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Endocrinology Vol. 143, No. 8 3017-3025
Copyright © 2002 by The Endocrine Society

ARTICLE

Identification of Intracellular Signaling Pathways that Induce Myotonic Dystrophy Protein Kinase Expression during Myogenesis

Marta Carrasco, Judith Canicio, Manuel Palacín, Antonio Zorzano and Perla Kaliman

Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain

Address all correspondence and requests for reprints to: Dr. Perla Kaliman, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain. E-mail: . perlak{at}porthos.bio.ub.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myotonic dystrophy (DM) is the most common inherited adult neuromuscular disorder. DM is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Decreased DMPK protein levels may contribute to the pathology of DM, as revealed by gene target studies. However, the postnatal regulation of DMPK expression and its pathophysiological role remain undefined. We studied the regulation of DMPK protein and mRNA expression during myogenesis in rat L6E9 myoblasts, mouse C2C12 myoblasts, and 10T1/2 fibroblasts stably expressing the myogenic transcription factor MyoD (10T1/2-MyoD). We detected DMPK as an 80-kDa protein mainly localized to the cytosolic fraction of skeletal muscle cells. DMPK expression and protein kinase activity were enhanced in IGF-II-differentiated cells. In L6E9 and C2C12 cells, DMPK expression was regulated through the same signaling pathways (i.e. phosphatidylinositol 3-kinase, nuclear factor-{kappa}B, nitric oxide synthase, and p38 mitogen-activated protein kinase) that had been described as being crucial for the myogenesis induced by either low serum or IGF-II. However, in 10T1/2-MyoD cells, p38 MAPK inhibition blocked cell fusion and caveolin-3 expression without affecting DMPK up-regulation. These results suggest that although DMPK is induced during myogenesis, its expression cannot be totally associated with the development of a fully differentiated phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MYOTONIC MUSCULAR dystrophy (DM) is an autosomal, dominant-inherited, neuromuscular disorder. Clinical expression of DM is extremely variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy, and insulin resistance (1).

The DM mutation has been identified as the expansion of an unstable CTG repeat in the 3'-untranslated region of a gene encoding the DM protein kinase (DMPK), at chromosome 19q13.3, and the age of onset and the severity of the disease correlate with the extent of expansion (2, 3, 4, 5). Several lines of evidence indicate that DM may be the result of distinct consequences of the CTG expansion, including DMPK insufficiency. In homozygous DMPK knockout mice, muscle weakness and myopathy are observed, indicating that alterations in DMPK expression may contribute to DM in skeletal muscle (6, 7). There is evidence for a delay in skeletal muscle maturation in DM patients, possibly as a consequence of a retarded rate of fusion of myoblasts to multinucleated myotubes. However, there are conflicting results on the level of total DMPK transcript in DM patients and the degree of differentiation of muscle cells overexpressing DMPK (8, 9, 10, 11, 12, 13).

The precise pathophysiological role of DMPK remains elusive, as little is known about its involvement in skeletal muscle cell differentiation or function. Available data regarding the regulation of DMPK in skeletal muscle cells reveal that its gene contains a low level promoter that operates in conjunction with an enhancer element in the first intron with conserved myogenic transcription factor MyoD-responsive E boxes (14). DMPK has been implicated in modulating the initial events of excitation-contraction coupling in skeletal muscle (15), and it was found to specifically associate with a small heat shock protein designated MKBP (DMPK-binding protein), which induces DMPK kinase activity in vitro and protects it from heat-induced inactivation (16).

