Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion, spreading, and migration

Paul A. Robinson*, Susan Brown*, Meagan J. McGrath, Imogen D. Coghill, Rajendra Gurung, and Christina A. Mitchell

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3168


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha 5beta 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-beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 4beta 1- and alpha 5beta 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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Fig. 1.   Endogenous skeletal muscle LIM protein 1 (SLIM1) localizes to the nucleus and focal adhesions in rat primary myoblasts. Primary rat myoblasts were plated on slides treated with fibronectin (15 µg/ml) for 3 h. Indirect immunofluorescence was performed using affinity-purified anti-SLIM1 (a) or preimmune serum (b). Myoblasts were costained with anti-SLIM1 (c) and phalloidin (d) with the merged image (e). Colocalization was also performed with anti-SLIM1 (f) and propidium iodide (g) with the merged image (h). Arrows indicate SLIM1 localization at stress fibers and focal adhesions. Scale bar represents 20 µm.

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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Fig. 2.   Recombinant hemaglutinin (HA)-SLIM1 and green fluorescent protein (GFP)-SLIM1 localize to the nucleus and focal adhesions in Sol8 myoblasts plated on fibronectin. A: Sol8 myoblasts transfected with HA-SLIM1, GFP-SLIM1, or HA-empty vector were plated on slides treated with fibronectin (15 µg/ml) for 3 h. HA-SLIM1 and HA were detected using anti-HA antibodies and visualized by confocal microscopy. Arrows indicate SLIM1 localization at focal adhesions. Scale bar represents 20 µm. B: HA-SLIM1-transfected myoblasts were costained with phalloidin or propidium iodide, as indicated, and colocalization is shown in the merged images. Scale bar represents 20 µm. C: Sol8 lysates from untransfected (lane 1), HA-empty vector-transfected (lane 2), or HA-SLIM1-transfected (lane 3) myoblasts were analyzed by 12.5% SDS-PAGE and immunoblotted with anti-HA antibodies. The migration of molecular mass markers is shown on left. D: Sol8 lysates from untransfected (lane 1), GFP-empty vector-transfected (lane 2), or GFP-SLIM1-transfected (lane 3) myoblasts were analyzed by 12.5% SDS-PAGE and immunoblotted with anti-GFP antibodies. E: HA-SLIM1-transfected Sol8 myoblasts were serum starved for 18 h and plated on fibronectin for 3 h in the absence of serum (-FCS). Alternatively, HA-SLIM1-transfected myoblasts were grown in the presence of 10% FCS for 18 h and plated on fibronectin for 3 h with FCS (+FCS). Cells were stained using anti-HA antibodies and visualized by confocal microscopy. Bars = 20 µm.

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 alpha 7beta 1- and alpha 1beta 1-integrins, respectively, distinct from alpha 4beta 1- and alpha 5beta 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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Fig. 3.   Integrin activation mediates HA-SLIM1 localization to the nucleus and focal adhesions. A: HA-SLIM1-transfected Sol8 myoblasts were plated for 3 h on laminin, collagen, or poly-L-lysine, as indicated. Cells were fixed and stained with anti-HA antibodies and visualized by confocal microscopy. Scale bar represents 20 µm. B: HA-SLIM1-transfected Sol8 myoblasts plated on poly-L-lysine for 3 h were fixed and labeled with anti-HA antibodies and costained with phalloidin or propidium iodide, as indicated and visualized by confocal microscopy. Colocalization with SLIM1 indicated by yellow staining in the merged image. Scale bar represents 20 µm. C: HA-SLIM1-transfected myoblasts were plated on fibronectin or poly-L-lysine and scored for the presence of HA-SLIM1 at focal adhesions, or in the nucleus. Data represent the percentage of HA-SLIM1-expressing cells demonstrating HA-SLIM1 in focal adhesions (shaded bars) or in the nucleus (hatched bars) on fibronectin vs. poly-L-lysine as indicated. Each bar represents the mean ± SE for 3 independent transfections in which 600 cells were scored.

The main receptors for fibronectin in myoblasts are alpha 5beta 1- and alpha 4beta 1-integrins. However, because activation of alpha 5beta 1 by fibronectin can induce protein kinase C (PKC)-mediated "inside-out" activation of alpha 4beta 1, alpha 5beta 1 ligand binding was blocked, and SLIM1 localization was determined (18). The alpha 5beta 1-integrin is activated by the RGD sequence of fibronectin; however, free RGD peptide in solution blocks binding of fibronectin to alpha 5beta 1 and subsequent alpha 5beta 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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Fig. 4.   RGD peptide inhibition of alpha 5beta 1-integrin activation decreases HA-SLIM1 localization in the nucleus and focal adhesions. A: Sol8 myoblasts were plated on slides treated with fibronectin (15 µg/ml) either without (-RGD) or with the indicated concentration of soluble RGD peptide (+RGD) in the media for 3 h. Sol8 myoblasts were fixed and labeled with anti-paxillin antibodies to detect focal adhesion formation. B: HA-SLIM1-transfected Sol8 myoblasts were pretreated with RGD peptide (50 µg/ml) for 20 min and then plated on fibronectin (15 µg/ml) for 3 h in the presence of RGD (50 µg/ml) in the media. HA-SLIM1-transfected cells were fixed, labeled with anti-HA, and costained with phalloidin or propidium iodide, and colocalization is shown in the merged images, indicated by yellow staining. Scale bar represents 20 µm. C: HA-SLIM1-transfected myoblasts were plated on fibronectin in the absence or presence of RGD peptide (50 or 500 µg/ml) as indicated, and the percentage of transfected cells demonstrating HA-SLIM1 in focal adhesions, or in the nucleus, was determined. Data represent the percentage of transfected cells demonstrating HA-SLIM1 in focal adhesions (shaded bars) or in the nucleus (hatched bars). Bars represent means ± SE of 3 independent transfections in which 200 cells, both with or without RGD, were scored.

