, RelA functionally
synergizes with SRF in the transactivation of a reporter construct
dependent only on the SRE site, indicating that the direct or
facilitated interaction between both proteins may neutralize the
inhibitory functions of the core domain of SRF.
B in the regulation of
p67SRF function. We demonstrate that NF-
B modulates the
transcriptional activity of the SRF induced by this GTPase.
Furthermore, this mechanism may also involve a cross-talking of RelA
and p50 NF-
B subunits with the transcription factor C/EBP
, which
is also able to bind to the SRF and regulate its transactivation
activity (43). Therefore, members of the NF-
B and C/EBP families of
transcription factors can interact and behave as accessory proteins in
the modulation of the transcriptional activity of the SRF induced by
the small GTPase RhoA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal, and the
-galactosidase assay as described previously (1).
453/+80)HIV-LUC contains the NF-
B sites of the HIV
enhancer/promoter and
NF-
B HIV-LUC contains a 3-base pair
substitution in each of the NF-
B-binding sites (46, 47). The
promoters of the different reporters contain the following specific
motifs: 4 × SRE-CAT, (4X) (AGGATGTCCATATTAGGACATCT) the sequence
of the SRE-binding site of the human c-fos gene 5' from a
minimum promoter harboring the TATA box (54); SREMUT-CAT promoter
contains the mutations (AGGATGTCAATACTAGGACATCT) that prevents binding
of the SRF, as previously reported (1); SRE-
-gal, the sequence of
the SRE-binding site of the human c-fos gene 5' from a
minimum promoter containing the TATA box; and 3D.A.CAT, a minimal
c-fos promoter SRE with a TCF-defective binding site
inserted 5' to a Xenopus
-actin TATA box (18).
pMEX-RelA(p65) and pMEX-p50 were provided by Dr. Bravo. pMSV-C/EBP
was generously provided by Dr. Pérez-Castillo and contains the
rat C/EBP
gene. pMSV-LIP was obtained by deletion of the 5' coding
region of the C/EBP
gene, digested with the NcoI enzyme
(48). The following plasmids have been previously described: pMEX
derived vector encoding for a truncated Vav protein (PJC7) (49); PCEV27
derived vector encoding for a truncated Ost protein (50); and
EXV-RasVal-12 (51) and pRcCMV-I
B
S32A/S36A
(52).
B activity
was performed by transfection of the reporter containing the wild-type
B sites of the HIV enhancer/promoter, (
453/+80)HIV-LUC, and the
one containing 3-base pair substitutions in each NF-
B site,
NF-
B HIV-LUC. Cells were harvested 24 h after transfection
and the protein extracts were prepared by three consecutive cycles of
freezing and thawing. The total amount of protein was determined with a
commercial kit based on the Bradford method (Bio-Rad). 2 µg of
protein were assayed for luciferase activity using a commercial kit
(Promega). For the SRE-binding site, both 4 × SRE-CAT and
3D.A.CAT reporters were used in this study. Protein extracts were
prepared and 10-50 µg were assayed for the chloramphenicol
acetyltransferase (CAT) activity as described previously (53).
Transfection efficiencies were corrected by the ratio of the luciferase
or CAT activity and the
-gal activity obtained in the same sample by
co-transfection of the pCMV-
-gal plasmid. None of the proteins used
in this study affected considerably the levels of transcriptional
activation of pCMV-
-gal because similar levels were always observed
when compared with the respective empty plasmids.
-gal plasmid
was performed as follows: NIH 3T3 cells were transfected with the
pSV2Neo vector along with a SRE-
-gal plasmid and selected for G418
resistance. Approximately 200 colonies obtained by serial dilution were
tested for
-galactosidase activity after serum stimulation. Four
selected clones were then transfected with the pCDNAIIIB-derived
expression vectors encoding for the constitutively activated Rho
proteins and cells were incubated in DMEM, 0.5% fetal calf serum for
the next 24 h. Cells were fixed in 1% glutaraldehyde and
incubated at 37 °C for
-gal activity staining, using
5-bromo-4-chloro-3-indoyl
-D-galactoside as substrate.
Efficiency of transfections were normalized by parallel transfections
using the pCMV-
-gal plasmid.
) (Santa Cruz
Laboratories). Immunocomplexes were visualized by enhanced
chemiluminescence detection (Amersham) using a biotinylated anti-rabbit
antibody and streptavidin peroxidase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B- and SRE-binding
Sites in COS-7 Cells--
Rho proteins are able to activate the
transcription factor NF-
B in diverse cell systems (1) and the serum
response factor in NIH 3T3 fibroblasts (18). We aimed to investigate
whether activation of NF-
B by Rho proteins was functionally related
to the activation of the SRE-dependent transcriptional
activity. We first identified a suitable cell system to carry out both
assays, NF-
B activation and SRE-dependent transcription,
under similar conditions. The NF-
B-dependent
(
453/+80)HIV-luciferase (HIV-LUC) plasmid was used (47) as a reporter
for NF-
B activity. This reporter contains two
B-binding sites
within its enhancer region, which are mostly responsible for the
transactivation of the HIV LTR. Fibroblast-like COS-7 cells were
co-transfected with the cDNAs encoding for the constitutively
activated forms of RhoA, Rac1, and Cdc42Hs (QL) proteins along with the
HIV-LUC plasmid. As shown in Fig.
