Nuclear Factor kappa B-inducing Kinase and Ikappa B Kinase-alpha Signal Skeletal Muscle Cell Differentiation*

Judith CanicioDagger , Pilar Ruiz-Lozano§, Marta CarrascoDagger , Manuel PalacínDagger , Kenneth Chien§, Antonio ZorzanoDagger , and Perla KalimanDagger

From the Dagger  Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain and the § Department of Medicine, University of California at San Diego, La Jolla, California 92093-0613

Received for publication, January 25, 2001, and in revised form, February 21, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa B (NF-kappa B)-inducing kinase (NIK), Ikappa B kinase (IKK)-alpha and -beta , and Ikappa Balpha are common elements that signal NF-kappa B activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappa B DNA-binding activity that correlated in time with the activation of IKKalpha , IKKbeta , and NIK. Moreover, the activation of IKKalpha , IKKbeta , and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the Ikappa Balpha (S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappa B activation and myoblast differentiation, indicating that phosphorylation of Ikappa Balpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The IGFs1 are the only known growth factors that are crucial to myogenesis (1). IGF-I and IGF-II switch on the myogenic program through the IGF-I receptor (2), activating the expression of myogenic transcription factors, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (3, 4). PI3K is an essential second messenger for myogenesis (5-9). We have recently described a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves PI3K activation, NF-kappa B activation, and inducible nitric-oxide synthase expression and activation (10). In this report, we further analyze the role of the NF-kappa B-activating signaling cascade in myogenesis. NF-kappa B transcription factors are key mediators of inflammatory responses, immune system functioning, transformation, oncogenesis, and anti-apoptotic signaling (11-13). NF-kappa B exists in the cytoplasm in an inactive form by virtue of its association with inhibitory proteins termed Ikappa B (11-15). NF-kappa B translocation to the nucleus and activation are most frequently achieved through the signal-induced proteolytic degradation of Ikappa B in the cytoplasm. Two kinases, IKKalpha and IKKbeta , which are contained in a high-molecular-weight multiprotein complex, show inducible Ikappa B kinase activity and play a key role in NF-kappa B activation by a variety of stimuli (16-19). Despite their high sequence similarity, IKKalpha and IKKbeta have different regulatory and functional roles. In mice lacking IKKbeta , the activation of NF-kappa B by cytokines is abolished, and mouse embryos die on days 12-13 of gestation due to massive liver apoptosis (20). In contrast, IKKalpha is dispensable for pro-inflammatory responses, but plays an essential role in embryonic development. Mice lacking IKKalpha exhibit defective proliferation and differentiation of epidermal keratinocytes and defective limb and skeletal patterning (21, 22). IKKalpha and IKKbeta are themselves phosphorylated and activated by one or more upstream kinases, like NIK, which is a member of the mitogen-activating protein kinase kinase kinase family (23-25).

We report here that Ikappa Balpha phosphorylation at Ser-32 and Ser-36 is required for both IGF-II-dependent NF-kappa B activation and differentiation in L6E9 myoblasts. We show that IKKalpha is involved in IGF-II-dependent multinucleated myotube formation and muscle-specific gene expression, whereas IKKbeta is not essential for these processes. Our data suggest that NIK activation triggers myogenin expression and multinucleated myotube formation in the absence of IGF-II.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- IGF-II was kindly given by Lilly. The rat skeletal muscle cell line L6E9 was kindly provided by Dr. B. Nadal-Ginard (Harvard University). Low-passage 293 cells were from Microbix (Ontario, Canada). The PI3K inhibitor LY294002 was from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). The NF-kappa B probe for electrophoretic mobility shift assay was kindly given by Dr. Jean-François Peyron (INSERM U364, Nice, France). The cDNAs encoding FLAG-IKKalpha , FLAG-IKKalpha (K44A), FLAG-IKKbeta , and FLAG-IKKbeta (K44A) were provided by Dr. D. Goeddel (Tularik, Inc.). The cDNA encoding GST-Ikappa Balpha -(1-54) was provided by Dr. M. Karin (University of California, San Diego, CA). Recombinant adenoviral vectors expressing Ikappa Balpha (S32A/S36A), kinase-proficient FLAG-tagged NIK, LacZ, and green fluorescent protein (GFP) were provided by Dr. Yibin Wang (University of Maryland, Baltimore, MD). Polyclonal antibody C38320 raised against caveolin-3 was from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibody MF20, which stains all sarcomeric myosin heavy chain isoforms, and mouse anti-rat myogenin monoclonal antibody F5D were from the Developmental Studies Hybridoma Bank. Polyclonal antibodies against Ikappa Balpha (C-15), IKKalpha , IKKbeta , and NIK were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-beta -actin (clone AC-15) and anti-FLAG M2 monoclonal antibodies were from Sigma.

