Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 Integrin, and its regulator ADAM12, but not TrkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK Inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 Integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of TrkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.