Insulin-like Growth Factor-II, Phosphatidylinositol 3-Kinase, Nuclear Factor-kappa B and Inducible Nitric-oxide Synthase Define a Common Myogenic Signaling Pathway*

Perla KalimanDagger §, Judith CanicioDagger , Xavier Testar, Manuel Palacín, and Antonio Zorzano

From the Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappa B (NF-kappa B) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappa B DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappa B inhibition. Both NF-kappa B and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappa B or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappa B, PI 3-kinase inhibition prevented NF-kappa B activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in Ikappa B-alpha protein content that correlated with a decrease in the amount of Ikappa B-alpha associated with p65 NF-kappa B.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Skeletal muscle cell differentiation is a highly ordered multistep process that involves the expression of myogenic transcription factors, followed by cyclin kinase inhibitor p21 protein induction, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (1-4). The commitment to differentiate into myotubes is influenced negatively by several factors. Treatment of myoblasts with fetal bovine serum, basic fibroblast growth factor 2, or transforming growth factor beta 1 is known to inhibit differentiation of myoblasts (5, 6). Myogenesis is also regulated negatively by oncogenes such as c-fos, Ha-ras, and E1a (7-9). The insulin-like growth factors (IGFs)1 are the only known growth factors that are crucial to myogenesis (10, 11). IGF expression is increased during myoblast differentiation in response to serum withdrawal (12-15). The amount of IGF-II secreted correlates with the rate of spontaneous differentiation that, in the absence of exogenous IGF-II, can be inhibited by antisense oligonucleotides complementary to IGF-II mRNA (16). Because of their myogenic actions, IGFs have been postulated as potential therapeutic tools in conditions characterized by muscle myopathy, atrophy, or muscle injury. In this context, IGFs have been implicated in the regulation of satellite cell function during regeneration, a characteristic response of adult muscle to injury (17, 18). However, IGFs are pleiotropic growth factors, and they also affect the growth of several tissues other than skeletal muscle. This has led to the analysis of the intracellular myogenic process initiated by IGFs. It is known that IGF-I and IGF-II switch on the myogenic program by activating the IGF-I receptor (19). During the last 2 years, the phosphatidylinositol (PI) 3-kinase has emerged as an essential second messenger for skeletal muscle cell differentiation (20-22). Moreover, by overexpressing a mutant p85 regulatory subunit of PI 3-kinase (Delta p85) lacking the ability to bind and activate the p110 catalytic subunit (L6E9-Delta p85), we showed that the heterodimeric p85-p110 is the PI 3-kinase isoform essential for IGF-induced myogenesis in L6E9 muscle cells (23). Currently, there is no information regarding the downstream signals activated by IGFs and PI 3-kinase or its PI 3-phosphate products during myogenesis, and we have recently shown that the serine/threonine p70 S6 kinase, a downstream element in several PI 3-kinase-dependent signaling cascades (24, 25), is not involved in the myogenic actions of IGFs in rat, mouse, or human cells (26).

Among the signaling events involved in myogenesis, the induction of chick embryonic myoblast fusion in low serum conditions requires NO production and NF-kappa B activity (27, 28). However, the mechanisms that trigger NF-kappa B and NOS activation in differentiating myoblasts and the involvement of these molecules in biochemical differentiation (i.e. expression of structural and functional muscle markers) remain to be defined. In the present study, we attempted to further characterize the myogenic intracellular pathway depending on IGF-II and PI 3-kinase. We describe here a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves (i) PI 3-kinase activation, (ii) Ikappa B-alpha degradation and dissociation from p65 NF-kappa B, (iii) NF-kappa B activation, and (iv) iNOS expression and activation. Moreover, we show the ability of the NOS substrate L-Arg to induce myogenesis in the absence of IGFs in both rat and human skeletal muscle cells.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- IGF-II was kindly given by Eli Lilly (Indianapolis, IN). L6E9 rat skeletal muscle cell line was kindly provided by Dr. B. Nadal-Ginard (Harvard University). Human muscle biopsies were obtained from the Departament de Neurologia, Hospital Universitari de la Santa Creu i de Sant Pau, Barcelona. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and pancreatin were from Whittaker (Walkersville, Belgium). PI 3-kinase inhibitor LY294002 (29) was from BioMol Research Laboratories (Plymouth, MA). The NF-kappa B probe for electrophoretic mobility shift assay was kindly given by Dr. Jean-François Peyron (Inserm U364, Nice, France).