Cell Culture-- Rat L6E9 myoblasts were cultured as described previously (5). Subconfluent myoblasts were differentiated by serum depletion in DMEM plus antibiotics with or without IGF-II (40 nM) in the absence or presence of other compounds, as indicated for each experiment. Cells were photographed after staining the nuclei with Mayer's hemalum solution for microscopy (Merck, Darmstadt, Germany), and cell fusion was quantified by counting nuclei in myotubes from a total of at least 1000 nuclei from 10-20 randomly selected microscope fields for each condition. For adenoviral transduction, subconfluent L6E9 myoblasts were transduced at a multiplicity of 50-100 particles/cell and then cultured for an additional 36 h before inducing differentiation with or without IGF-II. To compare the impact of IKKalpha and IKKbeta on myoblast differentiation, experimental conditions were selected to ensure similar levels of expression of IKKalpha and IKKbeta constructs (data not shown).

Construction of Recombinant Adenoviruses-- Recombinant adenoviruses expressing FLAG-tagged versions of either wild-type or dominant-negative mutant K44A human IKKalpha and IKKbeta were generated by homologous recombination as described by Graham and Prevec (38). cDNAs were cloned into the shuttle plasmid pAdl1/RSV and cotransfected with pJM17 into 293 cells to achieve homologous recombination. Individual plaques were isolated and checked for recombinant protein expression after infection of 293 cells. Recombinant adenoviruses were further amplified in 293 cells; purified by cesium chloride gradient centrifugation; dialyzed against 1 mM MgCl2, 10 mM Tris (pH 7.4), and 10% glycerol; and stored at -80 °C (39). Viral stocks were titrated by infecting 293 cells with serial dilutions of the preparation and observing the cytopathic effect on the cells 48 h after infection. An infectious titer was given assuming that a multiplicity of infection of 10 is required to cause a complete cytopathic effect at 48 h. In addition, the A260 of the preparation was measured to estimate the particle titer (1 A260 unit = 1012 particles/ml).

Electrophoresis and Immunoblotting of Membranes-- Cells were lysed for 30 min at 4 °C in 50 mM Tris (pH 7.5), 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 20 mM NaF, 0.1% phenylmethylsulfonyl fluoride, and 0.1% aprotinin supplemented with 1% Nonidet P-40. Cell extracts were centrifuged at 10,000 × g for 15 min at 4 °C, and 50 µg of the solubilized proteins was loaded. SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed as described previously (5).

Immunoprecipitation and Kinase Assays-- Cells were washed in PBS and resuspended at 106 cells/10 µl in hypotonic solution (10 mM Hepes (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 15 mM Na4P2O7, 20 mM NaF, 0.1% phenylmethylsulfonyl fluoride, and 0.1% aprotinin). After 10 min at 4 °C, Nonidet P-40 was added to 1%, and cells were centrifuged in a microcentrifuge at 10,000 × g for 15 min. The supernatant containing the cytoplasmic fraction was recovered.

For immunoprecipitation, antibodies were preadsorbed on protein G-Sepharose at 4 °C for 1 h and washed twice in hypotonic solution and 1% Nonidet P-40 before being incubated with the protein extracts for 2 h at 4 °C. The immunopellets were washed six times in the same buffer and once in kinase buffer (20 mM Hepes, 10 mM MgCl2, 100 µM Na3VO4, 20 mM beta -glycerophosphate, 2 mM dithiothreitol, and 50 mM NaCl (pH 7.5)). Kinase reactions were carried out for 30 min at 30 °C using 5 µCi of [gamma -32P]ATP and GST-Ikappa Balpha -(1-54) as substrate (except for NIK kinase assays, in which autophosphorylation of immunoprecipitated NIK was analyzed). The reaction products were analyzed on 10% polyacrylamide gels and revealed by autoradiography.

Electrophoretic Mobility Shift Assay-- NF-kappa B DNA-binding activity was analyzed in total cell extracts made in Totex lysis buffer (20 mM Hepes, 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% phenylmethylsulfonyl fluoride, and 0.1% aprotinin) (10). Supernatants from a 15-min 10,000 × g centrifugation were collected. The NF-kappa B probe was a synthetic double-stranded oligonucleotide containing the NF-kappa B-binding site of the interleukin-2 gene promoter (5'-GATCCAAGGGACTTTCCATG-3').

