"Cells of myogenic lineage respond to nerve Growth Factor (NGF) in vitro: a novel role for NGF".

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 neurotrophins. Previously we have shown rhat the addition of NGF to the medium of L6 cells (a rat myogenic cell line) increases the percentage of fusion. In the present study we show that NGF: (1) similarly increases myoblast fusion and proliferation in a primary culture Of adult human myoblasts; (2) does not affect a rhabdomyosarcoma cell line; (3) the addition of a neutralizing antibody anti-NGF or inhibitors of TrKA (the transducing receptor for NGF) induces changes in the proliferation, fusion and morphology in the three types of myogenic cells tested. MATERIALS AND METHODS. L6 cells, primary human myoblasts and a human rhabdomyosarcoma cell line (TE67I) were used. NGR TrKA and p75 were analyzed by western blot and immunocytochemistry. Several concentrations (from 5 to 800 ng/m1) of differenr preparations of NGF (7S, 2.5S and Beta) were added into the medium at every change. SimiUrty, the myogenic cells were treated with several concentrarion of a neurralizing anti-NGF antibody (from 1 to 200 ng/ml IgG) or with two inhibitors of TrKA, K252a(from 10 to 200 nM) or Tyrphostin AG879 (from 0.5 to 50 pM).The cells were counted for their fusion percentage (May-Grunwald staining) or analyzed at immunocytochemical level (MHC andVimentin) RESULTS AND DISCUSSION. The three types of cells showed detectable levels Of NGR TrKA and p75. Addition of NGF increased the percentage of fusion both in L6 and primary myoblasts, as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the endogenously produced NGF with a neutralizing anribody decreased the percentage of fusion both in L6 and primary myoblasts. Conversely, neither NGF nor the neutralizing antibody affected the rhabdomyosarcoma cells. pharmacological inhibition of TrKA signal transduction with K252aand AG879 resulted in a dramatic dose-dependent decrease in proliferation, inhibition of the fusion as well as morphological changes. These data suggest that NGF expression in skeletal muscle is associated not only with a classical target-derived function for peripheral neurons, but also with an autocrine action on developing myoblasts. These results define a novel biological role for NGF and suggest that pharmacological interference with NGF signaling transduction could be beneficial in the therapy of rhabdomyosarcoma.