The stromelysins are a family of secreted metalloproteinases that degrade various components of the extracellular matrix, and in particular, components of basal laminae (BL). Members of the stromelysin gene family have been implicated, moreover, in the degradation of BL which accompanies tumor cell invasiveness in metastasis, as well as during various phenomena during normal development. In previous work we found that the mRNA encoding stromelysin-1 (ST-1) is induced in PC12 cells at the same time that these cells undergo neuronal differentiation. Recently, we have found that the ST-1 protein is also expressed and released into the culture medium. In this application, we propose to test the hypothesis that expression of ST-1 confers the ability of PC12 cell neurites to penetrate BL. To this end, we have established an in vitro assay for neurite invasiveness which involves plating PC12 cells atop a Nuclepore filter coated with a film of the artificial BL, Matrigel. By scoring the numbers of neurites which penetrate this Matrigel-coated filter, quantitative analyses of neurite invasiveness can be made. We now propose to use a combination of experimental regimes which decrease or increase ST-1 activities, and then observe how these perturbations affect neurite invasiveness. Our hypothesis would predict that those regimes which decrease ST-1 would decrease neurite invasiveness, including (i) the addition of metalloproteinase inhibitors to the culture medium, (ii) selecting for ST-1 non-producing variant subclones of PC12, (iii) transfecting cells with ST-1 anti-sense oligonucleotides, and (iv), generating stably transfected PC12 cells lines expressing ST-1 anti-sense RNA. A second prediction of this hypothesis is that ST-1 should be released from the growth cones of PC12 cells. We propose to examine this possibility using immunostaining protocols with existing antibodies, as well as with additional antibodies we propose to raise. We will also examine whether ST-1 is released at the growth cone using various in situ substrate degradation experiments. We also propose to test whether ST is expressed during normal nervous system development, playing a similar role in conferring neurite invasiveness through BL. First, we will examine the tissue-specific and developmental state-specific expression of ST-1 mRNA and immunoreactivity during normal rat embryogenesis. We will then test whether alterations in ST-1 expression in normal cultured neurons changes neurite invasiveness. Finally, we will use a novel in vivo technique for testing the role of ST-1 in neurite invasiveness and nervous system morphogenesis. This technique involves infecting dissected neural tubes from quail embryos with a retroviral vector which causes the expression of both beta-galactosidase activity (as a marker) and ST-1, and the grafting portions of these transfected neural tubes into host chicken embryos in ovo. All grafted cells can be identified by the quail nucleolar marker and the infected graft cells can be identified by their expression of beta- galactosidase activity. In this way the in vivo effects of ST-1 expression on nervous system development can be determined.