Samples (70 µg) were incubated for 10 min at 4 °C with 30 ng of poly(dI·dC) and 5 µl of 5× reaction buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 5 mM dithiothreitol, 5 mM EDTA, 20% glycerol, and 0.4 mg/ml salmon sperm DNA) in a final volume of 25 µl. The end-labeled probe was added for a further incubation of 25 min at 25 °C. The specificity of the bands detected was verified by adding a 10-100-fold excess of competing unlabeled NF-kappa B probe. NF-kappa B-unrelated oligonucleotide probe controls did not show any specific binding activity (data not shown).

Immunofluorescence-- Cells grown on coverslips were fixed for 20 min with 3% paraformaldehyde in PBS, washed three times in PBS, and then treated as follows: (a) 10 min in PBS containing 50 mM NH4Cl, (b) 10 min in PBS containing 20 mM glycine, and (c) 30 min in PBS containing 10% fetal bovine serum. Subsequently, coverslips were incubated with primary antibodies (anti-NIK polyclonal antibodies, 1 µg/ml; anti-myogenin monoclonal antibody, undiluted culture supernatant) for 1 h at room temperature. After washing in PBS, coverslips were incubated with fluorochrom-conjugated antibodies (Oregon Green or Texas Red) for 45 min. Cells were washed three times in PBS and then mounted in immunofluor medium (ICN Biomedicals Inc., Aurora, OH). Images were obtained using a Leica TCS 4D laser confocal fluorescence microscope with a 40× objective.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IGF-II-induced Skeletal Muscle Cell Differentiation Involves NF-kappa B Signaling Cascade Activation-- IGF-II induces NF-kappa B DNA-binding activity as an early event during L6E9 myoblast differentiation (10). Most extracellular stimuli that activate the NF-kappa B pathway induce phosphorylation of the NF-kappa B repressor Ikappa Balpha at Ser-32 and Ser-36 as a requisite for its degradation (19). However, alternative pathways for NF-kappa B activation have been described (26, 27). To analyze the mechanism required by IGF-II for NF-kappa B activation during differentiation, L6E9 myoblasts were transduced with an adenovirus expressing an Ikappa Balpha mutant with Ser-32 and Ser-36 replaced by alanine residues (adv/Ikappa Balpha (S32A/S36A)). This mutant inhibits NF-kappa B in pathways involving serine phosphorylation and subsequent proteasome degradation of Ikappa Balpha . L6E9 myoblasts differentiated for 24 h with IGF-II exhibited an induction of NF-kappa B DNA-binding activity (Fig. 1A), which was blocked in myoblasts overexpressing Ikappa Balpha (S32A/S36A) (Fig. 1B). Myoblasts infected with an adenovirus expressing green fluorescent protein (adv/GFP) were used as control (Fig. 1B). Next, we analyzed the impact of Ikappa Balpha phosphorylation on IGF-II-dependent myoblast differentiation. After 4 days of IGF-II treatment, the expression of muscle-specific proteins such as myosin heavy chain and caveolin-3 was highly decreased in myoblasts overexpressing the Ikappa Balpha (S32A/S36A) mutant (50 ± 12%, n = 3) compared with non-transduced control cells or cells transduced with adv/GFP, whereas the expression of the non-muscle-specific protein beta -actin was similar under all conditions (Fig. 1C). Moreover, cells overexpressing the Ikappa Balpha (S32A/S36A) mutant did not fuse to myotubes, whereas control cells (adv/GFP) showed 82% of the nuclei in myotubes from a total of 1661 nuclei randomly counted (Fig. 1D). These data suggest that IGF-II requires NF-kappa B activation through a mechanism that involves Ikappa Balpha phosphorylation to trigger skeletal muscle cell differentiation.