1A, an efficient
transactivation of the HIV promoter could be readily observed. This
effect was dependent on the
B-binding sites since the activation was
completely avoided when a HIV-LUC reporter containing 3-base pair
substitutions in each
B motif was used (data not shown). Under
similar conditions, Rho proteins were also able to induce the
transactivation of a 4 × SRE-CAT reporter in the same cell line
(Fig. 1B). The 4 × SRE-CAT plasmid contains the CAT
gene under the control of a minimum promoter composed of four copies of
the sequence of the SRE of the human c-fos gene along with a
TATA box (54). Similar results were obtained when both constitutively
activated forms of two Rho family related exchange factors, Vav and Ost
(55), were overexpressed (Fig. 1B). Although there is a
clearly positive and reproducible response with a 3-4-fold induction
by serum, this is reduced compared with other cell systems. We also
were able to demonstrate a more than 10-fold activation by serum when
the NIH 3T3 cell system was used instead of the COS-7 cell sytem using
the same reporter vectors (data not shown). These results demonstrate
that these three members of the Rho family of GTPases mediate in the
signal transduction pathways implicated in the activation of the
transcription factor NF-
B and in the up-regulation of intracellular
cascades which promote gene expression through the SRE-binding site in the simian fibroblast-like COS-7 cells.
View larger version (31K):
[in a new window]
Fig. 1.
Rho proteins activate transcription through
the B and the SRE binding motifs in COS-7
cells. A, Rho proteins induce the transcriptional
activation of the HIV-LUC reporter. COS-7 cells were co-transfected
with 0.5 µg of (
453/+80)HIV-LUC and 1 µg of pCMV-
-gal per
60-mm plate along with 3 µg of the plasmid pCDNAIIIB or the
derived vectors expressing RhoA QL, Rac1 QL, or Cdc42Hs QL. Cells
transfected with pCDNAIIIB were stimulated with TNF
(10 ng/ml)
(vector + TNF
) 5 h before harvesting. Luciferase activity was
determined 24 h after transfection. Data represent the mean of a
single experiment performed in triplicate ± S.D. Results are
expressed as fold induction considering as 1 the luciferase activity of
the cells transfected with the empty vector. Same results were observed
in three independent experiments. B, Rho proteins induce the
transcriptional activation of the 4 × SRE-CAT reporter. COS-7
cells were co-transfected with 3 µg of 4 × SRE-CAT and 1 µg
of pCMV-
-gal per 60-mm plate along with 3 µg of pCDNAIIIB or
the derived vectors expressing RhoA QL, Rac1 QL, or Cdc42Hs QL, pMEX or
pMEX-vav, pCEV27, or pCEV27-ost. Cells transfected with pCDNAIIIB
were stimulated with 20% fetal bovine serum (vector + 20%
FBS) 5 h before harvesting. CAT activity was determined
24 h after transfection. Data represent the mean of a single
experiment performed in triplicate ± S.D. Results are expressed
as fold induction considering as 1 the CAT activity of the cells
transfected with the empty vector. Same results were observed in three
independent experiments. C, Rho proteins induce the
-galactosidase activity of SRE-
-gal stable transfectants. SRE-
-gal derived NIH 3T3
cells were transfected with 3 µg of the plasmid pCDNAIIIB or the
derived vectors expressing RhoA QL, Rac1 QL, or Cdc42Hs QL and starved
from serum for 48 h. Cells transfected with pCDNAIIIB were
stimulated with 20% fetal bovine serum (vector + 20% FBS)
5 h before harvesting.
-Galactosidase activity was determined
48 h after transfection. Data represent the mean of a single
experiment performed in triplicate ± S.D. Results are expressed
as fold induction considering as 1 the
-galactosidase activity of
the cells transfected with the corresponding empty vector. Similar data
were observed in other three SRE-
-gal derived clones. Three
independent experiments were performed for each derived clone with
similar results.
-gal plasmid and selected for G418 resistance. Over 200 different resistant colonies obtained by serial
dilution were isolated and tested for
-galactosidase activity after
serum stimulation. Four of them that scored close to 100% staining
when stimulated by serum were selected for further analysis. Cells were
transiently transfected with the pCDNAIIIB-derived expression
vectors encoding for the constitutively activated forms of RhoA, Rac1,
and Cdc42Hs GTPases and cells expressing the
-galactosidase were
scored as positive. Efficiency of transfection for each plasmid preparation was normalized by parallel transfections using the
-galactosidase gene placed under control of the CMV promoter, allowing for constitutive expression of the
-galactosidase enzyme. As shown in Fig. 1C, the three GTPases were able to promote
transactivation through the chromosomal SRE site in one of the selected
clones by at least 2-fold. Cdc42Hs was much more efficient, suggesting additional signaling pathways activated by Cdc42 to those activated by
RhoA and Rac1, in agreement with the results reported by Alberts et al. (56). Similar results were observed when the other
three independent clones were used (data not shown). Thus, these
results provide a strong support to the implication of Rho GTPases in signal transduction pathways which determine the activation of transcription due to the SRE sequences under physiological conditions and establishes that the COS-7 cell system is appropriate to
carry out the proposed experiments.