Here we studied the molecular signaling pathways that regulate DMPK protein and mRNA expression during myogenesis in three models of skeletal muscle: rat L6E9 myoblasts, mouse C2C12 myoblasts, and 10T1/2 fibroblasts stably expressing MyoD (10T1/2-MyoD). We analyzed the regulation of DMPK expression during IGF-induced myogenesis. IGFs play a crucial role in myogenesis. IGF-I and IGF-II are potent stimulators of muscle differentiation, and they are potential candidates for regulation of satellite cell function during regeneration, a characteristic response of adult muscle to injury (17). Moreover, IGF expression increases during myoblast differentiation in response to serum withdrawal and switches on the myogenic program through the IGF-I receptor, activating the expression of myogenic transcription factors, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (18, 19, 20, 21, 22, 23). Among the intracellular pathways that signal myogenesis, the phosphatidylinositol 3-kinase (PI 3-kinase) has emerged as an essential element (24, 25, 26, 27). Moreover, it has been described that myogenic signaling cascades initiated by IGF-II or low serum also require NF-{kappa}B activation, inducible nitric oxide synthase (NOS) expression and activation and p38 MAPK activation (28, 29, 30, 31, 32, 33). We show that DMPK expression is up-regulated during differentiation induced by either des(1,3)IGF-I or IGF-II. DMPK mRNA and protein expression induced by either IGFs or serum deprivation is tightly regulated by a myogenic signaling pathway that involves PI 3-kinase, NF-{kappa}B, NOS, and p38 MAPK activities. We show that MyoD overexpression in 10T1/2 cells triggers DMPK expression through a signaling pathway involving PI 3kinase, NF-{kappa}B, and NOS, but not p38 MAPK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
IGF-II and des(1,3)IGF-I were donated by Eli Lilly \|[amp ]\| Co. (Indianapolis, IN) and Genentech, Inc. (South San Francisco, CA), respectively. The L6E9 rat skeletal muscle cell line was provided by Dr. B. Nadal-Ginard (Harvard University, Boston, MA). 10T1/2 cells transfected with MyoD were obtained from Dr. Vicente Andrés (Centro de Biomedicina, Valencia, Spain). C2C12 cells were obtained from ATCC (Manassas, VA). A rabbit polyclonal antibody against ß1-integrin was donated by Dr. Carles Enrich (University of Barcelona, Barcelona, Spain). Mouse full-length DMPK cDNA was provided by Dr. B. Wieringa (University of Nijmegen, Nijmegen, The Netherlands). Monoclonal antibodies against DMPK (MANDM1 and MANDM5) were provided by Dr. G. E. Morris (North East Wales Institute, Wrexham, UK).

The PI 3-kinase inhibitor LY294002 was purchased from BIOMOL Research Laboratories, Inc. (Plymouth, MA). SB202190 was obtained from Calbiochem (La Jolla, CA). Sodium salicylate was purchased from Merck \|[amp ]\| Co. (Darmstadt, Germany). N{omega}-Nitro-L-arginine was obtained from Sigma (St. Louis, MO). Polyclonal antibody C38320 against caveolin-3 was obtained from Transduction Laboratories, Inc. (Lexington, KY). Mouse monoclonal antibody MF 20, which stains all sarcomeric myosin heavy chain isoforms, was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA). Anti-ß-actin (clone AC-15) monoclonal antibody was purchased from Sigma.

Cell culture
Cells were grown in monolayer culture in DMEM containing 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) antibiotics (10,000 U/ml penicillin G and 10 mg/ml streptomycin; BioWhittaker, Walkersville, MD). Confluent myoblasts were differentiated in DMEM plus antibiotics with or without the addition of IGF-II (20 nM), 2% FBS (L6E9 cells), 2% horse serum (HS; C2C12 cells) or 5% HS (10T1/2-MyoD cells) in the absence or presence of other compounds, as indicated for each experiment. 3T3L1 cells were cultured and differentiated as described previously (34).

RNA isolation and Northern blot analysis
Total RNA from cells was extracted using the phenol/chloroform method (35). All samples had a 260/280 absorbance ratio above 1.7. After quantification, total RNA (30 µg) was denatured at 65 C in the presence of formamide, formaldehyde, and ethidium bromide (36). RNA was separated on a 1.8% agarose/formaldehyde gel and blotted on Hybond N+ filters. The RNA in gels and filters was visualized with ethidium bromide and photographed by UV transillumination to ensure the integrity of RNA, to check the loading, and to confirm proper transfer. RNA was transferred in 10x standard saline citrate (0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Blots were incubated with a fluorescein-labeled probe prepared with the Gene Image random prime labeling module and were detected with the CDP-Star detection module (Amersham Pharmacia Biotech, Little Chalfont, UK). The probe for DMPK was 2.9-kb mouse DMPK cDNA.

