Kevin Hannon

Genesis of olfactory receptor neurons in vitro: Regulation of progenitor cell divisions by fibroblast growth factors

Olfactory receptor neurons are produced continuously in mammalian olfactory epithelium in vivo, but in explant cultures neurogenesis ceases abruptly. We show that in vitro neurogenesis is prolonged by fibroblast growth factors (FGFs), which act in two ways. FGFs increase the likelihood that immediate neuronal precursors (INPs) divide twice, rather than once, before generating neurons; this action requires exposure of INPs to FGFs by early G1. FGFs also cause a distinct subpopulation of explants to generate large numbers of neurons continually for at least several days. The data suggest that FGFs delay differentiation of a committed neuronal transit amplifying cell (the INP) and support proliferation or survival of a rare cell, possibly a stem cell, that acts as a progenitor to INPs.

Ectopic expression of IGF-I and Shh by skeletal muscle inhibits disuse-mediated skeletal muscle atrophy and bone osteopenia in vivo

The loss of normal weight‐bearing activity, which occurs during bed rest, limb immobilization, and spaceflight, stimulates a catabolic response within the musculoskeletal system, which results in a loss of skeletal muscle mass and bone mineral. The mechanism by which loading of muscle and bone is sensed and translated into signals controlling tissue formation remains a major question in the field of musculoskeletal research. In this investigation, we have examined the ability of two potentially anti‐atrophic proteins, IGF‐I and Shh, to inhibit disuse atrophy within muscle and bone, when electroporated into skeletal muscle. We have found that electroporation and ectopic expression of IGF‐I and/or Shh within the gastrocnemius/soleus muscle significantly stimulated muscle fiber hypertrophy and increases in muscle size. In addition, we report that electroporation and ectopic expression of IGF‐I and/or Shh within the gastrocnemius/soleus muscle attenuated the lost of muscle fiber area, muscle mass, and muscle mass density that normally occurs during disuse muscle atrophy. Finally, we found that ectopic expression of IGF‐I and Shh within the gastrocnemius/soleus muscle inhibits parameters of osteopenia within the tibia and fibula associated with hindlimb unloading. These results support the theory that skeletal muscle can regulate bone maintenance and could offer potentially novel and efficient therapeutic options for attenuating muscle and bone atrophy during aging, illness and spaceflight.

Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-kappaB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal‐regulated kinase (ERK) 1/2 pathway, but not p38 or c‐Jun NH2‐terminal kinase (JNK), is preferentially activated in fast‐twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow‐twitch fiber type‐specific reporter activity and repressed those associated with the fast‐twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow‐twitch fiber‐specific protein accumulation while repressing those characteristic of fast‐twitch fibers. Overexpression of MAP kinase phosphatase‐1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time‐dependent manner. Conversion to the slower phenotype was confirmed by up‐regulation of slow reporter gene activity and down‐regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up‐regulation of fast‐twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast‐twitch fiber phenotype with a concomitant repression of slow‐twitch fiber program.—Shi, H., Scheffler, J. M., Pleitner, J. M., Zeng, C., Park, S., Hannon, K. M., Grant, A. L., Gerrard, D. E. Modulation of skeletal muscle fiber type by mitogen‐activated protein kinase signaling. FASEB J. 22, 2990–3000 (2008)

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7+ and Myf5−) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.

Are fibroblast growth factors regulators of myogenesis in vivo

Recent advances in understanding of skeletal muscle differentiation implicate fibroblast growth factors (FGFs) as regulators of myogenesis; however, the identity and actions of factors that repress myogenesis in vivo remain to be established. This review will focus on the fibroblast growth factor family and the evidence for its role in regulating myogenesis in culture and in vivo.

Insulin-like growth factor I stimulates myoblast expansion and myofiber development in the limb

Insulin‐like growth factor I (IGF‐I) is expressed in the anterior and posterior mesodermal cells of the developing limb. However, a definite role for IGF‐I during early limb organogenesis is unknown. To determine the inherent participation of IGF‐I during limb organ development, a retroviral delivery system (RCAS) was used to overexpress IGF‐I throughout the developing hind limb of stage 24 chicken embryos. The area of the belly of the external gastrocnemius muscle in the IGF‐I infected limb was an average of 160, 90, 70, and 80% larger than the contralateral control muscle belly, 4, 5, 6, and 7 days postinjection, respectively (all differences P < 0.01). In comparison to the contralateral control muscles, there were a significantly greater number of muscle fibers in the IGF‐I infected muscles (P < 0.05), confirming that the majority of IGF‐I–mediated muscle enlargement was due to an increase in total fiber numbers (hyperplasia). Four days postinjection, there was a 32% increase in myoblast to myofiber ratio in the muscle of injected limbs compared with the muscle in the contralateral noninjected control limbs (P < 0.05). This result demonstrates that IGF‐I acts to expand the undifferentiated myoblast population, and as a result, more myofibers subsequently develop, and the muscles expressing ectopic IGF‐I are enlarged by means of hyperplasia. There was no difference in tibiotarsus and fibula length or diameter between the IGF‐I injected and control limb, suggesting that ectopic IGF‐I expression within the mesoderm was not a nonspecific growth stimulant of all tissues of the developing limb, but specifically enhanced skeletal muscle development and growth. Ectopic IGF‐I expression had no significant effect on myostatin mRNA concentrations. Our results support a model where mesodermally expressed IGF‐I acts to regulate the number of primary myofibers, and, therefore, size of skeletal muscles, which form during the initial events of limb myogenesis.

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS‐FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin‐expressing, connective‐tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked‐cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

FGFR1 inhibits skeletal muscle atrophy associated with hindlimb suspension.

BackgroundSkeletal muscle atrophy can occur under many different conditions, including prolonged disuse or immobilization, cachexia, cushingoid conditions, secondary to surgery, or with advanced age. The mechanisms by which unloading of muscle is sensed and translated into signals controlling tissue reduction remains a major question in the field of musculoskeletal research. While the fibroblast growth factors (FGFs) and their receptors are synthesized by, and intimately involved in, embryonic skeletal muscle growth and repair, their role maintaining adult muscle status has not been examined.MethodsWe examined the effects of ectopic expression of FGFR1 during disuse-mediated skeletal muscle atrophy, utilizing hindlimb suspension and DNA electroporation in mice.ResultsWe found skeletal muscle FGF4 and FGFR1 mRNA expression to be modified by hind limb suspension,. In addition, we found FGFR1 protein localized in muscle fibers within atrophying mouse muscle which appeared to be resistant to atrophy. Electroporation and ectopic expression of FGFR1 significantly inhibited the decrease in muscle fiber area within skeletal muscles of mice undergoing suspension induced muscle atrophy. Ectopic FGFR1 expression in muscle also significantly stimulated protein synthesis in muscle fibers, and increased protein degradation in weight bearing muscle fibers.ConclusionThese results support the theory that FGF signaling can play a role in regulation of postnatal skeletal muscle maintenance, and could offer potentially novel and efficient therapeutic options for attenuating muscle atrophy during aging, illness and spaceflight.