miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

[Prospective evaluation of intrasphincteric injections of autologous muscular cells in patients with stress urinary incontinence following radical prostatectomy]. / Évaluation prospective du traitement de l'incontinence urinaire post-prostatectomie par injections intrasphinctériennes de cellules musculaires autologues.

PURPOSE: Cell therapy for urinary incontinence management has been experienced in animals with encouraging results, but studies in human beings are lacking. Our primary objective was to assess the safety of intrasphincteric injections of autologous muscular cells in patients with postprostatectomy incontinence (PPI). Secondary objectives focused on complications efficacy. METHODS: We conducted an open, prospective study in a single center on 12 patients presenting PPI. Patients underwent intrasphincteric injections of autologous muscular cells isolated from a biopsy of deltoid muscle. The primary endpoint was the Q(max) variation at the three month visit in order to assess potential bladder outlet obstruction. Secondary endpoints assessed side effects and efficacy parameters based on symptoms, quality of life score, voiding diary, pad-test, and urethral pressure profile at one, two, three, six and 12 months after injection. RESULTS: No immediate complication occurred and no significant variation was noted on Q(max). The only side effects possibly product-related were three cases of urinary tract infection treated by antibiotics. An acceptable safety and tolerability of the procedure whatever the injected dose of muscular cells was demonstrated. Results on efficacy after one year were heterogeneous, with 4/12 patients describing reduced urine leakage episodes, 1/12 patient presenting increased maximal closure pressure, and 8/12 patients showing improvement on pad-test. CONCLUSIONS: Cell therapy consisting of intrasphincteric injections of autologous muscular cells in patients with PPI was a feasible and safe procedure. The results point out that some subjects may positively respond to this procedure, but clinical efficacy remains to be confirmed.

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative 'real-time' reporter gene for cell therapy preclinical studies.

An automatic image analysis approach to quantify stained cell cultures.

Counting cells in culture is a common task in biotechnology research and production. This process should be automated to provide fast and objective quantification. Flow cytometry is adapted to count cells in suspension. However, the morphological information and the spatial organisation of adherent cells are lost when cells are removed from culture. This paper proposes a methodology based on image analysis to quantify stained nuclei in culture. The protocol is composed of several steps: cell staining, automatic microscopy imaging, segmentation by an automatic algorithm including a classification approach, and computation of quantitative data that characterizes the growth of cells. An evaluation shows that the automatic process of counting provides results similar to human manual counting. The major interests of the proposed approach are the fully automated processing and preservation of cell shapes and positions in culture. More than two thousand culture conditions have been measured by this tool for various applications including optimization of cell culture media, improvement of the culture processes and measurement of drug toxicity.

Transforming growth factor-beta inhibition of insulin-like growth factor-binding protein-5 synthesis in skeletal muscle cells involves a c-Jun N-terminal kinase-dependent pathway.

Transforming growth factor-beta (TGF-beta) and insulin-like growth factors (IGFs) play critical roles in the control of myogenesis. Insulin-like growth factor-binding protein-5 (IGFBP-5), by regulating the bioavailability of IGFs, is involved in controlling IGF-dependent differentiation. We investigated the effects of TGF-beta on the IGFBP-5 production induced by IGFs in mouse myoblasts. TGF-beta leads to a decrease in IGFBP-5 synthesis at both transcript and protein levels, and blocked muscle differentiation. The Smad proteins and the c-Jun N-terminal kinase (JNK) have been shown to be involved in TGF-beta signaling pathways. We provide evidence that the JNK pathway, rather than Smad proteins, is involved in the response of muscle cells to TGF-beta. This factor failed to stimulate the GAL4-Smad 2/3 transcriptional activities of the constructs used to transfect myoblasts. Moreover, stable expression of the antagonistic Smad7 did not abolish the inhibitory effect of TGF-beta on IGFBP-5 production whereas expression of a dominant-negative version of MKK4, an upstream activator of JNK, did. We also showed, using a specific inhibitor, that the p38 mitogen-activated protein kinase (p38 MAPK) was not involved in the inhibition of IGFBP-5 production. Thus, TGF-beta-mediated IGFBP-5 inhibition is independent of Smads and requires activation of the JNK signaling pathway.

