Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.

Computational EST database analysis identifies a novel member of the neuropoietic cytokine family.

A novel member of the neuropoietic cytokine family has been cloned and the protein expressed and characterized. In an effort to identify novel secreted proteins, an algorithm incorporating neural network algorithms was applied to a large EST database. A full-length clone was identified that is 1710 bp in length and has a single open reading frame of 225 amino acids. This new cytokine is most homologous to cardiotrophin-1, having a similarity and an identity of 46 and 29%, respectively, and therefore we have named it cardiotrophin-like cytokine (CLC). Northern hybridization analysis identified a 1.4-kb messenger RNA that is highly expressed in spleen and peripheral leukocytes. Purified recombinant CLC induced the activation of NFkappaB and SRE reporter constructs in the TF-1, U937, and M1 cell lines. Furthermore, the signal transduction pathway for CLC was characterized in the neuroblastoma cell line SK-N-MC and found to involve tyrosine phosphorylation of gp130 and STAT-1.

Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1.

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.

Identification and characterization of a novel member of the fibroblast growth factor family.

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.