Sodium salicylate was from Merck (Darmstadt, Germany); L-arginine monohydrochloride was from Fluka (Buchs, Switzerlamd); L-lysine monohydrochloride and Nomega -nitro-L-arginine were from Sigma; pyrrolidinedithiocarbamic acid was from Alexis Corporation (Laufelfingen, Switzerland). Nitrate/nitrite colorimetric assay kit was from Cayman Chemical Company (Ann Arbor, MI).

The polyclonal antibody OSCRX was raised against the C terminus of GLUT4 (30). A rabbit polyclonal antibody against beta 1-integrin was kindly given by Dr. Carles Enrich (University of Barcelona) (31). Polyclonal antibody C38320 against caveolin 3 was from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibody MF 20, which stains all sarcomeric myosin heavy chain isoforms, was from the Developmental Studies Hybridoma Bank (Baltimore, MD). Polyclonal antibodies against p21 (C-19), alpha -Ikappa B (C-15), and p65 NF-kappa B (C-20) were from Santa Cruz Biotech (Santa Cruz, CA). The nitric-oxide synthase antibody set containing iNOS, cNOS, and eNOS polyclonal antibodies was from Calbiochem (La Jolla, CA).

Cell Culture-- Rat L6E9 myoblasts were grown in monolayer culture in DMEM containing 10% (v/v) fetal bovine serum and 1% (v/v) antibiotics (10,000 units/ml penicillin G and 10 mg/ml streptomycin). Confluent 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. Images shown are representative of 10-20 randomly selected microscope fields from each one of at least 10 independent experiments. Cells were photographed after staining the nuclei with Mayer's hemalum solution for microscopy (Merck). Cell fusion was quantified by counting the percentage of nuclei in myotubes from a total of at least 2500 nuclei from 15-25 independent randomly selected microscope fields for each condition tested.

Human muscle normal biopsies were minced into small pieces and cultured in Eagle's minimal essential medium (Mediatech, Reston, VA) supplemented with 10% heat-inactivated fetal calf serum (MA Bioproducts, Springville, MD), 2% chick embryo extracts (Life Technologies, Inc., Bethesda, MD), 50 mM glutamine and gentamycin, as described previously (26). Cultures were grown to subconfluence and examined to assess the development of myotubes after 6 days in DMEM and antibiotics without or with IGF-II (40 nM), together with other compounds when indicated. Images shown are representative of 10-20 randomly selected microscope fields from each one of three independent experiments, in which each condition was tested in duplicate.

Immunofluorescence Analysis-- For immunofluorescence labeling, cells were grown on 0.1% gelatin-pretreated glass coverslips. Myoblasts or 2-day-old IGF-II induced myotubes were fixed on coverslips and incubated with 30 µl of a 1:100 dilution of primary antibody in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin for 1 h at room temperature. Coverslips were washed three times in PBS, the last one for 15 min, before incubating with the rodamine-conjugated secondary antibody (1:100 dilution in 0.5% bovine serum albumin) for 1 h at room temperature. Nonspecific controls were carried out in the absence of primary antibody (data not shown). Coverslips were then washed three times in PBS, the last one for 15 min. Finally, coverslips were mounted with immunofluorescence medium.

Electrophoresis and Immunoblotting of Membranes-- Cells were lysed for 30 min at 4 °C in a buffer containing 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, 0.1% aprotinin, supplemented with 1% Nonidet-P40. Cell extracts were centrifuged for 15 min at 10,000 × g at 4 °C, and 50 µg of the solubilized proteins was loaded. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (32). Gels were blotted into Immobilon in buffer consisting of 20% methanol, 200 mM glycine, 25 mM Tris, pH 8.3. Following transfer, the filters were blocked with 5% nonfat dry milk in PBS for 1 h at 37 °C and then incubated overnight at 4 °C with primary antibodies in PBS containing 1% nonfat dry milk and 0.02% sodium azide. Proteins were detected by ECL chemiluminiscence system (Amersham Pharmacia Biotech) except for beta 1-integrin, which was detected using 125I-protein A for 3 h at room temperature. Quantification of protein expression was performed by scanning densitometry of at least three independent experiments for each condition.

alpha -Ikappa B/p65 NF-kappa B Association-- Cells were scraped and solubilized for 30 min at 4 °C in a buffer containing 50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM vanadate, 0.1% phenylmethylsulfonyl fluoride, 0.1% aprotinin, supplemented with 1% Nonidet P-40. The cell lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and 0.5 mg of the supernatants were immunoprecipitated with 10 µg of anti-p65 NF-kappa B or nonimmune controls (not shown). Antibodies were preadsorbed on protein G-Sepharose at 4 °C for 1 h and washed twice in 50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1% Nonidet P-40, before being incubated with the solubilized proteins for 2 h at 4 °C. The immunopellets were washed three times in the same buffer before being resuspended in SDS-polyacrylamide gel electrophoresis sample buffer and analyzed by Western blot using polyclonal antibodies against anti-Ikappa B-alpha , as described above.

