Department of Zoological Cell Biology, The Wenner-Gren Institute,
Stockholm University, SE-106 91 Stockholm, Sweden
* Present address: Laboratory for Developmental Biology, Cell and Molecular
Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden
Author for correspondence (e-mail:
anki.ostlund{at}cellbio.su.se
)
Accepted 26 April 2002
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Summary |
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Key words: BRG1, SWI/SNF, Actin filament organisation, RhoA, Rho kinases
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Introduction |
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Several studies in BRG1- and BRM-deficient cell lines, such as the human
adrenal adenocarcinoma cell line SW13 and a human cervix carcinoma cell line
C33A (Muchardt and Yaniv,
1993; Wang et al.,
1996b
; Wong et al.,
2000
), have shown that the BRG1 and the BRM proteins are involved
in the regulation of proliferation, differentiation and cell growth.
Expression of the BRG1 or the BRM protein in SW13 cells induces growth arrest
and a change in morphology to flat cells
(Dunaief et al., 1994
;
Strober et al., 1996
;
Shanahan et al., 1999
). These
effects have been linked to an interaction with the retinoblastoma protein
(Rb) (Dunaief et al., 1994
;
Singh et al., 1995
;
Strober et al., 1996
), an
important cell cycle control protein and tumour suppressor (for reviews, see
Knudsen et al., 1998
;
Dyson, 1998
). It has been
suggested that the association of Rb with the BRG1 or BRM protein is needed
for full repression of the transcription of genes under the control of the E2F
transcription factor (Trouche et al.,
1997
). BRG1 expression in SW13 cells reduces the level of proteins
such as cdc2, cyclin A and cdk2, which are responsible for cell cycle
progression (Zhang et al.,
2000
). These cell cycle proteins are also reduced in C33A cells if
BRG1 is expressed together with the Rb-protein
(Strobeck et al., 2000
;
Zhang et al., 2000
).
Furthermore, the BRG1 protein introduced into SW13 cells interacts with the
G1/S-phase-specific cyclin E, which inactivates the BRG1 protein by
phosphorylation (Shanahan et al.,
1999
). The phosphorylation of BRG1 excludes it from chromatin in
late G2/M-phase and inactivates the SWI/SNF complex
(Muchardt et al., 1996
;
Sif et al., 1998
). The BRM
protein is also phosphorylated, which causes it to be not only excluded from
chromatin but also degraded (Muchardt et
al., 1996
; Muchardt et al.,
1998
). Further evidence for a role of SWI/SNF complexes in the
control of proliferation is the finding that BRM knockout mice are larger than
normal (Reyes et al., 1998
).
The BRG1 protein is essential for early development and Brg1 null
homozygots die before the preimplantation stage
(Bultman et al., 2000
).
Many cellular processes are involved in cell cycle progression. The
morphology of cells changes during the cell cycle, a process that involves
rearrangement of the actin filaments (reviewed by
Van Aelst and D'Souza-Schorey,
1997). Actin filaments are responsible for the shape of
differentiated cells, spreading, anchorage, cell movement, and cytokinesis
(reviewed by Van Aelst and
D'Souza-Schorey, 1997
; Small
et al., 1999
). Actin filaments are highly dynamic structures, and
rearrangements are essential for these cellular processes. Therefore,
regulation of the organisation of actin filaments is highly controlled and
several signal transduction pathways affect the actin filament organisation.
The family of small Rho GTPases, which includes RhoA, Rac and cdc42, mediates
many of the external signals and the effects of the substratum by switching to
the active GTP-bound state upon stimulation (reviewed by
Hall, 1998
;
Small et al., 1999
;
Bishop and Hall, 2000
). Most
studies have been conducted on fibroblasts, in which the activation of
individual Rho family members results in different actin filament
organisation; RhoA induces stress-fibre formation and the assembly of focal
contacts, Rac induces lamellipodia, and cdc42 induces filopodia
(Small et al., 1999
;
Hall, 1998
). The activated
Rho-GTPases bind to and activate in turn downstream effectors. The formation
of stress fibres in fibroblasts requires at least two classes of the RhoA
effectors, Dia and Rho associated kinase (Rho-kinase/ROCK) (reviewed by
Bishop and Hall, 2000
).
