Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3168
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ABSTRACT |
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The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and its expression is downregulated significantly in dilated human cardiomyopathy. However, the function of SLIM1 is unknown. In this study, we investigated the intracellular localization of SLIM1. Endogenous and recombinant SLIM1 localized to the nucleus, stress fibers, and focal adhesions in skeletal myoblasts plated on fibronectin, collagen, or laminin. However, after inhibition of integrin signaling either by plating on poly-L-lysine or by soluble RGD peptide, SLIM1 localized diffusely in the cytosol, with decreased nuclear expression. Disruption of the actin cytoskeleton by cytochalasin D did not inhibit nuclear localization of SLIM1 in integrin-activated cells. Green fluorescent protein-tagged SLIM1 shuttled in the nucleus of untransfected NIH 3T3 cells, in a heterokaryon fusion assay. Overexpression of SLIM1 in Sol8 myoblasts inhibited cell adhesion and promoted cell spreading and migration. These studies show SLIM1 localizes in an integrin-dependent manner to the nucleus and focal adhesions where it functions downstream of integrin activation to promote cell spreading and migration.
myoblast; integrins; LIM proteins; migration
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INTRODUCTION |
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LIM DOMAINS ARE cysteine-rich double-zinc-finger motifs that mediate protein binding (17). LIM domains occur in transcription factors and signaling and cytoskeletal proteins. In the nucleus, LIM domains scaffold the interaction of transcription factors to regulate transcription and perform critical roles in embryonic development and tissue differentiation (2). In the cytoplasm, LIM proteins bind signaling proteins, and many have been shown to link protein kinases and phosphatases with the cytoskeleton. LIM proteins perform critical roles in striated muscle differentiation and function. Targeted deletion of muscle LIM protein (MLP) in mice causes marked disruption of myocardial cytoarchitecture, leading to a dilated cardiomyopathy and death resulting from cardiac failure (1). Similarly, ablation of the LIM-PDZ domain protein, Cypher, results in a severe skeletal myopathy (50).
The skeletal muscle LIM protein 1 (SLIM1) is a member of the four-and-a-half LIM (FHL) domain protein family (29). The other FHL proteins include SLIM2/FHL3, SLIM3/FHL2/DRAL, FHL4, and ACT [activator of the cAMP response element modulator (CREM) in the testis]. Each FHL protein contains a single NH2-terminal zinc finger, representing one-half of a LIM domain, followed by four complete LIM domains (29). Recently, several FHL proteins, with the notable exception of SLIM1, have been shown to activate transcription factors, including the cAMP response element binding protein (CREB), CREM, and the androgen receptor (19, 30). SLIM1 has two alternatively spliced isoforms, KyoT2 and SLIMMER/FHL1B (7, 44). The murine isoform KyoT2 contains the NH2-terminal two-and-a-half LIM domains of SLIM1 followed by 27 novel COOH-terminal amino acids that bind the DNA-binding protein RBP-J (44). By competing for binding to RBP-J, KyoT2 inhibits Notch and Epstein Barr Virus nuclear antigen-activated transcription. SLIMMER contains the NH2-terminal three-and-a-half LIM domains of SLIM1, followed by a novel sequence encoding nuclear localization and nuclear export signals and the RBP-J binding domain (7).
SLIM1 is most highly expressed in adult skeletal muscle, with intermediate expression in cardiac muscle and lower expression in a wide range of tissues (7). In the embryo, SLIM1 localizes to the somites and in the developing heart strongly localizes to the cardiac outflow tract (6). Microarray analysis has detected significantly reduced SLIM1 mRNA levels in ischemic dilated cardiomyopathy, a finding confirmed by decreased SLIM1 protein on immunoblot analysis (49). In contrast, SLIM1 levels are increased significantly in human congenital hypertrophic cardiomyopathy and in two mouse models of cardiomyopathy, dilated cardiomyopathy resulting from targeted deletion of MLP and in hypertrophic cardiomyopathy induced by transverse aortic constriction (12, 28). The level of SLIM3/FHL2/DRAL, which is also highly expressed in cardiac muscle, does not change in these models of cardiomyopathy. These findings suggest that SLIM1 may perform an important structural or regulatory role in the cytoskeleton of human heart muscle; however, little is known about the normal cellular function of SLIM1.
In this study, we investigate the intracellular localization of SLIM1 and demonstrate that integrin activation of skeletal myoblasts promotes SLIM1 localization in the nucleus and focal adhesions. SLIM1 inhibits integrin-mediated myoblast adhesion and promotes spreading and migration. Therefore, SLIM1 regulates integrin-mediated cytoskeletal rearrangement.
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EXPERIMENTAL PROCEDURES |
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Materials.