Electrophoretic Mobility Shift Assay-- NF-kappa B-DNA binding activity was analyzed in total cellular 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, 0.1% aprotinin) (33). 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'). Totex extract samples (70 µg) were incubated for 10 min at 4 °C with 30 ng of poly(dI·dC) and 5 µl of 5× NF buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM dithiothreitol, 5 mM EDTA, 20% glycerol 0.4 mg/ml salmon sperm DNA) in a final volume of 25 µl. The end-labeled probe was then added for a further incubation of 25 min at 25 °C. The specificity of the bands detected was verified by adding 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).

Nitrite/Nitrate Assay-- The NO production was determined by assaying for nitrite and nitrate accumulation in the culture media. Briefly, following treatment of the cells with the indicated agents, culture media were filtered with 0.2-µm filters, and 80 µl of each sample was treated with nitrate reductase and its cofactors to convert all of the nitrate to nitrite before applying 100 µl of the Griess reagent (0.5% naphthylethylenediamine dihydrochloride, 1% sulfanylamidein, 2.5% phosphoric acid). Absorbance was measured at 543 nm, and nitrite concentration was determined using a standard curve of sodium nitrite concentrations ranging from 0 to 50 nmol/ml.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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IGF-II Induces NF-kappa B and iNOS Activation as Early Events during Myogenesis-- During the process of L6E9 myoblast differentiation, IGF-II caused the induction of skeletal muscle protein markers such as the insulin-sensitive glucose transporter GLUT4, the isoform 3 of caveolin, which is a component of the distrophin complex, and the myosin heavy chain (Fig. 1a). The expression of these proteins increased with the time of exposure to IGF-II, being maximal at day 4, when a fully morphological differentiation is achieved (23). We observed that IGF-II induced as an early myogenic marker the expression of iNOS (Fig. 1a). At differentiation day 1, IGF-II induced 2.3 ± 0.2-fold (n = 3) increase in iNOS expression compared with myoblasts maintained in DMEM alone, and a progressive decrease in its expression was observed from day 1 to day 4. As a control, we examined the expression levels of beta 1-integrin, a nonmuscle-specific plasma membrane protein, that remained unaltered during IGF-II-induced differentiation (Fig. 1a).


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Fig. 1.   IGF-II induces NF-kappa B and iNOS activation as early events during myogenesis. a, confluent L6E9 myoblasts were allowed to differentiate in serum-free medium (DMEM) for 4 days in the absence or presence IGF-II (40 nM). iNOS and muscle-specific protein expression (caveolin 3, insulin-sensitive glucose transporter GLUT 4, and myosin heavy chain (MHC) were analyzed by Western blot of total cell lysates (50 µg) from each condition. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein beta 1-integrin. b, total cell extracts from cells differentiated in the absence (day 1) or presence of IGF-II (days 1-4) were incubated with a 32P-labeled NF-kappa B probe corresponding to the kappa B site in the interleukin-2 gene promoter and analyzed by electrophoretic mobility shift assay. The specificity of the bands was verified by adding 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). c, total cell extracts from cells differentiated for 24 h in the absence (DMEM) or presence of IGF-II with or without 10 mM NaSal were incubated with a 32P-labeled NF-kappa B probe corresponding to the kappa B site in the interleukin-2 gene promoter and analyzed by electrophoretic mobility shift assay. d, iNOS expression was analyzed in total cell lysates from myoblasts differentiated for 1 day in the absence or presence of IGF-II with or without NF-kappa B inhibitors NaSal (10 mM) or PDTC (100 µM) (upper panel). NOS activity was determined as nitrite and nitrate accumulation in the culture media at differentiation day 1 in the absence (bar a) or presence of IGF-II without (bar b) or with NaSal (10 mM, bar c) or PDTC (100 µM, bar d) and expressed as percentage of nitrite/nitrate accumulation in serum-free medium (DMEM) (lower panel).