B Activity Is Required for Transcriptional Activation of the
SRE by RhoA--
It has been previously reported that the different
Rel/NF-
B proteins are able to interact physically and functionally
with members of other families of transcription factors such as AP1, Sp1, Stat6, ATF, HMG/Y, the glucocorticoid receptor, TBP, TFIIB, and
C/EBP (57-66). Cross-talkling between subunits of different families
of transcription factors seems to be a relevant general event in the
regulation of gene expression. In particular, previous studies
suggested that NF-
B might also participate in the regulation of the
transcriptional activity of the serum response factor
(p67SRF), and a physical interaction between RelA and this
protein had been observed in vitro (42, 67, 68). Thus, we
explored whether the activation of NF-
B by Rho proteins could play
any role in the cascades controlling transcription through the
SRE-binding site.
B activity, the double
mutant I
B
S32A/S36A was used. I
B
is a member of the I
B family, having the ability to retain the NF-
B complexes in the cytoplasm in their inactive state. The mutation in the residues Ser32 and Ser36 avoids the inducible
proteolytic degradation of the protein in response to external stimuli
(52, 69-73). As previously reported, activation of NF-
B by RhoA,
Rac1, and Cdc42Hs GTPases is efficiently blocked by the overexpression
of I
B
S32A/S36A (1). Simian COS-7 cells were co-transfected with
the 4 × SRE-CAT reporter and the expression vector corresponding
to the constitutively activated RhoA along with increasing doses of the
cDNA of the double mutant I
B
S32A/S36A. As shown in Fig.
2A, inhibition of the NF-
B
activity efficiently interfered in the transactivation of the
SRE-dependent promoter induced by RhoA, in a
dose-dependent manner. A similar assay coexpressing the
activated version of each GTPase (RhoA QL, Rac1 QL, or Cdc42Hs QL)
along with the I
B
S32A/S36A protein was then performed. While the
transactivation of the SRE-dependent promoter induced by
RhoA could be inhibited by I
B
S32A/S36A, it had no
effect on the activation of the SRE due to Rac1 or Cdc42Hs overexpression (Fig. 2B). However, activation of the 4 × SRE-CAT reporter by serum was not affected by the overexpression of
the I
B
S32A/S36A mutant, an indication of a rather specific
effect. Thus, the requirement of the NF-
B activity for the SRE
transcriptional activation can be by-passed by alternative signaling
pathways also promoted by Rac1 and Cdc42Hs.
View larger version (34K):
[in a new window]
Fig. 2.
RhoA-induced transcriptional activation
through the SRE site depends on NF- B
activity. A, dose-dependent inhibition of
the RhoA-induced transcriptional activation of the 4 × SRE-CAT
reporter by I
B
S32A/S36A. COS-7 cells were co-transfected with 3 µg of 4 × SRE-CAT and 1 µg of pCMV-
-gal per 60-mm plate
along with 3 µg of pCDNAIIIB or the derived vector expressing
RhoA QL and 0, 0.1, 0.4, or 2 µg of pRcCMV-I
B
S32A/S36A. CAT
activity was determined 24 h after transfection. Data represent
the mean of a single experiment performed in triplicate ± S.D.
Results are expressed as percentage of maximal induction related to the
stimulation obtained with each plasmid alone. Similar results were
observed in three independent experiments. B, the mutant
I
B
S32A/S36A inhibits the transcriptional activation of the 4 × SRE-CAT reporter induced by RhoA, but not Rac1 or Cdc42Hs. COS-7
cells were co-transfected with 3 µg of 4 × SRE-CAT and 1 µg
of pCMV-
-gal per 60-mm plate along with 3 µg of pCDNAIIIB or
the derived vectors expressing RhoA QL, Rac1 QL, or Cdc42Hs QL and 2 µg of pRcCMV or pRcCMV-I
B
S32A/S36A. Cells transfected with
pCDNAIIIB were stimulated with 20% fetal bovine serum
(vector + 20% FBS) 5 h before harvesting. CAT activity
was determined 24 h after. Data represent the mean of a single
experiment performed in triplicate ± S.D. Results are expressed
as percentage of maximal induction related to the stimulation obtained
with each plasmid alone. Similar results were observed in three independent experiments.
C, the mutant I
B
S32A/S36A inhibits the transcriptional
activation of the 4 × SRE-CAT reporter induced by RhoA-related
exchange factors. COS-7 cells were co-transfected with 3 µg of 4 × SRE-CAT and 1 µg of pCMV-
-gal per 60-mm plate along with 3 µg
of pCDNAIIIB, pCDNAIIIB-rhoA QL, pMEX, pMEX-vav, pCEV27, or
pCEV27-ost and 2 µg of RcCMV or pRcCMV-I
B
S32A/S36A. CAT
activity was determined 24 h after transfection. Data represent
the mean of a single experiment performed in triplicate ± S.D.
