The important intracellular events that regulate astrocyte proliferation, a fundamental issue of neurobiology, are not well understood. Recently, it has been determined that the endogenous neuropeptides, atrial natriuretic peptide (ANP) and endothelin (ET), are anti-growth factor and growth factors, respectively, for cultured fetal diencephalic astrocytes. The expression of ANP and ET and their receptors in the developing fetal hypothalamus, a time of astrocyte growth, supports the idea that these peptides might play a role in vivo to modulate glial proliferation. Investigation of a common mechanism through which these two peptides might act identified the immediate early gene/protein, Tis 8 and the autologous growth factor, bFGF, as the key proteins through which ANP (negatively) and ET (positively) modulate astrocyte proliferation. There is good supporting data to hypothesize that this same pathway mediates the growth promoting actions of other endogenous glial growth factor/mitogens, as well. This proposal seeks to determine the specific steps, from transmembrane binding, to nuclear regulation, through which ANP and ET modulate astrocyte proliferation, and eventually to extend these findings as a general pathway for glial growth modulators. First, it will be determined whether protein kinase C(PKC) is the initial signal through which ET and ANP, positively and negatively, modulate the growth schema.The investigators will then show that the target protein for PKC is a specific member of the MAP-kinase cascade, consistent with their preliminary data that ANP inhibits and ET stimulates the activity of this growth-related cascade. They will investigate the precise mechanism as to how ANP inhibits the MAP-K pathway, postulating the activation of a specific phosphatase, shown through dephosphorylation assays. They will prove the definitive role for the MAP kinase pathway in the growth (anti- growth) paradigm, using anti-sense inhibition of MAP-K protein production. The role of MAP-K to increase Tis 8 transcription in response to ET, through phosphorylating a trans activating protein for the Tis 8 promoter will be shown, using DNase footprinting, gel shift and reporter gene functional studies. They will show that ANP inhibits this process and mechanism. It will then be determined through similar analysis, the mechanism through which ET-stimulated and ANP-inhibited Tis 8 protein production modulates the transcription of bFGF; for instance, the activational state of Tis 8, and what promoter sites are involved. A critical issue is whether bFGF then is secreted to bind its transmembrane receptor and trigger the growth cascade, or whether it directly translocates to the nucleus to induce the cell cycle. This will be determined by transfecting the astrocytes with a tyrosine kinase domain-mutated bFGF receptor-1 and/or receptor-3 constructs, which will inhibit wild type bFGF-R by dominant negative heterodimerization of FGFR1 and FGFR3, found on the astrocytes. They will then see if ET can cause mitogenesis in the FGF-R dysfunctional glia. Finally, induction of the cell cycle by growth factors involves the activation/production of G1 phase cyclins and kinases, or inhibition of inhibitory proteins which regulate the activity or the cyclin/kinase complex (CDI). They will determine the effects of ANP and ET on key G1 phase protein production and activity, to understand the critical events through which neuropeptides modulate astrocyte proliferation.
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