In this study we investigate the role of the BRG1 protein in the
organisation of actin filaments in SW13. BRG1 expression induces morphological
changes in SW13 cells and we present evidence that BRG1 expression induces the
formation of thick actin filament bundles in the cell bodies
(stress-fibre-like structures) by affecting the RhoA signalling pathway. We
present evidence that the RhoA GTPase is not directly affected, but
BRG1-expression results in an elevated protein level of ROCK1, one of the
isoforms of Rho-kinase/ROCK. The balance between Rho-kinases/ROCK and Dia
proteins is important for proper stress-fibre formation in fibroblasts
(Watanabe et al., 1999), and
we suggest that the elevated ROCK1 protein level affects this balance,
coupling RhoA activation to stress-fibre formation.
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Materials and Methods |
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Vector constructs and DNA cloning
Standard molecular biology cloning techniques were used for all cloning
steps. All vector constructs were verified by extensive sequencing.
The pORSVI-BRG1 expression vector was constructed by ligating a
SalI/SpeI fragment from pSVhSNF2ß
(Chiba et al., 1994) into the
SpeI/XhoI sites of pORSVI. pORSVI-BRG1-K798R was constructed
by exchanging a XhoI/BglII fragment with the same fragment
taken from the expression vector pBJ5-BRG1-K798R
(Khavari et al., 1993
).
A specific BRG1 antisense vector was constructed by ligating a 1625 base-pair, blunt-ended, N-terminal PCR fragment from the brg1 cDNA, in the antisense direction into the EcoRV site of the pORSVI expression vector of the LacSwitch system (Stratagene).
PCR primer 1: ATG CAT GCG GGA TCC CAG ACC CAC CCC TGG GCG GAA CTC C.
PCR primer 2: CGT ACG TAG GGG ATC CCC CTT CTG GTC GAT GAG CTT GCG GTA CCC C.
GST-Rhotekin-RBD (rho-binding-domain) was constructed by ligating a PCR-generated fragment of the human rhotekin cDNA, corresponding to amino acids 24-106, into the EcoRI/XhoI sites of the pGEX-4T-1 vector (Amersham/Pharmacia), generating an in-frame fusion protein. The human rhotekin fragment corresponds to amino acids 8-89 of the mouse rhotekin. cDNA was generated from total RNA isolated from untreated SW13 cells using MMLV reverse transcriptase and random primers (Life Technologies) according to the manufacturer's instructions.
PCR primer 1: ATGC ATGC GAA TTC GCA CAC CCC TGC CTT CCT CTC.
PCR primer 2: ATGC ATGC CTC GAG GCT TGT CTT CCC CAG CAC CTG.
Cell cultures
SW13 cells were cultivated in 5% fetal calf serum in 50% Dulbecco's
modified Eagle's medium (DMEM)/ 50% Ham's F12 medium supplemented with 50
µg/ml each of streptomycin and penicillin.
Transfection of cells
SW13 cells were transfected with the pBJ5 vector containing the cDNA for
BRG1 or a form of BRG1 with a non-functional ATPase
(BRG1-K798R, with a lysine to arginine mutation at position 783 in
the human BRG1 cDNA (Khavari et
al., 1993) corresponding to position 798 in the SWI2/SNF2
cDNA in the ATPase motif), together with a vector containing the gene for
neomycin/G418 resistance. The transfection was performed using
Lipofectamin plus (Life Technologies). SW13 cells were also transfected with
the BRG1 or the BRG1-K798R cDNA in the pORSVI expression
vector. Selection with 0.3 mg/ml of G418 was started 2 days after transfection
and colonies were collected 2-3 weeks after selection had been started.
Transient transfections were performed using Lipofectamin plus and cells were lysed for immunoblots, or fixed for immunofluorescence from 6 to 72 hours after transfection, as described below.