Sol8, COS-7, and NIH 3T3 cell lines were purchased from the American
Type Culture Collection. Anti-peptide anti-SLIM1 antibodies have been
characterized previously in our laboratory (7). The pEGFP-C2 vector was from CLONTECH Laboratories, and the pCGN vector was
a gift from Dr. Tony Tiganis (Monash University). Lipofectamine reagent
was from Life Technologies. Monoclonal antibodies to hemaglutinin (HA)
were obtained from Covance and to green fluorescent protein (GFP) from
CLONTECH. Polyclonal antibodies to HA were from UBI. Monoclonal
antibodies to -tubulin were obtained from Zymed.
Tetramethylrhodamine isothiocyanate (TRITC) and FITC-conjugated
anti-rabbit IgG and anti-mouse IgM antibodies were from Silenus.
TRITC-conjugated phalloidin and propidium iodide were from
Sigma-Aldrich. Fibronectin, collagen, and poly-L-lysine
were from Sigma-Aldrich, and laminin was from GIBCO-BRL. RGDS peptide
was purchased from Auspep. Transwell micropore six-well plates for
migration assays were from Corning. The GFP-Rev construct was a kind
gift from Dr. Mark Lamm (Monash University).
Isolation of primary rat skeletal myoblasts. All experiments were performed according to National Health and Medical Research Council guidelines, Monash University animal ethics number BAM/B/2000/17. Eight 1-day-old Sprague-Dawley rat pups were killed humanely, and skeletal muscle was dissected from the hindlegs. Muscle samples were digested in 0.25% trypsin in 10 ml dissociation buffer (116 mM NaCl, 5 mM KCl, 23 mM NaHCO3, 8 mM Na2PO4, 1 mM EDTA, 1 g/l glucose, and 10 mg/l phenol red, pH 7.4) for 40 min at 37°C. Cells were passed through 70-µm Falcon cell strainers. The cell mixture was centrifuged at 380 g for 15 min. To eliminate contaminating fibroblasts, the pelleted cells were resuspended in 15 ml Ham's F-10 media with 10% horse serum and plated for 1.5 h at 37°C. The myoblast-rich media were removed and plated on an 80-cm2 tissue culture flask and incubated overnight at 37°C. Primary myoblasts were trypsinized and replated on coverslips coated with 15 µg/ml fibronectin for 3 h in DMEM with 20% FBS.
Transient expression of recombinant HA-tagged and GFP-tagged SLIM1 in Sol8 myoblasts. cDNA encoding the open-reading frame of SLIM1 was PCR amplified and cloned in the pCGN and pEGFP-C2 vectors. The sequence of all constructs was verified by dideoxynucleotide sequencing. HA- or GFP-tagged SLIM1 was transiently transfected in Sol8 murine skeletal myoblasts using lipofectamine, according to the manufacturer's instructions.
Intracellular localization of SLIM1. Plates were treated with either fibronectin (1, 5, or 15 µg/ml), laminin (10 µg/ml), collagen (0.3%), or poly-L-lysine (0.01%) diluted in PBS for 1 h at room temperature. Plates were washed in PBS and then blocked with 1% BSA for 1 h. Primary rat myoblasts, or HA-SLIM1- or GFP-SLIM1-transfected Sol8 myoblasts, were plated on the indicated surface for 3 h before fixation. In experiments examining the effect of serum starvation, cells were maintained in serum-free media for 18 h before plating and for 3 h while plated on fibronectin. The cells were washed in PBS and fixed and permeabilized in PBS, with 3.6% formaldehyde and 2% Triton X-100. Preimmune or affinity-purified anti-SLIM1 sera were added to the cells, and staining was detected with FITC-conjugated anti-rabbit IgG antibodies (7). HA-SLIM1 was detected using a monoclonal anti-HA antibody diluted 1:1,000. For colocalization, cells were pretreated with RNase A (10 mg/ml) for 1 h followed by addition of propidium iodide (0.2 mg/ml) for 10 min. Colocalization was also performed by addition of 1:500 TRITC-conjugated phalloidin for 1 h, and cells were visualized by confocal microscopy.
Immunoblot analysis. Proteins were separated by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using either monoclonal anti-HA antibodies or polyclonal anti-GFP antibodies, as indicated. The secondary antibodies were horseradish peroxidase-conjugated antibodies diluted to 1:10,000. Immunoblots were developed using enhanced chemiluminescence.
Inhibition of 5
1-integrin
activation with RGDS peptide.
Transfected Sol8 myoblasts were preincubated with soluble RGDS peptide
(50 µg/ml) for 20 min, before replating on fibronectin for 3 h,
in the presence of soluble RGDS peptide (50 or 500 µg/ml).
Cytochalasin D and nocodazole treatment.
HA-SLIM1-transfected Sol8 myoblasts were plated on fibronectin or
poly-L-lysine for 2 h before treatment with
cytochalasin D (2 µM) for 1 h or nocodazole (10 µg/ml) for
2 h. Cells were then washed, fixed, and stained.