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Fig. 1.   IGF-II-induced skeletal muscle cell differentiation requires Ikappa Balpha phosphorylation at Ser-32 and Ser-36 for NF-kappa B activation. A, total cell extracts from L6E9 myoblasts differentiated with or without 40 nM IGF-II for 24 h were incubated with a 32P-labeled NF-kappa B probe corresponding to the NF-kappa B-binding site in the interleukin-2 gene promoter and analyzed by electrophoretic mobility shift assay. The specificity of the bands was verified by adding a 50-fold excess of competing unlabeled NF-kappa B probe (cold probe) or unrelated oligonucleotide probes (data not shown). B, total cell extracts from L6E9 myoblasts transduced with adv/Ikappa Balpha (S32A/S36A) (adv/Ikappa Balpha AA) or adv/GFP and differentiated with IGF-II (40 nM) for 24 h were incubated with a 32P-labeled NF-kappa B probe and analyzed by electrophoretic mobility shift assay. C, expression of Ikappa Balpha , myosin heavy chain (MHC), and caveolin-3 (Cav-3) was analyzed by immunoblotting of total cell lysates from non-transduced myoblasts (nt) or myoblasts transduced with adv/GFP or adv/Ikappa Balpha (S32A/S36A) and differentiated for 4 days with IGF-II. beta -Actin content was analyzed as a control of relative amounts of proteins in each sample. D, L6E9 myoblasts non-transduced or transduced with adv/GFP or adv/Ikappa Balpha (S32A/S36A) were grown to confluence and then allowed to differentiate in the presence of IGF-II for 4 days. Morphological differentiation was assessed by myotube formation. Images shown are representative of 15-25 microscope fields taken at random. The scale is the same for all panels. All infections were performed in duplicate, and the results shown are representative of three independent experiments.

IGF-II Induces PI3K-dependent IKKalpha and IKKbeta Activation during Myoblast Differentiation-- The data presented above indicating that Ikappa Balpha phosphorylation was required by IGF-II to induce myoblast differentiation led us to analyze the activity and expression of IKKalpha and IKKbeta during this process. L6E9 myoblasts exhibited a peak of IKKalpha activity after 24 h in IGF-II-containing differentiation medium (Fig. 2A, upper panel). The kinetics of IKKalpha activation was consistent with that of IGF-II-dependent NF-kappa B DNA-binding activation, which we have previously shown to be dependent on PI3K activity (10). To analyze whether IGF-II-dependent IKKalpha activation involves PI3K, L6E9 myoblast differentiation was induced with or without IGF-II in the absence or presence of the PI3K inhibitor LY294002. The PI3K inhibitor (20 µM) blocked the ability of IKKalpha to phosphorylate GST-Ikappa Balpha -(1-54) in response to IGF-II (Fig. 2B, upper panel). Neither IGF-II nor LY294002 altered IKKalpha protein expression (Fig. 2C, upper panel); however, the 24-h delay between the start of the IGF-II treatment and the activation of IKKalpha seemed to indicate that IKKalpha was not the direct target of the IGF-induced phosphorylation cascade. This appears to be the case, as IKKalpha activation by IGF-II was blocked in the presence of 5 µg/ml cycloheximide (Fig. 2D, upper panel, CH), indicating that it depends on de novo protein synthesis.


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Fig. 2.   Endogenous IKK activation during IGF-II-induced myoblast differentiation. A, cell extracts (50 µg) from L6E9 myoblasts differentiated with IGF-II (40 nM) were immunoprecipitated (IP) with anti-IKKalpha (upper panel) or anti-IKKbeta (lower panel) antibodies (10 µg) preadsorbed on protein G-Sepharose beads, and kinase assay was performed as described under "Experimental Procedures" using GST-Ikappa Balpha -(1-54) as a substrate. Nonspecific kinase activity was analyzed using a nonimmune antibody (data not shown). B, kinase assay was performed as described for A with cells differentiated for 24 h in the absence or presence of IGF-II (40 nM) with or without LY294002 (20 µM). C, IKK expression was analyzed by immunoblotting (IB) of cell lysates obtained from myoblasts differentiated for 24 h in the absence or presence of IGF-II with or without LY294002. The relative amounts of proteins in each sample were checked by expression of the non-muscle-specific protein beta -actin. D, subconfluent L6E9 myoblasts were differentiated for 24 h in DMEM in the absence or presence of IGF-II (40 nM) with or without cycloheximide (CH; 5 µg/ml). Kinase assay was performed as described above. The results shown are representative of three to four independent experiments.

Phosphorylation of the GST-Ikappa Balpha -(1-54) substrate by IKKbeta was also induced 24 h after triggering differentiation with IGF-II (Fig. 2A, lower panel). IKKbeta activation by IGF-II was blocked by the PI3K inhibitor LY294002 (Fig. 2B, lower panel). Neither IGF-II nor LY294002 treatment modified the level of IKKbeta expression in differentiating myoblasts (Fig. 2C, middle panel), indicating that changes in activity are caused by activation of the kinase rather than variations in IKKbeta protein content. As for IKKalpha , the activation of IKKbeta by IGF-II was blocked by cycloheximide (Fig. 2D, lower panel).