Preparation of cytosol and membrane fractions from muscle cells
Cells were homogenized by 20 strokes with a Dounce A homogenizer in 3 vol ice-cold buffer containing 20 mM HEPES, 250 mM sucrose, 2 mM EGTA, 5 mM NaN3, 0.2 mM phenylmethylsulfonylfluoride, 1 µM leupeptin, and 1 µM pepstatin, pH 7.4. Homogenates were centrifuged at 760 x g for 10 min at 4 C. The supernatants were then centrifuged at 200,000 x g for 90 min at 4 C to obtain the membrane and cytosolic fractions. The membrane pellets were resuspended in homogenization buffer and repeatedly passed through a 25-gauge needle before storage at -20 C. Proteins were measured by the method of Bradford (37), and 30 µg total proteins from each experimental series were subjected to SDS-PAGE.

Electrophoresis and immunoblotting of membranes
Cells were lysed for 30 min at 4 C in a buffer containing 20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 0.1% phenylmethylsulfonylfluoride, and 0.1% aprotinin, supplemented with 1% Nonidet P-40. Cell extracts were centrifuged for 15 min at 10,000 x g at 4 C, and 50 µg of the solubilized proteins were loaded. Extracts from rat adipocytes were prepared as previously described (34). SDS-PAGE was performed according to the Laemmli method (38). Gels were blotted into Immobilon in buffer consisting of 20% methanol, 200 mM glycine, and 25 mM Tris, pH 8.3. After 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 an enhanced chemiluminescence system (Amersham Pharmacia Biotech). ß1-Integrin was detected after incubating the membranes with [125I]protein A for 3 h at room temperature. Protein expression was quantified by scanning densitometry.

DMPK immunoprecipitation and protein kinase activity
L6E9 myoblasts or myotubes (differentiated for 4 d with IGF-II) were scraped in ice-cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 2 mM sodium orthovanadate, 100 mM NaF, 10% glycerol, and 1 mM ß-glycerophosphate supplemented with 1% Nonidet P-40 and protease inhibitors), solubilized on ice for 1 h, and centrifuged for 20 min at 10,000 x g. Cell lysates (2.5 mg) were immunoprecipitated for 2 h at 4 C with protein G-bound MANDM1 monoclonal antibody (1:100). Immunopellets were rinsed three times in lysis buffer and once in kinase buffer [25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM MgCl2, 5 mM ß-glycerophosphate, and 2.5 mM dithiothreitol]. Assays for protein kinase activity were carried out for 1 h at 37 C in a volume of 40 µl kinase buffer containing 0.1 mM [{gamma}-32P]ATP (1.5 mCi/µmol; Amersham Pharmacia Biotech) and myelin basic protein as exogenous substrate (1 µg; Sigma). Reactions were stopped with Laemmli sample buffer, and samples were loaded onto 15% acrylamide-sodium dodecyl sulfate gels. Gels were dried and developed by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of DMPK expression during skeletal muscle cell differentiation
During L6E9 myoblast differentiation, exogenous addition of IGF-II causes 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 dystrophin complex, and the myosin heavy chain. The expression of these proteins increases with the time of exposure to IGF-II, and is maximal on d 4, when a fully morphological differentiation is achieved (28).

