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

Mario Rende, Emanuela Brizi, Jim Conner, Susan Treves, Kathrin Censier, Carlo Provenzano, Giulio Taglialatela, Pietro Paolo Sanna, Rosario Donato

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

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 neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle 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 line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform 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 the fusion rate of both primary and L6 myoblasts; as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low μM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing 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 NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

Original languageEnglish (US)
Pages (from-to)869-885
Number of pages17
JournalInternational Journal of Developmental Neuroscience
Volume18
Issue number8
DOIs
StatePublished - 2000
Externally publishedYes

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Nerve Growth Factor
Myoblasts
Cell Proliferation
Rhabdomyosarcoma
Cell Line
Muscle Cells
Skeletal Muscle Fibers
Nerve Growth Factors
Nerve Growth Factor Receptor
Peripheral Nervous System
In Vitro Techniques
Neutralizing Antibodies
Signal Transduction
Pharmacology
Tyrphostins
Nerve Growth Factor Receptors
Neurons
Spiders
Intermediate Filaments
Receptor Protein-Tyrosine Kinases

Keywords

  • Differentiation
  • Myoblasts
  • Myotubes
  • NGF
  • Rhabdomyosarcoma

ASJC Scopus subject areas

  • Developmental Biology
  • Developmental Neuroscience

Cite this

Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA. / Rende, Mario; Brizi, Emanuela; Conner, Jim; Treves, Susan; Censier, Kathrin; Provenzano, Carlo; Taglialatela, Giulio; Sanna, Pietro Paolo; Donato, Rosario.

In: International Journal of Developmental Neuroscience, Vol. 18, No. 8, 2000, p. 869-885.

Research output: Contribution to journalArticle

Rende, Mario ; Brizi, Emanuela ; Conner, Jim ; Treves, Susan ; Censier, Kathrin ; Provenzano, Carlo ; Taglialatela, Giulio ; Sanna, Pietro Paolo ; Donato, Rosario. / Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA. In: International Journal of Developmental Neuroscience. 2000 ; Vol. 18, No. 8. pp. 869-885.
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T1 - Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA

AU - Rende, Mario

AU - Brizi, Emanuela

AU - Conner, Jim

AU - Treves, Susan

AU - Censier, Kathrin

AU - Provenzano, Carlo

AU - Taglialatela, Giulio

AU - Sanna, Pietro Paolo

AU - Donato, Rosario

PY - 2000

Y1 - 2000

N2 - 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 neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle 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 line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform 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 the fusion rate of both primary and L6 myoblasts; as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low μM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing 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 NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

AB - 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 neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle 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 line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform 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 the fusion rate of both primary and L6 myoblasts; as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low μM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing 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 NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

KW - Differentiation

KW - Myoblasts

KW - Myotubes

KW - NGF

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