Results are expressed as percentage of maximal induction related to the
stimulation obtained with each plasmid alone. Similar results were
observed in three independent experiments.
B
S32A/S36A. Ost and Vav proteins are two members
of the Dbl family of specific exchange factors for the Rho family of
small GTPases. In addition to the in vitro guanine nucleotide exchange activity exhibited by these proteins, several in vivo approaches have allowed to adscribe to these
molecules certain specificity for the different Rho GTPases (reviewed
in Ref. 74). In our assays, the Vav oncogene is able to activate the
transcription factor NF-
B in a signaling pathway which involves Rac1
GTPase, but not RhoA or Cdc42Hs. On the contrary, activation of the
HIV-LUC reporter due to Ost overexpression is efficiently inhibited by
RhoA-Asn19 and Cdc42Hs-Asn17 dominant negative
mutants, showing that RhoA and Cdc42, but not Rac1, mediate in the
activation of NF-
B induced by the Ost exchange factor (26).
B
S32A/S36A plasmid and the 4 × SRE-CAT reporter. In
agreement with the results described above, activation of the SRE due
to Vav overexpression was not significatively inhibited by the
expression of the I
B
S32A/S36A protein, showing that Rac1 activates transcription through the SRE-binding site by additional pathways (Fig. 2C). However, this inhibitor was able to
block the SRE-dependent transcription induced by the Ost
oncogene, suggesting the activation of complementary signals that make
Ost dependent on RhoA but not Cdc42 for the activation of SRF.
From these experiments, we can conclude that activation of the
SRE-binding site by RhoA critically involves a
NF-
B-dependent cascade.
B
S32A/S36A Mutant Blocks
the Activation of SRE-dependent Transcription by
Lysophosphatidic Acid--
It has been described that activation of the SRE
elicited by several extracellular agonists may be signaled by different
Rho GTPases. Indeed, expression of the C3 component of the C. botulinum toxin is able to inhibit the transcriptional activity of
the SRF induced by serum, LPA, and AlF4- (an activator of
heterotrimeric G proteins), indicating that these stimuli activate SRF
by RhoA-dependent pathways (18). It is also reported that
this GTPase is involved in the endothelin-1-induced nuclear signaling
to the c-fos SRE (75). On the other hand, the dominant
negative mutant Rac1-Asn17 blocks the activation through
this binding site in response to EGF and hydrogen peroxide (20,
21).
B activity (Fig. 2A), we
investigated whether the expression of the I
B
S32A/S36A mutant had
any effect in the activation of the SRE by LPA, a physiological
activator of the RhoA signaling cascade. To that end, COS-7 cells were
co-transfected with the pRcCMV or pRcCMV-I
B
S32A/S36A plasmid
along with the (
453/+80)HIV-LUC or the 4 × SRE-CAT reporter.
After removal of the precipitate, cells were treated with LPA for
5 h and total extracts were assayed for luciferase activity
(NF-
B assay) or CAT activity (SRE assay). We observed that this
physiological stimulus was able to efficiently induce the
transactivation of both promoters in these cells (Fig.
3). Overexpression of the I
B
S32A/S36A mutant inhibited the transactivation of both NF-
B- and SRE-dependent reporters by LPA. These results indicate
that activation of NF-
B is a relevant event in the physiological
regulation of the SRE transcriptional activity induced by a
RhoA-dependent extracellular stimulus.
View larger version (20K):
[in a new window]
Fig. 3.
LPA-induced transcriptional activation
through the B and SRE-binding sites is
inhibited by the
I
B
S32A/S36A
mutant. COS-7 cells were co-transfected with 1 µg of
pCMV-
-gal and 2 µg of pRcCMV or pRcCMV-I
B
S32A/S36A along
with either 0.5 µg of (
453/+80)HIV-LUC or 3 µg of 4 × SRE-CAT, per 60-mm plate. Cells were stimulated with (10 or 30 µM) LPA 5 h before harvesting. Luciferase and CAT
activities were determined 24 h after transfection. Data represent
the mean of a single experiment performed in triplicate ± S.D.
Results are expressed as fold induction considering as 1 the activity
of the cells transfected with the empty vector. Same results were
observed in three independent experiments.
B
affected the transactivation through the SRE was then investigated. We
had previously described that both RelA/p50 and p50/p50 dimers
translocate to the nucleus due to RhoA overexpression (1). Thus, we
explored whether these subunits could cooperate with RhoA in the
activation of the SRE-dependent reporter. To that end, we
used the corresponding expression vectors of the RelA and p50
transcription factors. Overexpression of both proteins was achieved by
transient transfection of the corresponding plasmids into COS-7 cells
(Fig. 4A). Both subunits were
able to potentiate the transactivation of the HIV-LUC reporter induced by RhoA (Fig. 4B).
View larger version (22K):
[in a new window]
Fig. 4.