Taking into account that iNOS expression is tightly regulated by the transcription factor NF-kappa B, we analyzed the DNA binding activity of NF-kappa B on total cell extracts from myoblasts differentiated in the absence or presence of IGF-II. Fig. 1b shows that the NF-kappa B DNA binding activity was increased by IGF-II in a way that correlated in time with the profile of iNOS expression. Maximal activation was detected at 24 h, when the level of NF-kappa B DNA binding activity induced by IGF-II was 2.6 ± 0.3-fold (n = 3) over controls (DMEM-treated cells). No significant differences in NF-kappa B DNA binding activity were observed at shorter differentiation times (data not shown and see Fig. 3a). NF-kappa B activation by IGF-II was sensitive to inhibitors of Ikappa B phosphorylation or degradation such as sodium salicylate (10 mM) (Fig. 1c) or PDTC (100 µM) (data not shown) (34, 35). By using these NF-kappa B inhibitors, we determined that IGF-II-induced iNOS expression was totally dependent on NF-kappa B activation (Fig. 1d, upper panel). Moreover, IGF-II-induced iNOS expression at day 1 correlated with IGF-II-dependent NO production, as measured by nitrite/nitrate accumulation in the cell culture media; the IGF-II-induced NO production was dependent on NF-kappa B activity, because it was completely blocked by NaSal or PDTC (Fig. 1d, lower panel).

IGF-II Requires NF-kappa B and iNOS Activation to Signal Myogenesis-- Results presented above indicate that NF-kappa B-dependent iNOS expression and activity are early events during myogenesis induced by IGF-II. We next tested the relevance of this IGF-II-induced pathway for muscle differentiation. L6E9 myoblast differentiation was induced with IGF-II (40 nM) in the absence or presence of NF-kappa B or NOS inhibitors, and after 4 days the expression of differentiation parameters was analyzed (Fig. 2, a-c). The IGF-II induction of muscle markers such as GLUT4 or caveolin 3 was inhibited in a dose-dependent manner by NF-kappa B inhibitors PDTC (Fig. 2a) or NaSal (Fig. 2b) and by NOS inhibitors NNA (Fig. 2c) or NG-nitro-L-arginine-methyl ester (data not shown).


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Fig. 2.   IGF-II-induced myogenesis is blocked by NF-kappa B and NOS inhibitors. a-c, confluent L6E9 myoblasts were differentiated for 4 days in the absence (-) or presence (+) of 40 nM IGF-II without or with increasing amounts of PDTC (a), NaSal (b), or NNA (c). The content of the skeletal muscle-markers GLUT4 and caveolin 3 was analyzed by Western blot in cell lysates from each condition (50 µg). Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein beta 1-integrin. d, L6E9 myoblasts were grown to confluence in a 10% fetal bovine serum-containing medium (Mb) and then allowed to differentiate in a serum-free medium (DMEM) or DMEM supplemented with 40 nM IGF-II in the absence or presence of NF-kappa B inhibitors PDTC (100 µM) or NaSal (10 mM) or NOS inhibitor NNA (5 mM). After 4 days in each condition, morphological differentiation was assessed by myotube formation. Images shown are representative of 15-25 microscope fields taken at random from each one of at least 10 independent experiments. e, human myoblasts (Mb) derived from skeletal muscle normal biopsies were grown to subconfluence and examined to assess the development of myotubes after 6 days in DMEM without or with IGF-II (40 nM) in the absence or presence of NaSal (10 mM). Images are representative of 10-20 microscope fields analyzed at random from each one of three independent experiments, in which each condition was tested in duplicate. Scale bars, 30 µm (the scale is the same for all panels).

At the morphological level, subconfluent myoblasts incubated for 4 days in a medium containing DMEM supplemented with IGF-II formed large multinucleated myotubes (Fig. 2d). In these conditions, the percentage of nuclei in myotubes induced by IGF-II was 66 ± 3% (a total of 2555 nuclei were counted from 19 randomly selected microscopic fields), whereas in the presence of DMEM alone only 9 ± 2% nuclei were in myotubes (a total of 2538 nuclei were counted from 21 randomly selected microscopic fields). When IGF-II-induced cell fusion was analyzed in the presence of NF-kappa B inhibitors such as NaSal (10 mM) or PDTC (50 µM) or in the presence of nitric-oxide synthase inhibitors such as NNA (5 mM) or NG-nitro-L-arginine-methyl ester (data not shown), a total blockade of the IGF-II myotube formation capacity was observed (no myotube formation was seen in any of at least 10 independent experiments performed under each of these conditions) (Fig. 2d). The involvement of NF-kappa B activation in IGF-II-induced myogenesis was also detected in myoblast primary cultures derived from human skeletal muscle biopsies; human myoblasts responded to IGF-II by activating cell fusion, and this effect was blocked by the addition of sodium salicylate (Fig. 2e) or PDTC (data not shown).

