Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro

J Neurosci Res. 2004 Jul 15;77(2):229-39. doi: 10.1002/jnr.20160.


Matrix metalloproteinase-9 (MMP-9) is a basal-lamina-degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP-9 colocalizes with growth-associated protein GAP-43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP-9, MMP-9-neutralizing antibody, and a broad-spectrum MMP inhibitor (Ro 31-9790) on sprout formation, elongation, and branching. Quantitative phase-contrast microscopy showed that MMP-9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP-43, a marker for growth cones, and alpha-tubulin, a marker for axonal microtubules, showed that MMP-9-treated cells had increased distribution of alpha-tubulin but no effect on GAP-43. Western blot analyses of cell lysates demonstrated that the NGF-induced increase in GAP-43 was unchanged with MMP-9 treatment or inhibition, confirming that MMP-9 had no effect on new sprout formation. However, Ro 31-9790 reduced GAP-43 levels to those seen in untreated cells, suggesting that an MMP other than MMP-9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM-p), a marker for regenerative elongation, was induced with MMP-9 treatment and was inhibited by the anti-MMP-9 antibody treatment, confirming the role of MMP-9 in axonal elongation. NFM-p colocalized with MMP-9 in regenerating sciatic nerve fibers. These findings suggest that MMP-9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / pharmacology
  • Biomarkers
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology
  • Female
  • GAP-43 Protein / metabolism
  • Growth Cones / drug effects*
  • Growth Cones / metabolism
  • Growth Cones / ultrastructure
  • Matrix Metalloproteinase 9 / metabolism
  • Matrix Metalloproteinase 9 / physiology*
  • Matrix Metalloproteinase Inhibitors
  • Nerve Growth Factor / metabolism
  • Nerve Growth Factor / pharmacology*
  • Nerve Regeneration / drug effects*
  • Nerve Regeneration / physiology
  • Neurofilament Proteins / antagonists & inhibitors
  • Neurofilament Proteins / metabolism
  • PC12 Cells
  • Peripheral Nerve Injuries
  • Peripheral Nerves / drug effects*
  • Peripheral Nerves / growth & development*
  • Phosphorylation / drug effects
  • Rats
  • Rats, Sprague-Dawley
  • Tubulin / metabolism
  • Up-Regulation / drug effects
  • Up-Regulation / physiology


  • Antibodies
  • Biomarkers
  • Enzyme Inhibitors
  • GAP-43 Protein
  • Matrix Metalloproteinase Inhibitors
  • Neurofilament Proteins
  • Tubulin
  • neurofilament protein M
  • Nerve Growth Factor
  • Matrix Metalloproteinase 9