BackgroundThe treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-β (TGF-β) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients’ functional decline.MethodsA murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-β ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody’s effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin.ResultsBoth the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement.ConclusionsWe demonstrated that the potent anti-myostatin antibody mRK35 and its clinical analog, domagrozumab, were able to induce muscle anabolic activity in both rodents, including the mdx mouse model of DMD, and non-human primates. A Phase 2, potentially registrational, clinical study with domagrozumab in DMD patients is currently underway.

Reactive oxygen and nitrogen species inhibit or promote cell proliferation by modulating the cell signaling pathways that dictate decisions between cell survival, proliferation, and death. In the growth factor-dependent pathways that regulate mitogenesis, numerous positive and negative effectors of signaling are influenced by physiological fluctuations of oxidants, including receptor tyrosine kinases, small GTPases, mitogen-activated protein kinases, protein phosphatases, and transcription factors. The same mitogenic pathways that are sensitive to oxidant levels also directly regulate the expression of cyclin D1, a labile factor required for progression through the G1 phase on the cell cycle. Because the transition from G0 to G1 is the only phase of the cell cycle that is not regulated by cyclin-dependent kinases, but rather by redox-dependent signaling pathways, expression of cyclin D1 represents a primary regulatory node for the dose-dependent effects of oxidants on the induction of cell growth. We suggest that expression of cyclin D1 represents a useful marker for assessing the integration of proliferative and growth inhibitory effects of oxidants on the redox-dependent signaling events that control reentry into the cell cycle.

NADPH oxidases produce reactive oxygen species (ROS) that serve as co-stimulatory signals for cell proliferation. In mouse lung epithelial cells that express Nox1, Nox2, Nox4, p22(phox), p47(phox), p67(phox), and Noxo1, overexpression of Nox1 delayed cell cycle withdrawal by maintaining AP-1-dependent expression of cyclin D1 in low serum conditions. In cycling cells, the effects of Nox1 were dose dependent: levels of Nox1 that induced 3- to 10-fold increases in ROS promoted phosphorylation of ERK1/2 and expression of cyclin D1, whereas expression of Nox1 with Noxo1 and Noxa1 (or expression of Nox4 alone) that induced substantial increases in intracellular ROS inhibited cyclin D1 and proliferation. Catalase reversed the effects of Nox1 on cyclin D1 and cell proliferation. Diphenylene iodonium, an inhibitor of NADPH oxidase activity, did not affect dosedependent responses of ERK1/2 or Akt to serum, but markedly inhibited the sequential expression of c-Fos and Fra-1 required for induction of cyclin D1 during cell cycle re-entry. These results indicate that Nox1 stimulates cell proliferation in actively cycling cells by reducing the requirement for growth factors to maintain expression of cyclin D1, whereas during cell cycle re-entry, NADPH oxidase activity is required for transcriptional activation of Fos family genes during the immediate early gene response.

Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G 0 -to-G 1 transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1-and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G 1 . Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G 0 -to-G 1 transition of the cell cycle.Quiescent cells that reenter the cell cycle in response to mitogens execute a series of cell signaling cascades that activate transcription factors required for the expression of genes involved in cell cycle progression. The activation of receptor tyrosine kinases by growth factors in quiescence signals to downstream effector kinase cascades which induce the immediate-early gene (IEG) response (25, 36). Transcription factors encoded by these early response genes regulate the expression of delayed-response genes, and proteins encoded by these genes in turn support chromatin remodeling, gene transcription, reorganization of the cytoskeleton, and other modifications required for cell cycle progression (19). Hence, the IEG response represents a classical regulatory lattice in which mitogenic signals are amplified and dispersed to multiple targets. Understanding the dynamics of the regulation of the IEG products is critical to understanding how mitogenic signals regulate cell cycle reentry.A critical regulator of the IEG response following mitogenic stimulation is the extracellular signal-regulated kinase (ERK) cascade. The ERK 1 and 2 (ERK1,2) isoforms initiate the IEG response by activating c-Fos transcription within minutes after mitogenic stimulation (25, 54), but sustained ERK1,2 activity is required for cell cycle reentry (6, 13, 53). Newly synthesized c-Fos dimerizes with Jun (c-Jun, JunB, and JunD) family proteins to form AP-1 transcription factor complexes (49). These Fos-containing AP-1 dimers enter the nucleus, where c-Fos undergoes a series of phosphorylation events that enhance its transcriptional potential. Initial phosphorylation events on the C terminus...