Nocodazole-treated cells were costained with polyclonal anti-HA
(1:1,000) and monoclonal anti--tubulin antibodies (1:1,000).
Heterokaryon fusion assay. The heterokaryon fusion assay was a modified version used previously (42). GFP-SLIM1-transfected COS-7 cells were mixed with untransfected NIH 3T3. To promote membrane fusion of the two cell populations, 50% polyethylene glycol (PEG) 6000 was added to the cells for 2 min. The cells were washed in PBS and replated on fibronectin overnight. The cell nuclei were stained with Hoechst 33258 dye and visualized by fluorescence microscopy.
Leptomycin B inhibition of nuclear export. HA-SLIM1-transfected Sol8 myoblasts were plated on fibronectin or poly-L-lysine for 3 h in the presence or absence of leptomycin B (10 ng/ml). In control experiments, GFP-Rev, which has a functional nuclear export sequence (20), was transfected in Sol8 myoblasts, and its localization was determined in the presence or absence of leptomycin B.
Adhesion assay. GFP-SLIM1- or GFP-empty vector-transfected Sol8 myoblasts were plated for 15 min on 1, 5, or 15 µg/ml fibronectin. The percentage of transfected adherent cells was determined and divided by the transfection efficiency for each construct to determine adhesion efficiency. The results represent the means of three separate transfection and adhesion assays (means ± SE) in which a total of 600 cells was counted for each construct on each fibronectin concentration.
Spreading assay. GFP-SLIM1- or GFP-empty vector-transfected Sol8 cells were grown to 50-60% confluency, trypsinized, and replated on coverslips coated with fibronectin (15 µg/ml) for 1, 2, or 3 h. At each time point, the cells were fixed and visualized by confocal microscopy. The percentage of transfected cells, which had spread, was determined at each of the time points in three separate transfections. Cells were counted as spread if the area of the cytosol was equal to or greater than two times the area of the nucleus. The results represent the mean of three separate transfection and spreading assays for each of the time points (mean ± SE) in which a total of 200 cells in three separate transfection experiments for each GFP-SLIM1 and GFP-empty vector was counted. The P value was determined by a Student's t-test.
Migration assay. Migration assays were performed as previously described (34). GFP-empty vector- or GFP-SLIM1-transfected Sol8 myoblasts were placed in the upper well of a Transwell chamber (Corning) and allowed to migrate for 12 h through a membrane with 8-µm pores to a lower chamber. Cells were trypsinized from both sides of the membrane, and the percentage of transfected cells in the lower chamber (migrating cells) and upper chamber (nonmigrating cells) was counted. To determine whether the transfected constructs promoted or inhibited cell migration, the percentage of transfected cells in the lower chamber was divided by the percentage of transfected cells in the upper chamber. If the construct had no effect on migration, the transfection efficiency in the upper and lower chambers should be the same. If the transfected construct promoted migration, the percentage of transfected cells in the lower chamber should be significantly greater than in the upper chamber. If the construct inhibited migration, the percentage of transfected cells in the lower chamber should be significantly less than in the upper chamber. The results presented represent the means of three separate transfection and migration assays (means ± SE) in which a total of 500 cells was counted. The P value was determined by a paired Student's t-test.
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RESULTS |
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SLIM1 is predominantly expressed in skeletal and cardiac muscle
(7, 29). We have previously reported the intracellular localization of endogenous and recombinant SLIM1 in Sol8 myoblasts to
be diffusely cytosolic with low-level nuclear staining and exclusively
cytosolic in Sol8 myotubes (7). However, when GFP-SLIM1 was expressed in COS-7 cells, a more variable localization of the
recombinant protein was noted, with SLIM1 detected in the nucleus of
some cells and in other cells localized predominantly at focal
adhesions and stress fibers (7). We speculated that this
variable localization in COS-7 cells, and the absence of SLIM1 focal
adhesion localization in myoblasts, may relate to different states of
integrin activation of the plated cells. Because focal adhesions are
integrin-rich sites, we proposed that integrin activation would augment
focal adhesion formation and may thereby promote SLIM1 localization to
focal adhesions and/or the nucleus. To investigate this hypothesis, we
placed primary skeletal myoblasts, which express high levels of
endogenous SLIM1, on a fibronectin matrix that specifically activates
4
1- and
5
1-integrins in myoblasts
(18). Indirect immunofluorescence was performed utilizing a previously characterized affinity-purified anti-SLIM1 anti-peptide antibody that is directed against a unique peptide sequence located in
the fourth LIM domain of SLIM1. In Sol8 myoblasts plated on fibronectin, SLIM1 localized to focal adhesions, with some staining of
actin stress fibers (Fig. 1a).
It is noteworthy that SLIM1 also localized strongly to the nucleus.
