The Activity of Guanine Exchange Factor NET1 Is
Essential for Transforming Growth Factor-
-mediated Stress Fiber
Formation*
Xing
Shen,
Jianming
Li,
Patrick Pei-chih
Hu,
David
Waddell,
Ji
Zhang, and
Xiao-Fan
Wang
From the Department of Pharmacology and Cancer Biology, Duke
University Medical Center, Durham, North Carolina 27710
Received for publication, October 18, 2000, and in revised form, January 18, 2001
 |
ABSTRACT |
To examine signaling pathways underlying
transforming growth factor-
(TGF-
)-mediated changes in cell
morphology, we used a microarray system to identify downstream target
genes that may play a role in this process. Through this approach, we
found that the NET1 gene was induced upon TGF-
treatment in several cell types. NET1 is a guanine nucleotide exchange
factor for RhoA whose activity has been implicated in stress fiber
formation. In the Swiss 3T3 cell line, TGF-
induces NET1
expression, and this correlated with an increase in stress fiber
formation. Overexpression of the wild type NET1 gene
increases stress fiber formation, and overexpression of a dominant
negative NET1 mutant (L392E) prevented TGF-
dependent
increase in stress fiber formation. Furthermore, treatment of the cells
with a RhoA kinase inhibitor Y-27632 blocks TGF-
-induced stress
fiber formation. By using a stable cell line expressing dominant
negative Smad3, we found that the Smad signaling pathway is essential
for the induction of NET1, which in turn leads to the
increase of Rho activity. Taken together, those data suggest that
induction of NET1 is important for the increase of Rho
activity upon TGF-
treatment, which may represent the critical trigger for a variety of downstream events in different cells. Our
results support the presence of a novel signaling pathway by
which TGF-
may regulate the formation of stress fibers and reorganization of cytoskeletal structures.
 |
INTRODUCTION |
TGF-
1 is a growth
factor with a diverse range of biological activities such as growth
inhibition, cellular migration, wound healing, immune regulation, and
bone remodeling (1-4). One important but relatively unexplored pathway
is the ability of TGF-
to modulate cell morphology and motility.
Cell morphologic change and elevated migration in response to TGF-
have been observed in a number of cell types (5-9), but the mechanism
underlying this TGF-
induced process remains largely unknown.
Cell morphologic change and migration are processes that involve
dynamic cytoskeleton reorganization. In addition to providing a
structural framework around which cell shape and polarity are defined,
actin cytoskeleton provides a driving force for the cells to move and
to divide (10). Recent works demonstrate that the Rho family of small
GTPases are key regulatory molecules linking surface receptors to the
organization of actin cytoskeleton (10-12). The Rho family consists of
Rho, Rac, and Cdc42 subfamilies. Among them, Rho is the molecular
switch for stress fiber formation, and Rac and Cdc42 regulate
lamellipodia and filopodia formation, respectively (10, 13-16).
Rho GTPase can bind and hydrolyze guanosine nucleotides. The exchange
of GDP for GTP results in a conformational change that unmasks
structural domains involved in the binding of Rho to its downstream
target proteins. This mechanism allows Rho GTPase to exchange between
active GTP-bound and inactive GDP-bound forms, a cycling process that
is regulated by three main classes of proteins as follows: 1) guanine
nucleotide exchange factors (GEFs), which stimulate the exchange of
bound GDP for GTP, leading to activation of GTPase; 2)
GTPase-activating proteins (GAPs), which promote the inactivation of
GTPase by increasing the rate of GTP hydrolysis; and 3) GDP
dissociation inhibitors (GDIs), which can inhibit the dissociation of
GDP from the GTPase, thus keeping GTPase in an inactive state (10, 11,
17). Although TGF-
can induce cell morphologic change and stress
fiber formation, it is still unclear how the TGF-
signal regulates
these pathways.