During myogenesis, the DMPK protein level was increased in response to IGF-II (20 nM; Fig. 1AGo). Compared with undifferentiated myoblasts (d 0), IGF-II induced an increase of 6.8 ± 0.5-fold (n = 3) in DMPK protein expression on differentiation d 4. As a control we examined the expression level of ß1-integrin, a nonmuscle-specific plasma membrane protein, which remained unaltered during IGF-II-induced differentiation. IGF-II-induced DMPK was mainly localized to the cytosol fraction of differentiated myotubes, and no accumulation of DMPK protein was detected in the membrane fraction of differentiated cells (Fig. 1BGo). IGFs induced DMPK expression in a dose-dependent manner, and the IGF-I analog des(1,3)IGF-I was at least 15-fold more potent than IGF-II for this effect (Fig. 1Go, C and D), indicating that both peptides induced DMPK expression through activation of the IGF-I receptor as described for other myogenic markers (22, 25).



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  		Figure 1. DMPK expression during L6E9 myoblast differentiation. A, Expression of DMPK was determined by immunoblot using MANDM1 mAb in total cell lysates from L6E9 myoblasts differentiated in the presence of 20 nM IGF-II for 2 or 4 d or left undifferentiated (d 0). The ß1-integrin content was analyzed as a control of relative amounts of proteins in each sample. B, Membrane (M) and cytosolic (C) fractions from L6E9 myoblasts (Mb) or myotubes (Mt) were analyzed by immunoblot using MANDM1 mAb. Confluent L6E9 myoblasts (d 0) were allowed to differentiate in serum-free medium for 2 d in the absence or presence of increasing concentrations of IGF-II (0–5 nM; C) or des(1,3)IGF-I (0–0.3 nM; D). DMPK content ({blacksquare}) was analyzed by immunoblotting 30 µg solubilized proteins from the different experimental groups. ß-Actin ({bullet}) was used as a control of the relative amounts of proteins in each sample. Protein content after 2 d in serum-free medium in the absence of IGFs was considered as basal expression, and data are expressed as the fold increase over basal. Data shown are representative from three independent experiments.

 
We verified that the 80-kDa protein induced by IGF-II during myogenesis and detected with the monoclonal antibody MANDM1 corresponded to DMPK, as it was specifically detected in myotubes, whereas in nonmuscle cells (HeLa cells, 3T3L1 adipocytes, and rat isolated adipocytes) we only detected a 72-kDa band that has been previously described as a cross-reacting protein (CRP; Fig. 2AGo) (39). Moreover, the electrophoretic mobility of rat DMPK detected with MANDM1 corresponded to that of DMPK from human skeletal muscle detected by either MANDM1 or the human-specific MANDM5 monoclonal antibody (Fig. 2BGo). DMPK mRNA was also induced on differentiation d 4 by either IGF-II (20 nM) or serum deprivation (2% FBS; Fig. 2CGo).



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  		Figure 2. DMPK protein and mRNA expression in skeletal muscle cells. A, Total extracts from L6E9 myoblasts and myotubes, HeLa cells, rat isolated adipocytes, and 3T3L1 adipocytes were analyzed by immunoblot using MANDM1 mAb. B, DMPK electrophoretic mobility was compared in total cell extracts from rat L6E9 myoblasts or myotubes (obtained after 4 d in IGF-II-containing differentiation medium) and from human muscle. MANDM1 mAb recognizes both human and rat DMPK, and MANDM5 mAb specifically recognizes human DMPK. C, Confluent L6E9 myoblasts (Mb) were allowed to differentiate for 4 d in presence of IGF-II (20 nM) or 2% FBS. Total RNA was obtained from the different experimental groups, and 30 µg RNA were laid on gels. After blotting, DMPK mRNA was detected by chemiluminescence following hybridization with a mouse full-length DMPK cDNA probe. The integrity and relative amounts of RNA in each sample were checked by ethidium bromide staining (28S rRNA). D, DMPK catalytic activity was measured after immunoprecipitation with MANDM1 mAb of the endogenous protein from L6E9 myoblasts (Mb) or myotubes (Mt) obtained after 4 d of differentiation in IGF-II-containing medium. Kinase reactions used MBP as the in vitro substrate (upper panel). DMPK protein expression in immunoprecipitated samples was analyzed in parallel by Western blot using MANDM1 mAb (lower panel). Data from representative experiments are shown.