Preimmune serum was nonreactive (Fig. 1b). SLIM1 colocalized
with phalloidin staining of actin stress fibers with prominent staining
at the ends of stress fibers, consistent with focal adhesion
localization (Fig. 1, c-e). SLIM1 also colocalized with
propidium iodide, which stains the nucleus (Fig. 1, f-h).
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To confirm the intracellular localization of SLIM1, recombinant
NH2-terminal HA- and GFP-tagged SLIM1 were expressed in the mouse skeletal myoblast Sol8 cell line. In transiently transfected Sol8
myoblasts plated on fibronectin, HA-SLIM1 and GFP-SLIM1 were localized
in the nucleus, actin stress fibers, and focal adhesions, consistent
with the localization of the endogenous protein (Fig. 2A). In control studies, HA
alone was detected diffusely in the cytosol (Fig. 2A).
HA-SLIM1 (Fig. 2B) and GFP-SLIM1 (data not shown)
colocalized both with phalloidin staining of actin stress fibers and
focal adhesions and with propidium iodide staining of the nucleus.
Immunoblots of transfected Sol8 lysates, using anti-HA or anti-GFP
antibodies, confirmed that HA-SLIM1 (predicted molecular mass 32 kDa;
Fig. 2C) and GFP-SLIM1 (predicted molecular mass 58 kDa;
Fig. 2D), respectively, were expressed intact, with minimal
proteolysis. A minor 47-kDa proteolytic fragment of GFP-SLIM1 was also
detected (Fig. 2D). We also investigated other signaling pathways that may regulate the localization of SLIM1. To determine the
effect of growth factor stimulation, HA-SLIM1 transiently transfected
Sol8 myoblasts were starved of serum for 18 h and then plated,
still in serum-free conditions, on fibronectin. In the majority
(>80%) of serum-starved cells plated on fibronectin, HA-SLIM1
localized strongly to the nucleus and to focal adhesions (Fig.
2E). There was no significant difference between the
localization of HA-SLIM1 in serum-starved vs. serum-treated myoblasts.
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To further investigate whether specific integrin receptors mediate
SLIM1 intracellular localization, HA-SLIM1-transfected myoblasts were
plated on laminin, collagen, or poly-L-lysine. In
myoblasts, laminin and collagen activate predominantly
7
1- and
1
1-integrins, respectively, distinct from
4
1- and
5
1-integrins activated by fibronectin
(15, 18, 23). Conversely, poly-L-lysine nonspecifically inhibits integrin activation and thereby blocks focal
adhesion formation (3). HA-SLIM1 localized to the nucleus, focal adhesions, and actin stress fibers in Sol8 myoblasts plated on
either laminin or collagen (Fig.
3A). Therefore, the
localization of SLIM1 in the nucleus and focal adhesions is not
dependent on activation of a specific integrin receptor, although we
cannot exclude cross-talk between the various integrins. On
poly-L-lysine, HA-SLIM1 was diffusely localized in the
cytosol and was not detected in the nucleus in the majority of
transfected cells (Fig. 3, A and B). After
plating on poly-L-lysine, there was complete inhibition of
stress fiber and focal adhesion formation, as shown by costaining with
phalloidin (Fig. 3B). In some cells plated on
poly-L-lysine, HA-SLIM1 concentrated in a perinuclear ring
(Fig. 3B). Colocalization with propidium iodide confirmed
that HA-SLIM1 was not localized in the nucleus in myoblasts plated on
poly-L-lysine (see merged image in Fig. 3B). The
localization of HA-SLIM1 in myoblasts plated on
poly-L-lysine was significantly different from that
demonstrated in myoblasts plated on the integrin ligands fibronectin,
laminin, or collagen. To quantitate this difference, in three separate experiments, transfected cells plated on either fibronectin or poly-L-lysine were counted for the presence of HA-SLIM1 at
focal adhesions and/or in the nucleus. The proportion of transfected cells demonstrating HA-SLIM1 localization at focal adhesions was decreased significantly in the myoblasts plated on
poly-L-lysine compared with myoblasts plated on fibronectin
(11 vs. 79%, respectively; Fig. 3C). This is consistent
with previous observations that poly-L-lysine, by
inhibiting integrin activation, inhibits focal adhesion formation (3). In addition, plating on poly-L-lysine
compared with fibronectin significantly decreased the proportion of
myoblasts demonstrating HA-SLIM1 in the nucleus (22 vs. 80%,
respectively; Fig. 3C). Collectively, these results suggest
that integrin activation is required for SLIM1 localization both in the
nucleus and at focal adhesions.
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The main receptors for fibronectin in myoblasts are
5
1- and
4
1-integrins. However, because activation
of
5
1 by fibronectin can induce protein
kinase C (PKC)-mediated "inside-out" activation of
4
1,
5
1
ligand binding was blocked, and SLIM1 localization was determined
(18). The
5
1-integrin is
activated by the RGD sequence of fibronectin; however, free RGD peptide
in solution blocks binding of fibronectin to
5
1 and subsequent
5
1 activation (39, 48).