To understand better the mechanism by which TGF-
modulates the
change in cell morphology, in this study we used a microarray system to
search for candidate genes associated with this
TGF-
-dependent activity. We found one gene in
particular, NET1, whose mRNA and protein levels increase
upon treatment with TGF-
. NET1 is a specific guanine nucleotide
exchange factor for RhoA (18). By increasing RNA and protein levels of
NET1, more RhoA may be in its GTP-bound active state to induce stress
fiber formation. Furthermore, the expression of NET1 appears
to be controlled by Smads, as the presence of a dominant negative
form of Smad3 blocks the TGF-
-mediated induction of NET1.
Our results suggest that NET1 may be a critical link between the
activation of RhoA and the TGF-
and Smad-dependent induction of stress fiber formation.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Human TGF-
1 was a generous gift from R & D
Systems. ROCK inhibitor Y-27632 was kindly provided by Yoshitomi
Pharmaceutical Ind., Ltd. Rhodamine-labeled phalloidin was purchased
from Molecular Probes (Eugene, OR). Ribonuclease Protection Assay kit
RPAII and Ribo-probe DNA for mouse cyclophilin, JunB, were from Ambion
Inc. (Austin, TX). Human cDNA Expression Array I was from
CLONTECH Laboratories Inc. (Palo Alto, CA).
Anti-NET1 antibody (N-17) and anti-Smad2/3 antibody (H-2) were from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Smad2 antibody
was from Upstate Biotechnology, Inc. (Lake Placid, NY).
Cell Culture--
Human HaCaT cells were a generous gift from
Drs. P. Baukamp and N. Fusenig (Institute of Biochemistry and Molecular
Biology, Heidelberg, Germany) and were maintained in minimum
Eagle's medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Life Technologies, Inc.). The
HaCaT stable cell line overexpressing a Smad3 mutant (D407E), a
generous gift from Dr. M. Kato (Japanese Foundation for Cancer
Research, Tokyo, Japan) (19), was cultured in MCDB153 medium (Sigma)
supplemented with 0.1 mM CaCl2, 5% dialyzed fetal bovine serum, 5 ng of epidermal growth factor, and 300 µg/ml G418. Swiss 3T3, A-549, and PANC-1 cells were from American Type Culture Collection (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. BJ
human diploid fibroblast was a gift from Dr. C. Counter (Duke University, Durham, NC).
Plasmids--
Human NET1 cDNA construct was a
generous gift from Dr. T. Miki at the NCI, National Institutes of
Health. NET1 cDNA was cloned into the Bluescript vector
to generate pNet1-1. pNet1-1 was linearized with StuI and
used to make riboprobe with T7 RNA polymerase. FLAG-tagged NET1
constructs were generated by polymerase chain reaction and cloned into
the pFLAG-CMV2 vector from Eastman Kodak Co. The NET1 mutation (L392E)
was created by a standard polymerase chain reaction-mediated mutagenesis method.
Ribonuclease Protection Assays (RPA)--
RNAs from
TGF-
-treated or untreated cells were extracted with the RNeasy mini
kit from Qiagen Inc. (Valencia, CA). Riboprobes were labeled with
[
-32P]UTP using T7 RNA polymerase and eluted at
37 °C overnight. 8 µg of total RNA was used for one reaction, and
a standard RPA protocol from the manufacturer was followed. Briefly,
total RNA was co-precipitated with radiolabeled probes and hybridized
overnight at 56 °C. The next day, reactions were treated with RNase
A/T1 at room temperature for 1 h to digest unhybridized RNA. Final products were separated on a 6% polyacrylamide gel containing 7 M urea, and the gel was exposed to film at
80 °C.
Immunostaining and Western Blot Analysis--
Swiss 3T3 cells
were plated on cover slides and grown to sub-confluency. The cells were
washed and grown in serum-free medium for 48 h before treatment
with TGF-
for 4 h. Then the cells were fixed in 4%
paraformaldehyde for 10 min at room temperature and incubated with
1:100 fluorescence-labeled phalloidin diluted in 2% bovine serum
albumin in phosphate-buffered saline solution in the dark for 30 min.
