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Am J Physiol Heart Circ Physiol 278: H1736-H1743, 2000;
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Vol. 278, Issue 6, H1736-H1743, June 2000

beta 1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal alpha -actin promoter in myoblasts

Lei Wei, Wei Zhou, Lu Wang, and Robert J. Schwartz

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c-fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal alpha -actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal alpha -actin promoter activity in myoblasts. We found that extracellular matrix proteins and the beta 1-integrin stimulated RhoA-dependent activation of the alpha -actin promoter. The muscle-specific isoform beta 1D selectively activated the alpha -actin promoter in concert with RhoA but inhibited the c-fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the alpha -actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited alpha -actin promoter activity induced by RhoA. These results suggest that RhoA, beta 1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

RhoA signaling; beta 1D-integrin; focal adhesion kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SMALL GTPase RhoA is involved in many actin-based cellular processes including cell adhesion, cytokinesis, stress fiber formation, and smooth muscle contractility (reviewed in Refs. 14 and 37). RhoA also modulates transcriptional activity of serum response factor (SRF), a homodimeric MADS box-containing protein (17). SRF has been shown to be required for expression of the skeletal and cardiac alpha -actin genes and smooth muscle alpha - and gamma -actin genes, which are among the earliest markers for mesoderm-derived skeletal, cardiac, and smooth muscle differentiation (4, 8, 28). We have previously shown that RhoA activates skeletal alpha -actin transcription in C2C12 myoblasts through SRF and that stable expression of a dominant negative mutant of RhoA reduces terminal differentiation, in part, through its effect on SRF activity (40). A recent report has also shown that inhibition of Rho family proteins by the GDP dissociation inhibitor RhoGDI suppresses myogenesis in C2C12 myoblasts (34).

One way to investigate the mechanisms by which RhoA is involved in myogenesis is to determine whether RhoA interacts with signal pathways previously shown to be important in muscle differentiation. RhoA has been shown to function both upstream and downstream of integrins in the context of focal adhesion formation and stress fiber assembly (reviewed in Ref. 5). All integrins are heterodimeric transmembrane receptors consisting of an alpha -subunit associated with a beta -subunit and mediating association between extracellular matrix (ECM) and cytoskeleton elements. Experimental evidence for the importance of integrin interaction with ECM proteins during muscle differentiation was elucidated in fibronectin null mice, which are defective in forming somites (10). Myotube formation and myogenic differentiation were also blocked in myoblast cultures treated with antibodies to integrins (25). In addition, mice lacking focal adhesion kinase (FAK), an important mediator of integrin signaling, displayed a general defect in mesoderm formation, a phenotype similar to that observed in fibronectin-deficient mice (18). Moreover, levels of expression of several beta 1-integrins such as alpha 4beta 1, alpha 5beta 1, and alpha 7beta 1 and their isoforms such as alpha 7A, alpha 7B, beta 1A, and beta 1D are developmentally regulated in skeletal muscle and heart (reviewed in Ref. 13), suggesting that integrin switches may be involved in myogenesis and heart development. In contrast to the common beta 1A-isoform that is widely expressed and prominent in prefusion myoblasts, the beta 1D-isoform is restricted to skeletal muscle and heart and becomes the major isoform in these tissues (3, 38), suggesting that it may have an important function in these tissues.

Another potential signaling pathway interacting with RhoA during muscle differentiation is signaling through phosphatidylinositol (PI) 3-kinase. Recent studies have shown that PI 3-kinase plays an important role in muscle differentiation and is required for insulin-like growth factor (IGF)-induced muscle differentiation (19-21). In addition, integrin-mediated signaling pathways also activate PI 3-kinase that is translocated to the cytoskeleton, which involves specific interactions of p85 subunit with actin filament and FAK (12). Moreover, PI 3-kinase functions downstream of RhoA in platelet and Swiss 3T3 cells (22, 42).