IGF-1 receptor as an alternative receptor for metabolic signaling in insulin receptor-deficient muscle cells.

We have derived skeletal muscle cell lines from wild-type (wt) and insulin receptor (IR) knockout mice to unravel the metabolic potential of IGF-1 receptor (IGF-1R). Both wt and IR(-/-) myoblasts differentiated into myotubes with similar patterns of expression of muscle-specific genes such as MyoD, myogenin and MLC1A indicating that IR is not required for this process. Binding of 125I-IGF-1 on wt and IR(-/-) myotubes was similar showing that IGF-1R was not upregulated in the absence of IR. Stimulation of IR(-/-) myotubes with IGF-1 (10(-10) to 10(-7) M) increased glucose uptake and incorporation into glycogen, induced IRS-1 phosphorylation and activated PI 3-kinase and MAP kinase, two enzymes of major signaling pathways. These effects were comparable to those obtained with wt myotubes using insulin or IGF-1 or with IR(-/-) myotubes using insulin at higher concentrations. This study provides a direct evidence that IGF-1R can represent an alternative receptor for metabolic signaling in muscle cells.

Cell density-dependent induction of endogenous myogenin (myf4) gene expression by Myf5.

Transcription factors Myf5 and MyoD are critical for myoblast determination. Myogenin is a direct transcriptional target of these factors and its expression is associated with commitment to terminal differentiation. Here, we have used myogenic derivatives of human U20S cells expressing Myf5 or MyoD under control of a tetracycline-sensitive promoter to study expression of endogenous myogenin (myf4). We find that Myf5-mediated induction of myogenin shows striking dependence on cell density. At high cell density, Myf5 is a potent inducer of myogenin expression. At low cell density, Myf5 (unlike MyoD) is a poor inducer of myogenin expression, whilst retaining the capacity to direct expression of other muscle-specific genes. The permissive influence of high cell density on myogenin induction by Myf5 is not a consequence of serum depletion or cell cycle arrest, but is mimicked by a disruption adjacent to the basic region of Myf5 (Myf5/mt) which reduces its DNA binding affinity for E-boxes without compromising its ability to transactivate a reporter gene driven by the myogenin promoter. Coculture of cells expressing wild-type Myf5 and Myf5/mt leads to reduced myogenin induction in Myf5/mt cells. We propose that at low cell density Myf5 inhibits induction of myogenin.

Constitutive instability of muscle regulatory factor Myf5 is distinct from its mitosis-specific disappearance, which requires a D-box-like motif overlapping the basic domain.

Transcription factors Myf5 and MyoD play critical roles in controlling myoblast identity and differentiation. In the myogenic cell line C2, we have found that Myf5 expression, unlike that of MyoD, is restricted to cycling cells and regulated by proteolysis at mitosis. In the present study, we have examined Myf5 proteolysis through stable transfection of myogenically convertible U20S cells with Myf5 derivatives under the control of a tetracycline-sensitive promoter. A motif within the basic helix-loop-helix domain of Myf5 (R93 to Q101) resembles the "destruction box" characteristic of substrates of mitotic proteolysis and thought to be recognized by the anaphase-promoting complex or cyclosome (APC). Mutation of this motif in Myf5 stabilizes the protein at mitosis but does not affect its constitutive turnover. Conversely, mutation of a serine residue (S158) stabilizes Myf5 in nonsynchronized cultures but not at mitosis. Thus, at least two proteolytic pathways control Myf5 levels in cycling cells. The mitotic proteolysis of Myf5 is unlike that which has been described for other destruction box-dependent substrates: down-regulation of Myf5 at mitosis appears to precede that of known targets of the APC and is not affected by a dominant-negative version of the ubiquitin carrier protein UbcH10, implicated in the APC-mediated pathway. Finally, we find that induction of Myf5 perturbs the passage of cells through mitosis, suggesting that regulation of Myf5 levels at mitosis may influence cell cycle progression of Myf5-expressing muscle precursor cells.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects.

Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci.

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5' non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired S phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with S phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying S phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events.

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

Novel isoforms of the fragile X related protein FXR1P are expressed during myogenesis.

