Loss of skeletal muscle mass and function in humans is associated with significant morbidity and ... more Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases. We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice. The human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mi...
Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanism... more Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude tha...
Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion... more Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathwa...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2003
Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are e... more Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The ...
While a number of growth factors have been described that are highly specific for particular cell... more While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function.
Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabo... more Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabolic (or atrophic) processes. Previously, no link between the molecular mediators of atrophy and hypertrophy had been reported. We demonstrate a hierarchy between the signals which mediate hypertrophy and those which mediate atrophy: the IGF-1/PI3K/Akt pathway, which has been shown to induce hypertrophy, prevents induction of requisite atrophy mediators, namely the muscle-specific ubiquitin ligases MAFbx and MuRF1. Moreover, the mechanism for this inhibition involves Akt-mediated inhibition of the FoxO family of transcription factors; a mutant form of FOXO1, which prevents Akt phosphorylation, thereby prevents Akt-mediated inhibition of MuRF1 and MAFbx upregulation. Our study thus defines a previously uncharacterized function for Akt, which has important therapeutic relevance: Akt is not only capable of activating prosynthetic pathways, as previously demonstrated, but is simultaneously and dominantly able to suppress catabolic pathways, allowing it to prevent glucocorticoid and denervation-induced muscle atrophy.
Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candi... more Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.
We have exploited a battery of approaches to address several controversies that have accompanied ... more We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a "preferred" ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their "preferred" ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their "nonpreferred" neurotrophin ligands.
We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk ... more We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.
To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar gra... more To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-El6 chicks, were radiolabeled with [s%]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, 11, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, 11, N-cadherin, and integrin also did not impair neuron binding to glia. References Altman, J., and Bayer, S. A. (1978). Prenatal development of the cerebellar system in the rat. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. J. Comp. Neural. 779, 23-48. Antonicek, H., Persohn, E., and Schachner, M. (1987). Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration. J. Cell. Biol. 704, 1587-1595.
Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2),... more Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2), was previously reported to induce severe insulin sensitivity, leading to early postnatal death. In the previous study, the targeting construct left the first eighteen exons encoding Inppl1 intact, generating a Inppl1 EX19-28−/− mouse, and apparently also deleted a second gene, Phox2a. We report a new SHIP2 knockout (Inppl1 −/− ) targeted to the translation-initiating ATG, which is null for Inppl1 mRNA and protein. Inppl1 −/− mice are viable, have normal glucose and insulin levels, and normal insulin and glucose tolerances. The Inppl1 −/− mice are, however, highly resistant to weight gain when placed on a high-fat diet. These results suggest that inhibition of SHIP2 would be useful in the effort to ameliorate diet-induced obesity, but call into question a dominant role of SHIP2 in modulating glucose homeostasis.
Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachex... more Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.
Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactiv... more Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.
Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, p... more Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, predominantly in conditions of fasting and diabetes. Given these prior studies, we sought to determine whether Trb3 plays a major role in modulating hepatic insulin sensitivity. To answer this question, we produced mice in which a lacZ reporter was knocked into the locus containing the gene Trib3, resulting in a Trib3 null animal. Trib3 expression analyses demonstrated that the Trib3 is expressed in liver, adipose tissues, heart, kidney, lung, skin, small intestine, stomach, and denervated, but not normal, skeletal muscle. Trib3(-/-) mice are essentially identical to their wild-type littermates in overall appearance and body composition. Phenotypic analysis of Trib3(-/-) mice did not detect any alteration in serum glucose, insulin, or lipid levels; glucose or insulin tolerance; or energy metabolism. Studies in Trib3(-/-) hepatocytes revealed normal Akt and glycogen synthase kinase- 3beta phosphorylation patterns, glycogen levels, and expressions of key regulatory gluconeogenic and glycolytic genes. These data demonstrate that deletion of Trib3 has minimal effect on insulin-induced Akt activation in hepatic tissue, and, as such, they question any nonredundant role for Trb3 in the maintenance of glucose and energy homeostasis in mice.
The receptor tyrosine kinase, MuSK, is required for the formation of the neuromuscular junction (... more The receptor tyrosine kinase, MuSK, is required for the formation of the neuromuscular junction (NMJ) where MuSK becomes phosphorylated when exposed to neuronally synthesized isoforms of agrin. To understand better the mechanisms by which MuSK mediates the formation of the NMJ, we have examined how MuSK expression is regulated during development in the embryo, by neuromuscular injury in the adult and by agrin in vitro. Here we show that MuSK is associated with the earliest observable AChR clusters at the developing motor endplate and that MuSK and AChRs codistribute throughout the development of the NMJ. These two proteins are also coordinately regulated on the surfaces of cultured myotubes where MuSK and AChRs colocalize both in spontaneous and agrin-induced clusters. While MuSK is normally restricted to the motor endplate in adult muscle, denervation results in its extrajunctional expression, although a discernible concentration of MuSK remains localized to the motor endplate even 14 days after denervation. Extrajunctional MuSK is first apparent 3 days after denervation and is sharply reduced upon reinnervation. Muscle paralysis also markedly alters the expression of MuSK in adult muscle and results in increased expression of MuSK as well as increased transcription of MuSK mRNA by extrasynaptic myonuclei. Together, these findings demonstrate that MuSK expression is highly regulated by innervation, muscle activity, and agrin, while the distribution of MuSK is precisely coordinated with that of the AChR.
Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such ... more Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1 À/À mice are treated with DEX, the MuRF1 À/À animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.