Cells were washed 4 times with phosphate-buffered saline solution
before mounting on glass slides. Photographs were taken with a Zeiss
confocal microscope. The immunostaining on Smad2/Smad3 was performed as
described previously.
The transfection of COS cells was performed by using a standard
DEAE-dextran protocol, and routine Western blots were performed as
described before. Swiss 3T3 cells were transfected with LipofectAMINE PLUS reagent by Life Technologies Inc.
 |
RESULTS |
TGF-
-mediated Cell Morphologic Change Requires Protein Synthesis
in HaCaT Cells--
TGF-
modulates cell morphologic changes in many
cell types (5-9). In the spontaneously immortalized human keratinocyte
cell line, HaCaT, TGF-
treatment leads to a dramatic morphologic
change after 24 h (Fig. 1,
A and B). Contrary to the normal compact
appearance for keratinocytes, TGF-
treatment leads to a bigger and
less compact cell shape in HaCaT cells. In addition, TGF-
-treated cells have significantly more membrane ruffling compared with untreated
cells (data not shown). When cells were treated with cycloheximide, a
protein synthesis inhibitor, the dramatic morphologic change of HaCaT
cells induced by TGF-
was no longer observed (Fig. 1C).
Since cycloheximide blocks protein synthesis, the result suggests that
TGF-
may cause morphologic change of these cells through inducing
the expression of certain TGF-
downstream target genes.

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Fig. 1.
Protein synthesis is required for
TGF- -mediated changes in cell morphology.
HaCaT cells were either untreated or TGF- -treated for 24 h,
with or without the protein synthesis inhibitor, cycloheximide. The
cells were subsequently fixed in 4% paraformaldehyde and stained using
a Dif-Quik staining kit (DADE, Behring). Pictures were taken
using an Olympus microscope. A, no treatment. B,
TGF- -treated for 24 h. C, TGF- plus 10 mg/ml
cycloheximide for 24 h.
|
|
TGF-
Induces NET1 Expression--
To better understand how
TGF-
modulates cell morphology and to identify downstream genes
critical for this process, we performed experiments using a microarray
system containing about 600 genes. mRNA extracted from treated and
untreated HaCaT cells were reverse-transcribed and hybridized with the
microarray system. In the samples treated with TGF-
for 1 h,
several genes were found to be significantly up-regulated (data not
shown). Among these genes, several of them (e.g.
c-jun and junB) have been previously reported
(20, 21). One novel gene that fit our search criteria was
NET1, a guanine nucleotide exchange factor (GEF) that was
previously shown to increase specifically the activity of the small
GTPase RhoA (18). In other studies, NET1 was isolated in a yeast
two-hybrid screen using RhoA as bait, and overexpressed NET1 was shown
to transform NIH 3T3 cells, cause stress fiber formation, and
lead to JNK activation (22).
We next performed RPA to confirm the up-regulation of NET1
messenger RNA levels by TGF-
. HaCaT cells, grown in both
serum-starved and normal serum conditions, were treated with TGF-
for different times, and total RNA samples were extracted. As shown in
Fig. 2, A and B,
TGF-
induced the expression of NET1 mRNA in both serum-free and normal serum conditions. The induction started at 30 min
following TGF-
treatment and peaked at 2 h. As a positive control, the cyclin-dependent kinase inhibitor (CDKI) p15
was also induced by TGF-
treatment (23) but with slower kinetics. Consistent with an increase in mRNA level, NET1 protein levels were
also increased in TGF-
-treated cells (Fig. 2C).

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Fig. 2.
TGF- induces
NET1 expression in HaCaT cells. A,
HaCaT cells were grown under normal serum conditions and treated with
TGF- from 30 min to 24 h. After the treatment, total RNAs were
extracted from these cells for the RPA. Radiolabeled NET1,
p15 (positive control), and GAPDH (internal loading control) riboprobes
were included in the assay. Protected RNAs were analyzed by 6%
denatured gel followed by fluorography. B, identical
experiments were performed as in A except that the cells
were grown in serum-free medium. Three riboprobes, NET1, p15, and
GAPDH, were used for RPAs. C, HaCaT cells were grown under
normal serum conditions and treated with TGF- for different time
points. Cell lysates were made, and NET1 protein level was analyzed by
Western blot using a specific antibody (Santa Cruz Biotechnology).