L-Arginine Mimics IGF-II-induced Morphological and Biochemical Skeletal Muscle Cell Differentiation-- As very little or no expression of iNOS is detected in L6E9 cells in the absence of IGF-II, we analyzed whether L6E9 cells expressed other NOS isoforms because both cNOS and the eNOS isoforms have been detected in other skeletal muscle models (36, 37). We detected the expression of cNOS in both L6E9 myoblasts and myotubes by immunofluorescence (Fig. 3a), whereas very low levels of type III NOS (eNOS) were detected (data not shown). As a control, we analyzed the expression of p21 cyclin-dependent kinase (Cdk) inhibitor expression, which as expected, was only detected in nuclei from myotubes (Fig. 3a). Based on these observations, we analyzed whether the NOS substrate L-Arg was able to mimic IGF-II ability to induce biochemical and morphological myoblast differentiation. We incubated subconfluent L6E9 myoblasts with the NOS substrate, L-Arg (5 mM), to generate NO in the absence of exogenous IGF-II addition. We observed that after 24 h of incubation, L-Arg induced a NO production similar to that induced by IGF-II, which was totally blocked by the NOS inhibitor NNA (Fig. 3b).


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Fig. 3.   L-Arginine mimics IGF-II-induced morphological and biochemical skeletal muscle cell differentiation. a, cNOS expression in L6E9 cells. Cells were grown to subconfluence on gelatin-treated glass coverslips and differentiated or not (myoblasts or myotubes, respectively) for 2 days in serum-free medium supplemented with 40 nM IGF-II. For immunofluorescence detection of cNOS and Cdk inhibitor p21 expression, cells were fixed with methanol and incubated with anti-cNOS or anti p21 antibodies. Finally, cells were incubated with a rodamine-conjugated secondary antibody, as described under "Materials and Methods." Scale bars, 10 µm. b, confluent L6E9 myoblasts were allowed to differentiate for 1 day in serum-free medium alone (DMEM) or supplemented with (i) 40 nM IGF-II, (ii) 5 mM L-Arg, or (iii) 5 mM L-Arg plus 5 mM NNA. NOS activity was estimated as nitrite and nitrate accumulation in the culture media and expressed as percentage of nitrite/nitrate accumulation in serum-free medium (DMEM). c, L6E9 myoblasts were grown to confluence in a 10% fetal bovine serum-containing medium and then allowed to differentiate in a serum-free medium supplemented either with 10 mM L-Arg or 10 mM L-Lys. After 4 days in each condition, morphological differentiation was assessed by myotube formation. Images shown are representative from 15-25 microscope fields taken at random from each one of at least 10 independent experiments. Scale bars, 75 µm (the scale is the same for all panels). d, human myoblasts derived from skeletal muscle normal biopsies were grown to subconfluence and then allowed to differentiate for 6 days in a serum-free medium supplemented with either 10 mM L-Arg or 10 mM L-Lys. Images are representative of 10-20 microscope fields analyzed at random from each one of three independent experiments in which each condition was tested in duplicate. Scale bars, 30 µm (the scale is the same for all panels). e, confluent L6E9 myoblasts were allowed to differentiate for 4 days in serum-free medium alone DMEM or supplemented with either (i) 40 nM IGF-II, (ii) 5 mM L-Arg, (iii) 5 mM L-Lys, or (iV) 5 mM L-Arg plus 5 mM NNA. Muscle-specific protein expression (caveolin 3, insulin-sensitive glucose transporter GLUT 4, and myosin heavy chain) was analyzed by Western blot of total cell lysates (50 µg) from each condition. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein beta 1-integrin.

L-Arginine (5 mM) induced a high level of morphological differentiation of L6E9 cells (46 ± 4% of nuclei into myotubes from a total of 2099 nuclei in 18 randomly selected microscopic fields) (Fig. 3c). This effect was specific for the NOS substrate, because in the same conditions the basic amino acid L-Lys did not induce additional cell fusion to that observed with DMEM alone (12 ± 2% of nuclei into myotubes from a total of 3158 nuclei in 22 randomly selected microscopic fields). Similar effects were observed in myoblast primary cultures derived from human skeletal muscle biopsies, where L-Arg but not L-Lys induced multinucleated myotube formation (Fig. 3d). L-Arg also led to the expression of muscle-specific protein markers such as caveolin 3, myosin heavy chain, and GLUT4, at levels comparable with those observed with IGF-II (IGF-II and L-Arg induced 2.6 ± 0.3- and 2.2 ± 0.4-fold increase over DMEM controls, respectively, in muscle-specific protein expression, n = 3) (Fig. 3e). Moreover, the effect of L-Arg on biochemical differentiation was dependent on NOS activity because it was totally blocked by the NOS inhibitor NNA, whereas no induction of muscle-specific proteins was observed in the presence of L-Lys (5 mM) in the same conditions (Fig. 3e).

