No abstract

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.

Extracts of bovine hypothalamus were found to contain a significant level of mitogenic activity when tested in a Swiss 3T3 cell [3HlThd incorporation assay and in a human umbilical vein endot elial cell growth assay. The mitogenic activity responsible for 3T3 cell activity was purified and characterized as a fibroblast growth factor (FGF)like mitogen. Neither the biologically active FGF-like mitogen purified from the hypothalamus extracts nor FGF purified from bovine pituitary glands was mitogenic when added to human endothelial cells in vitro, suggesting the presence of more than one mitogen in the hypothalamic extracts. The 3T3 and endothelial cell biological activities of hypothalamic extracts were both found to be inactivated by trypsin, subtilisin, and heat treatment, but were stable to dialysis. The endothelial cell growth factor activity could be efficiently separated from the FGF activity by gel exclusion chromatography. The endothelial cell mitogen possessed a molecular weigt of approximately 75,000, whereas that of FGF was approximately 15,000:'The endothelial cell growth factor activity was found to be inactivated with reducing agents whereas the 3T3 cell mitogenic activity was stable after incubation with 2-mercaptoethanol. Significant levels of endothelial cell mitogenic activity were also found in extracts of bovine brain and pituitary glands. The vascular endothelium exists as a functional monolayer interface between blood and tissue (1, 2) that has been previously characterized in vivo as a population of cells with a low mitotic index (3). Factors that influence the growth and survival of vascular endothelial cells in vitro have important implications not only in the elucidation of normal and pathological states such as thrombus formation (4), the generation of granulation tissue, wound regeneration (5), and tumor growth (6), but also in providing the necessary in itro systems for the study of these processes (7).Previous investigations have shown that human and bovine vascular endothelial cells could be grown and maintained in culture if the cell culture medium was supplemented with high concentrations of bovine brain or pituitary fibroblast growth factor (FGF) (5). These studies eventually led to the establishment of a bovine vascular endothelial cell line that has a strict requirement for FGF (7). Additional experiments revealed that the mitogenic effect of FGF in vascular endothelial cell culture can be potentiated by the addition of purified human thrombin (5). It was suggested that the enhancement of FGF action by thrombin in the vascular endothelial cell system does not proceed through a mechanism involving the proteolytic digestion or modification of FGF, but as a result of an interaction between the vascular endothelial cells and thrombin (5).Our studies indicate that human umbilical vein va'scular endothelial cells do not respond at low seed density to supplementation with high concentrations of biologically active FGF or human thrombin or both. However, an excellent human endo...

Heparin-binding growth factor-1 (HBGF-1) is an angiogenic polypeptide mitogen for mesoderm- and neuroectoderm-derived cells in vitro and remains biologically active after truncation of the amino-terminal domain (HBGF-1 alpha) of the HBGF-1 beta precursor. Polymerase chain reaction mutagenesis and prokaryotic expression systems were used to prepare a mutant of HBGF-1 alpha lacking a putative nuclear translocation sequence (amino acid residues 21 to 27; HBGF-1U). Although HBGF-1U retains its ability to bind to heparin, HBGF-1U fails to induce DNA synthesis and cell proliferation at concentrations sufficient to induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression. Attachment of the nuclear translocation sequence from yeast histone 2B at the amino terminus of HBGF-1U yields a chimeric polypeptide (HBGF-1U2) with mitogenic activity in vitro and indicates that nuclear translocation is important for this biological response.