NFI transcriptionally represses CDON and is required for SH-SY5Y cell survival.

Nuclear Factor One (NFI) family of transcription factors regulate proliferation and multiple aspects of differentiation, playing analogous roles in embryonic development and various types of cancer. While all NFI family members are expressed in the developing brain and are involved in progression of brain cancers, their role in neuroblastoma has not been studied. Here we show that NFIB is required for the survival and proliferation of SH-SY5Y neuroblastoma cells, assessed by viability and colony formation assays. Cdon, an Ig superfamily member, is a SHH dependence receptor that acts as a tumor suppressor in neuroblastoma. In the absence of NFI, Cdon is upregulated in the developing mouse brain, however the mechanisms by which its transcription is regulated remains unknown. We report CDON as a downstream target of NFIs in SH-SY5Y cells. There are three putative NFI binding sites within the one kb CDON promoter, two of which are occupied by NFIs in SH-SY5Y cells and human neural stem cells. In dual-luciferase assays, Nfib directly represses CDON proximal promoter activity. Moreover, silencing NFIB leads to upregulation of CDON in SH-SY5Y cells, however, decreased cell proliferation in NFIB silenced cells could not be rescued by concomitantly silencing CDON, suggesting other molecular players are involved. For instance, p21, an NFI target in glioblastoma and breast cancer cells, is also upregulated upon NFIB knock-down. We propose that NFIB is indispensable for SH-SY5Y cells which may involve regulation of apoptosis inducer proteins CDON and p21.

Absence of the transcription factor Nfib delays the formation of the basilar pontine and other mossy fiber nuclei.

Transcription factors of the Nuclear Factor I (Nfi) family are important for the development of specific neuronal and glial populations in the nervous system. One such population, the neurons of the basilar pontine nuclei, expresses high levels of Nfi proteins, and the pontine nuclei are greatly reduced in mice lacking a functional Nfib gene. Pontine neurons, along with other precerebellar neurons that populate the hindbrain, arise from precursors in the lower rhombic lip and migrate anteroventrally to reach their final location. Using immunohistochemistry, we find that NFI-B expression is specific for mossy fiber populations of the precerebellar system. Analysis of the Nfib(-/-) hindbrain indicates that the development of the basilar pontine nuclei is delayed, with pontine neurons migrating 1-2 days later than in control animals, and that significantly fewer pontine neurons are produced. While the mossy fiber nuclei of the caudal medulla do form, they also exhibit a developmental delay. Nfia and Nfix null mice exhibit no apparent pontine phenotype, implying specificity in the action of NFI family members. Collectively, these data demonstrate that Nfib plays an important role in the generation of precerebellar mossy fiber neurons, and may do so at least in part by regulating neurogenesis.

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity.

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.

Isolation and culture of adult and fetal rabbit bladder smooth muscle cells and their interaction with biopolymers.

PURPOSE: The aim of this study was to test the feasibility of isolation and culture of adult and fetal rabbit bladder smooth muscle cells (SMCs) and comparison of their interactions with different types of biodegradable biopolymers in cell culture. METHODS: Bladder SMCs isolated from adult and fetus rabbits were identified by immunostaining for smooth muscle alpha-actin and myosin. Growth kinetics of SMCs were estimated using population doubling time (PDT) and thymidine labeling index (TLI). Poly (D, L-lactide-co-glycolide; PLGA) copolymers were synthesized at 85:15 and 75:25 monomer ratios. The porous scaffolds prepared from these polymers were seeded with SMCs. The study compared the effectiveness of adsorbing fibronectin and fetal calf serum (FCS) on these biopolymers. The cells grown on these polymers were quantified using a neutral red uptake assay. RESULTS: Over 90% of the 2 cell populations stained positive for SMC marker proteins. Fetal SMCs were seen to emerge from the tissue after 3 to 4 days, whereas adult SMCs were seen after 5 to 6 days. However, estimated PDT of fetal and adult SMCs was 85.2 and 54.5 hours, respectively, and TLI of adult SMCs was also higher than with fetal SMCs. Proliferation on 75:25 PLGA was better than for 85:15 and for both biopolymers; adsorption of FCS significantly affected cell attachment relative to fibronectin. CONCLUSIONS: Although fetal SMCs were shown to emerge from explants early after seeding onto dishes, doubling time and S-phase fraction of adult bladder SMCs were markedly higher than of fetal derived cells. Adsorption of serum proteins significantly enhances the attachment of both fetal and adult SMCs to biopolymers.