Immunofluorescence
Cells were fixed with 2% formaldehyde (Sigma) for 20 minutes at 37°C
and lysed in 0.2% Triton X-100 for 20 minutes at 37°C. Actin filaments
were visualised using 0.6 µM TRITC- or FITC- conjugated phalloidin (Sigma)
in phosphate-buffered saline (PBS). The BRG1 protein was visualised using
affinity-purified rabbit antibodies against an N-terminal fragment of the
human BRG1 protein (a kind gift from Emma Lees, DNAX Research Institute,
Uppsala, Sweden) or against a C-terminal fragments of the rat BRG1 protein
(Östlund Farrants et al.,
1997). The myc-tagged RhoA proteins were detected using antimyc 9E
(Santa Cruz) and the Rho protein was detected using monoclonal anti-RhoA
(Santa Cruz). TRITC- and FITC-conjugated secondary antibodies used for
visualisation were obtained from Jackson Immunochemical. The specimens were
mounted in Vectashield (Vector Laboratories). Immunofluorescence images were
recorded using a Leitz Aristoplan microscope and confocal images were recorded
using a Leica confocal laser scanning microscope, with an objective lens of of
magnification 63x and a numerical aperture of 1.4.
Immunoblots
The cells were lysed in 20 mM Tris-Cl at pH 8.0, 0.7 M NaCl, 0.5% Nonident
40, frozen and thawed, and then centrifuged at 11,700 g for 20
minutes to remove DNA and cell debris. The proteins of cell lysates (20 µg)
were separated by SDS-polyacrylamide gel electrophoresis and transferred to an
Immobilone membrane (Millipore). The membrane was probed with anti-BRG1 serum,
monoclonal anti-ß-actin (Sigma), monoclonal anti-RhoA (Cytoskeleton),
anti-ROCK1 antibodies (Santa Cruz), anti-ROCK2 antibodies (Santa Cruz),
anti-Dia1 antiserum (Immunoglobe), anti-histone deacetylase 2 antibodies
(Abcam), anti p65 of NFB antibodies (Abcam) and monoclonal
anti-
-tubulin (Amersham-Pharmacia).
RNA analysis
Total RNA was prepared using Trizol (Life Technologies) and 2 µg of
total RNA was converted to cDNA using 200 U MMLV reverse transcriptase and 100
ng random primer (Life Technologies). One tenth of the reaction volume was
used for target-specific PCR reactions to detect the presence of BRG1, GAPDH,
ROCK1, ROCK2 and Dia1 mRNAs.
RhoA activation assay
The GST pull-down assay was performed essentially as described
(Ren and Schwartz, 2000),
except that the RhoA-binding-domain from human rhotekin was used instead of
that from the mouse. They differ by seven amino acids in the N-terminal
portion of the protein. In order to load equal amounts of total protein for
each pull-down and input control, the cells were grown in single dishes at
equal densities (
60% confluence) and then the protein concentrations of
the crude lysates were measured.
Protein analysis
The protein concentration was determined using Bradford reagent
(Bio-Rad).
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Results |
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In the BRG1 and the BRG1-K798R-expressing clones, but not in native SW13
cells, a clear BRG1 protein signal was observed, which was located in the
nucleus (compare Fig. 1A and D with
1F). The BRG1-positive clones that were isolated formed thick
actin filament bundles stretching mostly from the plasma membrane into the
cell body (Fig. 1B,C), but
could also appear in short stretches within the cell body. These
stress-fibre-like actin structures were absent both in clones that expressed
the BRG1-K798R protein (Fig.
1E) and in native SW13 cells
(Fig. 1G). Instead, a thin
actin filament network was seen in the cell bodies of these cells, and actin
filaments were abundant at the edges (Fig.
1E,G). The BRG1-expressing cells developed flat cell morphology
and grew larger than native SW13 cells and cells expressing the BRG1-K798R
protein after 4-5 days of growth, although some of the cells in the BRG1-K798R
clone were slightly larger than native SW13 cells
(Fig. 1E). Furthermore, SW13
cells that expressed the BRG1 protein were thinner (approximately 5 µm)
than native SW13 cells (approximately 20 µm), measured by viewing the depth
of focus in the confocal microscope. The expression of the BRG1 protein in the
clones was confirmed by immunoblots (Fig.
1H), while no expression of the BRG1 protein was detected in SW13
(Fig. 1H). The change in actin
organisation observed in the BRG1-expressing clones was not caused by
differences in -actin levels, since native SW13 cells and the
BRG1-expressing clones contained approximately the same levels of ß-actin
(Fig. 1H).
|
Many of the large BRG1-expressing cells had multiple nuclei (not shown), indicating a disturbance in mitosis and cytokinesis. However, multinuclear cells were observed not only in cells that expressed the BRG1 protein, but also in cells expressing the mutated BRG1-K798R protein (Fig. 1D). These cells were also larger than mononuclear cells, but they were without any thick actin filament bundles in the cell body. This suggests that some of the effects caused by the BRG1 protein are achieved without the ATPase activity.