IGF-II Requires IKKalpha , but Not IKKbeta , to Induce Skeletal Muscle Cell Differentiation-- To determine whether the activation of IKKs by IGF-II is functionally linked to myogenic differentiation, we generated replication-deficient adenoviral vectors expressing FLAG-tagged wild-type (adv/FLAG-IKKalpha and adv/FLAG-IKKbeta ) or dominant-negative (adv/FLAG-IKKalpha (K44A) and adv/FLAG-IKKbeta (K44A) forms of IKKalpha and IKKbeta . Subconfluent L6E9 myoblasts were transduced with the different recombinant adenoviruses using adv/GFP as a control; and 36 h later, differentiation was induced by placing the cells in an IGF-II-containing medium.

Expression and kinase activity of the transduced proteins were evaluated 24 h after inducing differentiation with IGF-II. Cells transduced with adv/FLAG-IKKalpha exhibited GST-Ikappa Balpha -(1-54) phosphorylation activity in immunoprecipitates with an anti-FLAG monoclonal antibody, whereas no specific Ikappa Balpha phosphorylation activity was detected in immunoprecipitates from cells transduced either with adv/GFP or adv/FLAG-IKKalpha (K44A) (Fig. 3A, upper panel). Under these conditions, similar levels of wild-type and dominant-negative forms of IKKalpha were expressed, as verified by immunoblotting using anti-FLAG antibody (Fig. 3A, lower panel). After 4 days of differentiation in the presence of IGF-II, cells overexpressing FLAG-IKKalpha (K44A) remained highly undifferentiated, as measured by caveolin-3 and myosin heavy chain expression, compared with cells transduced with either adv/GFP or adv/FLAG-IKKalpha . The expression of the non-muscle-specific protein beta -actin was not altered by FLAG-IKK(K44A) overexpression (Fig. 3B).


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Fig. 3.   IGF-II requires IKKalpha (but not IKKbeta ) activation to induce skeletal muscle cell differentiation. Subconfluent L6E9 myoblasts were transduced with adv/GFP, adv/FLAG-IKKalpha , or adv/FLAG-IKKalpha (K44A) (A and B) or with adv/GFP, adv/FLAG-IKKbeta , or adv/FLAG-IKKbeta (K44A) (C and D). After 36 h, cells were induced to differentiate with IGF-II. A and C: upper panels, kinase activity of the transduced proteins was measured in anti-FLAG immunoprecipitates (IP) after 24 h in IGF-II differentiation medium by in vitro phosphorylation of GST-Ikappa Balpha -(1-54) fusion protein. Nonspecific (ns) Ikappa Balpha phosphorylation activity was analyzed using a nonimmune antibody. Lower panels, expression of transduced proteins was checked by immunoblotting using anti-FLAG antibody. B and D, shown is the muscle-specific protein expression in total cell lysates from undifferentiated myoblasts (mb) or from myoblasts differentiated for 4 days with IGF-II 36 h after being transduced with adv/GFP, adv/FLAG-IKKalpha , adv/FLAG-IKKalpha (K44A), adv/FLAG-IKKbeta , or adv/FLAG-IKKbeta (K44A) or left non-transduced (nt). The relative amounts of proteins in each sample were checked by expression of the muscle-specific protein beta -actin. All infections were done in duplicate, and the results shown are representative of three independent experiments. MHC, myosin heavy chain.

To analyze the role of IKKbeta activity in myoblast differentiation, cells were transduced with adv/FLAG-IKKbeta or adv/FLAG-IKKbeta (K44A). Both proteins were expressed to similar levels as detected on anti-FLAG immunoblots (Fig. 3C, lower panel), and adv/FLAG-IKKbeta (K44A)-transduced cells exhibited no Ikappa Balpha kinase activity on anti-FLAG immunoprecipitates (upper panel). In contrast to IKKalpha , IKKbeta does not seem to play an essential role in myoblast differentiation, as cells transduced with adv/FLAG-IKKbeta (K44A) expressed skeletal muscle-specific proteins as efficiently as non-transduced cells or cells transduced with adv/GFP or adv/FLAG-IKKbeta (Fig. 3D). At the morphological level, overexpression of FLAG-IKKbeta (K44A) did not alter the ability of myoblasts to fuse in response to IGF-II (78% of nuclei in myotubes from a total of 1488 nuclei randomly counted) compared with non-transduced control cells (78%, total of 1616), cells transduced with adv/GFP (82%, total of 1661), or cells transduced with adv/FLAG-IKKbeta (80%, total of 1011) (Fig. 4; arrows show large accumulations of nuclei in myotubes). Conversely, when FLAG-IKKalpha (K44A) was overexpressed, only thin, spindle-like myotubes were formed compared with the large myotubes observed in non-transduced cells, cells transduced with adv/GFP, or cells transduced with adv/FLAG-IKKalpha (Fig. 4). Indeed, after 4 days in IGF-II-containing differentiation medium, only 25% of the nuclei (total of 1367) in cells overexpressing FLAG-IKKalpha (K44A) were in myotubes with >10 nuclei. Under the same culture conditions, 77% of the nuclei from cells overexpressing FLAG-IKKalpha (total of 2987) were in myotubes with >10 nuclei. Taken together, these results suggest that IKKalpha plays a relevant role in IGF-II-dependent morphological and biochemical differentiation of skeletal muscle cells, whereas IKKbeta is not essential to this process.