Integrin ligand binding and subsequent integrin activation result in
integrin clustering and the recruitment of signaling and actin-binding
proteins to form focal adhesions, whereby clustered integrins form a
link between actin stress fibers and the extracellular matrix
(14). Therefore, focal adhesion formation is a marker of
integrin activation. Inhibition of integrin activation by RGD was
confirmed by demonstrating inhibition of focal adhesion formation. In
myoblasts plated on fibronectin, there was extensive focal adhesion
formation, as demonstrated by paxillin localization to focal adhesions
(Fig. 4A).
However, in myoblasts plated on fibronectin in the presence of 50 µg/ml RGD in the media, paxillin staining at focal adhesions
decreased, and in the presence of 500 µg/ml RGD was not detected,
indicating that focal adhesion formation and hence integrin activation
were inhibited (Fig. 4A; see Ref. 9). This
higher RGD concentration is equivalent to that previously shown to
inhibit focal adhesion formation in myoblasts, as measured by complete
inhibition of focal adhesion kinase (FAK) phosphorylation
(18). In the majority of Sol8 cells plated on fibronectin,
with 50 µg/ml RGD in the media, HA-SLIM1 localized diffusely in
the cytosol and was not detected in the nucleus (Fig. 4B).
The absence of nuclear HA-SLIM1 localization was confirmed by
counterstaining with propidium iodide.
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To quantitate the effect of RGD peptide blocking on the localization of SLIM1, the percentage of transfected Sol8 myoblasts plated on fibronectin in which HA-SLIM1 localized at focal adhesions or in the nucleus was determined in the absence of or presence of 50 or 500 µg/ml RGD in the media. In the absence of RGD, HA-SLIM1 localized to focal adhesions in 80% of transfected Sol8 cells plated on fibronectin (Fig. 4C). In contrast, with 50 µg/ml RGD, 31% of transfected cells and, with 500 µg/ml RGD in the media, only 5% of transfected cells plated on fibronectin demonstrated HA-SLIM1 localization to focal adhesions. The presence of RGD in the media resulted in a similar decrease in cells, demonstrating HA-SLIM1 localization in the nucleus. Without RGD in the media, HA-SLIM1 localized to the nucleus in 95% of transfected cells plated on fibronectin. However, HA-SLIM1 localized to the nucleus in 40% of transfected myoblasts with 50 µg/ml RGD and to the nucleus in only 11% of myoblasts with 500 µg/ml RGD in the media (Fig. 4C).
Integrin activation results in extensive remodeling of the actin
cytoskeleton with formation of focal adhesions and actin stress fibers.
To determine whether integrin-mediated nuclear localization of SLIM1 is
dependent on an intact cytoskeleton, HA-SLIM1-transfected myoblasts
were treated with cytochalasin D, which inhibits actin polymerization
and thereby disrupts the actin cytoskeleton (22). In
myoblasts plated on fibronectin and treated with cytochalasin D (2 µM), HA-SLIM1 remained in the nucleus despite disruption of the
cytoskeleton, as demonstrated by phalloidin staining (Fig.
5A).
HA-SLIM1 colocalized with propidium iodide in the nucleus of
cytochalasin D-treated cells. In addition, cytochalasin D treatment of
HA-SLIM1-transfected Sol8 cells plated on poly-L-lysine,
demonstrated that HA-SLIM1 remained localized diffusely in the cytosol
and did not relocalize to the nucleus (Fig. 5B). These
studies indicate that cytoskeletal sequestration is not the mechanism
via which SLIM1 is excluded from the nucleus in non-integrin-activated
myoblasts plated on poly-L-lysine. Furthermore, the
localization of SLIM1 was not dependent on intact microtubules. In Sol8
myoblasts, plated on fibronectin, nocodazole treatment disrupted the
microtubules, as demonstrated by -tubulin immunofluoresence; however, HA-SLIM1 remained localized in the nucleus and at focal adhesions (Fig. 5C). Therefore, nuclear localization of
SLIM1 in integrin-activated cells is not disrupted by dismantling of either the actin cytoskeleton or microtubules.
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SLIM1 demonstrated an integrin-mediated dual localization in the
nucleus and in the cytoplasm, specifically at focal adhesions and
stress fibers. Therefore, using a modified transient interspecies heterokaryon formation assay, we investigated SLIM1 cytoplasmic-nuclear shuttling (42). COS-7 cells were transiently transfected
with GFP-SLIM1 and plated on fibronectin. GFP-SLIM1 localized to the nucleus of transfected COS-7 cells (Fig.
