ROLE OF THE PROTEASE STROMELYSIN IN AXON INVASIVENESS

The stromelysins are a family of secreted metalloproteinases that degrade
various components of the extra-cellular matrix, and in particular,
components of basal laminae (BL). Members of the stomelysin 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 related 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 rise. 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 stage-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 then 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.