 
In IGF-II-differentiated cells the up-regulation of DMPK protein expression correlated with increased levels of DMPK Ser/Thr kinase activity toward myelin basic protein (MBP), which is a well characterized substrate of the recombinant protein (40) (Fig. 2DGo). In undifferentiated myoblasts, only low levels of MBP phosphorylation were detected in DMPK immunoprecipitates. Upon differentiation, DMPK kinase activity was strongly induced (upper panel), and this correlated with higher levels of DMPK protein expression in immunoprecipitated samples (lower panel).

Intracellular signaling pathways that regulate DMPK expression in skeletal muscle cells
We next analyzed the intracellular elements involved in the up-regulation of DMPK during skeletal muscle differentiation. We have previously shown that PI 3-kinase, NF-{kappa}B, and NOS define a common myogenic signaling pathway initiated by IGF-II (28). Moreover, there is evidence that p38 MAPK- and PI-3 kinase-dependent pathways act in parallel to signal IGF-induced myogenic differentiation (33).

Here we analyzed DMPK protein and mRNA expression in L6E9 myoblasts induced to differentiate by either IGF-II or serum deprivation in the absence or presence of specific inhibitors of these pathways in skeletal muscle cells (28, 33). We used LY294002 to selectively inhibit PI 3-kinase activity, N{omega}-nitro-L-arginine (NNA) to impair NOS activity, sodium salicylate (NaSal) to block NF-{kappa}B activation, and SB202190 to inhibit p38 MAPK activity. On differentiation d 4, the expression of DMPK was increased in parallel to the induction of muscle-specific proteins, such as myosin heavy chain and caveolin-3 (Fig. 3AGo), and correlated with the development of a fully differentiated phenotype measured as multinucleated myotube formation (data not shown). The expression of DMPK protein was blocked when cells were differentiated in the presence of LY294002, SB202190, NaSal, or NNA, indicating that PI 3-kinase, p38 MAPK, NF-{kappa}B, and NOS are required for DMPK expression. As a control, we analyzed in every experiment the cell content of a protein that is not regulated by differentiation (i.e. ß-actin). We observed that in the presence of drugs that inhibited myogenesis, the expression of DMPK, myosin heavy chain, and caveolin-3 was blocked, whereas no changes were observed in ß-actin cell content. These results indicated that the decrease in DMPK expression was due to the inhibition of myogenesis and not to cellular damage induced by the antimyogenic drugs. Moreover, no morphological evidence of cell death was observed for any of the drugs at the doses used (data not shown).



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  		Figure 3. Intracellular myogenic signaling pathways that regulate DMPK expression in L6E9 cells. Confluent L6E9 myoblasts (Mb) were differentiated for 4 d in the presence of 20 nM IGF-II or 2% FBS with or without inhibitors of PI 3-kinase (LY294002, 20 µM), NF-{kappa}B (NaSal, 10 mM), p38 MAPK (SB202190, 10 µM), or NOS (NNA, 10 mM). A, The contents of DMPK and skeletal muscle markers myosin heavy chain (MHC) and caveolin-3 were analyzed by Western blot in cell lysates from each condition. Relative amounts of proteins in each sample were checked by the expression of the nonmuscle-specific protein ß-actin. B, Total RNA was obtained from the different experimental groups, and 30 µg RNA were laid on gels to analyze DMPK mRNA expression. The integrity and relative amounts of RNA in each sample were checked by ethidium bromide staining (28S rRNA). Data from representative experiments are shown.

 
The regulation of DMPK protein expression correlated with data obtained by Northern blot analysis. DMPK mRNA was not detected in undifferentiated myoblasts, was highly up-regulated after 4 d of differentiation induced by either IGF-II or low serum, and was totally blocked when PI 3kinase, NF-{kappa}B, NOS, and p38 MAPK were inhibited (Fig. 3BGo). The delay between the initiation of IGF-II treatment and the detection of DMPK protein and mRNA, and the fact that identical results were obtained when cells were differentiated with low serum or IGF-II medium, suggested an indirect action of IGF-II on DMPK expression, probably through the activation of additional elements involved in the differentiation program.