PI 3-Kinase Is Required for IGF-II Activation of NF-kappa B and iNOS-- We have recently reported that the p85/p110 phosphatidylinositol 3-kinase is an essential mediator of IGF-II-myogenic actions (23). Indeed, either overexpression of a dominant negative mutant of p85 PI 3-kinase regulatory subunit or treatment of L6E9 myoblast with the PI 3-kinase inhibitor LY294002 blocked the IGF-II-dependent myogenic program. Results presented above indicate that NF-kappa B and iNOS activation are also critical for IGF-II-induced myogenic program. Therefore, we next evaluated whether the differentiation cascade involving IGF-II/NF-kappa B/iNOS was dependent on PI 3-kinase activity. To this end, L6E9 myoblast differentiation was induced with IGF-II in the absence or presence of the PI 3-kinase inhibitor LY294002. Fig. 4 shows that the presence of the PI 3-kinase inhibitor (20 µM) blocked the IGF-II-induced peak of NF-kappa B activation (Fig. 4a), the IGF-II induction of iNOS expression (Fig. 4b), and the IGF-II-induced NO production (Fig. 4c). In all, these results indicate that IGF-II requires PI 3-kinase to induce NF-kappa B and iNOS activation.


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Fig. 4.   PI 3-kinase is required for IGF-II induction of NF-kappa B DNA binding activity, iNOS expression, and NOS activity. a, total cell extracts were obtained at different times (12, 24, and 48 h) after differentiation in the absence or presence of 40 nM IGF-II without or with 20 µM LY294002. Cell extracts were incubated with a 32P-labeled NF-kappa B probe corresponding to the kappa B site in the interleukin-2 gene promoter and analyzed by electrophoretic mobility shift assay. The specificity of the bands was verified by adding 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). b, confluent L6E9 myoblasts were allowed to differentiate in serum-free medium (DMEM) for 24 h in the absence or presence 40 nM IGF-II without or with 20 µM LY294002. iNOS protein expression was analyzed by Western blot of total cell lysates (50 µg) from each condition. c, NOS activity was determined as nitrite and nitrate accumulation in the culture media at differentiation day 1 in the absence (DMEM) or presence of IGF-II without or with 20 µM LY294002 and expressed as percentage over nitrite/nitrate accumulation in serum-free medium (DMEM).

IGF-II Induces Ikappa B-alpha Degradation and Dissociation from p65 NF-kappa B through a PI 3-Kinase-dependent Pathway-- We next analyzed the mechanism involved in the activation of NF-kappa B by IGF-II and PI 3-kinase. A primary level of control for NF-kappa B is through interaction with the inhibitory protein Ikappa B, and most of NF-kappa B activators tested induced Ikappa B-alpha degradation (38). Therefore, we analyzed the effect of IGF-II on Ikappa B-alpha protein levels in L6E9 myoblasts. We observed an up-regulation in the expression of both Ikappa B-alpha and the p65 subunit of NF-kappa B after 12 h of IGF-II treatment compared with cells incubated in DMEM alone. The IGF-II-induced increase in protein levels was 2.0 ± 0.1- and 1.5 ± 0.1-fold (n = 3) for Ikappa B-alpha and p65, respectively (Fig. 5a, IGF-II versus DMEM at 12 h).


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Fig. 5.   IGF-II decreases alpha -Ikappa B protein levels and Ikappa B-alpha dissociation from p65 NF-kappa B through a PI 3-kinase-dependent pathway. a, confluent L6E9 myoblasts were allowed to differentiate in serum-free medium (DMEM) for 1 day in the absence or presence IGF-II (40 nM). Ikappa B-alpha and p65 NF-kappa B protein content was analyzed at different times by Western blot of total cell lysates (50 µg) from each condition. b, confluent L6E9 myoblasts were allowed to differentiate for 24 h in serum-free medium supplemented with 40 nM IGF-II without or with 20 µM LY294002. Ikappa B-alpha protein content was analyzed at different times by Western blot of total cell lysates (50 µg) from each condition. c, confluent L6E9 myoblasts were differentiated for 24 h in serum-free medium supplemented with 40 nM IGF-II without or with 20 µM LY294002. Cells were solubilized and proteins (0.5 mg) were incubated with 10 µg of antiserum against p65 NF-kappa B and protein G-Sepharose. Immune complexes were analyzed by Western blot with an antibody against Ikappa B-alpha .

