Neurite penetration into collagen gels requires Ca2+-dependent metalloproteinase activity

Cultured sympathetic neurons from neonatal rats release a Ca2+-dependent metalloproteinase capable of degrading native and denatured type I collagen. A large fraction of the metalloproteinase activity is released from growth cones of growing neurites, which suggests that this proteinase may be involved in neurite penetration into collagen-rich extracellular matrices during development. In the present study, we investigated the possibility that neurite penetration into 3-dimensional collagen gels requires the Ca2+-dependent metalloproteinase. Sympathetic neurons were plated on top of collagen gels and the neuronal metalloproteinase activity was inhibited with the collagenase inhibitor HSCH2CH[CH2CH(CH3)2]-CO-Phe-Ala-NH2 (HS-LFA). HS-LFA inhibited the Ca2+-dependent metalloproteinase activity, penetration of neurites into collagen gels and release of 3H from 3H-collagen gels in a dose-dependent manner over very similar concentration ranges (EC50 = 150-200 nM). Highly significant correlations existed between neurite penetration into collagen gels and metalloproteinase activity (r = 0.99), and metalloproteinase activity and release of 3H from 3H-collagen gels (r = 0.99). Although HS-LFA inhibited the growth of neurites within collagen gels, it had no effect on neurite outgrowth on collagen films. Thus, the neuronal Ca2+-dependent metalloproteinase appears to be required for neurite penetration into and growth within 3-dimensional collagen matrices, but not for growth along a 2-dimensional collagen substrate.