6). Hoechst 33258 staining of COS-7 cells
demonstrated a diffuse nuclear staining pattern. In contrast,
untransfected mouse fibroblast NIH 3T3 cells demonstrated a
characteristic punctate nuclear staining with Hoechst 33258, with no
detectable GFP expression. The differential Hoechst staining of the
nuclei of the two distinct cell populations was used to differentiate
the COS-7 nuclei from the NIH 3T3 nuclei. The GFP-SLIM1-transfected COS-7 and untransfected NIH 3T3 cells were mixed and fused with 50%
PEG 6000 and then plated on fibronectin in the presence of cycloheximide to block further protein synthesis. In the fused cells,
GFP-SLIM1 was now detected in the nontransfected NIH 3T3 nuclei,
identified by their punctate nuclear Hoechst staining (Fig. 6). This
study indicates that GFP-SLIM1 had been exported from the COS-7 nuclei
into the cytoplasm and subsequently imported into the NIH 3T3 nuclei.
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At 32 kDa, SLIM1 is small enough to passively diffuse in and out of the
nucleus (16). The sequence of SLIM1 does not contain any
consensus nuclear localization or leucine-rich nuclear export signals
(7, 33). However, SLIM1 may be imported actively in and
exported from the nucleus by binding proteins, which themselves contain
nuclear import and/or export signals. In integrin-activated cells,
SLIM1 at focal adhesions may have been either actively exported from
the nucleus or translocated directly from the cytosol. To determine if
SLIM1 undergoes CRM-1-mediated nuclear export to focal adhesions after
integrin engagement, GFP-SLIM1-transfected cells plated on fibronectin
were plated in the presence or absence of leptomycin B. Previous studies have demonstrated that leptomycin B, a fungal
metabolite, inhibits CRM-1-dependent nuclear export (20).
In myoblasts plated on fibronectin, GFP-SLIM1 localized to the nucleus
and focal adhesions both in the presence and absence of leptomycin B
(10 ng/ml; Fig. 7A). This
localization was confirmed by colocalization with the focal adhesion
protein, paxillin, and propidium iodide in the nucleus. In control
experiments, leptomycin B blocked the nuclear export of GFP-Rev, which
contains a functional nuclear export sequence (Fig. 7B; see
Ref. 20). The finding that leptomycin B did not inhibit
the localization of SLIM1 at focal adhesions indicates that most of the
SLIM1 translocated to focal adhesions after integrin activation is
unlikely to have been actively exported from the nucleus and therefore
must translocate from the cytosol.
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Despite inhibition of nuclear export by leptomycin B, in the absence of integrin activation, SLIM1 did not accumulate in the nucleus. In non-integrin-activated cells plated on poly-L-lysine, SLIM1 is predominantly cytoplasmic. GFP-SLIM1 was localized diffusely in the cytoplasm of myoblasts plated on poly-L-lysine in both the presence and absence of leptomycin B (Fig. 7C). Therefore, in non-integrin-activated cells, SLIM1 is unlikely to be actively exported from the nucleus, and passive export of SLIM1, which at 32 kDa is small enough to pass through the nuclear pore, may occur.
Integrin-mediated focal adhesion formation and signaling to the
cytoskeleton regulate cell adhesion, spreading, and migration (14). The 5
1-integrin
mediates myoblast attachment and spreading on fibronectin
(18). To investigate the role SLIM1 plays in regulating
integrin-mediated cell adhesion, we determined the proportion of
GFP-SLIM1-expressing Sol8 myoblasts adhering to fibronectin, relative
to the overall transfection efficiency. If GFP-SLIM1 inhibited cell
adhesion, the percentage of adherent cells should be lower than the
transfection efficiency. Conversely, if GFP-SLIM1 promoted cell
adhesion, the percentage of adherent cells should be greater than the
transfection efficiency. These experiments were initially performed on
plates treated with 15 µg/ml fibronectin, the same concentration as
in the earlier experiments examining the intracellular localization of
SLIM1, and also at lower fibronectin concentrations (1 and 5 µg/ml)
previously used to demonstrate FAK-mediated inhibition of adhesion
(47). On plates treated with 15 µg/ml fibronectin, the
percentage of adherent GFP-SLIM1-transfected cells did not differ
significantly from the transfection efficiency (1.03 ± 0.22 relative to transfection efficiency), indicating that GFP-SLIM1 had no
effect on cell adhesion at this fibronectin concentration (Fig.
8). However, the proportion of
GFP-SLIM1-transfected myoblasts adhering to plates treated with 1 or 5 µg/ml fibronectin was reduced significantly relative to the GFP-SLIM1
transfection efficiency (0.23 and 0.29, respectively, relative to
transfection efficiency; Fig. 8). In control experiments, the
percentage of adherent GFP-empty vector-transfected cells was the same
as the transfection efficiency of the GFP-empty vector on all
fibronectin concentrations, confirming that GFP itself had no effect on
cell adhesion. Therefore, SLIM1 inhibits myoblast adhesion at lower
fibronectin concentrations (1 and 5 µg/ml) but not at high
fibronectin concentrations (15 µg/ml), suggesting the increased
strength of the integrin-ligand interaction at higher fibronectin
concentrations may be resistant to the effects of SLIM1 overexpression.