From 12 to 24 h of treatment, IGF-II induced a 39 ± 4% (n = 3) decrease in Ikappa B-alpha total cell content without any detectable alteration in the expression levels of p65 (Fig. 5a, IGF-II, 24 h versus 12 h). IGF-II-induced decrease in Ikappa B-alpha levels was inhibited when differentiation was carried out in the presence of the PI 3-kinase inhibitor LY294002. Thus, at 24 h of IGF-II treatment, IkB-alpha protein content was 1.7 ± 0.3-fold (n = 3) higher in the presence than in the absence of PI 3-kinase inhibitor (Fig. 5b, IGF-II+LY294002 versus IGF-II at 24 h).

Immunoprecipitation of myoblast lysates with an anti-p65 antibody co-immunoprecipitated Ikappa B-alpha (Fig. 5c). After 16 h of IGF-II treatment, the amount of Ikappa B-alpha associated with p65 was similar in the absence or presence of LY294002 (data not shown). However at 24 h, the amount of Ikappa B-alpha associated with p65 was 1.8 ± 0.2-fold (n = 3) higher in the presence of IGF-II and LY294002 than in the presence of IGF-II alone.

It is interesting to note that the increase in Ikappa B-alpha and p65 expression detected at 12 h of IGF-II treatment remained unaffected by LY294002 in three independent experiments (for Ikappa B-alpha , Fig. 5, a and b, 12 h lanes; for p65, data not shown). These results suggest that during the first 12 h of treatment, IGF-II up-regulated the expression of Ikappa B-alpha and p65 NF-kappa B subunit in a PI 3-kinase-independent manner (Fig. 5, a and b, 12 h lanes); during the following 12 h, IGF-II induced a decrease in Ikappa B-alpha protein content and the activation of NF-kappa B by dissociation of p65 from Ikappa B-alpha through a PI 3-kinase-dependent pathway (Fig. 5, b and c, 24 h lanes).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Results presented here define a model for the IGF-II-induced myogenic pathway, in which PI 3-kinase, NF-kappa B, and NOS activities are critical elements. Our data show that IGF-II-induced myogenesis can be blocked by either PI 3-kinase inhibitors, NF-kappa B inhibitors, or NOS inhibitors. Several lines of evidence indicate that PI 3-kinase, NF-kappa B, and iNOS are elements of a common myogenic cascade: (i) IGF-II-induced NF-kappa B activation was blocked by PI 3-kinase inhibition, (ii) IGF-II-induced iNOS expression correlated in time with the activation of NF-kappa B and was blocked by either PI 3-kinase or NF-kappa B inhibitors, and (iii) IGF-II-induced iNOS expression was accompanied by an IGF-II-induced NO production that was blocked by PI 3-kinase, NF-kappa B, or NOS inhibitors.

An early event in the IGF-II-induced differentiation program was the increase in Ikappa B-alpha and the p65 NF-kappa B subunit protein content. These data are consistent with the evidence that NF-kappa B and Ikappa B are components of a mutual regulatory system in which the levels of one component control the activity or quantity of the other (39). After 24 h of IGF-II treatment, a transient peak of NF-kappa B DNA binding activity was observed that correlated with a decrease in Ikappa B-alpha protein levels. It has been reported that differentiation of skeletal muscle cells in a medium containing low serum concentration induced a down-regulation of the activity of NF-kappa B and other proliferating transcription factors (40). The authors did not consider the effects of IGFs in their systems, and they established from their data that there was a causative relation between NF-kappa B down-regulation and myogenesis. However, the authors did not examine whether the NF-kappa B activity detected after 24 h in a low serum differentiation medium, even if it was lower than that observed during proliferation, was necessary for differentiation. Indeed, this appears to be the case, because consistent with our observations for rat and human myoblasts differentiated with IGF-II, Lee et al. (28) reported that chick embryonic myoblasts require NF-kappa B activity to fuse in a low serum-containing differentiation medium.

The effect of IGF-II in modulating NF-kappa B activity in L6E9 myoblasts appears to be tightly regulated by PI 3-kinase activity. Our results indicate that PI 3-kinase is required for NF-kappa B activation during myoblast differentiation through a mechanism that involves Ikappa B-alpha degradation. The peak of NF-kappa B DNA binding activity after 24 h of IGF-II treatment was blocked by PI 3-kinase inhibition, and this correlated in time with higher amounts of Ikappa B-alpha protein and higher amounts of inactive p65 NF-kappa B complexed with Ikappa B-alpha in the presence of LY294002 than those detected in control myoblasts.