Cooperation between
NF- B and RhoA in the SRE-dependent
transcriptional activation. A, overexpression of p50
and RelA NF-
B subunits. COS-7 cells were transiently transfected
with 1 µg of pMEX, pMEX-p50, or pMEX-RelA per 60-mm plate. Total
lysates were subjected to Western blot analysis after SDS-PAGE and
immunoblotted with the corresponding antiserum (anti-p50 or anti-RelA;
Santa Cruz Laboratories). B, p50 and RelA cooperate with
RhoA in the transactivation of the HIV-LUC reporter. COS-7 cells were
co-transfected with 0.5 µg of (
453/+80)HIV-LUC and 1 µg of
pCMV-
-gal, 1 µg of pCDNAIIIB or pCDNAIIIB-rhoA QL, along
with 0.1 or 0.5 µg of pMEX, pMEX-RelA, or pMEX-p50 per 60-mm plate.
Luciferase activity was determined 24 h after transfection. Data
represent the mean of a single experiment performed in triplicate ± S.D. Results are expressed as fold induction over the luciferase
activity obtained with the corresponding empty vector. Similar results
were observed in two independent experiments. C and
D, RelA and p50 cooperate with RhoA in the transactivation
of the 4 × SRE-CAT reporter. COS-7 cells were co-transfected
with 3 µg of 4 × SRE-CAT, 1 µg of pCMV-
-gal, 1 µg
of pCDNAIIIB or pCDNAIIIB-rhoA QL, along with 0.1 or 0.5 µg
of pMEX, pMEX-RelA (C), or pMEX-p50 (D) per 60-mm
plate. CAT activity was determined 24 h after transfection. Data
represent the mean of a single experiment performed in triplicate ± S.D. Results are expressed as fold induction over the CAT activity
obtained with the corresponding empty vector. Same results were
observed in three independent experiments.
promoter. By contrast, both RelA and p50 potentiated
the activation of the 4 × SRE-CAT reporter by RhoA (Fig.
4D). As control, a similar experiment was performed using
the 3.DA.CAT plasmid, which contains the CAT gene under the regulation
of a minimal promoter with the c-fos-derived SRF-binding
site but defective for TCF binding (18). The same effect was observed
regarding the cooperation between RhoA and both RelA and p50 proteins,
verifying that the NF-
B-dependent activation of the SRE
is mediated by the SRF-linked pathway (data not shown). Finally, this
effect was specific for the SRE site, since no competition was observed
when a similar construct carrying the NF-
B site was used (Ref. 1,
and data not shown). All these results corroborate that RhoA and
NF-
B complexes are placed in the same signal transduction cascade to
activate the SRE-binding site.
B-dependent Activation of the SRE-binding Site by
RhoA Involves the C/EBP Family of Transcription Factors--
Binding
of C/EBP
dimers to the c-fos SRE within a region
overlapping and immediately 3' to its CArG box has been shown to be
required for maximal contribution to c-fos activation (43, 76, 77). C/EBP
is included in the C/EBP family of transcription factors, characterized by the presence of a basic region involved in
DNA binding and a leucine zipper motif which allows protein dimerization (bZIP motif) (78, 79). C/EBP
, C/EBP
, C/EBP
, C/EBP
, C/EBP
, and CHOP-10 have been identified as members of this
family, binding to specific DNA sequences as distinct homo- or
heterodimers. Different members of the C/EBP and NF-
B families of
transcription factors are able to synergize efficiently in the
transcriptional regulation of certain promoters and, particularly, physical interactions between C/EBP
and RelA or p50 have been described (57, 68, 80-84). All these data and the results reported by
Stein et al. (59) that suggested an increase of gene
expression mediated by c-fos SRE in the presence of C/EBP
and NF-
B transcription factors, prompted us to further investigate
whether such cooperation was involved in the mechanism induced by RhoA
regarding SRE activation.
proteins and
the 4 × SRE-CAT reporter, which contains in its core the
C/EBP-binding site (Fig. 5A).
A cooperative effect in the promoter transactivation was observed when
the RhoA GTPase was coexpressed with C/EBP
alone. Moreover, the
combined expression of RhoA, RelA, and C/EBP
proteins potentiated
further the transcriptional activation through the SRE-binding site. By
contrast, the same promoter carrying a mutated SRE site but an intact
C/EBP
site was not functional after expression of the RelA or
C/EBP
proteins (Fig. 5B), an indication that both RelA
and C/EBP
were unable to stimulate transcription by themselves and
require a functional SRE site. Similar results were obtained when RhoA
and C/EBP
were coexpressed along with the p50 subunit (Fig.
5C and data not shown). Thus, activation of RhoA triggers
the translocation of NF-
B dimers to the nucleus, promoting their
binding to the
B-specific sequences and also enabling the
cooperation with the C/EBP
protein in the transcriptional regulation
of the SRE site in an SRF-dependent manner. Therefore,
cross-talking between NF-
B and C/EBP
transcription factors is an
intracellular event regulated by a RhoA-dependent signal
transduction pathway. These results also suggest that NF-
B and
C/EBP
can act as accessory factors for SRE-dependent
transcription, which could liberate the SRF from its own inhibited
transcriptional capability. On the contrary, a cooperation among H-Ras,
NF-
B, and C/EBP
was not observed (data not shown). Thus, the
latter transcription factors may play a role in conjunction with the SRF in the TCF-independent pathway for the transcriptional activation through the SRE.