Arrow indicates the approximate position of NET1 in 8%
SDS-polyacrylamide gel.
|
|
To test if NET1 expression is subject to TGF-
-mediated
regulation in a broader range of cell types, we examined several other TGF-
-responsive cell lines for an increase in NET1
mRNA expression. As shown in Fig.
3A, human lung carcinoma A549
and human diploid fibroblast BJ cell lines were found to increase
NET1 expression after 2 h of TGF-
treatment. A more
moderate level of induction of NET1 expression was also
observed in a mouse fibroblast cell line, Swiss 3T3, after 2 h of
TGF-
treatment (Fig. 3B). The human pancreatic carcinoma
cell line, PANC-1, was found to express NET1 at a very low
basal level, and it is not further induced by TGF-
(Fig.
3A). Taken together, we conclude that the up-regulation of
NET1 expression by TGF-
is not restricted to the human
keratinocyte HaCaT cells, but instead is a general response shared by
other TGF-
-responsive cell lines.

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Fig. 3.
NET1 mRNA level is increased
in additional cell types. A, using HaCaT cells as a
positive control, several TGF- -responsive human cell lines, A549,
BJ, and PANC-1, were examined for NET1 induction after
TGF- treatment. RNA samples from the indicated cell lines, either
untreated or TGF- -treated for 2 h, were prepared and analyzed
by RPA. B, RNA from Swiss 3T3, a mouse fibroblast cell line,
was extracted after 2 h TGF- treatment or no treatment and analyzed
by RPA. Three riboprobes, NET1, JunB (positive control), and
cyclophilin (internal loading control) were included in the
reaction.
|
|
TGF-
Induces Stress Fiber Formation in Swiss 3T3 Cells--
As
a guanine exchange factor, NET1 is known for its specific activity
toward RhoA (18). Since RhoA activity is critical in modulating the
process of stress fiber formation and up-regulation of NET1 expression
is likely to increase RhoA activity, we decided to investigate the role
of NET1 in TGF-
-mediated stress fiber formation in a suitable
system. We examined the cell lines in which NET1 is induced by TGF-
to see if TGF-
could also enhance stress fiber formation in these
cells. In HaCaT, A549, and PANC-1 cells, stress fibers were difficult
to visualize both before and after TGF-
treatment. A significant
amount of stress fiber was seen in BJ fibroblast cells, but the basal
level of stress fiber was too high to allow an assessment of the
TGF-
effect on the process in these fibroblasts even with 2 days of
growing the cells under serum-starved conditions. Finally, we found the
Swiss 3T3 cell line to be an ideal model system for this study. As
shown in Fig. 4A, upon TGF-
treatment for 30 min, a significant nuclear accumulation of two
effector proteins of the TGF-
signaling pathway, Smad2 and Smad3,
was observed, suggesting that those cells are responsive to TGF-
treatment and the TGF-
-Smad signaling pathway is intact. In normal
serum-containing media, the presence of high levels of stress fibers in
Swiss 3T3 cells was probably due to the activation of RhoA pathway by
various growth factors in the serum. In contrast, a much lower level of
stress fibers was seen when the cells were cultured in medium without
serum for 48 h and stained with rhodamine-labeled phalloidin (Fig.
4B). After TGF-
was added into the medium and incubated
for 4 h prior to staining, a dramatic increase in stress fiber
formation was observed with phalloidin staining (Fig. 4B).
The formation of stress fibers could be observed as early as 2 h
post-TGF-
treatment.

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Fig. 4.
TGF- -mediated stress
fiber formation in Swiss 3T3 cells is RhoA-dependent.