BackgroundIdentifying translatable, non-invasive biomarkers of muscular dystrophy that better reflect the disease pathology than those currently available would aid the development of new therapies, the monitoring of disease progression and the response to therapy.ObjectiveThe goal of this study was to evaluate a panel of serum protein biomarkers with the potential to specifically detect skeletal muscle injury.MethodSerum concentrations of skeletal troponin I (sTnI), myosin light chain 3 (Myl3), fatty acid binding protein 3 (FABP3) and muscle-type creatine kinase (CKM) proteins were measured in 74 Duchenne muscular dystrophy (DMD), 38 Becker muscular dystrophy (BMD) and 49 Limb-girdle muscular dystrophy type 2B (LGMD2B) patients and 32 healthy controls.ResultsAll four proteins were significantly elevated in the serum of these three muscular dystrophy patient populations when compared to healthy controls, but, interestingly, displayed different profiles depending on the type of muscular dystrophy. Additionally, the effects of patient age, ambulatory status, cardiac function and treatment status on the serum concentrations of the proteins were investigated. Statistical analysis revealed correlations between the serum concentrations and certain clinical endpoints including forced vital capacity in DMD patients and the time to walk ten meters in LGMD2B patients. Serum concentrations of these proteins were also elevated in two preclinical models of muscular dystrophy, the mdx mouse and the golden-retriever muscular dystrophy dog.ConclusionsThese proteins, therefore, are potential muscular dystrophy biomarkers for monitoring disease progression and therapeutic response in both preclinical and clinical studies.

Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC-MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.

Gain-of-function mutations in SHP-2͞PTPN11 cause Noonan syndrome, a human developmental disorder. Noonan syndrome is characterized by proportionate short stature, facial dysmorphia, increased risk of leukemia, and congenital heart defects in Ϸ50% of cases. Congenital heart abnormalities are common in Noonan syndrome, but the signaling pathway(s) linking gain-of-function SHP-2 mutants to heart disease is unclear. Diverse cell types coordinate cardiac morphogenesis, which is regulated by calcium (Ca 2؉ ) and the nuclear factor of activated T-cells (NFAT). It has been shown that the frequency of Ca 2؉ oscillations regulates NFAT activity. Here, we show that in fibroblasts, Ca 2؉ oscillations in response to FGF-2 require the phosphatase activity of SHP-2. Conversely, gain-of-function mutants of SHP-2 enhanced FGF-2-mediated Ca 2؉ oscillations in fibroblasts and spontaneous Ca 2؉ oscillations in cardiomyocytes. The enhanced frequency of cardiomyocyte Ca 2؉ oscillations induced by a gain-of-function SHP-2 mutant correlated with reduced nuclear translocation and transcriptional activity of NFAT. These data imply that gain-of-function SHP-2 mutants disrupt the Ca 2؉ oscillatory control of NFAT, suggesting a potential mechanism for congenital heart defects in Noonan syndrome.calcium signaling ͉ cardiomyocytes ͉ FGF ͉ receptor tyrosine kinase signaling ͉ tyrosine phosphatases T he ubiquitously expressed src homology 2 (SH2)-containing protein tyrosine phosphatase (PTP), SHP-2 (PTPN11), regulates numerous intracellular signaling cascades that control cell proliferation, differentiation, cell survival, migration, adhesion, and apoptosis (1). SHP-2 contains two NH 2 terminus SH2 domains, a PTP domain, and a COOH terminus containing two tyrosyl phosphorylation sites (1). It is now well established that SHP-2 is required for activation of the extracellular-regulated kinases (ERKs) 1 and 2 in response to the activation of receptor tyrosine kinase (RTK) and cytokine receptors (1). The SH2 domains of SHP-2 mediate not only binding to RTKs but also scaffold proteins such as Gab-1, IRS-1, and FRS-2 (1). In virtually all cases, stimulation of SHP-2 catalysis is required for downstream signaling. The SH2 domains of SHP-2 also regulate its activation. Engagement of the NH 2 SH2 domain of SHP-2 with its cognate phosphotyrosyl protein results in its activation. The mechanism of this activation involves displacement of the NH 2 SH2 domain from the PTP domain which in the basal (unbound SH2 domain) state occludes the PTP active site. Upon NH 2 SH2 domain binding, a conformational relief of this inhibitory state is achieved and the phosphatase becomes active. Insights from the crystallographic structure of SHP-2 (2) resulted in the generation of engineered mutations at residues critical for the maintenance of the basal inactivated state of SHP-2. These mutations, within the NH 2

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle–specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]–SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK–SHP-2–null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.