We and others have previously shown the specificity in skeletal muscle cells of the chemical inhibitors used for this study (24, 28, 29, 33). We verified here that DMPK expression in response to IGF-induced differentiation was inhibited by LY294002, SB202190, NaSal, or NNA in a concentrationdependent manner and that these inhibitors exhibited 50% inhibitory doses for the inhibition of DMPK expression that correlated with those described for the specific inhibition of the signaling pathways analyzed (LY294002, 1 µM; SB202190, 25 nM; NNA, 1 mM; NaSal, 2 mM; Fig. 4Go, A–D) (41, 42, 43).



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  		Figure 4. Dose-response inhibition of DMPK expression by LY294002, NNA, NaSal, and SB202190. Confluent myoblasts (Mb) were allowed to differentiate in serum-free medium for 4 d in the presence of 20 nM IGF-II at increasing concentrations of LY294002 (0–5 µM; A), NNA (0–2 mM; B), NaSal (0–5 mM; C), or SB202190 (0–0.1 µM; D). DMPK ({bullet}), caveolin-3 ({blacksquare}), or myosin heavy chain (MHC; {blacksquare}) and ß-actin ({blacktriangleup}) content was analyzed by immunoblotting 30 µg solubilized proteins from the different experimental groups. Quantification of protein expression was performed by scanning densitometry. In all cases, protein content after 4 d in serum-free medium in the presence of IGF-II was considered 100% expression, and data are expressed as a percentage of the maximum. Data from representative experiments are shown.

 
Moreover, we confirmed results obtained in rat L6E9 myoblasts in a different cell model of skeletal muscle, mouse C2C12 myoblasts. As observed in L6E9 cells, in C2C12 myoblasts DMPK protein was induced after 4 d of differentiation with either IGF-II or 2% HS (Fig. 5AGo). Incubation of C2C12 cells with LY294002, SB202190, NaSal, or NNA during differentiation revealed that in mouse myoblasts, the induction of DMPK expression by low serum or IGF-II was also dependent on the previously characterized myogenic signaling pathways.



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  		Figure 5. Intracellular myogenic signaling pathways that regulate DMPK expression in C2C12 cells. A, Confluent C2C12 myoblasts (Mb) were differentiated for 4 d in the absence (DMEM) or presence of 20 nM IGF-II or 2% HS with or without inhibitors of PI 3-kinase (LY294002, 20 µM), NF-{kappa}B (NaSal, 10 mM), p38 MAPK (SB202190, 10 µM), or NOS (NNA, 10 mM). The contents of DMPK and skeletal muscle marker caveolin-3 were analyzed by Western blot in cell lysates from each condition. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein ß-actin. Data from representative experiments are shown.

 
DMPK expression in 10T1/2-MyoD cells
10T1/2 fibroblasts stably transfected with MyoD expressed skeletal muscle-specific proteins and fused into myotubes when induced to differentiate in a medium containing either IGF-II or 5% HS (Fig. 6Go, A and C). After 4 d of differentiation in these conditions, 10T1/2-MyoD cells also expressed DMPK protein and mRNA (Fig. 6Go, A and B). In differentiating 10T1/2-MyoD cells, DMPK and caveolin-3 expression was regulated by a myogenic pathway inhibited by LY294002, NaSal, or NNA, indicating the involvement of PI 3-kinase, NF-{kappa}B, and NOS, respectively (Fig. 6AGo). However, when p38 MAPK activity was blocked by SB202190, caveolin-3 and cell fusion were inhibited (Fig 6Go, A and C), whereas DMPK protein and mRNA were expressed as in nontreated cells (5% HS or IGF-II; Fig. 6Go, A and B). These data showed that in MyoD-overexpressing cells, DMPK did not require p38 MAPK to be up-regulated and demonstrated a dissociation between DMPK expression and the development of a fully differentiated phenotype.