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To determine whether SLIM1 regulates cell spreading, Sol8 myoblasts
were transiently transfected with GFP-empty vector or GFP-SLIM1 and
plated on fibronectin for 1, 2, or 3 h (Fig.
9A). At each time point, the
percentage of spread cells, defined as cells in which the surface area
of the cytoplasm was at least two times the surface area of the
nucleus, was determined. At 1 h, 54% of GFP-SLIM1-transfected
cells were spread, compared with 27% of GFP-empty vector-transfected
cells and at 2 h 68 vs. 37%, and at 3 h 75 vs. 42% (Fig.
9B), respectively. Therefore, at each time point, GFP-SLIM1
significantly enhanced myoblast spreading.
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The effect of GFP-SLIM1 overexpression on cell migration was also
investigated using a modified Transwell migration assay (34). Sol8 myoblasts transiently transfected with either
GFP-SLIM1 or GFP-empty vector were plated in the upper chamber of a
Transwell plate on a cell-permeable membrane and incubated for 12 h. The lower surface of the membrane was coated with fibronectin. The relative proportion of GFP-SLIM1-transfected cells migrating to the
lower chamber (migrating cells) was compared with cells remaining in
the upper chamber (nonmigrating cells). The proportion of
GFP-SLIM1-transfected cells in the migrating population was twofold
greater than in the nonmigrating cells, suggesting SLIM1 promotes
myoblast migration on fibronectin (Fig.
10). In control experiments, the
proportion of GFP-empty vector-transfected cells was the same in the
migrating and nonmigrating cells, confirming that GFP had no effect on
cell migration. In addition, virtually no cells migrated if the
permeable membrane was coated with poly-L-lysine,
confirming that the migration of GFP-SLIM1-expressing cells was
mediated by integrin activation (data not shown).
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DISCUSSION |
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SLIM1 is a member of an emerging family of FHL domain proteins. SLIM1 is implicated in the pathogenesis of cardiomyopathy, as suggested by recent studies showing SLIM1 is significantly downregulated in human dilated cardiomyopathy and upregulated in hypertrophy (12, 28, 49). However, despite these studies, little is known about the function of SLIM1 in muscle. The studies reported here demonstrate that overexpression of SLIM1 promotes myoblast spreading and migration, suggesting that SLIM1 regulates integrin-dependent myoblast cytoskeletal rearrangement.
We have shown an integrin-dependent localization of SLIM1 in the nucleus and cytoskeleton, specifically at focal adhesions and stress fibers. In previous studies, we reported that SLIM1 localized diffusely to the cytosol with low-level nuclear expression in Sol8 myoblasts, and in COS-7 cells SLIM1 localized to the nucleus and focal adhesions in a variable manner (7). The activation status of integrin receptors in this previous study may account for the observed difference in intracellular localization to that reported here.
Inhibition of integrin activation, either by soluble RGD peptide or nonspecifically by plating on poly-L-lysine, caused SLIM1 to be localized diffusely in the cytoplasm in the majority of myoblasts. The localization of SLIM1 at focal adhesions is likely to be mediated by one or more of its LIM domains, since SLIM1 does not contain any other protein-binding motifs. This has been well described for other LIM domain proteins. The LIM domains of paxillin and zyxin mediate focal adhesion localization (5, 35). Serine phosphorylation of LIM3 of paxillin, by an associated kinase, promotes paxillin localization to focal adhesions and promotes spreading (5).
The results presented here suggest a model in which SLIM1 focal adhesion localization is dependent on translocation from the cytosol rather than nuclear-cytoskeletal translocation, since inhibition of nuclear export using leptomycin B did not inhibit the localization of SLIM1 at focal adhesions. Therefore, nuclear SLIM1 is unlikely to be actively transported to the cytoplasm or cytoskeleton; however, we cannot exclude the possibility that a small pool of SLIM1 shuttles between the nucleus and focal adhesions. SLIM1 nuclear localization is regulated by integrin signaling. However, the protein sequence of SLIM1 does not contain any consensus nuclear import sequences, unlike its spliced isoform SLIMMER (7). Therefore, the nuclear import of SLIM1 is likely to be regulated by protein-binding partners, which move with SLIM1 into the nucleus, as has been shown for other LIM domain-containing proteins. For example, the LIM domains of Ajuba mediate nuclear localization although they do not contain a recognizable nuclear localization signal (26). The nuclear localization of SLIM1 suggests it may, like other related FHL proteins, regulate transcription factors, although this has yet to be shown (19, 30).