A, Swiss 3T3 cells were grown in normal serum and untreated
or treated with TGF- for 30 min. Cells were then stained with
-Smad2/3 (Santa Cruz Biotechnology) and fluorescence-labeled
secondary antibody to determine Smad2/3 localization. B,
Swiss 3T3 cells, after growing in serum-free medium for 48 h, were
treated with TGF- for 4 h or left untreated. Stress fibers were
visualized using a fluorescence-labeled phalloidin, and pictures were
taken under a Zeiss confocal microscope.
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|
NET1 Is Involved in TGF-
-mediated Stress Fiber
Formation--
Since overexpression of NET1 was previously shown to be
sufficient to cause stress fiber formation, it is possible that NET1 serves as a link between the activation of TGF-
signaling pathway and the activation of RhoA and consequently stress fiber formation. To
test this, we first determined if overproduction of NET1 could also
lead to stress fiber formation in Swiss 3T3 cells. We constructed expression plasmids containing either a FLAG-tagged wild type NET1 or a mutant NET1 with a point mutation on
the conserved Dbl domain (L392E) which abolishes its guanine exchange
activity toward RhoA. The two constructs express equal amounts of NET1
protein in transfected COS cells, as shown by Western blot analysis on Fig. 5A. We then introduced
the NET1 constructs by transient
transfection into Swiss
3T3 cells and subsequently stained cells with rhodamine-labeled phalloidin. As shown in Fig. 5B, the NET1 wild
type construct-transfected cells, indicated by the co-transfected GFP
marker, have significantly more stress fibers when compared with the
surrounding non-transfected cells or GFP alone-transfected cells,
suggesting that increased expression of NET1 is sufficient to cause
enhancement of stress fiber formation in Swiss 3T3 cells. TGF-
treatment did not lead to further increase in stress fiber formation
(data not shown). Most important, transfection of the cells with the
mutant NET1 construct led to significantly less stress
fibers upon TGF-
treatment (Fig. 5C), suggesting that the
function of NET1 is critical in the TGF-
-mediated stress fiber
formation.


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Fig. 5.
NET1 is critical in
TGF- -mediated stress fiber formation.
A, FLAG-tagged NET1 constructs, both the wild type and the
mutant, were transfected into COS cells using a DEAE-dextran method.
Two days after transfection, total cell lysates were made and analyzed
by Western blot using a -FLAG antibody lane 1, no
transfection; lane 2, wild type-NET1; and lane 3,
mutant NET1). B, wild type NET1 construct was transfected
into Swiss 3T3 cells using the LipofectAMINE Plus kit (Life
Technologies, Inc.). 12 h after transfection, cells were switched
to serum-free medium for 48 h. Cells were stained with
rhodamine-labeled phalloidin after the treatment as in Fig.
4B. Pictures were taken in both green channel
(for GFP) and red channel (for rhodamine). Arrows
indicate transfected cells (shown by GFP staining). Pictures from the
green channel are shown on the left side, and the
same views from red channel are on the right
side. C, NET1 mutant construct was transfected into
Swiss 3T3 cells as in B. Cells were treated with TGF- for
4 h following the 48-h serum starvation. Pictures were taken in
both green channel and red channel as in
B. Left row of photos were taken in
green channel and identical views from red
channel were shown on the right. White arrows
indicate transfected cell (shown by GFP staining). D, as in
Fig. 4B, serum-starved Swiss 3T3 cells were treated with
TGF- in the presence of a Rho kinase inhibitor, Y27632. Staining was
performed as in Fig. 4B.
|
|
To confirm the involvement of RhoA in TGF-
-mediated stress fiber
formation, we used a specific inhibitor Y27632 for Rho kinase (ROCK), a
Rho downstream kinase that is required for RhoA-mediated stress fiber
formation. We found that Y-27632 potently inhibited TGF-
-dependent stress fiber formation (Fig.
5D). Taken together, our results suggest that in Swiss 3T3
cells NET1 induction is both necessary and sufficient for stress fiber
formation to occur, probably through increasing RhoA activity.