The formation of thick actin filaments in the cell body in
BRG1-expressing cells depends on the BRG1 protein
In order to confirm that the formation of actin filament bundles in the
cell body in BRG1-expressing clones depends upon the expression of the BRG1
protein, we used a vector co-expressing a specific antisense BRG1-RNA fragment
and the GFP (Green Fluorescent Protein). The expression of the antisense BRG1
reduced the protein level in BRG1-expressing cells by approximately 50% 72
hours after transfection, but did not affect the protein level of
-tubulin (Fig. 2A). The
BRG1-antisense vector at amounts higher than 5 µg was toxic and resulted in
cell death, and no further reduction could be detected in cells transfected
with the vector above these amount. The expression of the antisense BRG1-RNA
fragment in BRG1-expressing cells abolished the thick actin bundles in the
cell body (Fig. 2B). The
expression of a GFP protein expressed from the same vector was used as a
marker for cells expressing the antisense BRG1-RNA fragment
(Fig. 2C). The same vector
expressing only the GFP protein was used as a control, and these cells still
had stress-fibre-like actin filaments (Fig.
2D,E). The disappearance of thick actin filament bundles in
BRG1-expressing cells in response to a reduced level of BRG1 protein strongly
suggests that this protein is responsible for the induction of thick actin
bundles in the cell body.
|
Several protein complexes in addition to the BRG1-containing SWI/SNF
complexes can remodel chromatin and may be involved in transcriptional
regulation. Modification of the histone tails by acetylation alters the
structure of chromatin, and is required for many cellular processes
(Kingston and Narlikar, 1999).
Therefore, we next examined whether chromatin remodelling by acetylation in
the absence of the BRG1 protein resulted in a similar actin bundle formation
as that found in BRG1-expressing cells. SW13 cells were treated with the
histone deacetylase inhibitor trichostatin A (TSA) at concentrations of 50
ng/ml, 150 ng/ml and 450 ng/ml for 24 hours, which leads to hyperacetylation
of the histones. None of these concentrations induced stress-fibre-like actin
filament bundles in SW13 cells, but the actin filaments at the edges were
affected at the 150 ng/ml (Fig.
3A). Several protrusions resembling retraction fibres were
observed. TSA at high concentrations is toxic and, as expected, the highest
concentration caused cell death. BRG1-expressing cells treated with TSA at the
same concentrations for 24 hours had a larger number of thick actin bundles in
their cell bodies than BRG1-expressing cells, indicating that hyperacetylation
of histones increases the effect of the BRG1 protein on the actin filament
organisation in the cell body (compare Fig.
3C with 3B).
|
Thick actin filament bundles appear between 24 hours and 48 hours
after BRG1 transfection of SW13 cells
We next examined the relationship in time between BRG1 expression and the
appearance of thick actin bundles in the cell body in SW13 cells by
transfecting SW13 cells with either the pBJ5-BRG1 or the pBJ5-BRG1-K798R
expression vector. Samples were taken at 10, 24, 30, 36 and 48 hours after
transfection. The BRG1 and the ATP-deficient BRG1-K798R proteins were first
detected in immunoblots 10 hours after transfection, with a peak at 30 hours
(Fig. 4A). The protein levels
expressed from both vectors were in the range of the levels found in HeLa
cells and NIH 3T3 cells (not shown). Actin filament bundles in the cell bodies
could be clearly detected by immunofluorescence in many of the BRG1-expressing
cells at 24 hours, and at 48 hours after transfection 48% of the
BRG1-expressing cells had thick actin bundles in the cytoplasm
(Fig. 4B). Few cells with actin
bundles in the cytoplasm were detected in cells expressing the BRG1-K798R
protein, and the level did not increase with time
(Fig. 4B). There was no clear
difference in the morphology of the filaments after 24 hours and 48 hours, but
the number of filaments was lower at 24 hours (compare
Fig. 4C and D with 4E and
F).