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Fig. 4.   Overexpression of dominant-negative FLAG-IKKalpha (K44A) in L6E9 myoblasts impairs IGF-II-induced myotube formation. Subconfluent myoblasts transduced with adv/GFP, adv/FLAG-IKKalpha , adv/FLAG-IKKalpha (K44A), adv/FLAG-IKKbeta , or adv/FLAG-IKKbeta (K44A) were differentiated with IGF-II for 4 days. Cells were photographed after nuclear staining, and myotube formation was quantified. Proliferating myoblasts are also shown. Images are representative of 25 microscope fields taken at random from each of at least five independent experiments. The scale is the same for all panels.

NIK Is Activated in Differentiating Myoblasts-- NIK is a common mediator in the NF-kappa B signaling cascades, and IKKalpha has been reported to be a better substrate than IKKbeta for phosphorylation by NIK (24). The endogenous kinase activity and protein expression of NIK in differentiating L6E9 myoblasts were studied. Induction of NIK autophosphorylation activity was detected after 24 h in IGF-II-containing differentiation medium (Fig. 5A). As observed for NF-kappa B DNA-binding activation (10) and IKKalpha and IKKbeta activities (Fig. 2B), the activation of NIK was totally blocked by LY294002, indicating that IGF-II requires PI3K to activate NIK in differentiating myoblasts (Fig. 5A). No changes were detected in NIK protein expression in response to IGF-II or LY294002 (Fig. 5B). As for IKKalpha and IKKbeta , NIK activation by IGF-II was blocked by cycloheximide (Fig. 5C), indicating that it requires de novo protein synthesis.


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Fig. 5.   NIK activation during IGF-II-induced differentiation. A, cell extracts (50 µg) from L6E9 myoblasts differentiated for 24 h in DMEM in the absence or presence of IGF-II (40 nM) with or without LY294002 (20 µM) were immunoprecipitated (IP) with anti-NIK antibody (5 µg) preadsorbed on protein G-Sepharose beads. Autophosphorylation assay was performed as described under "Experimental Procedures," and nonspecific (ns) NIK activity was tested using a nonimmune antibody. B, NIK expression was analyzed by Western blotting of cell lysates obtained at 24 h of differentiation, and the relative amounts of proteins in each sample were checked by expression of beta -actin. C, subconfluent L6E9 myoblasts were differentiated for 24 h in DMEM in the absence or presence of IGF-II (40 nM) with or without cycloheximide (CH; 5 µg/ml). Kinase assay was performed as described above. D, subconfluent L6E9 myoblasts were differentiated for 24 h with IGF-II (40 nM) with or without LY294002 (20 mM), added either for the whole 24 h or during the last 6 h or the last 1 h of IGF-II treatment. Kinase assay was performed at 24 h as described above. The data shown are representative of three independent experiments.

Interestingly, LY294002 inhibited NIK activation only when it was present together with IGF-II during the whole 24-h treatment. In contrast, when LY294002 was added during the last 6 h or the last 1 h of IGF-II incubation (at 18 or 23 h of differentiation, respectively), no inhibition of NIK activity was observed (Fig. 5D). These results suggest that PI3K is most probably involved in the de novo synthesis of the factor(s) required for NIK activation rather than being a direct upstream element of the NIK-activating cascade.