We examined whether beta 1-integrin-, FAK-, and PI 3-kinase-mediated myogenic signaling pathways cross talk with the RhoA signaling pathway to regulate SRF-dependent muscle gene expression such as skeletal alpha -actin promoter activity. In the present study, we found that ECM proteins and the beta 1-integrin affect RhoA-dependent activation of the skeletal alpha -actin in myoblasts. Interestingly, the muscle-specific beta 1D-integrin potentiates RhoA-dependent activation of the skeletal alpha -actin promoter and inhibits RhoA-dependent activation of the c-fos promoter, suggesting that RhoA regulates these two SRF-dependent promoter activities through different signaling pathways. Moreover, FAK and PI 3-kinase are required for RhoA-dependent activation of the alpha -actin promoter. A dominant negative mutant of FAK [FAK-related non-kinase (FRNK)] inhibits activation of the alpha -actin promoter by RhoA. Both wortmannin and a dominant negative mutant of PI 3-kinase (Delta p85) inhibit alpha -actin promoter activity induced by RhoA. In addition, RhoA increases PI 3-kinase activity associated with exogenous p85 subunit. These results suggest that RhoA, beta 1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle-specific gene expression.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid constructs. The reporter plasmid SK-luc contains the avian skeletal alpha -actin promoter from -398 to +25 bp linked to the firefly luciferase reporter gene (24). The reporter plasmid c-fos-SRE-luc contains the c-fos serum response element (SRE) region (-318 to -291 bp) (24). The expression plasmids pCGN-RhoA and pCGN-V14-RhoA were constructed as previously described (40). The cDNA constructs for full-length avian FAK and the truncated COOH-terminal FAK (FRNK) were cloned into the pCMV-Myc vector and were kindly provided by Dr. J. Thomas Parsons (31). The expression plasmids pCMVIL2R, pCMVIL2Ralpha 5, and pCMVIL2Rbeta 1 containing the human interleukin-2 receptor extracellular and transmembrane domains in combination with the intracellular domain for either alpha 5- or beta 1-integrin were kindly provided by Drs. Susan LaFlamme and Kenneth Yamada (23). The full-length cDNA constructs for human beta 1A- and beta 1D-integrins cloned into the pECE vector were the kind gift of Dr. Alexey Belkin (3). The expression plasmids SRalpha -p85 and SRalpha -Delta p85, containing the wild type (p85) and the mutant bovine p85alpha -subunit lacking the binding site for the p110 catalytic subunit of PI 3-kinase (Delta p85), respectively, were kindly provided by Dr. Masato Kasuga (15).

Tissue culture, plasmid DNA transfection, and reporter gene assays. C2C12 mouse myoblasts (27) were maintained in DMEM (GIBCO-BRL) with 10% fetal bovine serum (FBS). Cells were plated at a density of 3 × 105 cells in 60-mm tissue culture plates and were transfected after 24 h. Cells were transfected with ~1 µg of total plasmid DNA containing the indicated reporter plasmid (0.1 µg of SK-luc or c-fos-SRE-luc) with expression plasmids and balanced with parental expression vector. Transfections were performed using lipofectamine (GIBCO-BRL) according to the manufacturer's instructions. Cells were placed in DMEM with 10% FBS and harvested 48 h posttransfection. Luciferase activity and protein content were measured as previously described (24). Luciferase activity was normalized to the total protein, and data were expressed as luciferase activity normalized to baseline reporter gene. All experiments were performed in duplicate and repeated three to five times. For experiments evaluating effects of ECM proteins on skeletal alpha -actin promoter activity, plates were coated overnight with rat tail collagen, fibronectin (20 µg/ml), and poly-L-lysine (10 µg/ml) and then blocked with 2% BSA for 2 h before plating. For experiments evaluating PI 3-kinase activity, cells were placed in medium with or without wortmannin (1 µM) and harvested 48 h posttransfection. The medium was replaced every 8 h because of the instability of wortmannin.

Protein expression analysis. Expression levels of cDNA expression plasmids were examined by Western blot analysis. Myoblasts on 60-mm plates were transiently transfected with 2 µg of each expression plasmid using lipofectamine. After 48 h, the cells were solubilized with lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 100 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 50 µg/ml antipain) for 30 min with shaking at 4°C. Cell lysates were then cleared by centrifugation. In the detection of Myc-tagged FAK and FRNK, whole cell protein extracts (50 µg) were run on an 8% SDS-PAGE gel, transferred to Immobilon membrane (Millipore), and probed with anti-Myc monoclonal antibody (Oncogene). In the detection of HA-tagged p85 or Delta p85, cell extracts (200 µg) were incubated with anti-HA monoclonal antibody (2 µg/ml) at 4°C for 1 h. Immunoprecipitates were then collected on protein A/G-Sepharose (Santa Cruz), washed three times with lysis buffer, and separated on an 8% SDS-PAGE gel. The blots were then probed with polyclonal anti-p85 antibodies (Upstate Biotechnology). Enhanced chemiluminescence (Amersham) was used as the detection system.

PI 3-kinase assay. HA-tagged p85 was immunoprecipitated from cell extracts of transiently transfected myoblasts as described above. PI 3-kinase activity was assayed by TLC as described previously (41). Briefly, immunoprecipitates were incubated with 50 µl of reaction buffer containing PI (20 µg), 20 mM Tris (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 50 µM ATP, and [gamma -32P]ATP (10 µCi) for 15 min with constant agitation at 37°C. The reaction was terminated by the addition of 20 µl of 6 N HCl, and lipids were extracted by the addition of 160 µl of CHCl3-CH3OH (1:1) and separated by TLC in CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2). The TLC plate was dried and autoradiographed, and radioactivity of 32P-labeled PI 3-phosphate spots was determined by PhosphorImaging analysis (Molecular Dynamics).