View larger version (19K):
[in a new window]
Fig. 5.
C/EBP and
NF-
B cooperate for RhoA-induced
transcriptional activation through the SRE motif in an
SRF-dependent manner. COS-7 cells were co-transfected
with 3 µg of 4 × SRE-CAT (A and C) or a
mutated SRE site as described under "Experimental Procedures,"
SREMUT-CAT (B) along with 1 µg of pCMV-
-gal, 1 µg of
pCDNAIIIB or pCDNAIIIB-rhoA QL along with 0.5 µg of pMSV or
pMSV-C/EBP
, and 0.1 µg of pMEX or pMEX-RelA (A) or
pMEX-p50 (B), per 60-mm plate. CAT activity was determined
24 h after transfection. Data represent the mean of a single
experiment performed in triplicate ± S.D. Results are expressed
as fold induction over the CAT activity obtained with the corresponding
empty vector. Similar results were observed in two (B) or
three independent experiments (A and C).
gene encodes for three
in-frame methionines which can potentially give rise to three
translation products of 38, 35, and 20 kDa (rat or murine genes). p38
and p35 (LAP) are active proteins, while p20C/EBP
(also known as
LIP) behaves as a competitive inhibitor of transcription because it
lacks the N-terminal transactivation domain (48). Sealy et
al. (43) have reported that both p35 and p20 homodimers as well as
p35/p20 heterodimers contribute to the SRE complex at the
c-fos promoter in NIH 3T3 cells, while overexpression of
p20C/EBP
(LIP) is able to inhibit its activation by serum
stimulation. In order to corroborate that C/EBP
was implicated in
the regulation of SRF by RhoA, we used LIP as a specific repressor of
the activity of this transcription factor.
. These results suggest that RhoA regulates the SRE through a signaling cascade which
also involves the transcription factor C/EBP
.
View larger version (32K):
[in a new window]
Fig. 6.
C/EBP is involved in
the transcriptional activation through the SRE motif regulated by
RhoA. A, LIP repressor inhibits the transactivation of
the 4 × SRE-CAT reporter induced by RhoA. COS-7 cells were
co-transfected with 3 µg of 4 × SRE-CAT and 1 µg of
pCMV-
-gal, 1 µg of pCDNAIIIB or pCDNAIIIB-rhoA QL along
with 0.3 µg of pMSV-LIP and 0.5 µg of pMSV-C/EBP
per 60-mm
plate. Cells were stimulated with 30 µM LPA (vector + LPA) 5 h before harvesting. CAT activity was determined 24 h
after transfection. Data represent the mean of a single experiment
performed in triplicate ± S.D. Results are expressed as
percentage of maximal induction related to the stimulation obtained
with each plasmid alone. Same results were observed in three
independent experiments. B, RhoA induces the expression of
C/EBP
. NIH 3T3 cells were transfected with 5 µg of pCDNAIIIB
or pCDNAIIIB-rhoA QL per 60-mm plate. Cells tranfected with the
control vector were stimulated for 1, 3, and 5 h with 10 µM forskolin (FK) (Sigma). Total lysates were
subjected to Western blot analysis after SDS-PAGE and immunoblotted
with a C/EBP
-specific antiserum (Santa Cruz Laboratories).
Protein--
The above
results indicate that RhoA is able to modulate the
SRE-dependent transcription by a mechanism that depends
upon the function of C/EBP
. The activity of C/EBP
is controlled
at the transcriptional level, by direct activation of its expression (85-87) or by post-translational modifications (76, 88-90). When RhoA
(QL) was transiently overexpressed in NIH 3T3 fibroblasts, an increase
of the level of the C/EBP
protein was observed (Fig. 6B).
Under similar conditions, treatment with forskolin, an activator of
PKA, also induced a time-dependent increase in the levels
of the C/EBP
protein, as described previously (87). Although we cannot exclude the possibility that RhoA could also affect C/EBP
function by other mechanisms such as post-translational modifications, these results strongly support the evidence, provided in this study, of
the regulation of the SRE through the participation of NF-
B,
C/EBP
, and SRF proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in diverse
cell types (1) by at least two alternative pathways (26). Hill et
al. (18) have recently demonstrated that in NIH 3T3 cells, the
serum response factor (p67SRF) is regulated by
Rho-dependent signaling pathways. This activation is
achieved by mechanisms independent of the signal transduction cascades
related to TCFs. Although it is not clear how Rho proteins modulate the
transcriptional activation of the SRE, it has been suggested that this
mechanism is dependent on a second accessory protein targeted by the
Rho family of GTPases. Such a molecule could associate with the
DNA-bound SRF and relieve the transactivation domain of the SRF from
its own inhibitory regulation (3, 18, 23-25). In this study, we have
explored this signaling pathway and found that the transcriptional
activation through the SRE induced by RhoA, but not Rac1 or Cdc42Hs, is
dependent on the activity of the transcription factor NF-
B.
