Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro.
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.
Shubayev
Veronica I
VI
San Diego VA Healthcare System, San Diego, California, USA . vshubayev@ucsd.edu
Myers
Robert R
RR
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015415:Biomarkers; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019922:GAP-43 Protein / Q000378:metabolism; D020439:Growth Cones / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism / Q000502:physiology*; D061965:Matrix Metalloproteinase Inhibitors; D020932:Nerve Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D009416:Nerve Regeneration / Q000187:drug effects* / Q000502:physiology; D016900:Neurofilament Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D016716:PC12 Cells; D059348:Peripheral Nerve Injuries; D010525:Peripheral Nerves / Q000187:drug effects* / Q000254:growth & development*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D014404:Tubulin / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D000906:Antibodies; D015415:Biomarkers; D004791:Enzyme Inhibitors; D019922:GAP-43 Protein; D061965:Matrix Metalloproteinase Inhibitors; D016900:Neurofilament Proteins; D014404:Tubulin; C061195:neurofilament protein M; D020932:Nerve Growth Factor; D020780:Matrix Metalloproteinase 9