Statistics. Data are expressed as means ± SE, relative to values for parallel cultures of control vector-transfected cells. Student's t-test was used for data comparison, with a significance level of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ECM proteins regulate activation of the skeletal alpha -actin promoter by RhoA. We have shown that V14-RhoA, a constitutively active RhoA mutant, activates skeletal alpha -actin promoter up to seven times the basal level, whereas N19-RhoA, a dominant negative mutant, represses the basal level (control vector-transfected cells) up to 40% in a dose-dependent manner in myoblasts (40). We have shown that activation of the skeletal alpha -actin promoter by RhoA depends on an intact SRE and a functional SRF (40). To investigate the possible interactions between integrin and RhoA signaling pathways during muscle differentiation, we first examined the role of integrin-independent (poly-L-lysine) and -dependent (collagen and fibronectin) cell adhesion on skeletal alpha -actin promoter activity in concert with RhoA. As shown in Fig. 1, activation of the skeletal alpha -actin promoter by V14-RhoA was significantly increased in myoblasts attached to collagen (P = 0.046) or fibronectin (P = 0.007) but was decreased in cells attached to poly-L-lysine compared with myoblasts plated on uncoated dishes. These results indicate that certain ECM proteins may potentiate RhoA-mediated activation of the skeletal alpha -actin promoter.


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  		Fig. 1.   Extracellular matrix proteins enhance activation of skeletal alpha -actin promoter by RhoA. C2C12 myoblasts were plated onto uncoated dishes or dishes coated with poly-L-lysine (poly-Lys), collagen, or fibronectin. Transfection was carried out with 0.1 µg of SK-luc reporter plasmid together with 0.25 µg of control vector plasmid or expression plasmid encoding V14-RhoA. After 48 h, cells were harvested and assayed for luciferase activity. Luciferase activity was normalized to protein content in cell lysates. Values are means ± SE and are expressed relative to values for control vector transfected cells. The effect of each transfection dose on activity of the reporter gene was measured in duplicate or triplicate, and results indicated are representative of 3-5 independent experiments. *Significantly different from control vector-transfected cells (P < 0.05).

Disruption of beta 1-integrin signaling inhibits skeletal alpha -actin promoter activity stimulated by RhoA. Collagen and fibronectin interact with the beta 1-subfamily of integrins that have been shown to play a critical role in muscle differentiation (10, 25). We examined whether beta 1-integrin signaling mediates RhoA-dependent activation of the skeletal alpha -actin promoter. A chimeric protein (IL2R-beta 1) composed of the extracellular and transmembrane domains of interleukin-2 receptor and the cytoplasmic domain of beta 1A-integrin was used to disrupt beta 1-integrin signaling (Fig. 2A) (23). A similar construct (IL2R-alpha 5) containing the cytoplasmic domain of alpha 5-integrin and the expression vector served as controls. Our results show that IL2R-beta 1 significantly reduced the activity of the promoter in V14-RhoA-transfected cells (P = 0.027) with no effect on the basal activity of the promoter (P = 0.382). The noninteractive IL2R-alpha 5 control had no significant effect on the activity of the promoter in both V14-RhoA-transfected (P = 0.338) and control vector-transfected cells (P = 0.171) (Fig. 2B). Thus results of this experiment indicate that disruption of beta 1-integrin-mediated signaling interferes with RhoA-dependent activation of the skeletal alpha -actin promoter in myoblasts.


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  		Fig. 2.   RhoA-dependent activation of skeletal alpha -actin promoter is reduced by alteration of beta 1-integrin signaling. A: schematic diagram of beta 1- and alpha 5-integrin chimeras. IL2R, interleukin-2 receptor. B: C2C12 myoblasts were transfected with 0.1 µg of SK-luc together with 0.75 µg of control vector plasmid or 0.25 µg of V14-RhoA, 0.5 µg of IL2Rbeta 1, or 0.5 µg of IL2Ralpha 5 alone or in combination as indicated. Data are presented as described in Fig. 1 legend. *Significantly different from control vector-transfected cells (P < 0.05).

Muscle-specific beta 1D-isoform potentiates activation of the skeletal alpha -actin promoter by RhoA. The beta 1A- and beta 1D-integrin isoforms are very similar and differ by only 13 amino acids in their cytoplasmic tail (3, 38). We examined whether these two integrin isoforms have different regulatory roles in skeletal alpha -actin promoter activity. Expression of either of these integrins alone in myoblasts did not result in increased activity of the skeletal alpha -actin promoter. Coexpression of beta 1D-integrin, but not beta 1A-integrin, with V14-RhoA significantly increased RhoA-dependent activation of the SK-luc reporter plasmid (P = 0.034) (Fig. 3A), indicating a selective role in mediating RhoA signaling in regulating skeletal alpha -actin gene expression. It is worth noting that exogenous expression of beta 1A-integrin had no effect on the activation of the skeletal alpha -actin promoter by RhoA, whereas a dominant negative beta 1A-isoform inhibited this activation. These results suggest that the endogenous level of beta 1A-integrin is not a limiting factor for activation of the alpha -actin promoter by RhoA in myoblasts.


