The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles.

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.     

Redundancy of myostatin and growth/differentiation factor 11 function

Background  

Myostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor β (TGFβ) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.     

Differential phenotypic expression induced in cultured rat astroblasts by acidic fibroblast growth factor, epidermal growth factor, and thrombin

We compared the effects of three growth factors, acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), and thrombin, on rat astroblast proliferation, morphology, glutamine synthetase-specific activity, and phenotypic expression of proteins. In vitro experiments were made on 20-day-old primary cultures. Astroblast proliferation was stimulated transiently (after 48 h treatment) by the three growth factors, while the cell glutamine synthetase activity began to increase significantly only after 3 days of treatment. Acidic FGF and EGF, but not thrombin, modified the cell morphology. The effects on phenotypic expression were first determined after 5 days of treatment to minimize the mitogenic effect of the factors. Proteins synthesized during the last 18 h of the treatments were separated by two-dimensional polyacrylamide gel electrophoresis. About 600 spots were compared, 54 were modulated by the various treatments, 13 were altered similarly by all three factors, 28 by aFGF and EGF, 7 by only aFGF, 3 by only EGF, and 3 by only thrombin. These results indicate a large similarity of effects between aFGF and EGF (41 proteins) and show that these factors elicit a more extended modulation of the phenotypic expression than thrombin (13 proteins). Each of the three factors has a few specific effects, which suggests that even for aFGF and EGF, which are supposed to elicit their effects through membrane receptor-associated tyrosine kinase activity, some specificity appears in their mechanism of action. A model is proposed to suggest that cell maturation is characterized by the modulation of the synthesis of many proteins which can be grouped into classes. Each class appears to be under the control of one regulatory element. The specificity of the effect of a growth factor should result from the activation of a specific combination of such regulatory elements. Analysis of the proteins after only 18 h of treatment, when neither proliferation nor maturation were significantly affected, showed that 11 proteins were regulated only at that time. These proteins could be related to intermediate steps of the growth factor signal transduction.     

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.     

Distribution of cholinergic neuronal differentiation factor/leukemia inhibitory factor binding sites in the developing and adult rat nervous system in vivo

Cholinergic neuronal differentiation factor/leukemia inhibitory factor (CDF/LIF) is a multi-functional cytokine that affects neurons as well as many other cell types. Toward elucidating its neural functions in vivo, we previously investigated the distribution of CDF/LIF binding sites with iodinated native CDF/LIF in embryonic to postnatal day 0 (P0) rats. In the present study, we have extended our examination to postnatal ages and find that specific CDF/LIF binding sites are present at defined developmental stages in additional brain regions not previously exhibiting binding by P0. High levels of binding are detected in all P7 sensory and autonomic ganglia examined, but only in restricted postnatal central nervous system structures. Cranial motor and mesencephalic trigeminal neurons maintain high levels throughout, while binding to spinal motor neurons, which decreases to low levels at P0, reappears by P14 and increases with age. Most other structures, which show detectable binding by P0, exhibit higher levels at postnatal ages, including the red, deep, ventral cochlear, trapezoid, superior olivary, vestibular, ventral tegmental, and ventral posterior thalamic nuclei as well as the glomerular layer of the olfactory bulb. High levels are also detected in several structures for the first time after P0, including the cerebellar cortex (molecular and Purkinje cell layers), lateral reticular nucleus of the medulla and reticular formation, as well as the reticulotegmental, medial geniculate, solitary (rostral, dorsomedial, and commissural regions), medial septal, lateral mammillary, and lateral habenular nuclei. These results not only identify regions of potential CDF/LIF-responsive neurons and glia throughout development but suggest new CDF/LIF roles in the nervous system. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 163–192, 1997.     

Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.     

Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation

Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation.     

Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles

Mdx mice uniquely recover from degenerative dystrophic lesions by an intense myoproliferative (regenerative) response. To investigate a potential role of endogenous basic fibroblast growth factor (bFGF) in injury-repair processes, we investigated its localization in several striated muscles of mdx and control mice using immunofluorescence labeling with specific antibodies. Basic FGF was localized consistently to the myofiber periphery and nuclei of intact myofibers, as well as in single, dystrophin-positive cells in close association with the myofibers (potential myosatellite cells). In mdx mice, actively degenerating skeletal or cardiac muscle fibers presented intense cytoplasmic anti-bFGF staining prior to mononuclear infiltration. Small regenerating fibers in mdx skeletal muscle exhibited greater bFGF accumulation than adjacent larger myofibers. Strong nuclear anti-bFGF immunolabeling was frequently observed in mdx cardiac myocytes at the borders of necrotic regions. In agreement with differences in intensity of immunolabeling, extracts from slow-twitch muscles contained higher levels of bFGF compared to those from fast-twitch muscles, in both control and mdx mice. In addition, bFGF levels were consistently higher in extracts from all mdx tissues compared to those derived from their control counterparts. Our data suggest that bFGF participates in the degenerative and regenerative responses of striated muscle to dystrophic injury and also indicate a potential involvement of this factor with the physiology of different striated muscles.     

Role of fibroblast growth factor 8 in growth and progression of hormonal cancer

Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

Mannose 6-Phosphate/Insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886-20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF available in vivo.     

Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating, and denervated chicken fast skeletal muscles

Using monoclonal antibodies (McAbs) which can distinguish between breast- and leg-type troponin T (TnT), we studied the spatial distribution of TnT isoforms in adult chicken fast skeletal muscles. The breast (pectoralis major) and leg (iliotibialis posterior) muscles were composed predominantly of homogeneous fibers containing breast- and leg-type TnT, respectively. The posterior latissimus dorsi muscle was composed of heterogeneous fibers of at least two types, namely breast and leg types. In developing and regenerating fast muscles, only leg-type TnT was expressed at early stages, and later breast-type TnT appeared either transiently or permanently. This led ultimately to several distinct adult fast muscle breast/leg TnT isoform profiles. Since both types of TnT were synthesized in embryonic and regenerating muscles with nerves intact as well as in regenerating muscles with nerves resected, the switching on of their expression during fast muscle development appears to be independent of nerves. However, its full development ("fine tuning" of the protein isoform distribution within the fast fiber types) and the maintenance of the adult state are presumed to be dependent on the nerves, since, although regenerating fibers in denervated muscles could exhibit the early and then the later embryonic stainabilities, they again returned to the early embryonic state; further, the denervation of adult muscles caused the replacement of TnT isoform from the adult to the early embryonic state.     

Postnatal growth and differentiation in three hindlimb muscles of the rat

Summary The postnatal development, between 0 and 90 days, of three hindlimb muscles and diaphragm of the rat was investigated with respect to fiber types and diameter (histochemistry) and substrate oxidation rates and enzyme activities (biochemistry). The process of muscle fiber differentiation into mature patterns was evaluated by visual classification into 3 or 4 groups having different staining intensities for 3 enzyme-histochemical reactions, enabling 26 fiber types to be distinguished. These exhibited specific sizes and growth rates that varied among the muscles. One of the hindleg muscles (flexor digitorum brevis) remained much more immature than soleus and extensor digitorum longus.The histochemical and biochemical findings correlated well. The capacity for pyruvate and palmitate oxidation, and the activities of cytochrome c oxidase and citrate synthase, increased markedly between 9 and 37 days in soleus and extensor digitorum longus (except citrate synthase in the latter) but not in flexor digitorum brevis. Creatine kinase activity increased in all hindlimb muscles. Both the capacity and the activity of pyruvate oxidation (determined in homogenates and intact isolated muscles, respectively), were in accordance with the fiber type composition. In contrast to oxidation capacity, the activity of pyruvate oxidation decreased after birth until the mature stage, when a value of 18–42% of that of early postnatal muscles was recorded.     

Protein-tyrosine phosphatase PTPepsilon C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells

We engineered and expressed both a wild-type and mutant cytosolic isoform of PTPepsilon (PTPepsilonC) in murine M1 leukemic cells, which can be induced to growth arrest and monocytic differentiation by interleukin (IL)-6 and leukemia inhibitory factor (LIF). Forced expression of PTPepsilonC inhibited IL-6- and LIF-induced monocytic differentiation and apoptosis in M1 cells, whereas expression of PTPepsilonM, a transmembrane isoform of PTPepsilon, did not. PTPepsilonC expression resulted in lower levels of IL-6-induced tyrosine phosphorylation of Jak1, Tyk2, gp130, and Stat3 compared with parent cells. In M1 transfectants expressing an inactive mutant of PTPepsilonC, both tyrosine phosphorylation and apoptosis induced by IL-6 and LIF were potentiated rather than inhibited. These results suggest an important role for PTPepsilonC in negative regulation of IL-6- and LIF-induced Jak-STAT signaling.     

Isozymes of myosin in growing and regenerating rat muscles

Native myosin isozymes of rat muscles have been isolated by electrophoreses in non-dissociating conditions. Their mobilities were measured, using taenia coli myosin as an internal standard and their relative concentrations were determined by computer planimetry of the electrophoretograms. Three isozymes were observed in extensor digitorum longus (EDL), two in soleus (SOL), four in neonatal muscles three days before birth. Regenerates of minced EDL or SOL muscles in adult animals had no native myosin the third day after surgery; they were similar to neonatal muscles 15 days after surgery and to adult muscles 60 days after surgery.     

Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle

Hepatocyte growth factor (HGF) is the onlyknown growth factor that activates quiescent satellite cells inskeletal muscle. We hypothesized that local delivery of HGF may enhanceregeneration after trauma by increasing the number of myoblastsavailable for restoring normal tissue architecture. Injection of HGFinto muscle at the time of injury increases myoblast number but doesnot enhance tissue repair as determined using quantitative histologicalanalyses. Rather, depending on the dose and the timing of HGFadministration relative to the injury, regeneration can be inhibited.The greatest inhibitory effect is observed when HGF is administered onthe day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and earlydifferentiation normally occur. To establish a mechanism for thisinhibition, we show that HGF can act directly on primary muscle cellsto block differentiation. These results demonstrate that1) exogenous HGF synergizes withfactors in damaged muscle to increase myoblast number,2) regeneration is not regulatedsolely by myoblast number, and 3)HGF inhibits muscle differentiation both in vitro and in vivo.

     

Differential adaptation to weightlessness of functional and structural characteristics of rat hindlimb muscles

Soleus, vastus intermedius, tibialis anterior, and extensor digitorum longus muscles were removed from rats following space flight onboard the SLS-2 mission and from control animals. Muscle tissues were studied for their calcium and strontium activated tension characteristics and for structural changes. Muscles were also examined for myosin composition using electrophoresis. Results indicate that changes occurred in structural and functional muscle characteristics in both slow and fast muscle fiber types. These results are detailed and discussed.