Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-κB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.
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We thank J. R. Sanes and members of U.382 for valuable discussions and critical review of the manuscript. We are grateful to S. P. Hunt for anti Reg-2 antibody; J. R. Sanes for anti β-gal antibody; T. Jessell for Isl1 and Chat plasmids; H. Bödeker for purification of Reg-2 protein and rat Reg-2 plasmid; D. Pennica for CT-1 null mutant mice; C. Lasserre for mouse Reg-2 plasmid; C. J. Woolf for adenovirus coding LacZ; A. Garcès and J. Livet for the WM-ISH technique for spinal cord; V. Arce for technical suggestions; R. Goetz for genotyping the LIF/CNTF knockout embryo; and C. Moretti for confocal microscopy techniques. This work was funded by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Association Française contre les Myopathies (AFM), Institut pour la Recherche sur la Moelle Epinière (IRME), and European Commission BIO4 contract CT960433. H.N. was supported by JST Overseas Research Fellowship and a postdoctoral fellowship from NOVARTIS Foundation (Japan).
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