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  		Fig. 3.   Muscle-specific beta 1D-integrin isoform enhances activation of skeletal alpha -actin promoter by RhoA but inhibits activation of c-fos-SRE by RhoA. A: C2C12 myoblasts were transfected with 0.1 µg of SK-luc together with 0.75 µg of control vector plasmid or 0.25 µg of V14-RhoA, 0.5 µg of beta 1A-integrin, or 0.5 µg of beta 1D-integrin alone or in combination as indicated. B: transfection conditions were same as in A, except that c-fos-SRE-luc reporter plasmid was substituted for SK-luc plasmid. Data are presented as described in Fig. 1 legend. *Significantly different from control vector-transfected cells (P < 0.05).

We have previously observed that RhoA activates both skeletal alpha -actin and c-fos-SRE promoters in myoblasts (40). Interestingly, in contrast to its stimulatory effect on skeletal alpha -actin promoter activation by RhoA, beta 1D-integrin inhibited activation of c-fos-SRE by RhoA (P = 0.004) (Fig. 3B), indicating that RhoA regulates these two SRF-dependent promoter activities through different means.

FAK is required for skeletal alpha -actin promoter activity. We next examined whether FAK, an important downstream mediator of integrin signaling, would also be a mediator of RhoA signaling in skeletal alpha -actin promoter activation. A truncated mutant of FAK, FRNK (Fig. 4A), which contains the COOH-terminal domain of FAK without the kinase domain, was previously shown (31) to act as a negative inhibitor. Expression of FRNK alone reduced basal level of the skeletal alpha -actin promoter activity (P = 0.025). It also reduced activation of the alpha -actin promoter by V14-RhoA (P = 0.029) and coactivation by V14-RhoA and beta 1D-integrin (P = 0.026) (Fig. 4B), indicating that FAK may be required for efficient activation of the alpha -actin promoter. However, expression of exogenous FAK did not significantly stimulate the alpha -actin promoter in V14-RhoA-transfected (P = 0.427) and control vector-transfected cells (P = 0.469), perhaps indicating that the level of endogenous FAK might not be a limiting factor for the activation of the skeletal alpha -actin promoter under these conditions. Expression of exogenous FAK and FRNK was confirmed by Western analysis (Fig. 4C).


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  		Fig. 4.   A dominant negative mutant of focal adhesion kinase (FAK) reduces activation of skeletal alpha -actin promoter by RhoA. A: schematic diagram of FAK and FAK-related non-kinase (FRNK). B: C2C12 myoblasts were transfected with 0.1 µg of SK-luc together with 0.75 µg of control vector plasmid or 0.25 µg of V14-RhoA, 0.5 µg of FAK, or 0.5 µg of FRNK expression plasmid alone or in combination as indicated. Data are presented as described in Fig. 1 legend. *Significantly different from control vector-transfected cells (P < 0.05). C: expression of FAK and FRNK was analyzed by Western blot using monoclonal anti-Myc antibody.

PI 3-kinase is required for skeletal alpha -actin promoter activity. PI 3-kinase is another potential mediator of RhoA signaling in myogenesis. Two approaches were taken to determine whether PI 3-kinase is required for RhoA-mediated activation of the skeletal alpha -actin promoter. First, wortmannin was added to muscle cultures to inhibit PI 3-kinase activity (36). Second, PI 3-kinase activity was inhibited by expression of cotransfected plasmid expressing a dominant negative mutant of the p85 subunit (Delta p85) that lacks the binding site for the p110 catalytic subunit (15).

As shown in Fig. 5, 1 µM wortmannin significantly inhibited RhoA-mediated activation of the skeletal alpha -actin promoter. Another chemical inhibitor of PI 3-kinase, LY-294002, also was found to inhibit activation of the actin promoter by RhoA (data not shown). Because wortmannin may not be PI 3-kinase specific, we then evaluated the effect of Delta p85, a dominant negative PI 3-kinase mutant, on skeletal alpha -actin promoter activation (Fig. 6A). The expression plasmid SRalpha -Delta p85 was transfected into C2C12 myoblasts, and its effects were compared with the empty vector SRalpha and the expression plasmid SRalpha -p85 encoding the wild- type p85 (Fig. 6B). Expression of Delta p85 reduced the basal activity of skeletal alpha -actin promoter (P = 0.028) and the activity stimulated by V14-RhoA (P = 0.021), whereas the wild-type p85 had no significant effect on the promoter activity under these conditions (P = 0.125 or 0.345, respectively). Expression of these recombinant proteins was confirmed by Western blot analysis (Fig. 6C). These observations are consistent with the results obtained with wortmannin, indicating that PI 3-kinase may mediate RhoA-dependent activation of the skeletal alpha -actin promoter.


