Identification of CASZ1 NES reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma.

As a transcription factor, localization to the nucleus and the recruitment of cofactors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc-finger transcription factor CASZ1 to function as a neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation, we define a critical region at the CASZ1 N terminus (AAs 23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identified a nuclear export signal (NES) at the N terminus (AAs 176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1-dependent manner. An analysis of CASZ1 protein expression in a primary NB tissue microarray shows that high nuclear CASZ1 staining is detected in tumor samples from NB patients with good prognosis. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumor samples from patients with poor prognosis. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as NB tumorigenesis.

CASZ1, a candidate tumor-suppressor gene, suppresses neuroblastoma tumor growth through reprogramming gene expression.

Neuroblastoma (NB) is a common childhood malignant tumor of the neural crest-derived sympathetic nervous system. In NB the frequent loss of heterozygosity (LOH) on chromosome 1p raises the possibility that this region contains tumor-suppressor genes whose inactivation contributes to tumorigenesis. The human homolog of the Drosophila neural fate determination gene CASZ1, a zinc-finger transcription factor, maps to chromosome 1p36.22, a region implicated in NB tumorigenesis. Quantitative real-time PCR analysis showed that low-CASZ1 expression is significantly correlated with increased age (≥18 months), Children's Oncology Group high-risk classification, 1p LOH and MYCN amplification (all P<0.0002) and decreased survival probability (P=0.0009). CASZ1 was more highly expressed in NB with a differentiated histopathology (P<0.0001). Retinoids and epigenetic modification agents associated with regulation of differentiation induced CASZ1 expression. Expression profiling analysis revealed that CASZ1 regulates the expression of genes involved in regulation of cell growth and developmental processes. Specific restoration of CASZ1 in NB cells induced cell differentiation, enhanced cell adhesion, inhibited migration and suppressed tumorigenicity. These data are consistent with CASZ1 being a critical modulator of neural cell development, and that somatically acquired disruption of normal CASZ1 expression contributes to the malignant phenotype of human NB.

The MYCN oncogene is a direct target of miR-34a.

Loss of 1p36 heterozygosity commonly occurs with MYCN amplification in neuroblastoma tumors, and both are associated with an aggressive phenotype. Database searches identified five microRNAs that map to the commonly deleted region of 1p36 and we hypothesized that the loss of one or more of these microRNAs contributes to the malignant phenotype of MYCN-amplified tumors. By bioinformatic analysis, we identified that three out of the five microRNAs target MYCN and of these miR-34a caused the most significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines with MYCN amplification. Quantitative RT-PCR showed that neuroblastoma tumors with 1p36 loss expressed lower level of miR-34a than those with normal copies of 1p36. Furthermore, we demonstrated that MYCN is a direct target of miR-34a. Finally, using a series of mRNA expression profiling experiments, we identified other potential direct targets of miR-34a, and pathway analysis demonstrated that miR-34a suppresses cell-cycle genes and induces several neural-related genes. This study demonstrates one important regulatory role of miR-34a in cell growth and MYCN suppression in neuroblastoma.

Downregulation of Bim by brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from paclitaxel but not etoposide or cisplatin-induced cell death.

Chemoresistance and increased expression of TrkB and brain-derived neurotrophic factor (BDNF) are biomarkers of poor prognosis in tumors from patients with neuroblastoma (NB). Previously, we found BDNF activation of TrkB through PI3K/Akt protects NB from etoposide/cisplatin-induced cell death. In this study, the role of Bim, a proapoptotic protein, was investigated. Bim was involved in paclitaxel but not etoposide or cisplatin-induced cell death in NB cells. Pharmacological and genetic studies showed that BDNF-induced decreases in Bim were regulated by MAPK and not PI3K/Akt pathway. Both MAPK and PI3K pathways were involved in BDNF protection of NB cells from paclitaxel-induced cell death, while PI3K predominantly mediated BDNF protection of NB cells from etoposide or cisplatin-induced cell death. These data indicate that different chemotherapeutic drugs induce distinct death pathways and growth factors utilize different signal transduction pathways to modulate the effects of chemotherapy on cells.