|
Expression of BRG1 affects the RhoA pathway
To gain further insight into the effect of the BRG1 protein on the
formation of actin filament bundles, we investigated whether the BRG1 protein
affected the RhoA pathway, the major signalling pathway leading to
stress-fibre formation. We exposed SW13-cells and BRG1-expressing cells to LPA
(lysophosphatidic acid) and serum, two stimuli that are known to activate the
RhoA pathway and induce stress-fibre formation in fibroblasts
(Ridley and Hall, 1992). SW13
cells were unable to respond to LPA and serum treatment by forming
stress-fibrelike actin filament bundles. Because the SW13 cells were sensitive
to starvation, we starved these cells only for 6 hours
(Fig. 5A). No thick actin
filaments were induced by LPA concentrations of 0.1, 10 or 90 µM for 5
minutes, 20 minutes, 6 hours or 18 hours.
Fig. 5B shows cells exposed to
10 µM LPA for 20 minutes. Serum (5%) did not have an effect on the actin
filament organisation in native SW13 cells
(Fig. 1G), nor could other
stimuli that induce stress-fibres in fibroblasts, such as different substrata,
induce the formation of stress-fibre-like actin filament bundles (not shown).
BRG1-expressing cells that were starved for 18 hours had lost most of their
thick actin filament bundles in the cell body
(Fig. 5C), but in contrast to
native SW13 cells, clear filament bundles in the cell body reformed when the
cells were exposed to 5 µM LPA for 20 minutes
(Fig. 5D). Exposure to 5% serum
for 20 minutes resulted in a stronger effect, and the thick actin filament
bundles in the cell body were fully restored
(Fig. 5E). These results
indicate that BRG1 expression in SW13 establishes an active RhoA pathway that
can respond to stimuli and thereby induce the formation of stress-fibre-like
structures.
|
We next transfected BRG1-expressing cells with a vector containing a myc-tagged, dominant negative RhoA cDNA, RhoA(N19), which causes loss of stress-fibres in fibroblasts when expressed. Expression of RhoA(N19) in BRG1-expressing cells caused disassembly of the thick actin bundles in the cell body 18 hours after transfection (Fig. 5F), leaving behind diffusely stained cells. The cells that expressed the RhoA(N19) protein were detected with the myc-9E antibody (Fig. 5G).
The RhoA GTPase is activated in both SW13 cells and BRG1-expressing
cells
The different response between native SW13 cells and BRG1-expressing cells
to stimuli that activate the RhoA pathway prompted us to analyse directly the
activity state of the RhoA-protein in these cells. The human rhotekin-GST
fusion, which differs from the commonly used mouse rhotekin by 15 amino acids
in the N-terminal, was used, and Fig.
6A shows that it was specific for the RhoA-GTP form in a pull-down
assay. The rhotekin protein fragment bound the constitutively active RhoA(L63)
protein from cells lysate, but not the dominant negative RhoA(N19) protein
(Fig. 6A). When the RhoA-GTP
level was compared in SW13 cells and clones expressing the BRG1 protein or the
BRG1-K798R protein continuously grown in the presence of 5% serum, no
difference was detected between the cell populations
(Fig. 6B). Similarly, the RhoA
protein was activated to the same extent in native SW13 cells, BRG1-expressing
cells and cells expressing the K798R-BRG1 when exposed to 5% serum for 20
minutes after 18 hours of starvation (Fig.
6C). Thus, the RhoA GTPase was equally activated by serum in all
three cell types, but serum induced only thick actin filaments in the cell
body in BRG1-expressing cells (compare Fig.
5C and D with 5A and Fig.
1G). Therefore, the formation of thick actin filament structures
in BRG1-expressing cells cannot be caused by an upregulation of the amount of
RhoA-GTP, but must be regulated by effectors downstream of the
RhoA-GTPase.
|
The BRG1 protein increases the expression of the ROCK1 protein
The RhoA small GTPase has several downstream effector targets, at least two
of which are involved in the induction of stress-fibres in fibroblasts, the
Rho-kinase/ROCK (Leung et al.,
1996; Amano et al.,
1997
; Sahai et al.,
1998
) and the profilinbinding protein Dia
(Watanabe et al., 1997
;
Watanabe et al., 1999
). To
determine whether downstream effectors were involved in formation of thick
actin filament bundles in BRG1-expressing cells, we treated BRG1-expressing
cells with Y-27632, a specific inhibitor of the Rho-kinase/ROCK. Inhibiting
the Rho-kinase/ROCK by adding 10 µM of Y-27632 to the culture medium for 1
hour led to the disruption of the thick actin bundle formation in the
BRG1-expressing cells (compare Fig.