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  		Fig. 5.   Phosphatidylinositol (PI) 3-kinase inhibitor wortmannin inhibits activation of skeletal alpha -actin promoter by RhoA. C2C12 myoblasts were transfected with 0.2 µg of SK-luc together with 0.75 µg of control vector plasmid or varying amounts of V14-RhoA as indicated. After transfection, cells were placed in medium with or without 1 µM wortmannin. Data are presented as described in Fig. 1 legend. *Significantly different from control vector-transfected cells (P < 0.05).



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  		Fig. 6.   A dominant negative mutant of PI 3-kinase reduces activation of skeletal alpha -actin promoter by RhoA. A: schematic diagram of wild type (p85) and dominant negative mutant (Delta p85). B: C2C12 myoblasts were transfected with 0.1 µg of SK-luc together with 0.25 µg of V14-RhoA, 0.5 µg of p85, or 0.5 µg of Delta p85 expression plasmid alone or in combination as indicated. Data are presented as described in Fig. 1 legend. *Significantly different from control vector-transfected cells (P < 0.05). C: expression of p85 and Delta p85 was analyzed by immunoprecipitation of cell extracts using anti-hemagglutinin (HA) antibody followed by Western blot analysis using polyclonal anti-p85 antibodies.

Finally, to investigate the mechanism by which PI 3-kinase modulates RhoA activation of the skeletal alpha -actin promoter, we examined whether V14-RhoA increased PI 3-kinase activity in myoblasts. HA epitope-tagged p85 or Delta p85 was expressed with or without V14-RhoA in myoblasts. These exogenous proteins were then immunoprecipitated with anti-HA antibody, which does not affect catalytic activity of immunoprecipitated PI 3-kinase, and assayed for lipid kinase activity, which was normalized to the p85 content in the immunocomplex determined by Western blot analysis (Fig. 7A). The activity of PI 3-kinase activity associated with the wild- type p85 was significantly increased in the presence of V14-RhoA (P = 0.015, n = 3), whereas that associated with Delta p85 was not affected (Fig. 7B). One possible mechanism by which RhoA increases PI 3-kinase activity is to increase its association with FAK. However, by Western blot analysis, we found only a minor portion of the endogenous p85 subunit of PI 3-kinase associated with FAK (<10%), and this interaction was not increased by V14-RhoA. Similarly, PI 3-kinase activity coimmunoprecipitated with FAK was not increased by V14-RhoA. These observations suggest that PI 3-kinase might not be immediately downstream of FAK in regulating RhoA-dependent activation of the skeletal alpha -actin promoter.


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  		Fig. 7.   RhoA activates PI 3-kinase activity in myoblasts. A: C2C12 myoblasts were transfected with 2 µg of HA-p85 or HA-Delta p85 expression plasmid alone or together with 2 µg of HA-V14-RhoA expression plasmid. HA-p85 was immunoprecipitated from cell extracts and divided equally into 2 aliquots; 1 aliquot was assayed for PI 3-kinase activity by measuring 32P-labeled PI 3-phosphate level (PIP) by TLC (top), and the other aliquot was analyzed by Western blot for p85 expression level (middle) with anti-p85 antibody. V14-RhoA expression in cell extracts was also confirmed by Western blot with anti-HA antibody (bottom). B: PI 3-kinase activity was normalized to the p85 content in the immunocomplex. Values are means ± SE and are expressed relative to values for cells transfected with HA-Delta p85 without V14-RhoA. HA-Delta p85 is unable to associate with catalytic subunit. *Significantly different from HA-Delta p85-transfected cells (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Integrin-mediated signaling regulates RhoA-dependent activation of the skeletal alpha -actin promoter. We examined whether RhoA interacts with signaling pathways involved in muscle differentiation. The integrin-mediated signaling pathway was of particular interest because considerable amounts of evidence support a critical role for integrin-mediated signals in muscle differentiation, and functional interactions between RhoA and integrin signaling pathways have been extensively investigated in focal adhesion formation and stress fiber assembly. We showed several lines of evidence for functional interactions between RhoA and integrin-mediated signals in regulating muscle gene expression: 1) extracellular matrix proteins such as collagen and fibronectin enhanced RhoA-dependent activation of the skeletal alpha -actin promoter; 2) a dominant negative mutant of beta 1-integrin reduced activation of the alpha -actin by RhoA; and 3) the muscle-specific beta 1-integrin beta 1D-isoform was found to enhance RhoA-dependent activation of the skeletal alpha -actin promoter while repressing c-fos promoter activity.

In contrast to the common beta 1A-isoform, which is widely expressed, the beta 1D-isoform is restricted to skeletal muscle and heart and is the major isoform in these tissues, suggesting that it may have an important function in these tissues. Expression of beta 1D-isoform in C2C12 myoblasts represses myoblast replication, suggesting that beta 1D-isoform may play a role in myoblast withdrawal from the cell cycle (2). However, mice lacking the beta 1D-isoform (having only beta 1A-isoform) form normal mature muscle without obvious histological or ultrastructural abnormality (1). Our observation that beta 1D-isoform increased RhoA-dependent activation of the skeletal alpha -actin promoter, whereas beta 1A-isform was ineffectual, indicates selective functional interaction between RhoA and this muscle-specific isoform in regulating muscle-specific gene expression. Our finding that beta 1D-isoform inhibits activation of c-fos-SRE by RhoA indicates that RhoA may act through at least two different pathways to regulate skeletal alpha -actin and c-fos promoters.