Loss of skeletal muscle mass and function in humans is associated with significant morbidity and ... more Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases. We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice. The human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mi...
Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanism... more Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude tha...
Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion... more Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathwa...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2003
Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are e... more Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The ...
While a number of growth factors have been described that are highly specific for particular cell... more While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function.
Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabo... more Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabolic (or atrophic) processes. Previously, no link between the molecular mediators of atrophy and hypertrophy had been reported. We demonstrate a hierarchy between the signals which mediate hypertrophy and those which mediate atrophy: the IGF-1/PI3K/Akt pathway, which has been shown to induce hypertrophy, prevents induction of requisite atrophy mediators, namely the muscle-specific ubiquitin ligases MAFbx and MuRF1. Moreover, the mechanism for this inhibition involves Akt-mediated inhibition of the FoxO family of transcription factors; a mutant form of FOXO1, which prevents Akt phosphorylation, thereby prevents Akt-mediated inhibition of MuRF1 and MAFbx upregulation. Our study thus defines a previously uncharacterized function for Akt, which has important therapeutic relevance: Akt is not only capable of activating prosynthetic pathways, as previously demonstrated, but is simultaneously and dominantly able to suppress catabolic pathways, allowing it to prevent glucocorticoid and denervation-induced muscle atrophy.
Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candi... more Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.
We have exploited a battery of approaches to address several controversies that have accompanied ... more We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a "preferred" ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their "preferred" ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their "nonpreferred" neurotrophin ligands.
We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk ... more We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.
To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar gra... more To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-El6 chicks, were radiolabeled with [s%]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, 11, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, 11, N-cadherin, and integrin also did not impair neuron binding to glia. References Altman, J., and Bayer, S. A. (1978). Prenatal development of the cerebellar system in the rat. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. J. Comp. Neural. 779, 23-48. Antonicek, H., Persohn, E., and Schachner, M. (1987). Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration. J. Cell. Biol. 704, 1587-1595.
Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2),... more Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2), was previously reported to induce severe insulin sensitivity, leading to early postnatal death. In the previous study, the targeting construct left the first eighteen exons encoding Inppl1 intact, generating a Inppl1 EX19-28−/− mouse, and apparently also deleted a second gene, Phox2a. We report a new SHIP2 knockout (Inppl1 −/− ) targeted to the translation-initiating ATG, which is null for Inppl1 mRNA and protein. Inppl1 −/− mice are viable, have normal glucose and insulin levels, and normal insulin and glucose tolerances. The Inppl1 −/− mice are, however, highly resistant to weight gain when placed on a high-fat diet. These results suggest that inhibition of SHIP2 would be useful in the effort to ameliorate diet-induced obesity, but call into question a dominant role of SHIP2 in modulating glucose homeostasis.
Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachex... more Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.
Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactiv... more Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.
Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, p... more Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, predominantly in conditions of fasting and diabetes. Given these prior studies, we sought to determine whether Trb3 plays a major role in modulating hepatic insulin sensitivity. To answer this question, we produced mice in which a lacZ reporter was knocked into the locus containing the gene Trib3, resulting in a Trib3 null animal. Trib3 expression analyses demonstrated that the Trib3 is expressed in liver, adipose tissues, heart, kidney, lung, skin, small intestine, stomach, and denervated, but not normal, skeletal muscle. Trib3(-/-) mice are essentially identical to their wild-type littermates in overall appearance and body composition. Phenotypic analysis of Trib3(-/-) mice did not detect any alteration in serum glucose, insulin, or lipid levels; glucose or insulin tolerance; or energy metabolism. Studies in Trib3(-/-) hepatocytes revealed normal Akt and glycogen synthase kinase- 3beta phosphorylation patterns, glycogen levels, and expressions of key regulatory gluconeogenic and glycolytic genes. These data demonstrate that deletion of Trib3 has minimal effect on insulin-induced Akt activation in hepatic tissue, and, as such, they question any nonredundant role for Trb3 in the maintenance of glucose and energy homeostasis in mice.
The receptor tyrosine kinase, MuSK, is required for the formation of the neuromuscular junction (... more The receptor tyrosine kinase, MuSK, is required for the formation of the neuromuscular junction (NMJ) where MuSK becomes phosphorylated when exposed to neuronally synthesized isoforms of agrin. To understand better the mechanisms by which MuSK mediates the formation of the NMJ, we have examined how MuSK expression is regulated during development in the embryo, by neuromuscular injury in the adult and by agrin in vitro. Here we show that MuSK is associated with the earliest observable AChR clusters at the developing motor endplate and that MuSK and AChRs codistribute throughout the development of the NMJ. These two proteins are also coordinately regulated on the surfaces of cultured myotubes where MuSK and AChRs colocalize both in spontaneous and agrin-induced clusters. While MuSK is normally restricted to the motor endplate in adult muscle, denervation results in its extrajunctional expression, although a discernible concentration of MuSK remains localized to the motor endplate even 14 days after denervation. Extrajunctional MuSK is first apparent 3 days after denervation and is sharply reduced upon reinnervation. Muscle paralysis also markedly alters the expression of MuSK in adult muscle and results in increased expression of MuSK as well as increased transcription of MuSK mRNA by extrasynaptic myonuclei. Together, these findings demonstrate that MuSK expression is highly regulated by innervation, muscle activity, and agrin, while the distribution of MuSK is precisely coordinated with that of the AChR.
Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such ... more Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1 À/À mice are treated with DEX, the MuRF1 À/À animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.
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Papers by Trevor Stitt