INTRODUCTION: Neurotrophins are expressed in muscle cells both during development and in adults. However, only fragmentary and controversial information is available regarding the responsiveness Of muscle cells to neurorrophins. Previously, we have shown that, in a rat myogenic cell line (L6), the low-affinity neurotrophins receptor (p75) is expressed only at very early developmental stages and its level Of expression is associated with myoblast fusion into myotube. In the present study, we show that (i) TrKA, the high-affinity, transducing receptor for NGF, is expressed both in myoblasts and myotubes and (ii) adding NGF to the medium of L6 myoblast increases the fusion rate in a dose-dependent manner. MATERIALS AND METHODS: L6 myoblast cell line was used throughout and the cells were analyzed at day I, 3 and 6 after plating (day 0). NGF and TrKA were detected by immunohistochemistry and Western blotting. Several dosages Of ZSNGF, from 10 to 800 ng/ml, were added at every change of the medium. As a control for the biological effect of NGF, some untreated L6 cultures were conditioned by the addition to the medium of (i) anti NGF IgG (from I to 200 ng/ml) or (ii) K2S2a (200 nM), an inhibitor of TrKA activity. The L6 fusion rate was analyzed after May-Grunwald staining. RESULTS AND DISCUSSION: Untreated L6 cells showed a detectable level of NGF at day 3 and 6, mainly in myotubes. Similarly, TrkA was clearly detected at day I, 3 and 6, both in myoblasts and myotubes. The addition of NGF to the medium of L6 myoblast increases the fusion rate in a dose-dependent manner, with the highest effect at 10-20 ng/ml. Conversely, the addition of anti NCF IgG to the medium results in a dose-dependent decrease of the fusion rate, with the maximal effect at 50 ng/ml. The addition of K252a induced a block of the L6 proliferation and fusion. These data suggest that the expression of NGF in developing muscle may not only be associated with a classical, target-derived, neurotrophic function for the innervating peripheral nervous system neurons, but may have autocrine actions on developing myoblasts, influencing their differentiation and fusion into myotubes.
"Nerve growth factor (NGF) stimulates myoblast fusion into myotubes in a rat myogenic cell line (L6): a novel autocrine role for NGF in muscle development.
Bruno R.
1998-01-01
Abstract
INTRODUCTION: Neurotrophins are expressed in muscle cells both during development and in adults. However, only fragmentary and controversial information is available regarding the responsiveness Of muscle cells to neurorrophins. Previously, we have shown that, in a rat myogenic cell line (L6), the low-affinity neurotrophins receptor (p75) is expressed only at very early developmental stages and its level Of expression is associated with myoblast fusion into myotube. In the present study, we show that (i) TrKA, the high-affinity, transducing receptor for NGF, is expressed both in myoblasts and myotubes and (ii) adding NGF to the medium of L6 myoblast increases the fusion rate in a dose-dependent manner. MATERIALS AND METHODS: L6 myoblast cell line was used throughout and the cells were analyzed at day I, 3 and 6 after plating (day 0). NGF and TrKA were detected by immunohistochemistry and Western blotting. Several dosages Of ZSNGF, from 10 to 800 ng/ml, were added at every change of the medium. As a control for the biological effect of NGF, some untreated L6 cultures were conditioned by the addition to the medium of (i) anti NGF IgG (from I to 200 ng/ml) or (ii) K2S2a (200 nM), an inhibitor of TrKA activity. The L6 fusion rate was analyzed after May-Grunwald staining. RESULTS AND DISCUSSION: Untreated L6 cells showed a detectable level of NGF at day 3 and 6, mainly in myotubes. Similarly, TrkA was clearly detected at day I, 3 and 6, both in myoblasts and myotubes. The addition of NGF to the medium of L6 myoblast increases the fusion rate in a dose-dependent manner, with the highest effect at 10-20 ng/ml. Conversely, the addition of anti NCF IgG to the medium results in a dose-dependent decrease of the fusion rate, with the maximal effect at 50 ng/ml. The addition of K252a induced a block of the L6 proliferation and fusion. These data suggest that the expression of NGF in developing muscle may not only be associated with a classical, target-derived, neurotrophic function for the innervating peripheral nervous system neurons, but may have autocrine actions on developing myoblasts, influencing their differentiation and fusion into myotubes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