FAK is required for efficient activation of the skeletal alpha -actin promoter. FAK is an important downstream signaling molecule of integrins, because it has been found to be the most prominent of the tyrosine-phosphorylated proteins in cells responding to integrin activation (reviewed in Ref. 5). Functional interactions between RhoA and FAK have been demonstrated in the formation of the focal adhesion complex (9). It has been proposed that RhoA and FAK interact through the clustering of integrin induced by RhoA activation that may thereby activate FAK (29). This may occur through a direct interaction, because FAK has recently been found to associate with Graf, a Rho GTPase-activating protein (16). However, a role for FAK in muscle differentiation has not been reported. The present study shows that FAK modulates RhoA-dependent activation of the skeletal alpha -actin promoter, suggesting a functional relationship between RhoA and FAK in regulating muscle-specific gene expressions in myoblasts. In primary cultured chicken myoblasts, we observed that FRNK reduced myosin heavy chain expression in mature myotubes (unpublished results), also supporting a role for FAK in myodifferentiation.

PI 3-kinase is required for efficient activation of the skeletal alpha -actin promoter. Recent reports have shown that PI 3-kinase plays a vital role in myogenesis (19-21). Functional interactions between RhoA and PI 3-kinase have been observed between RhoA and PI 3-kinase in platelet and Swiss 3T3 cells, where RhoA is required for PI 3-kinase activation in response to extracellular stimuli (22, 42). Our results demonstrate that PI 3-kinase may be a mediator of the RhoA signaling pathway in regulating muscle-specific gene expression. Wortmannin, a known inhibitor of PI 3-kinase, and a dominant negative mutant of the p85 subunit of PI 3-kinase both inhibited RhoA-dependent activation of the skeletal alpha -actin promoter. PI 3-kinase activity associated with cotransfected p85 was also increased by V14-RhoA in myoblasts.

The mechanism by which PI 3-kinase interferes with RhoA signaling remains to be determined. It has been suggested that PI 3-kinase is involved in the integrin-mediated signal pathway, because PI 3-kinase is activated following cell attachment to ECM proteins and is then translocated to the cytoskeleton, which involves specific interactions of p85 subunit with actin filament and FAK (12). However, we observed that only a minor portion of the endogenous p85 subunit of PI 3-kinase was associated with FAK (<10%), and this interaction was not increased by V14-RhoA, suggesting that RhoA may activate the PI 3-kinase signaling pathway through other mechanisms. In addition, RhoA may also increase the level of the PI 3-kinase substrate PI 4,5-bisphosphate through activation of PI 4-phosphate 5-kinase (30). It has been shown that IGF-dependent signal pathways through association with insulin receptor substrate 1 activate PI 3-kinase (20). Whether IGF-dependent activation of PI 3-kinase is involved in regulating the RhoA-dependent signal pathway in muscle cells is under investigation.

RhoA regulates SRF-dependent gene expressions through different signaling pathways in myoblasts and fibroblasts. SRF mediates RhoA signaling to the skeletal alpha -actin promoter through the proximal SRE site of the promoter (40). Since the discovery of regulation of c-fos transcription by RhoA through SRF in fibroblasts, considerable progress has been made in identifying signaling pathways involved in RhoA activation of c-fos promoter. Regulators of actin dynamics such as LIM kinases (where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) (33), nuclear factor-kappa B (NF-kappa B) and CCAAT/enhancer-binding protein transcription factors (26), protein kinases C-alpha and -epsilon (32), and Rho kinases (7) have been shown to mediate RhoA signaling to c-fos SRE in fibroblasts. In addition, the PI 3-kinase inhibitor wortmannin had no significant effect on the c-fos promoter activation by RhoA (17), and PI 3-kinase was reported to function upstream RhoA to activate c-fos promoter (39) in fibroblasts. In C2C12 myoblasts, treatment with the Rho kinase inhibitor Y27632 (35) had no effect on myoblast differentiation and on activation of the skeletal alpha -actin promoter by RhoA (unpublished results). An NF-kappa B inhibitor, Ikappa Balpha S32A/S36A, had no effect on the activation of the skeletal alpha -actin by RhoA (unpublished results). We are currently evaluating whether there is a role for LIM kinases in RhoA signaling to skeletal alpha -actin in myoblasts. These observations suggest that RhoA signaling to c-fos promoter in fibroblasts may be different from RhoA signaling to skeletal alpha -actin promoter in myoblasts. We have previously shown that activation of cardiac alpha -actin promoter by SRF is regulated by the formation of a functional complex of SRF with other muscle-specific cofactors such as Nkx2.5 (6) and GATA4 (unpublished results). SRF also associates with myogenic factors such as myogenin and MyoD (11). These observations may explain the discrepancies observed for nonmuscle c-fos versus myogenic SRE-dependent activities and suggest that RhoA activation of SRF-dependent muscle gene expression may involve recruitment of muscle-specific accessory factors that are susceptible to other myogenic signals.