7A with Fig. 1B and
C). These cells displayed a thin network of actin filaments in the
cell body similar to that seen in untreated SW13 cells (compare
Fig. 7A with
Fig. 1G). The actin filament
structure in SW13 cells exposed to 10 µM of Y-27632 remained unaltered
(Fig. 7B). This indicated a
role for the Rho-kinase/ROCK in the formation of thick actin bundles in
BRG1-expressing cells, and we next determined the protein levels of RhoA
effectors involved in stress-fibre formation. The balance between the two RhoA
effectors, Rho-kinase/ROCK and Dia, is important for the formation and
appearance of the actin filament bundles
(Watanabe et al., 1999
). The
protein levels of the two Rho-kinase/ROCK isozymes, ROCK1 and ROCK2
(ROK
), and Dia1 were determined in cells transiently transfected with
either the BRG1 vector or the ATPase-deficient BRG1-K798R
vector, and samples were taken at 12, 24, 36, 48 and 60 hours after
transfection. The protein level of ROCK1 was slightly higher in
BRG1-expressing cells after 24 hours, whereas the BRG1-K798R-expressing cells
showed no increase (Fig. 7C).
The protein levels of ROCK2 and Dia1 remained essentially unaltered in all
three cell types (Fig. 7C), as
were those of actin and
-tubulin
(Fig. 7C). We also transfected
the BRG1-expressing clone with the vector from which the specific antisense
BRG1-RNA fragment was expressed in order to interfere with the BRG1
expression. The 0.6x106 cells were transfected with
increasing amounts of vector, 2.5, 5 and 10 µg, for 72 hours. The BRG1
expression decreased following the increase in antisense BRG1 vector
(Fig. 7D), and the same pattern
was displayed by ROCK1 and also by ROCK2. The protein levels of these proteins
were not affected by the vector without the antisense BRG1 fragment
(Fig. 7D). The protein level of
Dia1 remained unaltered, as did the protein levels of
-tubulin and
actin (Fig. 7D). Two proteins
unrelated to actin filament organisation, histone deactylase (HDAC) and p65 in
NF
B, remained unaltered by the BRG1-antisense expression
(Fig. 7D). We conclude that the
effect of BRG1-expression in SW13 cells affects the protein level of
Rho-kinase/ROCK, but seems to have a stronger effect on that of ROCK1.
|
We also determined the mRNA levels of ROCK1, ROCK2 and Dia1 in transient transfected SW13 cells (not shown), but there were no significant changes in mRNA levels between native SW13 cells, BRG1-expressing cells and BRG1-K798R-expressing cells, suggesting that the increase in ROCK1 protein is not caused by direct transcriptional regulation by the BRG1 action on the ROCK1 gene.
Transiently expressed ROCK1 and ROCK2 induce stress-fibre like
structures in SW13 cells
The elevated protein level of ROCK1 protein in BRG1-expressing cells would
result in an alteration in the balance between the Rho-kinase/ROCK and Dia,
which in turn leads to changes in actin filament organisation. To examine
whether an elevated Rho-kinase/ROCK activity induced stress-fibre-like
structures in SW13 cells, we expressed a myc-tagged fragment of the
catalytical kinase domain of ROCK2. SW13 cells that expressed the catalytical
fragment clearly formed thick actin filaments in the cell body
(Fig. 8A,B), indicating that an
increased Rho-kinase/ROCK activity was sufficient to form stress-fibre like
structures in these cells. The myctagged, wild-type protein of both isoforms
of Rho-kinase/ROCK, ROCK1 and ROCK2, were also expressed in SW13 cells, and
both proteins induced thick actin filaments
(Fig. 8C,E, respectively).
Cells expressing the proteins were detected with anti-9E-myc antibodies
(Fig. 8D,F). Furthermore,
BRG1-expressing cells were transfected with myc-tagged, dominant-negative
forms of ROCK1 or ROCK2. The ROCK1 construct (KD-IA) carried mutations in both
the RhoA binding site and the kinase catalytical site
(Ishizaki et al., 1997), and
the cells expressing this protein lost their actin stress-fibres
(Fig. 8G,H). Cells expressing
ROCK2 mutated in the RhoA binding site and with a deleted kinase domain
(pEFBOS-myc-RB/PH(TT) (Amano et al.,
1997
) also abolished stress-fibre-like structures when expressed
(not shown). These results suggest that the Rho-kinase/ROCK level in SW13 is
involved in regulating the formation of stress-fibre-like actin
structures.
