Recent studies in several neuronal lineages suggest that extrinsic factors such as polypeptide growth factors regulate various stages of neuronal development, from initial commitment of multipotent progenitors to induction of specific gene expression that is characteristic of terminal neuronal differentiation. In the present study, immortalized hypothalamic neurons of the GT1-1 lineage were used to analyze proliferative, as well as morphological and molecular differentiation actions of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I). These effects were compared with those induced by specific activators of protein kinase A and C pathways, which potently inhibited cell proliferation and gonadotropin-releasing hormone (GnRH) gene expression, but stimulated morphological neuronal maturation as determined by the length and number of neurite outgrowth. bFGF exerted a broad spectrum of stimulatory effects, increasing the rate of proliferation measured both by the incorporation of 3H-thymidine and by cell number, and parameters of terminal differentiation, such as neurite outgrowth and induction of gene expression. bFGF stimulated the expression of the hybrid transgene-containing portions of the rat GnRH promoter. In contrast, EGF, TGF-alpha, and IGF-I inhibited cell proliferation, while having subtle effects on neurite outgrowth. Thus, GT1-1 cells appear to be differentially responsive to distinct neurotrophic factors, providing a model for studying the specific effects of neurotrophic factors on functional differentiation, migration, and connectivity of hypothalamic neurons.

The formation of a new blood supply, angiogenesis, is an essential component of carcinogenesis and unrestricted tumor growth. A substance capable of inhibiting angiogenesis would be of considerable therapeutic potential in the treatment of cancer. We previously reported that the 16-kilodalton N-terminal fragment of rat PRL (16K rPRL) was a potent inhibitor of capillary endothelial cell proliferation via a novel receptor. We now report that the nanomolar concentrations of recombinant human 16K PRL inhibit basal and basic fibroblast growth factor- or vascular endothelial growth factor-stimulated growth of bovine brain capillary endothelial cells. 16K human (h) PRL also inhibits stimulation of human umbilical vein endothelial cell proliferation by basic fibroblast growth factor. The organization of endothelial cells into capillary-like structures in type I collagen gels is also prevented by 16K hPRL. Furthermore, in an in vivo assay, the chick embryo chorioallantoic membrane assay, 16K hPRL as well as 16K rPRL were potent inhibitors of capillary formation. 16K hPRL, like 16K rPRL, maintains its biological activity as a partial PRL agonist at PRL receptors on mammary gland epithelial cells. These data demonstrate for the first time that the biological activity of 16K rPRL is not unique and that similar fragments of hPRL are active. The antiangiogenic activity of these molecules is conserved across avian and mammalian species. That 16K hPRL is a potent antiangiogenic factor in in vitro and an in vivo assay raises the exciting potential of this peptide being capable of inhibiting tumor growth.

The N-terminal fragment of PRL (16K PRL) is an antiangiogenic factor that, in vitro, inhibits several components of angiogenesis including basic fibroblast growth factor (bFGF)-induced cell division, migration, and organization of capillary endothelial cells. An essential step in the regulation of angiogenesis is the activation of urokinase (urokinase type plasminogen activator, uPA), which in turn activates a cascade of proteases that play essential roles in endothelial cell migration and tissue remodeling. Treatment of bovine capillary endothelial cells (BBEC) with 16K PRL inhibited bFGF-stimulated urokinase activity in BBEC as detected by plasminogen substrate gel assay. 16K PRL did not appear to be acting via an effect on uPA expression because no change in messenger RNA levels were observed. However, protein levels of plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of urokinase, were increased by 16K PRL independent of the action of bFGF. The 16K PRL-induced increase in PAI-1 protein levels appear to be the result of increased expression of the PAI-1 gene. Increased production of PAI-1 induced by 16K PRL results in the formation of inactive PAI-1/uPA complexes, consistent with the observed decrease in uPA activity.

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