In conclusion, regulation of muscle differentiation by RhoA is a complex process under the control of several regulatory pathways. These signaling pathways involve integrin interactions with the ECM proteins, FAK-mediated signal transduction, and generation of phospholipids. RhoA, a signaling molecule implicated in a diverse set of cellular functions, regulates muscle gene expression through cross talk with these signal pathways. Because SRF activation by RhoA appears to be regulated by different signaling pathways in fibroblasts and myoblasts, we propose that activation of SRF-dependent muscle gene expression by RhoA depends on the presence and activity of muscle-specific cofactors. Further studies are necessary to establish the physiological significance of the RhoA signaling network in muscle development, regeneration, and hypertrophy.


    ACKNOWLEDGEMENTS

We thank Drs. Kenneth M. Yamada (National Institutes of Health, Bethesda, MD), Susan E. LaFlamme (Albany Medical College, Albany, NY), Alexey M. Belkin (American Red Cross Holland Laboratory, Rockville, MD), J. Thomas Parsons (University of Virginia, Charlottesville, VA), and Masato Kasuga (Kobe University, Kobe, Japan) for providing plasmids. We thank James E. Agan for help in the tissue culture of myoblasts.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-50422 and P01-HL-49953 (to R. J. Schwartz) and an American Heart Association-Texas Affiliate Beginning Grant-in-Aid Award (to L. Wei).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. J. Schwartz, Dept. of Molecular and Cellular Biology, Rm. 145E, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail: schwartz{at}bcm.tmc.edu).

Received 1 December 1999; accepted in final form 19 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baudoin, C, Goumans MJ, Mummery C, and Sonnenberg A. Knockout and knockin of the beta 1 exon D define distinct roles for integrin splice variants in heart function and embryonic development. Genes Dev 12: 1202-1216, 1998[Abstract/Free Full Text].

2.   Belkin, AM, and Retta SF. beta 1D integrin inhibits cell cycle progression in normal myoblasts and fibroblasts. J Biol Chem 273: 15234-15240, 1998[Abstract/Free Full Text].

3.   Belkin, AM, Zhidkova NI, Balzac F, Altruda F, Tomatis D, Maier A, Tarone G, Koteliansky VE, and Burridge K. beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells. J Cell Biol 132: 211-226, 1996[Abstract/Free Full Text].

4.   Boxer, LM, Prywes R, Roeder RG, and Kedes L. The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor. Mol Cell Biol 9: 515-522, 1989[Abstract/Free Full Text].

5.   Burridge, K, and Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signaling. Ann Rev Cell Dev Biol 12: 463-518, 1996[Web of Science][Medline].

6.   Chen, CY, and Schwartz RJ. Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha -actin gene transcription. Mol Cell Biol 16: 6372-6384, 1996[Abstract].

7.   Chihara, K, Amano M, Nakamura N, Yano T, Shibata M, Tokui T, Ichikawa H, Ikebe R, Ikebe M, and Kaibuchi K. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase. J Biol Chem 272: 25121-25127, 1997[Abstract/Free Full Text].

8.   Chow, KL, and Schwartz RJ. A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha -actin gene. Mol Cell Biol 10: 528-538, 1990[Abstract/Free Full Text].

9.   Flinn, HM, and Ridley AJ. Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin. J Cell Sci 109: 1133-1141, 1996[Abstract].

10.   George, EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, and Hynes RO. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119: 1079-1091, 1993[Abstract].

11.   Groisman, R, Masutani H, Leibovitch MP, Robin P, Soudant I, Trouche D, and Harel-Bellan A. Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins. J Biol Chem 271: 5258-5264, 1996[Abstract/Free Full Text].

12.   Guinebault, C, Payrastre B, Racaud-Sultan C, Mazarguil H, Breton M, Mauco G, Plantavid M, and Chap H. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha  with actin filaments and focal adhesion kinase. J Cell Biol 129: 831-842, 1995[Abstract/Free Full Text].

13.   Gullberg, D, Velling T, Lohikangas L, and Tiger CF. Integrins during muscle development and in muscular dystrophies. Front Biosci 3: D1039-D1050, 1998[Medline].

14.   Hall, A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol 10: 31-54, 1994[Web of Science].

15.   Hara, K, Yonezawa K, Sakaue H, Ando A, Kotani K, Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR., Hawkins PT, Dhand R, Clark AE, Holman GD, Waterfield MD, and Kasuga M. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci USA 91: 7415-7419, 1994[Abstract/Free Full Text].