The fragile X syndrome results from transcriptional silencing of the FMR1 gene and the absence of its encoded FMRP protein. Two autosomal homologues of the FMR1 gene, FXR1 and FXR2, have been identified and the overall structures of the corresponding proteins are very similar to that of FMRP. Using antibodies raised against FXR1P, we observed that two major protein isoforms of relative MW of 78 and 70 kDa are expressed in different mammalian cell lines and in the majority of mouse tissues. In mammalian cells grown in culture as well as in brain extracts, both P78and P70isoforms are associated with mRNPs within translating polyribosomes, similarly to their closely related FMRP homologues. In muscle tissues as well as in murine myoblastic cell lines induced to differentiate into myotubes, FXR1P78and P70isoforms are replaced by novel unpredicted isoforms of 81-84 kDa and a novel FXR1 exon splice variant was detected in muscle RNA. While P81-84isoforms expressed after fusion into myotubes in murine myoblast cell lines grown in culture are associated with polyribosomes, this is not the case when isolated from muscle tissues since they sediment with lower S values. Immunohistochemical studies showed coexpression of FMRP and FXR1P70and P78in the cytoplasm of brain neurons, while in muscle no FMRP was detected and FXR1P81-84were mainly localized to structures within the muscle contractile bands. The complex expression pattern of FXR1P suggests tissue-specific expression for the various isoforms of FXR1 and the differential expression of FMRP and FXR1Ps suggests that in certain types of cells and tissues, complementary functions may be fulfilled by the various FMRP family members.

Up-regulation of insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002.

Skeletal myoblast differentiation is stimulated by insulin-like growth factors (IGFs). The autocrine action of IGFs is mediated through the type-1 IGF receptor (IGFR-1) and modulated by IGF binding proteins (IGFBPs) secreted by the cells. The mouse C2 myoblast cell line stably transfected with a vector producing IGF-II antisense RNA was used to show that specific IGFBP expression changes with the state of the cells: high levels of IGFBP-2 messenger RNA (mRNA) were found only in proliferating myoblasts, whereas IGFBP-3 mRNA was induced in quiescent cells. Secretion of IGFBP5 was strongly stimulated during differentiation. Insulin and IGF dose-response experiments showed that up-regulation of IGFBP-5 resulted from IGFR-1 activation. Drugs interfering with IGFR-1 signaling and inhibiting myoblast differentiation had different effects on IGFBP-5 up-regulation. Two phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmaninn and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], failed to alter IGFBP-5 up-regulation, which persisted in the absence of differentiation. Rapamycin which indirectly prevents activation of the p70 ribosomal protein-S6 kinase (p70S6k), suppressed IGFBP-5 induction. Because the PI3-kinase inhibitors block p70S6k, neither kinase would be required for IGFR-1-dependent IGFBP-5 induction. In C2 anti-IGF-II myoblasts, IGFBP-5 induction is therefore rapamycin-sensitive and independent of differentiation.

Cell cycle-regulated expression of the muscle determination factor Myf5 in proliferating myoblasts.

Myf5 is the earliest-known muscle-specific factor to be expressed in vivo and its expression is associated with determination of the myoblast lineage. In C2 cells, we show by immunocytolocalization that Myf5 disappears rapidly from cells in which the differentiation program has been initiated. In proliferating myoblasts, the levels of Myf5 and MyoD detected from cell to cell are very heterogeneous. We find that some of the heterogeneity of Myf5 expression arises from a posttranscriptional regulation of Myf5 by the cell cycle. Immunoblotting of extracts from synchronized cultures reveals that Myf5 undergoes periodic fluctuations during the cell cycle and is absent from cells blocked early in mitosis by use of nocodazole. The disappearance of Myf5 from mitotic cells involves proteolytic degradation of a phosphorylated form of Myf5 specific to this phase of the cell cycle. In contrast, MyoD levels are not depleted in mitotic C2 cells. The mitotic destruction of Myf5 is the first example of a transcription factor showing cell cycle-regulated degradation. These results may be significant in view of the possible role of Myf5 in maintaining the determination of proliferating cells and in timing the onset of differentiation.

Increased expression of the LAZ3 (BCL6) proto-oncogene accompanies murine skeletal myogenesis.