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Control of the actin filament organisation is complex and several factors
and signal transduction pathways are involved. These include actin regulatory
proteins, cell adhesion signal pathways and signal transduction pathways that
are activated in response to growth factors. In particular, signal pathways
involving small GTPases of the Rho family play a major role in the
reorganisation of actin, and studies in fibroblasts have implicated RhoA in
stress-fibre formation. Our results suggest that BRG1 protein affects the RhoA
signalling pathway, but not by regulating the activity state of the RhoA
GTPase. The RhoA GTPase in native SW13 cells and in cells expressing the
ATP-deficient BRG1-K798R protein is equally activated when exposed to
stimulatory factors, such as serum, as the RhoA GTPase in BRG1-expressing
cells after starvation, but the activation of RhoA in native SW13 cells and in
those expressing the BRG1-K798R protein does not trigger formation of
stress-fibre-like structures. Uncoupling of activated RhoA from stress-fibre
formation has been observed in ras-transformed fibroblasts with a sustained
activated MAPK pathway, which have lost their stress-fibres
(Sahai et al., 2001;
Pawlak and Helfman, 2002
).
These cells have a downregulated protein level of the RhoA downstream effector
Rho-kinase/ROCK; both a downregulation of one of the isoforms, ROCK1,
accompanied by an altered subcellular distribution
(Sahai et al., 2001
), and a
downregulation of both ROCK1 and ROCK2
(Pawlak and Helfman, 2002
)
have been reported. We observed an upregulation of the ROCK1 protein in
BRG1-expressing cells, which gained the ability to form stress-fibres. We
could not find any difference in subcellular distribution of these proteins
between BRG1-expressing cells and SW13 cells, with RhoA, ROCK1, ROCK2 and
Dia1, another RhoA effector involved in stress-fibre formation, found in the
soluble Triton X-100 fraction (P.A. and A.-K.Ö. F., unpublished).
Both Rho-kinase/ROCK and Dia contribute to the formation of and to the
maintenance of the stress-fibres (Leung et
al., 1996; Amano et al.,
1996
; Watanabe et al.,
1997
; Sahai et al.,
1998
; Watanabe et al.,
1999
), and studies have shown that they co-operate in the
formation of RhoA-activated stress-fibres
(Watanabe et al., 1999
;
Nakano et al., 1999
). The
mechanisms of Rho-kinase/ROCK and Dia function are not fully understood. The
Rho-associated kinase/ROCK induce stress-fibres by activating myosin
(Leung et al., 1996
;
Amano et al., 1997
;
Chihara et al., 1997
;
Ishizaki et al., 1997
), most
probably both by a direct activating phosphorylation of the myosin light chain
(Amano et al., 1996
;
Kureishi et al., 1997
) and by
an inactivating phosphorylation of the myosin light chain phosphatase
(Kimura et al., 1996
;
Kawano et al., 1999
). In
addition, cofilin, with an actin-depolymerising activity, is inactivated by
phosphorylation by the Rho-kinase/ROCK downstream target LIM kinase
(Maekawa et al., 1999
;
Yang et al., 1998
). The Dia
protein is an FH protein that binds profilin and these proteins may work in
cooperation to induce stress-fibres upon activation
(Watanabe et al., 1997
). It
has been shown that different ratios of Rho-kinase/ROCK to Dia contribute to
different types of actin structures; overexpression of an active Dia1 gives
thin disorganised filaments, similar to those seen in untreated SW13 cells,
whereas overexpression of an active Rho-kinase/ROCK gives very thick condensed
fibres (Watanabe et al.,
1999
).