We have demonstrated that SLIM1 regulates integrin-mediated
cellular functions. SLIM1 overexpression may interfere with normal myoblast adhesion. Furthermore, increased SLIM1 promoted myoblast spreading and migration. Integrin signaling regulates muscle migration, proliferation, and differentiation (18). Myoblast
adhesion, spreading, and migration on fibronectin are regulated
specifically by the 5
1-integrin (4,
18). Overexpression of activated
5
1 increases myoblast adhesion but
inhibits migration (4). Cellular migration is optimized
when integrins are activated intermediately, since both weak and strong
integrin interactions lead to decreased rates of migration (10,
37). SLIM1 is unlikely to perform a stabilizing or structural
role within focal adhesions because promotion of strong focal adhesion
interactions with the extracellular matrix or with the actin
cytoskeleton inhibits cell migration. For example, overexpression of
any one of the structural focal adhesion proteins, such as
-actinin,
vinculin, or talin, decreases muscle cell migration (21, 40,
36). We have noted that overexpression of SLIM1 has similar
effects on cell spreading and migration, as has been shown for
overexpression or activation of kinases such as PKC or FAK
(47). Activation of specific PKC isoforms inhibits
adhesion and promotes spreading and migration (13, 34,
43). Furthermore, in a manner analogous to SLIM1, PKC-
translocates from the cytosol to both the nucleus and focal adhesions upon integrin activation of vascular smooth muscle cells
(24). Targeted deletion of FAK leads to increased focal
adhesion formation and decreased cell motility, whereas overexpression
of FAK promotes cell migration (8, 25). SLIM1 may
therefore have a similar dynamic regulatory role in focal adhesions,
possibly by linking a kinase or phosphatase with the cytoskeleton.
SLIM1 regulation of integrin signals to the cytoskeleton, and in the
nucleus, may contribute to the pathogenesis of cardiomyopathy and
cardiac failure. SLIM1 mRNA levels are decreased significantly in
dilated cardiomyopathy and increased in hypertrophic cardiomyopathy (12, 28, 49). In both dilated and hypertrophic
cardiomyopathy, cardiac muscle fibers are subject to altered tensile
forces (38, 45). Integrins are significant
mechanotransducers relaying signals between the extracellular matrix
and the intracellular cytoskeleton, and integrin receptor patterns are
altered in cardiomyopathy (45). Overexpression of
1-integrin results in a hypertrophic phenotype in vitro,
whereas inhibition of
1-integrin abrogates the
hypertrophic response to adrenergic stimulation (38, 41).
Pressure overload on the ventricle results in altered downstream
integrin signaling with activation and cytoskeletal recruitment of
kinases and adaptor proteins, including FAK, c-Src, and
p130Cas, and activation of ERK1/2
(32).
We have demonstrated that SLIM1 regulates integrin-mediated events,
including myoblast spreading and migration. SLIM1 may therefore
contribute to integrin-mediated hypertrophic signaling in the
myocardium. Interestingly, although targeted deletion of the
SLIM1-related FHL domain protein DRAL/FHL2/SLIM3 results in normal
cardiac development and function, DRAL knockout mice develop an
exaggerated cardiac hypertrophy in response to -adrenergic stimulation (11, 27). This suggests that DRAL may modify
the hypertrophic response in the heart via its direct interactions with
integrin receptors and in the nucleus with transcription factors. It is
noteworthy that, unlike SLIM1, DRAL mRNA levels are not altered in
human and mouse models of cardiomyopathy, suggesting both family
members may play distinct roles in cardiac function (12,
28). Moreover, both DRAL and FHL3, with the notable exception of
SLIM1, have been shown to be downstream transmitters of Rho-specific signals, implicating SLIM1 involvement in signaling pathways distinct from its other family members (31). Collectively, these
studies suggest that SLIM1 plays a significant regulatory role in
integrin-mediated signaling, and recent studies predict that this has
functional consequences in both hypertrophic and dilated cardiomyopathy.
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ACKNOWLEDGEMENTS |
---|
We thank Lisa Ooms, Mike De Niese, Tony Tiganis, and Mark Lamm for helpful discussions and technical advice.
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FOOTNOTES |
---|
* P. A. Robinson and S. Brown contributed equally to this work.
This work was funded by grants from the National Health and Medical Research Council of Australia. S. Brown is the recipient of a National Heart Foundation postdoctoral fellowship. M. McGrath is the recipient of a National Heart Foundation postgraduate scholarship.
Address for reprint requests and other correspondence: C. A. Mitchell, Dept. of Biochemistry and Molecular Biology. Monash Univ., Wellington Rd., Clayton, VIC, Australia, 3168 (E-mail: christina.mitchell{at}med.monash.edu.au).
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.
First published October 23, 2002;10.1152/ajpcell.00370.2002
Received 15 August 2002; accepted in final form 17 October 2002.
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