Smad Signaling Pathway Is Essential for NET1 Induction and Rho
Activation--
As transcription factors, Smad proteins have been
shown to mediate the transcriptional activation of a number of TGF-
target genes. To determine if Smads are also involved in the induction of NET1 by TGF-
, we took advantage of a HaCaT stable cell
line that overexpresses a Smad3 dominant negative mutant (D407E).
Earlier studies indicated that the phosphorylation of endogenous Smad2 and Smad3, as well as the inhibition of cell proliferation, in response
to TGF-
treatment was significantly blocked in this cell line (19).
To test if TGF-
is capable of inducing the expression of
NET1 in this cell line, we performed RPA analysis. As shown
in Fig. 6A, the induction of
NET1 in the Smad3D407E cell line was significantly lower
than that of the control HaCaT cell line after TGF-
treatment for
2 h. As a demonstration for the inhibition of Smad activity, the
phosphorylation of endogenous Smad2 in response to TGF-
was shown to
be blocked significantly in Smad3D407E by using a phospho-specific
antibody against Smad2 (Fig. 6B).

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Fig. 6.
Smad signaling pathway is essential for NET1
induction and Rho activation. A, control HaCaT and
Smad3D407E cells were treated with TGF- for 2 h, and total RNAs
were harvested for RPAs. 8 µg of total RNA from each treatment was
used for one RPA reaction. Radiolabeled human NET1 and GAPDH
riboprobes were included in the assay. Arrows indicated the
protected band of NET1 and GAPDH. B, the cell
lysates were harvested from control HaCaT and Smad3D407E cells
in the presence or absence of TGF- (30 min of treatment) and were
subjected to SDS-polyacrylamide gel electrophoresis and Western
blotting. Identical protein samples were blotted with
anti-phospho-Smad2 (top panel) and anti-Smad2/3
(bottom panel). Arrows indicate the right
molecular weight for these proteins. C, SRE-luc DNA was
transfected into control HaCaT, HaCaT-pCDNA3, and
HaCaT-Smad3D407E cells. After TGF- treatment for 24 h, cell
lysates were prepared and assayed for luciferase activity.
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|
To test if the inhibition of Smad signaling and induction of
NET1 by TGF-
lead to changes in downstream signaling, we
next studied whether the activation of Rho is altered in Smad3D407E stable line. To do this, we used a serum-responsive element luciferase reporter (SRE-luc) that has been used previously for assaying Rho
activity. As shown in Fig. 6C, TGF-
treatment for 24 h leads to a 10-fold induction of the SRE reporter gene in the control HaCaT cell line, indicating that a significant increase in Rho activity
was induced by TGF-
. Similar activation of Rho also occurred in
control stable line HaCaT-pCDNA3. However, in the Smad3D407E cell
line, it was unchanged upon TGF-
treatment, suggesting that Rho
activity may not be affected by TGF-
treatment in the absence of
functional Smads and the induction of NET1.
 |
DISCUSSION |
As a multifunctional growth factor, TGF-
regulates a wide range
of biological processes, including cell morphologic change and
migration, likely through the modulation of expression of downstream
target genes. To explore the signaling pathway that may mediate the
specific effect of TGF-
on changes in stress fiber formation, we
have identified the NET1 gene as a candidate target gene
whose function may be important for this TGF-
-induced process. We
have demonstrated that NET1, a specific GEF for RhoA, is rapidly
induced by TGF-
in several TGF-
-responsive cell lines. In Swiss
3T3 cells, TGF-
induces the formation of stress fibers in a
RhoA-dependent manner, a process correlated with the
induction of NET1 expression. To test if this increased
expression of NET1 is functionally important for
TGF-
-regulated stress fiber formation, we overexpressed a dominant
negative NET1 mutant in Swiss 3T3 cells and found
that it could block stress fiber formation induced by TGF-
. These
results, together with previous findings that implicate NET1 as an
activator of RhoA, support a model that TGF-
-dependent NET1 induction could lead to the activation of RhoA, which
in turn causes increased stress fiber formation.