Antineoplastic effects of rosiglitazone and PPARgamma transactivation in neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. In the present study, we evaluated the role of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone (RGZ) in two NB cell lines (SK-N-AS and SH-SY5Y), which express PPARgamma. Rosiglitazone decreased cell proliferation and viability to a greater extent in SK-N-AS than in SH-SY5Y. Furthermore, 20 microM RGZ significantly inhibited cell adhesion, invasiveness and apoptosis in SK-N-AS, but not in SH-SY5Y. Because of the different response of SK-N-AS and SH-SY5Y cells to RGZ, the function of PPARgamma as a transcriptional activator was assessed. Noticeably, transient transcription experiments with a PPARgamma responsive element showed that RGZ induced a three-fold increase of the reporter activity in SK-N-AS, whereas no effect was observed in SH-SY5Y. The different PPARgamma activity may be likely due to the markedly lower amount of phopshorylated (i.e. inactive) protein observed in SK-N-AS. To our knowledge, this is the first demonstration that the differential response of NB cells to RGZ may be related to differences in PPARgamma transactivation. This finding indicates that PPARgamma activity may be useful to select those patients, for whom PPARgamma agonists may have a beneficial therapeutic effect.

Ethanol inhibits brain-derived neurotrophic factor-mediated intracellular signaling and activator protein-1 activation in cerebellar granule neurons.

Developmental exposure to ethanol causes profound damage to the cerebellum, ranging from aberration in neuronal differentiation to cell loss. As a major neurotrophic factor, brain-derived neurotrophic factor (BDNF) and its receptor TrkB are expressed in the developing, as well as adult, cerebellum. Many neurotrophic effects of BDNF are mediated by gene transcription. We hypothesized that ethanol interfered with BDNF signaling and disrupted BDNF-regulated transcriptional activity. Using a transgenic mouse model expressing an activator protein-1 (AP-1) luciferase reporter construct, we demonstrated that BDNF stimulated AP-1 transactivation in cultured cerebellar granule neurons. This observation was validated by the study using a human neuronal cell line expressing inducible TrkB (TB8 neuroblastoma cells). BDNF induced AP-1 transactivation, as well as increased the binding activity of AP-1 protein complex to a DNA sequence containing AP-1 sites in TB8 cells. BDNF-mediated AP-1 activation was mediated by PI3K/Akt and JNK pathways; BDNF activated Akt and JNKs, and blocking these pathways significantly inhibited BDNF-stimulated AP-1 transactivation. More importantly, ethanol inhibited BDNF-mediated activation of PI3K/Akt and JNKs, and blocked BDNF-stimulated AP-1 activation. Since ethanol did not affect either the expression or autophosphorylation of TrkB, it could be concluded that the site of ethanol action was downstream of TrkB. The present study establishes that this AP-1 reporter transgenic mouse model is valuable for assessing AP-1 activity in the CNS neurons. Our results provide an insight into molecular mechanism(s) of ethanol action.

Retinoic acid decreases targeting of p27 for degradation via an N-myc-dependent decrease in p27 phosphorylation and an N-myc-independent decrease in Skp2.

Poor prognosis neuroblastoma (NB) tumors are marked by amplification and overexpression of N-myc. Retinoic acid (RA) decreases N-myc levels and induces cell cycle arrest in vitro and increases event-free survival in advanced stage NB patients. In this study, we investigated the mechanism(s) by which RA regulates cell cycle and how N-myc affects NB cell cycle progression. Constitutive N-myc overexpression stimulates increases in cyclin E-dependent kinase activity and decreases in p27 resulting in increased DNA synthesis. N-myc regulates p27 levels through an increase in targeting of p27 to the proteasome via cyclin E kinase-dependent phosphorylation of p27 and its ubiquitination. N-myc also stimulates an increase in proteasome activity. In RA-treated cells in which N-myc levels decline as p27 levels increase, degradation of p27 is also decreased. However, RA does not affect the activity of proteasome. The decrease in the degradation of p27 in RA-treated cells is due in part to a decrease in the N-myc stimulated phosphorylation of p27. However, RA also decreases Skp2 levels thus impairing the ability of p27 to be ubiquitinated. Thus, RA induces both N-myc-dependent and -independent mechanisms to minimize the degradation of p27 and arrest NB cell growth.

Increased expression of p27Kip1 arrests neuroblastoma cell growth.

BACKGROUND AND PROCEDURE: To investigate the molecular mechanisms by which retinoic acid (RA) alters cell growth, the expression and activity of components of the cell cycle machinery were analyzed. RESULTS AND CONCLUSIONS: Within 2 days of RA treatment, and prior to the arrest of NB cells in the G1 phase of the cell cycle, there was a complete downregulation of GI cyclin/cdk activities. Protein levels for the G1 cyclin/cdk were essentially unchanged during this time, although there was a decrease in the steady state levels of hyperphosphorylated Rb and p60N-MYC proteins. The cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR, while p15 INK4B and p16 INK4A mRNA were undetected. Within 24 hr of RA treatment, there was a 4-fold increase in the expression of p27Kip1, although p27 mRNA levels were unchanged. Levels of p21Cip1 were unaltered. Coincident with the decrease in kinase activity there was an increase in p27 bound to G1 cyclin/cdk. The increase in p27 was not due to an increase in transcription. In other cell systems, increased expression of c-MYC has been shown to lead to a decrease in p27 levels that is regulated at the post-transcriptional level (sequestration). To determine whether increased levels of N-MYC could affect the level of p27, we evaluated the expression of p27 in a series of N-MYC transfected cells and found that constitutive overexpression of N-MYC led to a decrease in the steady-state levels of p27 and in p27 bound to G1 cyclin/cdk complexes. Using adenoviral vectors expressing p27, we found that infection leads to increased p27 expression, which causes a decrease in cdk activity and an accumulation of cells in G1.

