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

Abstract

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 α‐tubulin, a marker for axonal microtubules, showed that MMP‐9‐treated cells had increased distribution of α‐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.