Although NET1 is the first gene so far identified in TGF-
signaling pathway whose function may be directly associated with the
process of cytoskeletal reorganization, we suspect that there may be
other related genes that can mediate the TGF-
effect involved in the
regulation of cell motility and morphologic changes. For example, we
have found that NET1 expression is not induced in primary
mouse fibroblasts (data not shown), even though TGF-
has an effect
on the migration of those cells, a process that involves dynamic
cytoskeletal change. This observation suggests that TGF-
may use
alternative pathways to activate RhoA, as small GTP-binding proteins
could be regulated by GEFs, GAPs, and GDIs. It is likely that in
different cell types, different GEFs, or GAPs and GDIs serve as targets
that can be regulated by the TGF-
signaling pathway.
On the other hand, activation of stress fiber formation may
not be the only function of NET1 induction. Consistent with
this notion, we failed to observe significant increases in stress fiber formation in certain types of cells, such as HaCaT cells that showed a
dramatic NET1 induction in response to TGF-
treatment. One possible consequence of NET1 induction is the activation
of the JNK pathway, as a previous report demonstrated that
overexpression of NET1 could lead to JNK activation (18). JNK
activation by TGF-
has been reported to occur in various cell types
(24, 25). In one recent study, TGF-
was found to induce JNK
activation in a two-wave kinetic manner as JNK kinase activity peaked
at an early time point (10 min) and a late time point (12 h) following TGF-
treatment (24). Although the pathway by which JNK is activated by TGF-
remains unknown, it was speculated that the immediate-early phase of JNK activation is probably a process independent of Smad proteins, known effectors of the TGF-
signaling pathway involved in
transcription regulation of target genes, whereas the later phase of
induction is Smad-dependent. Thus, the ability of NET1 to
activate JNK pathway may provide a possible link between TGF-
and
the later phase of JNK activation.
From the rapid time course of NET1 mRNA accumulation
(Fig. 2A), we noticed a close correlation between the
induction of NET1 expression and the known kinetics of
phosphorylation and nuclear accumulation of Smad2 and Smad3 (26, 27).
To examine if NET1 serves as one of the direct target genes
for these two Smads, we used recombinant adenoviruses to determine if
Smad3 and Smad4 overexpression would be able to stimulate the
expression of the endogenous NET1 gene. The result was
negative (data not shown), suggesting that Smad overexpression may not
be sufficient to activate the expression of NET1, and other
signaling events may have to be initiated by the TGF-
receptor
complex simultaneously. Alternatively, the overexpression of Smad
proteins clearly may not accurately reflect the signaling process of
Smad activation and action. To explore this possible link further, we
have used a HaCaT stable line overexpressing Smad3D407E, a mutant that
can block the phosphorylation of endogenous Smad2 and Smad3 upon
TGF-
treatment. The induction of NET1 in Smad3D407E cells
was dramatically decreased in comparison to that of normal HaCaT cells,
suggesting that Smads are essential for NET1 induction.
The detailed mechanism for the Smad-mediated transcriptional
regulation of NET1 expression will have to be revealed with
the cloning and characterization of the NET1 promoter in the future.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. M. Kato for the
Smad3D407E cell line; Dr. T. Miki for the NET1 cDNA construct; Dr.
H. Moses and B. Law for Smad3 dominant negative adenoviruses; and
Yoshitomi Pharmaceutical Ltd. for ROCK inhibitor Y27632. In addition,
we thank members of the Wang laboratory for helpful discussions and
Yong Yu for technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK45746 and CA75368 (to X. F. W.).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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Cancer Biology, Box 3813, Duke University Medical Center, Durham,
NC 27710. Tel.: 919-681-4861; Fax: 919-681-7152; E-mail: wang@galactose.mc.duke.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M009534200
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
NET1, neuro epithelioma transforming
gene 1;
GEF, guanine nucleotide exchange factor;
GAP, GTPase-activating
proteins;
GDI, GDP dissociation inhibitor;
RPA, Ribonuclease protection
assay;
JNK, c-Jun N-terminal kinase;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
 |
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