To test whether the activation of NIK by IGF-II was a key event during differentiation, we transduced L6E9 myoblasts with recombinant adenovirus expressing kinase-proficient FLAG-tagged wild-type NIK that exhibited a high degree of basal autophosphorylation activity on anti-FLAG or anti-NIK immunoprecipitates (Fig. 6A; the lower band that was detected with anti-NIK antibodies in cells transduced with adv/FLAG-NIK is probably due to cross-activation of endogenous NIK by the overexpressed kinase). After 2 days in the absence of IGF-II (DMEM), myoblasts infected with control adv/GFP fused poorly (12% from a total of 1713 nuclei counted at random were found in multinucleated myotubes) (Fig. 6B). In contrast, transduction with adv/FLAG-NIK promoted myoblast fusion (48% of nuclei in myotubes from a total of 1789 nuclei counted at random) (Fig. 6B). Co-immunofluorescence assays showed that NIK-overexpressing cells highly expressed myogenin in their nuclei (Fig. 6C). The number of nuclei expressing myogenin was 6-fold higher in cells overexpressing NIK (adv/NIK) than in control cells (adv/LacZ) (15 randomly selected fields were analyzed from each of two independent experiments with each condition performed in triplicate).


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Fig. 6.   NIK overexpression induces myoblast differentiation in the absence of IGF-II. A, 293 cells were transduced with adv/FLAG-NIK. Non-transduced (nt) cells were used as controls. Cell extracts obtained 24 h after infection were immunoprecipitated (IP) with antibodies against NIK or FLAG preadsorbed on protein G-Sepharose beads. Autophosphorylation assay was performed as described under "Experimental Procedures." Nonspecific (ns) NIK autophosphorylation activity was tested using nonimmune antibodies. B, subconfluent L6E9 myoblasts were transduced with adv/FLAG-NIK or control adv/GFP. 36 h after infection, cells were allowed to differentiate for 2 days in the absence of IGF-II (DMEM). Morphological differentiation was assessed by myotube formation, and cells were photographed after nuclear staining. Images shown are representative of 25 microscope fields taken at random from each of three independent experiments. The scale is the same for both panels. C, shown is myogenin expression in L6E9 cells transduced with adv/FLAG-NIK or control adv/LacZ. Cells were grown to subconfluence on glass coverslips and maintained for 2 days in serum-free medium in the absence of exogenous IGF-II (DMEM). For immunofluorescence detection, cells were fixed and simultaneously probed for myogenin and NIK as described under "Experimental Procedures." Cells showing myogenin nuclear staining were counted and averaged from a minimum of 15 randomly selected fields. All infections were done in triplicate, and the results shown are representative of two independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We describe a pathway by which IGF-II modulates skeletal muscle cell differentiation through activation of the IKK complex. The activation of the NF-kappa B cascade (NIK, IKKalpha , and IKKbeta activation; Ikappa B degradation; and NF-kappa B DNA-binding activation) was detected 24 h after placing subconfluent myoblasts in IGF-II-containing differentiation medium. These are early events during the differentiation program induced by IGF-II, but they differ greatly in their kinetics compared with the rapid activation of this cascade, which can be detected within minutes of exposure of cells to cytokines or other stimuli. These observations indicate that IKK might not be a direct target of the IGF-induced phosphorylation cascade, an assumption reinforced by the fact that IGF-mediated IKK and NIK activation requires de novo protein synthesis. The nature of the factor(s) induced by IGF-II to trigger NF-kappa B cascade activation during myogenesis remains undefined. Among the possibilities to be considered is that IGF-II could induce the secretion of an autocrine factor by differentiating myoblasts. In this context, we did not detect expression of tumor necrosis factor-alpha (a classical activator of NF-kappa B) by reverse transcription-polymerase chain reaction studies in myoblasts induced to differentiate by IGF-II (data not shown). Another possibility is that the newly synthesized protein is a differentiation-induced kinase that may be a target for IGF-II itself or for an alternative factor generated in response to IGF-II.

