Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as 'target- derived factors'. During differentiation, muscle cells also express some ne urotrophin receptors, such as the low-affinity p75 neurotrophin receptor, w hich binds all neurotrophins, and the high affinity tyrosine kinase recepto r TrKA, nerve growth factor (NGF) transducing receptor. The functional role s of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscl e cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell lin e TE-671 were used in this study. As expected, all the three cell types exp ressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts fol lowing differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was exp ressed in myoblasts but not in myotubes early after plating; while some pri mary human myoblasts expressed it at all the time-points tested. Some fusif orm cells of the TE-671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased th e fusion rate of both primary and L6 myoblasts; as well as the proliferatio n of the slowly dividing primary myoblasts. Consistently, blocking the acti on of endogenously produced NGF with a specific neutralizing antibody decre ased the percentage of fusion in both primary and L6 myoblasts. On the cont rary, blocking the binding of NGF to p75 did not affect the percentage of f usion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing anti bodies appeared to affect the rhabdomyosarcoma cells, which have a high pro liferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low muM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especiall y evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhi bitors also blocked fusion of L6 and primary myoblasts and induced morpholo gical changes characterized by the flattening of the cells and a 'spider-li ke' rearrangement of the intermediate filaments in all three cell lines wit h some minor differences. A transfection study showed that p75-overexpressi ng L6 cells do not fuse and present changes in their morphology similar to the TrKA-inhibitors treated L6 cells. These data support the notion that NG F expression in skeletal muscle is not only associated with a classical tar get-derived neurotrophic function for peripheral nervous system neurons, bu t also with an autocrine action which affects the proliferation, fusion int o myotubes, and cell morphology of developing myoblasts. The present data a lso suggest that these effects of NGF are mediated by TrKA receptors and th at a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myoge nic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggest s that pharmacological interference with NGF signal transduction could be e ffective in the control of these malignancies. (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.