B and a
SRE-dependent promoter in the simian COS-7 system. NIH
3T3-derived clones stably transfected by a SRE-
-gal plasmid were
used to demonstrate that RhoA, Rac1, and Cdc42Hs induce the
transcriptional activation through a chromosomal SRE motif. Alberts
et al. (56) have recently reported that RhoA, Rac1, and
Cdc42 regulate transcriptional activation by SRF (18). At least RhoA
and Cdc42 requires also H4 hyperacetylation as an additional signal for
the activation of SRF-regulated chromosomal templates. Furthermore,
while RhoA does not provide by itself the cooperating signals required
for the induction of H4 hyperacetylation, Cdc42 does. The partial discrepancies in the results of Alberts et al. (56) and ours may be due to differences in the sensitivity of the methodologies used,
microinjection versus transient transfections. However, both
reports are consistent with the fact that Cdc42 is more efficient than
RhoA for the activation of SRF-regulated chromosomal templates, although the basis for this difference is currently unknown.
B could modulate the activity of
p67SRF (42, 67). We investigated whether the activation of
the SRF by Rho GTPases could involve a NF-
B-dependent
cascade. Our results clearly demonstrate that the double mutant
I
B
S32A/S36A was able to block the activation of the 4 × SRE-CAT reporter induced by RhoA. At the same time, activation of the
4 × SRE-CAT induced by the stimulation of COS-7 with LPA was also
blocked by the ectopic expression of the I
B
S32A/S36A mutant. All
these results demonstrate that NF-
B is critically involved in the
signaling cascade triggered by RhoA to regulate
SRE-dependent transcription. The cooperative effect
observed when RhoA is co-expressed with the NF-
B subunits RelA or
p50 also agrees with this hypothesis. Similar results were obtained
with the 3D.A.CAT reporter which is lead by an SRE site defective for
TCF binding, suggesting that NF-
B is involved in the regulation of
the SRE by SRF. Indeed, Franzoso et al. (42) have found that
RelA is able to physically interact in vitro with the
DNA-binding region of SRF (coreSRF), through its Rel
homology domain. Thus, NF-
B subunits could act as accessory proteins
for the SRF, in order to regulate the activity of the SRE, liberating
the negative effect exerted by the DNA-binding and dimerization
domains, as other authors have previously suggested (18, 25, 42).
B dimers or as
monomers, although NF-
B is supposed to bind to other transcription
factors in its dimeric configuration (59). It has been described that
p50 and SRF are not able to interact physically and that they do not
cooperate functionally in SRE-driven transcription (42). In contrast, we have observed similar results by coexpression of RhoA along with the
RelA or p50 subunits. RhoA promotes the translocation of both RelA/p50
and p50/p50 dimers to the nucleus (1). Thus, it can be proposed that
RelA/p50 may be the NF-
B dimer involved in the regulation of SRF and
that the physical interaction with such a factor could be mediated by
the RelA subunit.
B acts as an accessory protein for SRF by
affecting its functions.
B
S32A/S36A mutant was not able to block the
activation of the 4 × SRE-CAT by Rac1 or Cdc42Hs or Vav, an
exchange factor specific for Rac1 in vivo (26), suggesting that the requirement for the NF-
B activity can be by-passed by alternative mechanisms. This effect can be explained by the fact that
both Rac1 and Cdc42Hs may regulate the activity of the SRE through the
TCF-dependent pathway, since they activate JNK/SAPK and p38
cascades (1, 45, 97), and such kinases have been found to phosphorylate
TCFs (15-17). The effects reported here were specific for the SRE
site, since no competition was observed using an NF-
B site and was
abolished by a mutated SRE construct.
, which belongs to the family of CCAAT box
enhancer-binding proteins, characterized by the presence of the bZIP
motif (78-79). These results are in agreement with others previously
described that suggest an increase of gene expression mediated by
c-fos SRE in the presence of C/EBP and NF-
B transcription factors (59). These authors had observed a strong synergistic stimulation of a SRE-dependent reporter due to the joint
overexpression of RelA(p65) NF-
B and C/EBP
. Different C/EBP and
NF-
B subunits are able to synergize for the transcriptional
regulation of certain promoters and, particularly, physical
interactions between C/EBP
and RelA or p50, through their bZIP and
Rel homology domains, respectively, have been described (57, 59, 68,
80-84, 105). It has been suggested that, at least, an additional
factor could contribute to the C/EBP dimer bound neighboring to the
SRE. Sealy et al. (43) have reported that approximately 50%
of the C/EBP complex which is bound to 32P-labeled SRE DNA
remains unaffected when competed by C/EBP
antibodies, although they
were not able to visualize any supershift when a RelA(p65)-specific
antibody was used. In keeping with this, two proteins of 64 and 43 kDa
have been described to associate with rNFIL-6 (C/EBP
) by
immunoprecipitation with a specific antibody, in cells stimulated with
forskolin (76).
(LIP) protein acts as a transdominant negative
regulator of the C/EBP activity (48). It is known that the regulation
of gene transcription by C/EBP
involves a precise balance of both
activator and inhibitor forms (43, 48, 106). When p20/C/EBP
(LIP)
was transfected along with RhoA, the activation of the SRE was
efficiently inhibited. Moreover, the full activity was recovered by
overexpression of the full-length C/EBP
, a demonstration of the
specificity of the inhibitory effect by LIP. All these results support
the idea of cooperation between NF-
B and C/EBP
subunits in order
to potentiate the transcriptional activation through the SRE, as shown
schematically in Fig. 7. This cooperation is dependent on SRF binding to the SRE site since it was lost when a
mutated SRE site was used and was not observed by expressing NF-
B or
c/EBP
alone or in combination, in the absence of RhoA.