The involvement of NF-kappa B in myogenic signaling has been described in rat, human, and chick embryonic myoblasts (10, 28, 29), although differentiation of C2C12 skeletal muscle cells in a 2% horse serum-containing medium seems to occur through a signaling cascade in which NF-kappa B plays a negative regulatory role (30). We have previously established that PI3K, NF-kappa B, and inducible nitric-oxide synthase are elements of a common myogenic cascade in which IGF-II induces, through a PI3K-dependent pathway, a decrease in Ikappa Balpha protein content that correlates with a decrease in the amount of Ikappa Balpha associated with p65 NF-kappa B, NF-kappa B DNA-binding activation, and NO production (10). PI3K is a key mediator of myogenesis (5-9), and the role of PI3K in NF-kappa B cascade activation during myogenesis is reinforced by data presented here showing that activation of both NIK and IKK by IGF-II in differentiating myoblasts is blocked by inhibiting PI3K. PI3K is known to be directly involved in the activation of NF-kappa B in processes like anti-apoptotic platelet-derived growth factor signaling (31) and tumor necrosis factor-mediated immune and inflammatory responses (32). However, in view of our results, PI3K does not seem to directly activate NIK phosphorylation (LY294002 inhibited NIK activation only when it was present together with IGF-II during the whole 24-h treatment, but not during the last 6 h or the last 1 h). These data suggest that PI3K is most probably involved in the de novo synthesis of protein(s) required for NIK activation rather than being an upstream element in the activation of NIK. In contrast, our results do not rule out a biphasic role for IGF-II: first, in initiating the de novo synthesis of a NIK-activating factor and, second, in promoting NIK activation by the newly synthesized protein that (in view of the results presented in Fig. 5D) could occur only through a PI3K-independent pathway.

Although the stimuli that activate IKKbeta and the substrates that mediate its biological activity are known, the stimuli and the relevant substrates for IKKalpha are less well characterized. IKKalpha and IKKbeta appear to exert different and non-interchangeable physiological roles. Gene targeting experiments revealed that although IKKalpha is not involved in the activation of NF-kappa B by pro-inflammatory stimuli, it is involved in morphogenesis (20-22). In this context, our results show that although IGF-II induced both IKKalpha and IKKbeta activities early during the differentiation program, the overexpression of a kinase-deficient mutant of IKKbeta did not alter the expression of muscle-specific proteins or the formation of multinucleated myotubes. The differentiation process was, however, blocked by a kinase-deficient mutant of IKKalpha , suggesting that endogenous IKKbeta cannot substitute for IKKalpha in myogenic signaling. Interestingly, skeletal muscle poorly expresses IKKbeta , whereas it is one of the tissues with the higher expression levels of IKKalpha (33).

Proliferation precedes differentiation in IGF-stimulated myogenesis, and the opposing early and late effects of IGF during myogenesis are reflected in the phosphorylation state of the cell cycle regulatory retinoblastoma protein in skeletal myoblasts (34, 35). Before exiting from the cell cycle, IGFs induce a last round of proliferation in which NF-kappa B is required to increase cyclin D1 expression and pRb hyperphosphorylation (33, 35, 37). Then, a decrease in NF-kappa B activity followed by a decrease in cyclin D1 levels seems to be required to allow the exit from the cell cycle that precedes differentiation (3, 36). A causative relation between NF-kappa B down-regulation and myogenesis was initially proposed, without considering that the NF-kappa B activity detected after 24 h in differentiation medium (even if it was lower than that observed during proliferation) could exert a myogenic role (36). Indeed, this appears to be the case, as, consistent with data reported here, NF-kappa B activity was shown to be required by human, mouse, and chicken myoblasts to fuse (10, 31, 32). We show here that IGF-II-dependent differentiation triggers a delayed induction of the NF-kappa B-activating cascade, which requires PI3K activity and de novo synthesis of still undefined factors. Our data suggest that the activation of NIK and IKKalpha and the subsequent phosphorylation of Ikappa Balpha at Ser-32 and Ser-36 are key events in skeletal muscle differentiation induced by IGF-II.

    ACKNOWLEDGEMENTS

We thank Drs. Yibin Wang and M. Hoshijima (University of California at San Diego, La Jolla, CA) for help in recombinant adenovirus construction and production, Drs. M. Camps and S. Castel (Servei Científico Tècnics, University of Barcelona) for expert advice on microscopy techniques, and Robin Rycroft for editorial assistance.

    FOOTNOTES

* This work was supported by Research Grant PB98/0197 from the Dirección General de Investigación Científica y Técnica, Research Grant 00/2101 from the Fondo de Investigación Sanitaria, the Fundació la Marató de TV3, and Research Grant SGR-0039 (1999) from the Generalitat de Catalunya, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda Diagonal 645, E-08028 Barcelona, Spain. Tel.: 34-3-4021547; Fax: 34-3-4021559; E-mail: perlak@porthos.bio.ub.es.

Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M100718200

    ABBREVIATIONS

The abbreviations used are: IGFs, insulin-like growth factors; PI3K, phosphatidylinositol 3-kinase; NF-kappa B, nuclear factor kappa B; IKK, Ikappa B kinase; NIK, NF-kappa B-inducing kinase; GST, glutathione S-transferase; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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