Structure and promoter analysis of the human unc-33-like phosphoprotein gene. E-box required for maximal expression in neuroblastoma and myoblasts.

The human unc-33-like phosphoprotein (hUlip/CRMP-4) is a member of a family of developmentally regulated genes that are highly expressed in the nervous system. Mutations in the C. elegans unc-33 gene lead to worms with abnormal movements. The hUlip gene encodes a 570-amino acid protein with 98% homology to its murine (Ulip) (Byk, T., Dobransky, T., Cifuentes-Diaz, C., and Sobel, A. (1996) J. Neurosci. 16, 688-701) and rat (CRMP-4) (Wang, L. H., and Strittmatter, S. M. (1996) J. Neurosci. 16, 6197-6207) counterparts (Gaetano, C., Matsuo, T., and Thiele, C. J. (1997) J. Biol. Chem. 272, 12195-12201). The hUlip gene was isolated from a human genomic library. It contains 15 exons, including an exon defined by an anaplastic oligodendroglioma expressed sequence tag, and spans at least 61.7 kilobases. hUlip lacks sequences corresponding to the first six exons found in unc-33. unc-33 exons correspond to homologous hUlip exons as follows: VII to 1 and 2, VIII to 3-9, IX to 10-12, and X to 13 and 14. Using the hUlip clone 1 phage, fluorescence in situ hybridization analysis indicates that the hybridization signal localizes to human chromosome 5q32. Deletion analysis of 5'-flanking sequences delineated the sequences sufficient to express a reporter gene in both neuroblastoma cells and myoblasts. A consensus MyoD/myogenin binding site is located in a region of the downstream promoter that is nearly identical to its mouse homologue. Mutagenesis shows that this conserved MyoD/myogenin site is necessary for full promoter activity in both myoblasts and neuroblastoma cells.

Up-regulation of insulin-like growth factor-II expression is a feature of TrkA but not TrkB activation in SH-SY5Y neuroblastoma cells.

The types of neurotrophin receptors that are expressed in neuroblastomas have different prognostic implications; trkA is a marker of good prognosis, whereas trkB expression is associated with poor prognosis. This suggests that either the signaling that is mediated via these receptors modulates the biological features of neuroblastoma cells differently, or that distinct lineages of sympathoadrenal precursors have been transformed. In this report, we evaluate the biological effects after activation of trkA or trkB by their major ligands in SH-SY5Y human neuroblastoma cells. Both trkA and trkB induce differentiation, inhibit growth, and promote the survival of cells under conditions of nutrient deprivation. However, the up-regulation of insulin-like growth factor-II (IGF-II) expression is a predominant feature of trkA activation by nerve growth factor (NGF). The growth inhibition induced by blocking the insulin-like growth factor-I receptor suggests that IGF-II is a component of the effector mechanism of trkA activation by NGF in trkA-transfected cells. Although trkA and trkB expression is associated with different prognoses in neuroblastoma, our study indicates that the effects mediated by these receptors in vivo may be quite similar for certain subsets of neuroblastomas.

Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein.