The structural alterations of the LAZ3 (BCL6) gene are one of the most frequent events found in non-Hodgkin lymphoma. LAZ3 encodes a transcriptional repressor with a POZ/zinc finger structure similar to several Drosophila development regulators and to the human promyelocytic leukemia-associated PLZF gene. Consistent with the origin of LAZ3-associated malignancies, LAZ3 is expressed in mature B-cells and required for germinal center formation. However, its ubiquitous expression, with predominant levels in skeletal muscle, suggests that it may act outside the lymphoid system. To study how LAZ3 could be involved in skeletal muscle differentiation, we examined its expression in the C2 muscle cells. We report here that LAZ3 is upregulated at both mRNA and protein levels during the differentiation of proliferating C2 myoblasts into post-mitotic myotubes. This rise in LAZ3 expression is both precocious and sustained, and is not reversed when myotubes are re-exposed to mitogen-rich medium, suggesting that irreversible evens occurring upon myogenic terminal differentiation contribute to lock LAZ3 upregulation. In addition, using two different models, we found that a "simple" growth-arrest upon serum starvation is not sufficient to induce LAZ3 upregulation which rather appears as a feature of myogenic commitment and/or differentiation. Finally, BrdU incorporation assays in C2 cells entering the differentiation pathway indicate that "high" LAZ3 expression strongly correlates with their exit from the cell cycle. Taken as a whole, these findings suggest that LAZ3 could play a role in muscle differentiation. Together with some results reported in other cell types, we propose that LAZ3 may contribute to events common to various differentiation processes, possibly the induction and stabilization of the withdrawal from the cell cycle.

Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.

Prosome cytodistribution relative to desmin and actin filaments in dividing C2.7 myoblasts and during myotube formation in vitro.

Prosomes constitute the multicatalytic proteinase (MCP) core of the 26S proteasomes, but were first observed as subcomplexes of untranslated mRNP; this suggests that they play a putative role in the control of protein biosynthesis in addition to their catabolic enzymatic function. In previous investigations it was shown that some prosomes colocalize with the intermediate filaments (IF) of the cytoskeleton, of the cytokeratin type in epithelial cells, and of the vimentin type in fibroblasts. Studies on adult rat muscle carried out with prosome-specific monoclonal antibodies (p-mAbs) have shown, surprisingly, that specific types of prosomes predominantly occupy a particular zone in between the M and the Z lines of the sarcomeric structure. The data presented here show that the subunit composition of prosomes changes when the dividing C2.7 myoblasts fuse into myotubes. We show furthermore that, in dividing C2.7 myoblasts, prosomes colocalize with the desmin network as well as with that of actin, in a distribution that changes with the subunit pattern of the prosomes investigated by individual p-mAbs. Surprisingly, when myogenic fusion is induced, specific types of prosomes move first to the nuclei; later on, they reappear in the cytoplasm. There, superimposing initially onto the reorganizing desmin filaments that run from one pole of the prefusion myoblast to the other, prosomes gradually colocalize with the actin fibers in the fusing myotubes, finally forming a "pearl on a string" pattern. These results are discussed in relation to parallel observations of prosome distribution between the actin and IF networks not only in epithelial cells but also in fusing muscle satellite cells, which made it possible to monitor the complete buildup of the sarcomeric structure.

Wortmannin inhibits IGF-dependent differentiation in the mouse myogenic cell line C2.

Evidence has accumulated that suggests that insulin-like growth factors (IGFs) exert a positive influence on myoblast differentiation. We have undertaken to study the signalling events required for differentiation resulting from type-1 IGF receptor stimulation in C2 myoblasts, where autocrine production of IGF-II was abolished by means of antisense RNA. Exposure of the cells to IGFs leads to a rapid and sustained activation of phosphatidyl-inositol 3-kinase followed by the expression of Myod, myogenin and differentiation. The fungal metabolite, wortmannin, inhibits both PI 3-kinase and muscle differentiation with an IC 50 in the nanomolar range. IGFs are also known to cause a rapid activation of MAP kinase. However, the synthetic inhibitor of MEK, PD098059, which prevents MAP kinase activation, does not affect myoblast differentiation. These results provide evidence that PI 3-kinase, but not MAP kinase, is required for insulin-like growth factor receptor-dependent differentiation of muscle cells.