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  		Figure 6. Intracellular myogenic signaling pathways that regulate DMPK expression in 10T1/2-MyoD cells. A, Confluent 10T1/2-MyoD cells (Mb) were differentiated for 4 d in the absence (DMEM) or presence of 20 nM IGF-II or 5% HS with or without inhibitors of PI 3-kinase (LY294002, 20 µM), NF-{kappa}B (NaSal, 10 mM), p38 MAPK (SB202190, 10 µM), or NOS (NNA, 10 mM). The contents of DMPK and skeletal muscle marker caveolin-3 were analyzed by Western blot in cell lysates from each condition. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein ß-actin. B, Confluent 10T1/2-MyoD cells (Mb) were differentiated for 4 d in the absence (DMEM) or presence of 20 nM IGF-II or 5% HS with or without inhibitor of p38 MAPK (SB202190, 10 µM). Total RNA was obtained, and 30 µg RNA were laid on gels to analyze DMPK mRNA expression. The integrity and relative amounts of RNA in each sample were checked by ethidium bromide staining (28S rRNA). C, Confluent 10T1/2-MyoD cells (Mb) were differentiated for 4 d in the absence (DMEM) or presence of 20 nM IGF-II with or without p38 MAPK inhibitor (SB; SB202190, 10 µM). Morphological differentiation was assessed by myotube formation. Images shown are representative of 30 microscopic fields taken at random from 3 independent experiments. Scale bars, 30 µm; the scale is the same for all panels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe intracellular signaling pathways that regulate DMPK protein and mRNA expression during myogenesis. Molecular masses for DMPK ranging from 45–85 kDa were reported, depending on the tissue or antibody used, until Lam et al. (39) generated a panel of monoclonal antibodies that allowed them to state that DMPK is an 80-kDa protein expressed almost exclusively in muscle and heart. Using a monoclonal antibody from that panel (MANDM1), we showed that DMPK protein is detected in skeletal muscle cells as an 80-kDa band that is induced during differentiation in parallel with other well-characterized skeletal muscle-specific proteins (i.e. myosin heavy chain and caveolin-3) and multinucleated myotube formation until a fully differentiated phenotype is observed. We further characterized the specificity of MANDM1 in skeletal muscle cells, as it also recognizes in many human tissues a 72-kDa CRP that is not DMPK (39). Our data indicate that the protein recognized by MANDM1 in skeletal muscle cells is DMPK, as it is detected in three skeletal muscle models (L6E9, C2C12, and 10T1/2-MyoD cells), and it is clearly distinguishable from the CRP detected in nonmuscle cells, such as HeLa cells, rat isolated adipocytes, or 3T3L1 adipocytes. The identification of the band detected with MANDM1 as DMPK is further supported by the following observations: 1) its expression pattern correlated with that of DMPK mRNA in Northern blot analysis; and 2) we specifically immunoprecipitated endogenous DMPK from L6E9 myotubes with MANDM1, and it exhibited Ser/Thr kinase activity toward MBP, which is a well characterized substrate of the DMPK recombinant protein (40). We observed a correlation between the induction of DMPK mass and activity during differentiation, suggesting that protein expression may be a major point of regulation for this kinase.

One of the first events during low serum-induced myogenesis is the secretion of IGF-II by the differentiating myoblasts (23). Here we presented experiments in which differentiation was induced by placing myoblasts in either low serum or IGF-II-containing medium. The fact that in all cell lines and conditions analyzed DMPK was only detected after 2 d of differentiation suggests that the effect of IGF-II on DMPK expression is indirect through the activation of additional elements involved in the differentiation program. Moreover, during this process, the increase in DMPK expression was specifically related to the activation of the IGF-I receptor, as the IGF-I potent analog des(1,3)IGF-I was 15-fold more effective than IGF-II for this effect.