Chromosomal translocations resulting in the expression of chimaeric transcription factors are frequently observed in tumour cells, and have been suggested to be a common mechanism in human carcinogenesis. Ewing sarcoma and related peripheral primitive neuroectodermal tumours share recurrent translocations that fuse the gene EWSR1 (formerly EWS) from 22q-12 to FLI1 and genes encoding other ETS transcription factors (which bind DNA through the conserved ETS domain). It has been shown that transduction of the gene EWSR1-FLI1 (encoding EWS-FLI1 protein) can transform NIH3T3 cells, and that mutants containing a deletion in either the EWS domain or the DNA-binding domain in FLI1 lose this ability. This indicates that the EWS-FLI1 fusion protein may act as an aberrant transcription factor, but the exact mechanism of oncogenesis remains unknown. Because ETS transcription factors regulate expression of TGFBR2 (encoding the TGF-beta type II receptor, TGF-beta RII; Refs 9,14), a putative tumour suppressor gene, we hypothesized that TGFBR2 may be a target of the EWS-FLI1 fusion protein. We show here that Ewing sarcoma [corrected] (ES) cell lines with the EWSR1-FLI1 fusion have reduced TGF-beta sensitivity, and that fusion-positive ES cells and primary tumours both express low or undetectable levels of TGFBR2 mRNA and protein product. Co-transfection of FLI1 and the TGFBR2 promoter induces promoter activity, whereas EWSR1-FLI1 leads to suppression of TGFBR2 promoter activity and FLI1-induced promoter activity. Introduction of EWSR1-FLI1 into cells lacking the EWSR1-FLI1 fusion suppresses TGF-beta RII expression, whereas antisense to EWSR1-FLI1 in ES cell lines positive for this gene fusion restores TGF-beta RII expression. Furthermore, introduction of normal TGF-beta RII into ES cell lines restores TGF-beta sensitivity and blocks tumorigenicity. Our results implicate TGF-beta RII as a direct target of EWS-FLI1.

Insulin-like growth factor-II expression is down-regulated in TrkA-transfected SK-N-AS neuroblastoma cells.

Expression level of trkA tyrosine kinase receptor for nerve growth factor is a major prognostic determinant of neuroblastoma, suggesting that defective trkA-mediated signaling is responsible for the tumorigenesis of this childhood malignancy. We investigated the biologic effect of trkA, with special reference to its effect on insulin-like growth factor-II (IGF-II) expression, in SK-N-AS human neuroblastoma cells transfected with human trkA cDNA. Nerve growth factor treatment of trkA-transfected cells promoted growth and changed the morphologic phenotype into a substrate-adherent, flatter phenotype (S-type), and down-regulated the mRNA expression of IGF-II. The effects on both growth and the morphologic differentiation of SK-N-AS cells differed significantly from those of previous studies, and implied that trkA effects can be diverse, depending on the phenotype of the individual neuroblastoma cells. Immunohistochemical screening of trkA and IGF-II expression in adrenal neuroblastomas (n = 25) also favored the nonoverlapping pattern of trkA and IGF-II expression (p < 0.05). Because IGF-II is believed to play a significant role in the tumorigenesis of neuroblastoma, the inverse relationship between trkA and IGF-II strongly suggests that a low level of trkA can be a feature of the pathogenetic mechanism of IGF-II expressing adrenal neuroblastomas.

Inflammatory cytokines and HIV-1-associated neurodegeneration: oncostatin-M produced by mononuclear cells from HIV-1-infected individuals induces apoptosis of primary neurons.

Neurologic abnormalities are common in HIV-1-infected patients and often represent the dominant clinical manifestation of pediatric AIDS. The neurological dysfunction has been directly related to CNS invasion by HIV-1 that is principally, if not exclusively, supported by blood-derived monocytes/macrophages and lymphocytes. By using primary long term cultures of human fetal sensory neurons as well as sympathetic precursors-like neuronal cells, we determined that blood-derived mononuclear cells from HIV-1-infected individuals spontaneously release soluble mediators that can potently inhibit the growth and survival of developing neurons as well as the viability of postmitotic neuronal cells by inducing apoptotic cell death. Analysis of the cytokines produced by lymphomonocytic cells, HIV-1 infected or activated, indicated that oncostatin M (oncM) is a major mediator of these effects. Since low TGF-beta1 concentrations were capable of enhancing oncM-mediated neuronal alterations, our data indicate that by acting in concert with other cytokines, oncM may induce neuronal demise in both the developing and the mature brain. Thus, this cytokine may contribute to the setting of the neuronal cell damage observed in HIV-1-infected individuals.

Basic fibroblast growth factor supports human olfactory neurogenesis by autocrine/paracrine mechanisms.

Throughout life, olfactory sensory neurons are renewed from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of olfactory neuronal precursors; however, evidence indicates that soluble mediators may play a central role in olfactory neurogenesis. To identify molecules that regulate olfactory self-renewal and differentiation, we have recently established, cloned and propagated in vitro primary long-term cell cultures from the human fetal olfactory neuroepithelium. Here we show that primary human olfactory neuroblasts synthesize and release biologically active basic fibroblast growth factor which, in turn, supports neuroblast growth by autocrine/paracrine mechanisms. The growth-promoting activity of basic fibroblast growth factor is dose dependent and is accompanied by morphological changes of the cells and by an increase in the expression of neuronal-related genes. These observations indicate that endogenous basic fibroblast growth factor participates in controlling olfactory self-renewal and suggest that this cytokine represents a key regulatory element of olfactory neurogenesis.

p27Kip1: a key mediator of retinoic acid induced growth arrest in the SMS-KCNR human neuroblastoma cell line.