Our results suggest a biphasic effect of IGF-II in modulating NF-kappa B activity in L6E9 myoblasts: during the first 12 h, IGF-II induced the up-regulation of Ikappa B-alpha and p65 expression in a PI 3-kinase-independent way, and from 12 to 24 h, IGF-II induced the dissociation of Ikappa B-alpha from p65 and the degradation of Ikappa B-alpha through PI 3-kinase-dependent mechanisms. We interpret the complexity of these results as an example of the diversity of signals that can be elicited when a growth factor activates a tyrosine kinase receptor and generates a set of distinct second messengers in the cell. In this particular case, the expression of p65 and IkB-alpha is induced by IGF-II through yet undefined signaling pathways that do not involve PI 3-kinase activity. In contrast, the degradation of IkB-alpha and its dissociation from p65 require PI 3-kinase activity. In this latter case, it is tempting to hypothesize that PI 3-kinase could be acting upstream from the IkB kinases responsible for NF-kappa B activation. Indeed, many if not all activators of NF-kappa B have been reported to induce degradation of Ikappa B-alpha (41, 42) by activating Ikappa B kinases that phosphorylate Ikappa B-alpha on serines 32 and 36 within the N-terminal regulatory domain (43-47). This is the most probable mechanism by which IGF-II and PI 3-kinase induce NF-kappa B activation in differentiating myoblasts, because we did not detect any IGF-II-induced Ikappa B-alpha tyrosine phosphorylation, which represents a proteolysis-independent mechanism for NF-kappa B activation (data not shown) (48). Potential kinases acting between PI 3-kinase and IkB kinases during IGF-II myogenesis could be the protein kinase B and some PKC isoforms that are activated by PI 3-kinase products (49-51). In this context, zeta PKC and epsilon PKC have been implicated in NF-kappa B activation (52, 53).

We show here that iNOS expression and activation are early events during IGF-II-induced myogenesis. Moreover, we describe here the ability of the NOS substrate L-Arg to induce myogenesis in the absence of IGFs in both rat and human skeletal muscle cells. The identification of direct targets for NO action in myogenesis is a matter of current interest, and one gene that may be regulated by NO in skeletal muscle cells is the Cdk inhibitor p21. It has been reported that NO blocks the cell cycle progression at the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that the p21 induction is involved in the Cdk2 inhibition (54). A causative role for NO action in nerve growth factor-induced cell cycle exit and differentiation of PC12 neuronal cells has been reported (55). It has been proposed that NO diffusion from the producer cell could promote cessation of growth in adjacent cells, contributing to synchronization of development from precursor cells. The expression of Cdk inhibitor p21 and cell cycle exit are prerequisites for myogenesis, and these events, which are both induced by IGF-II in L6E9 cells, may be mediated by NO (1, 26, 23). Results presented here suggest that IGF-II-dependent myoblast differentiation can be mimicked by L-Arg treatment, both at the biochemical and at the morphological level, and this ability of L-Arg to induce myogenesis in the absence of IGFs may contribute to the development of new strategies for the treatment of myopathies.

    ACKNOWLEDGEMENTS

We thank Robin Rycroft for editorial support and Dr. M. Camps, Dr. R. Casaroli, and S. Castel (Servei Científico Tècnics, University of Barcelona) for expert advice in microscopy techniques. We are grateful to Dr. I. Illa and E. Gallardo (Departament de Neurologia, Hospital Universitari de la Santa Creu i de Sant Pau, Barcelona) for providing human skeletal muscle biopsies and to Dr. J.-F. Peyron (Inserm U364, Nice, France) and Dr. T. Carbonell and Dr. Jesus Ródenas (Departament de Fisiologia Animal, Universitat de Barcelona) for helpful discussions.

    FOOTNOTES

* This work was supported by Dirección General de Investigación Científica y Técnica Research Grant PB95/0971, Fondo de Investigación Sanitaria Research Grant 97/2101, and Generalitat de Catalunya Research Grants 1995 SGR-537 and 1997 SGR-121.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.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 34-3-4021547; Fax: 34-3-4021559; E.mail: perlak{at}porthos.bio.ub.es.

    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; NOS, nitric-oxide synthase; iNOS, inducible NOS; cNOS, constitutive NOS; eNOS, endothelial NOS; NaSal, sodium salicylate; NNA, Nomega -nitro-L-arginine; L-Arg, L-arginine; L-Lys, L-lysine; PDTC, pyrrolidinedithiocarbamic acid; NF-kappa B, nuclear factor kappa B; PBS, phosphate-buffered saline.

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