16.   Hildebrand, JD, Taylor JM, and Parsons JT. An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol Cell Biol 16: 3169-3178, 1996[Abstract].

17.   Hill, CS, Wynne J, and Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159-1170, 1995[Web of Science][Medline].

18.   Ilic, D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, and Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377: 539-544, 1995[Medline].

19.   Jiang, BH, Zheng JZ, and Vogt PK. An essential role of phosphatidylinositol 3-kinase in myogenic differentiation. Proc Natl Acad Sci USA 95: 14179-14183, 1998[Abstract/Free Full Text].

20.   Kaliman, P, Canicio J, Shepherd PR, Beeton CA, Testar X, Palacin M, and Zorzano A. Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells. Mol Endocrinol 12: 66-77, 1998[Abstract/Free Full Text].

21.   Kaliman, P, Vinals F, Testar X, Palacin M, and Zorzano A. Phosphatidylinositol 3-kinase inhibitors block differentiation of skeletal muscle cells. J Biol Chem 271: 19146-19151, 1996[Abstract/Free Full Text].

22.   Kumagai, N, Morii N, Fujisawa K, Nemoto Y, and Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem 268: 24535-24538, 1993[Abstract/Free Full Text].

23.   LaFlamme, SE, Akiyama SK, and Yamada KM. Regulation of fibronectin receptor distribution. J Cell Biol 117: 437-447, 1992[Abstract/Free Full Text].

24.   MacLellan, WR, Lee TC, Schwartz RJ, and Schneider MD. Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. J Biol Chem 269: 16754-16760, 1994[Abstract/Free Full Text].

25.   Menko, AS, and Boettiger D. Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation. Cell 51: 51-57, 1987[Web of Science][Medline].

26.   Montaner, S, Perona R, Saniger L, and Lacal JC. Activation of serum response factor by RhoA is mediated by the nuclear factor-kappa B and C/EBP transcription factors. J Biol Chem 274: 8506-8515, 1999[Abstract/Free Full Text].

27.   Montarras, D, Pinset C, Chelly J, Kahn A, and Gros F. Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells. EMBO J 8: 2203-2207, 1989[Web of Science][Medline].

28.   Muscat, GE, and Kedes L. Multiple 5'-flanking regions of the human alpha -skeletal actin gene synergistically modulate muscle-specific expression. Mol Cell Biol 7: 4089-4099, 1987[Abstract/Free Full Text].

29.   Parsons, JT. Integrin-mediated signalling: regulation by protein tyrosine kinases and small GTP-binding proteins. Curr Opin Cell Biol 8: 146-152, 1996[Web of Science][Medline].

30.   Ren, XD, and Schwartz MA. Regulation of inositol lipid kinases by Rho and Rac. Curr Opin Genet Dev 8: 63-67, 1998[Web of Science][Medline].

31.   Richardson, A, and Parsons T. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 380: 538-540, 1996[Medline].

32.   Soh, JW, Lee EH, Prywes R, and Weinstein IB. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol 19: 1313-1324, 1999[Abstract/Free Full Text].

33.   Sotiropoulos, A, Gineitis D, Copeland J, and Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98: 159-169, 1999[Web of Science][Medline].

34.   Takano, H, Komuro I, Oka T, Shiojima I, Hiroi Y, Mizuno T, and Yazaki Y. The Rho family G proteins play a critical role in muscle differentiation. Mol Cell Biol 18: 1580-1589, 1998[Abstract/Free Full Text].

35.   Uehata, M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997[Medline].

36.   Ui, M, Okada T, Hazeki K, and Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci 20: 303-307, 1995[Web of Science][Medline].

37.   Van Aelst, L, and D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 11: 2295-2322, 1997[Free Full Text].

38.   Van der Flier, A, Gaspar AC, Thorsteinsdottir S, Baudoin C, Groeneveld E, Mummery CL, and Sonnenberg A. Spatial and temporal expression of the beta 1D integrin during mouse development. Dev Dyn 210: 472-486, 1997[Web of Science][Medline].

39.   Wang, Y, Falasca M, Schlessinger J, Malstrom S, Tsichlis P, Settleman J, Hu W, Lim B, and Prywes R. Activation of the c-fos serum response element by phosphatidyl inositol 3-kinase and rho pathways in HeLa cells. Cell Growth Differ 9: 513-522, 1998[Abstract].

40.   Wei, L, Zhou W, Croissant JD, Johansen FE, Prywes R, Balasubramanyam A, and Schwartz RJ. RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation. J Biol Chem 273: 30287-30294, 1998[Abstract/Free Full Text].

41.   Whitman, M, Kaplan DR, Schaffhausen B, Cantley L, and Roberts TM. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 315: 239-242, 1985[Medline].

42.   Zhang, J, King WG, Dillon S, Hall A, Feig L, and Rittenhouse SE. Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. J Biol Chem 268: 22251-22254, 1993[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 278(6):H1736-H1743
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


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