View larger version (13K):
[in a new window]
Fig. 7.
Model for the activation of
SRF-dependent transfection by RhoA.
Activation of the SRF-dependent transcription by RhoA
is mediated by NF- B subunits p50 and RelA/p65 as well as C/EBP
.
Activation of NF-
B by RhoA follows the conventional nuclear
translocation of RelA/p50 and p50/p50 dimers by phosphorylation and
proteolytic degradation of the inhibitory subunit I
B
(1, 26).
Activation of C/EBP
is achieved at least by transcriptional
up-regulation of the factor by an as yet unknown effector.
We have also observed an increase in the level of the C/EBP protein
induced by RhoA in NIH 3T3. Such an increase could be originated by the
transcriptional activation of the C/EBP
gene or the stabilization of
its mRNA. From our experiments, we cannot conclude which of these
mechanisms is regulated by RhoA. Regarding the transcription of the
C/EBP
gene, it is mostly controlled by two CREB sites situated close
to the TATA box (87). The CREB protein is functionally regulated by
phosphorylation, which can be mediated by PKA- or p38-linked signaling
cascades. RhoA is not capable of activating the p38 pathway in NIH 3T3
and it is also reported that LPA, a physiological activator of RhoA
signaling, induces a decrease in cAMP levels (97, 107). Thus, if RhoA increases the level of C/EBP
by activating transcription of the gene, this could be triggered by the regulation of CREB by a not yet
defined pathway or through other specific DNA sequences located in the
promoter. Such hypothesis deserves further investigation. Finally, the
c-Jun NH2-terminal kinase (JNK) pathway is involved in the
stabilization of interleukin-2 mRNA (108). Thus, it would also be
interesting to study whether RhoA could be implicated in a similar
process regarding the stabilization of the C/EBP
mRNA.
Mitogen stimulation induces cell division, triggering the transcription
of a wide range of immediate early genes, which relies most on
post-translational modification of pre-existing transcription factors.
As an example of such proteins, the NF-B dimers remain in the
cytoplasm in an inactive state and may elicit a rapid and efficient
transcriptional response after stimulation by different agonists.
Activation of NF-
B is an immediate early event after serum
stimulation of quiescent fibroblasts, suggesting that its activity is
important for G0-G1 transition (30). Therefore, NF-
B dimers may also regulate the transcriptional activity of other
factors activated in response to serum, promoting a synergistic effect
as we have observed with the ternary complex which bind to the
c-fos SRE.
Cross-talking between subunits of different families of transcription
factors was shown to be a critical event in the regulation of gene
transcription, and actually NF-B subunits can interact with
different members of other families of transcription factors. Regarding
the complexes involving the SRF, we cannot exclude that other proteins
could interact with NF-
B and/or SRF, leading to the formation of
higher transcription complexes, since other transcription coactivators
have already been shown to interact with NF-
B (109-112). Thus, the
picture of the mechanism by which Rho proteins regulate SRE-dependent transcription may still not be complete. In
fact, what has been demonstrated in this study for the interaction of SRF, RelA, p50, and C/EBP
cannot be fully applied to Rac1 and Cdc42Hs, two important prototype members of the same family of Rho
GTPases. Thus, each member of the Rho family of GTPases seems to serve
specific signaling wirings that although controlling similar targets
may exert different biological responses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Karin for the HIV-LUC plasmids;
S. Gutkind for the pCDNAIII vectors carrying the rho
genes; A. Israël for the IB
S32A/S36A mutant; R. Treisman
for the 3D.A.CAT reporter; R. Bravo for pMEX-RelA(p65) and pMEX-p50
plasmids; A. Pérez-Castillo for the pMSV-C/EBP
expression
vector. We also thank X. Bustelo and T. Miki for pMEX-vav
and PCEV27-ost plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported by Spanish Department of Health Grants FIS-96/2136 and FIS-98/0514 and Comunidad de Madrid Grants 07-114-96 and 08.1/0024/97.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this work.
§ Fellow from Comunidad de Madrid (CAM).
¶ Fellow from Fundación Ramón Areces.
To whom correspondence should be addressed: Instituto de
Investigaciones Biomédicas, Arturo Duperier, 4 28029 Madrid,
Spain. Tel.: 34-91-585-4607; Fax: 34-91-585-4606; E-mail:
jclacal{at}iib.uam.es.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SRE, serum response
element;
SRF, serum response factor;
TCF, ternary complex factor;
LPA, lysophosphatidic acid;
NF-B, nuclear factor-
B;
DMEM, Dulbecco's modified Eagle's medium;
HIV, human immunodeficiency
virus;
CAT, chloramphenicol acetyltransferase;
-gal,
-galactosidase;
PAGE, polyacrylamide gel electrophoresis;
CMV, cytomegalovirus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|