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 beta -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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Fig. 5.   Disruption of either the actin cytoskeleton or microtubules does not inhibit SLIM1 nuclear localization. HA-SLIM1-transfected Sol8 myoblasts were plated on fibronectin (A) or poly-L-lysine (B) for 2 h and then treated with cytochalasin D (2 µM) for 1 h to disrupt the actin cytoskeleton before fixation. HA-SLIM1-transfected Sol8 myoblasts were stained with anti-HA antibody. Cells were costained with phalloidin and propidium iodide as indicated and visualized by confocal microscopy. Colocalization is shown in the merged images as yellow staining. Scale bar represents 20 µm. C: HA-SLIM1-transfected Sol8 myoblasts were plated on fibronectin for 2 h and then left untreated (-nocodazole) or were treated with nocodazole (10 µg/ml; +nocodazole) for 2 h to disrupt the microtubules before fixation. HA-SLIM1-transfected Sol8 myoblasts were stained with anti-HA antibody. Cells were costained with anti-beta -tubulin antibody and propidium iodide as indicated and visualized by confocal microscopy. Scale bar represents 20 µm.

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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Fig. 6.   GFP-SLIM1 shuttles into the nucleus. A modified heterokaryon fusion assay was performed. GFP-SLIM1-transfected COS-7 and untransfected NIH 3T3 cells were costained with Hoechst 33258, and nuclear staining was visualized with fluorescence microscopy. Threefold excess of untransfected NIH 3T3 cells was mixed with GFP-SLIM1-transfected COS-7 cells, in the presence of polyethylene glycol (PEG) 6000, to promote plasma membrane fusion of the two cell populations in the presence of cycloheximide (100 µg/ml), and fused cells were visualized by fluorescence microscopy.

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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Fig. 7.   Leptomycin B inhibition of nuclear export does not alter the localization of GFP-SLIM1 in Sol8 myoblasts. A: GFP-SLIM1-transfected Sol8 myoblasts were plated on fibronectin for 3 h in the absence (-Lep B) or presence (+Lep B) of leptomycin B (10 ng/ml). Cells were fixed and counterstained with anti-paxillin antibodies, or propidium iodide, as indicated. Colocalization is shown by the yellow staining in the merged image. Scale bar represents 20 µm. B: GFP-Rev-transfected Sol8 myoblasts were plated in the absence (-Lep B) or presence (+Lep B) of leptomycin B (10 ng/ml), fixed, and visualized by confocal microscopy. C: GFP-SLIM-transfected myoblasts were plated on poly-L-lysine in the absence or presence of leptomycin B (10 ng/ml), fixed, and costained with propidium iodide as indicated and visualized by confocal microscopy. Colocalization is shown by yellow staining in the merged image.

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 alpha 5beta 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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Fig. 8.   Overexpression of GFP-SLIM1 inhibits Sol8 myoblast adhesion. GFP-SLIM1 or GFP-empty vector-transfected Sol8 myoblasts were plated on 1, 5, or 15 µg/ml fibronectin for 15 min, and the percentage of transfected adherent cells was counted and compared with the overall transfection efficiency for each construct. Data represent the percentage of GFP-empty vector (shaded bars)- vs. GFP-SLIM1-transfected cells (filled bars) adhering relative to the transfection efficiency for each construct at each fibronectin concentration, as indicated. Bars represent means ± SE of 3 separate transfection experiments and adhesion assays in which 600 cells were counted.

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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Fig. 9.   Overexpression of GFP-SLIM1 in enhanced Sol8 myoblast cell spreading. A: GFP-empty vector- or GFP-SLIM1-transfected cells were plated on fibronectin (15 µg/ml) for 1, 2, or 3 h and visualized by confocal microscopy, and the percentage of spread cells was determined for each of the constructs at the 3 time points. Cells were counted as spread if the area of the cytosol was greater than or equal to two times the area of the nucleus. A: representative GFP-empty vector- and GFP-SLIM1-transfected cells at 1 and 3 h after plating, imaged by confocal microscopy. B: data indicate the percentage of GFP-empty vector (shaded bars)- vs. GFP-SLIM1 (filled bars)-transfected myoblasts that have spread at each of the indicated time points. Bars represent the means ± SE of 3 independent transfection and spreading assays in which 200 cells were counted at each time point.

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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Fig. 10.   Overexpression of GFP-SLIM1 promotes cell migration. GFP-empty vector- or GFP-SLIM1-transfected cells were plated in the upper chamber of a Transwell micropore well and allowed to migrate through a porous membrane to a lower chamber. The cells on either side of the porous membrane were trypsinized, and the percentage of transfected cells that had migrated to the lower chamber (migrating cells) and the amount of transfected cells remaining in the upper chamber (nonmigrating cells) were determined. Data show the percentage of transfected cells, relative to the percentage of transfected nonmigrating cells for each construct, for the nonmigrating cells and migrating cells, as indicated. The GFP-empty vector-transfected cells are represented by the shaded bars, and the GFP-SLIM1-transfected cells are represented by the filled bars. Bars represent means ± SE of 3 independent transfection and migration assays in which 500 cells were counted.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 5beta 1-integrin (4, 18). Overexpression of activated alpha 5beta 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 alpha -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-alpha 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 beta 1-integrin results in a hypertrophic phenotype in vitro, whereas inhibition of beta 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 beta -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.


    ACKNOWLEDGEMENTS

We thank Lisa Ooms, Mike De Niese, Tony Tiganis, and Mark Lamm for helpful discussions and technical advice.


    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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