Retinoic acid (RA) treatment of SMS-KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal differentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR while p15INK4B and p16INK4A were not detected. RA induced an increase in the expression of p27Kip1 but not p21Cip1. Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27Kip1. These results indicate that changes in the level of p27Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.

Activation of three distinct RXR/RAR heterodimers induces growth arrest and differentiation of neuroblastoma cells.

Naturally occurring retinoids, like all-trans retinoic acid and 9-cis retinoic acid, are known to affect proliferation and differentiation of sensitive neuroblastoma cell lines. Cellular responsiveness to retinoic acid depends on its interaction with two distinct classes of receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). Both receptor classes have three different subtypes (RARalpha, RARbeta, and RARgamma and RXRalpha, RARbeta, and RARgamma) that act as ligand-dependent transcription factors. To examine the involvement of the different receptor classes and subtypes in the biological responses of neuroblastoma cells to retinoids, we analyzed the effects of a panel of receptor-selective retinoids on cell growth, differentiation, and gene expression on in vitro cultured KCNR cells. Any association of per se inactive RXR-selective with RAR-selective ligands efficiently regulates growth inhibition, differentiation (neurite extension), and expression of RARbeta, TrkB, and N-myc. SR11383 alone, a very potent retinoid, entirely reproduces the pattern of biological responses induced by naturally occurring retinoids. In contrast to other tumor cell lines, the growth of neuroblastoma cell lines is not altered using AP1-antagonistic retinoids. These studies raise the possibility that three distinct RXR/RAR heterodimers mediate the effects of retinoids on neuroblastoma cells through an AP-1 antagonism-independent mechanism.

HIV-1 infection and the developing nervous system: lineage-specific regulation of viral gene expression and replication in distinct neuronal precursors.

Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. Microglia and macrophages are major HIV-1 targets in the brain, whereas HIV-1 infected neurons or glial cells have been rarely reported. This suggests that indirect mechanisms may account for the severe neuronal damage observed in these patients. Nevertheless, immature, mitotically active neuronal and glial cells, which are present during fetal development, are susceptible to HIV-1 infection and replication in vitro, suggesting that HIV-1 infection during organ development may present unique features. To better characterize virus-host cells interactions in the developing CNS, we have examined the susceptibility of embryologically and biochemically distinct neuronal cell lines to HIV-1 infection. Here we show that mitotically active, immature neurons of distinct lineages, have different susceptibilities to HIV-1 infection and replication and different abilities to support viral gene expression. Mutational analysis of HIV-1 LTR reveals that a region of the viral promoter between nucleotide -255 to -166 is responsible for most quantitative and qualitative differences in viral transactivation among different neuroblasts. This suggests that specific regions of the viral promoter and cellular factors, either lineage- or differentiation-dependent, which bind to those regions, may contribute to control the levels of virus replication and possibly restrict the viral tropism in the developing brain. This may contribute to the establishment of a virus reservoir in the immature CNS and participate by either direct or indirect mechanisms to the severity of the AIDS-related pediatric neurological dysfunction.

Identification and characterization of a retinoic acid-regulated human homologue of the unc-33-like phosphoprotein gene (hUlip) from neuroblastoma cells.

A cDNA, 7G1, was isolated from retinoic acid (RA) differentiated neuroblastoma cells whose expression was high in human fetal brain and spinal cord mRNA but undetectable in adult brain or non-neuronal tissues. Sequence analysis indicates that 7G1 is homologous to the Caenorhabditis elegans gene unc-33. A 5.5-kilobase pair full-length cDNA from a human fetal brain cDNA library contains an 1710-base pair open reading frame. Because the predicted 570 amino acid sequence of 7G1 shares 98% identity with the murine Ulip gene product, an unc-33-like-phosphoprotein, we refer to 7G1 as the human Ulip (hUlip). hUlip is also similar to the bacterial enzyme D-hydantoinase and the recently described vertebrate gene products CRMP62, TOAD-64, CRMP1, CRMP2, and mUNC. RA stimulates an increase in hUlip mRNA that is transcriptionally regulated. RA stimulates an increase in polypeptides of 58, 60, 65, and 70 kDa with the 58- and 65-kDa species being dephosphorylated forms of the 60- and 70-kDa species. This study presents a model in which to study the regulation and expression of the hUlip gene, a member of an emerging family of molecules that potentially mediates signals involved in axonal outgrowth.