Among the signaling pathways activated by IGFs and required for myogenesis are those involving PI 3-kinase, NF-{kappa}B, NOS, and p38 MAPK (28, 33). The data presented here indicate that DMPK expression is negatively regulated in proliferating myoblasts, and its up-regulation during differentiation totally depends on activation of the PI 3-kinase-dependent signaling pathway, which also regulates crucial myogenic events such as myoblast fusion and myogenin, p21 cyclin-dependent kinase inhibitor, and caveolin-3 expression (24, 25, 26, 27, 28, 29, 44, 45). Our results were essentially identical in the three skeletal muscle models analyzed (rat L6E9, mouse C2C12 myoblasts, and 10T1/2 fibroblasts stably expressing MyoD). Although DMPK gene contains a low level promoter that operates in conjunction with a MyoD-responsive enhancer element in the first intron (14), results in 10T1/2-MyoD cells indicate that MyoD overexpression is not sufficient to trigger DMPK up-regulation when PI 3-kinase activation is blocked. Similar conclusions were reached when we studied the myogenic signaling cascade involving NF-{kappa}B and NOS activation; MyoD overexpression could not bypass the blockade of DMPK up-regulation imposed by NF-{kappa}B or NOS inhibitors. In contrast, when 10T1/2-MyoD cells were differentiated in the presence of the p38 MAPK inhibitor SB202190, DMPK up-regulation remained unaffected, whereas myogenic events such as cell fusion and caveolin-3 expression were totally blocked. These data indicate that the PI 3-kinase-dependent pathway is a key regulator of DMPK expression that probably acts downstream from MyoD, whereas p38 MAPK, although involved in DMPK expression during myogenesis, seems to act upstream from MyoD or in a functionally alternative myogenic pathway.

Specific inhibition of p38 MAPK blocked the myogenic effect of IGFs in different skeletal muscle models of both mouse and rat myoblasts (Ref. 33 and our present study). However, it has been shown that the fold increase and the kinetics of p38 MAPK activation during muscle differentiation are very similar in the absence or presence of IGFs (33). These observations suggest that both IGF-dependent (mainly involving PI 3-kinase and Akt) and IGF-independent (involving p38 MAPK) pathways are required for IGF-induced myogenic differentiation. One interesting question from these observations was whether the signals from the two pathways converged on the same targets during myogenesis. We present experimental data supporting a model in which both mechanisms are operative. In MyoD-overexpressing cells both pathways are required for the induction of caveolin-3, myosin heavy chain, and myotube formation, whereas the PI 3-kinase pathway, but not the p38 MAPK pathway, is required for DMPK expression.

Regarding the role of DMPK in myogenesis, our findings demonstrate that DMPK up-regulation is not sufficient to induce myogenesis, as in the presence of SB202190, 10T1/2-MyoD cells expressed DMPK, but did not differentiate. These data indicate that the expression of DMPK cannot be totally associated with the development of a fully differentiated phenotype. However, the specific regulation of DMPK expression and protein kinase activity during differentiation suggests a functional implication of DMPK in the generation and/or maintenance of the skeletal muscle. Experiments aimed at defining the intracellular elements interacting with or regulated by DMPK in differentiating skeletal muscle cells are underway.


   Acknowledgments
 
We thank Mr. Robin Rycroft for his editorial assistance.


   Footnotes
 
This work was supported by research grants from the Dirección General de Investigación, Ministerio de Ciencia y Tecnología (SAF-2001-3500; PB98/0197), Fondo de Investigación Sanitaria (00/0125), Fundació la Marató de TV3 (981310), and Generalitat de Catalunya (1999 SGR-0039), Spain.

M.C. is the recipient of fellowship from the Comissió Interdepartamental de Recerca i Tecnologia (Catalonia, Spain).

Abbreviations: CRP, Cross-reacting protein; DM, myotonic dystrophy; DMPK, myotonic dystrophy protein kinase gene; FBS, fetal bovine serum; HS, horse serum; MBP, myelin basic protein; NaSal, sodium salicylate; NNA, N{omega}-nitro-L-arginine; NOS, nitric oxide synthase; PI 3-kinase, phosphatidylinositol 3-kinase.

Received October 10, 2001.

Accepted for publication April 24, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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