We propose that the elevated ROCK1 level in BRG1-expressing cells couples
the activation of RhoA to stress-fibre formation by altering the balance
between the Rho-kinase/ROCK activity and the Dia activity. Although we could
detect only an increased ROCK1 protein level in BRG1-expressing cells, both
forms were downregulated when interfering with BRG1-expression, which
indicates that both are affected. However, the ROCK1 is affected more
strongly. The fact that increasing the Rho-kinase activity in SW13 cells by
transient expression of the kinase domain induced thick actin filament
formation further supports this. No specificity between the two isoforms of
Rho-kinase/ROCK could be seen when transiently expressing wild-type ROCK1 or
ROCK2, since both induced thick actin filaments that resemble those in
BRG1-expressing cells. In addition, both nonfunctional, dominant-negative
forms of ROCK1 and ROCK2 were able to abolish the thick actin filament
formation in BRG1-expressing clones. The lack of specificity could be
explained by the similarity between these isoforms. The kinase domains are 92%
identical on the amino acid level
(Nakagawa et al., 1996), and
they have the same targets. The elevated ROCK1 protein level observed in
BRG1-expressing cells would result in an increase in overall phosphorylation
of myosin and cofilin, which subsequently would lead to the formation of
stress-fibrelike actin filaments.
We could not find evidence for a transcriptional regulation of the
ROCK1 gene directly, since the mRNA levels in BRG1-expressing cells
were not significantly different from those in native SW13 cells. Instead, the
effect of BRG1 on the ROCK1 level may be at the post-transcriptional or the
post-translational level. Transcriptional downregulation has been reported for
ROCK2 in ras-transformed cells (Zuber et
al., 2000), but it has been suggested that post-transcriptional
mechanisms exist for ROCK1 and ROCK2 regulation. The ROCK1 protein that is
obtained when expressed exogenously in ras-transformed cells is significantly
lower than that found in the corresponding native cells, although the
mRNA-level increases (Sahai et al.,
2001
). In a separate study of ras-transformed fibroblasts, both
the ROCK1 and ROCK2 protein level were lower than in native cells, but no
decrease in the mRNA-levels could be observed
(Pawlak and Helfman, 2002
).
Our results suggest that the BRG1 protein affects the stability of the ROCK1
protein and, to a lesser extent, ROCK2. Since the ATPase domain of the BRG1
protein is required for the increase of ROCK1 protein level, it is tempting to
speculate that the BRG1 is involved in transcriptional regulation of
components involved in regulating protein stability.
TSA treatment, which leads to hyperacetylation of the histone tails by
inhibiting histone deacetylases, did not induce the thick actin bundles in the
cell body in SW13 cells. This means that the formation of thick actin filament
bundles in the cell body does not depend on histone acetyltransferases.
Nevertheless, TSA clearly potentiated the thick actin bundle formation in the
cell bodies of BRG1-expressing cells, indicating a co-operation between the
BRG1 protein and histone acetylation. Co-operation between histone acetylation
and ATP-dependent chromatin remodelling has been observed in gene activation
of several genes in yeast and Xenopus
(Sudarsanam et al., 1999;
Cosma et al., 1999
;
Li et al., 1999
;
Krebs et al., 1999
).
Interestingly, TSA treatment of HeLa cells, which express the BRG1 and BRM
proteins, increases the number of stress-fibres
(Hoshikawa et al., 1994
),
which could reflect a co-operation between the BRG1 protein as a component of
the SWI/SNF complex and histone acetyl transferases, similar to the effect
displayed in BRG1-expressing SW13 cells. Recently, it was shown that the BRG1
protein is a component of two nuclear complexes with slightly different
subunit compositions and chromatin remodelling activities, the BAF complex
(SWI/SNF A) and PBAF (SWI/SNF B) (Xue et
al., 2000
; Sif et al.,
2001
). Therefore, the effects of the BRG1 protein expressed in
SW13 cells may arise from its role in different nuclear complexes with
different functions. This may also explain the multiple effects of the BRG1
protein when expressed in SW13 cells, including the induction of multinuclear
cells (Zhang et al., 2000
).
However, we also found cells that expressed the ATP-deficient BRG1-K798R
protein that were multinuclear, indicating that these cells arise
independently of a functional ATPase activity. It remains to be clarified
which of the BRG1-containing SWI/SNF complexes is responsible for inducing the
change in actin filament organisation. The post-transcriptional mechanism
involved in regulating the ROCK1 protein level also needs to be characterised
in order to determine at which step or steps in the regulation of the actin
filament structure the BRG1 protein is directly involved.
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Acknowledgments |
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References |
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