Loss of Nkx3.1 leads to the activation of discrete downstream target genes during prostate tumorigenesis.

The expression of NKX3.1, a transcriptional regulator and tumor suppressor gene in prostate cancer, is downregulated during early stages of prostate tumorigenesis. However, little is known of the alterations in gene expression that occur as a result of this event. We combined laser capture microdissection and gene expression profiling to analyse the molecular consequences of Nkx3.1 loss during prostate cancer initiation using Nkx3.1-deficient mice. This analysis identified a cohort of genes (loss-of-Nkx3.1 signature) that are aberrantly overexpressed during loss-of-Nkx3.1-driven tumor initiation. We studied the expression of these genes in independent loss-of-Pten and c-myc overexpression prostate adenocarcinoma mouse models. Nkx3.1 expression is lost in prostate epithelial proliferation in both of these mouse models. However, Nkx3.1 loss is an early event of tumor development in the loss-of-Pten model, whereas it occurs at later stages in c-myc transgenic mice. A number of genes of the loss-of-Nkx3.1 signature, such as clusterin and quiescin Q6, are highly expressed in prostatic hyperplasia and intraepithelial neoplasia (PIN) lesions that also lack Nkx3.1 in the Pten-deficient prostate, but not in similar lesions in the c-myc transgenic model. Meta-analysis of multiple prostate cancer gene expression data sets, including those from loss-of-Nkx3.1, loss-of-Pten, c-myc overexpression and constitutively active Akt prostate cancer models, further confirmed that genes associated with the loss-of-Nkx3.1 signature integrate with PTEN-AKT signaling pathways, but do not overlap with molecular changes associated with the c-myc signaling pathway. In human prostate tissue samples, loss of NKX3.1 expression and corresponding clusterin overexpression are co-localized at sites of prostatic inflammatory atrophy, a possible very early stage of human prostate tumorigenesis. Collectively, these results suggest that the molecular consequences of NKX3.1 loss depend on the epithelial proliferative stage at which its expression is lost, and that alterations in the PTEN-AKT-NKX3.1 axis are important for prostate cancer initiation.

Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons.

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.

Dynamics of neural crest-derived cell migration in the embryonic mouse gut.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Neurturin signalling via GFRalpha2 is essential for innervation of glandular but not muscle targets of sacral parasympathetic ganglion neurons.

Neurturin, a member of the glial cell-derived neurotrophic factor familys of ligands, is important for development of many cranial parasympathetic ganglion neurons. We have investigated the sacral component of the parasympathetic nervous system in mice with gene deletions for neurturin or its preferred receptor, GFRalpha2. Disruption of neurturin signalling decreased cholinergic VIP innervation to the mucosa of the reproductive organs, but not to the smooth muscle layers of these organs or to the urinary bladder. Thus, neurturin and its receptor are involved in parasympathetic innervation of a select group of pelvic visceral tissues. In contrast, noradrenergic innervation was not affected by the gene ablations. The epithelium of reproductive organs from knockout animals was atrophied, indicating that cholinergic innervation may be important for the maintenance of normal structure. Cholinergic neurons express GFRalpha2 on their terminals and somata, indicating they can respond to neurotrophic support, and their somata are smaller when neurturin signalling is disrupted. Colocalisation studies showed that many peripheral glia express GFRalpha2 although its role in these cells is yet to be determined. Our results indicate that neurturin, acting through GFRalpha2, is essential for parasympathetic innervation of the mucosae of reproductive organs, as well as for maintenance of a broader group of sacral parasympathetic neurons.

GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development.

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.

RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons.

Sympathetic axons use blood vessels as an intermediate path to reach their final target tissues. The initial contact between differentiating sympathetic neurons and blood vessels occurs following the primary sympathetic chain formation, where precursors of sympathetic neurons migrate and project axons along or toward blood vessels. We demonstrate that, in Ret-deficient mice, neuronal precursors throughout the entire sympathetic nervous system fail to migrate and project axons properly. These primary deficits lead to mis-routing of sympathetic nerve trunks and accelerated cell death of sympathetic neurons later in development. Artemin is expressed in blood vessels during periods of early sympathetic differentiation, and can promote and attract axonal growth of the sympathetic ganglion in vitro. This analysis identifies RET and artemin as central regulators of early sympathetic innervation.

Frequent and early loss of the EGR1 corepressor NAB2 in human prostate carcinoma.

The transcription factor EGR1 is frequently overexpressed in human prostate cancer and regulates the expression of several genes important for tumor progression. In addition, mice lacking the Egr1 gene show a defect in prostate tumorigenesis. NAB2 is a novel corepressor molecule that modulates EGR1 activity and is induced by the same stimuli that induce EGR1. The human NAB2 gene has been localized to 12q13.3-14.1, within a chromosomal region that is thought to harbor a prostate tumor suppressor. We have examined the expression of NAB2 in human prostate carcinoma specimens. We show here that NAB2 protein expression is lost in a majority of primary prostate carcinoma specimens, including many samples that have high EGR1 levels. This loss occurs early in the tumorigenic process and is sustained, as it is seen in precursor prostatic intraepithelial neoplasia lesions as well as in metastases. Furthermore, loss of NAB2 did not correlate with the tumor grade or stage. Our findings suggest that high levels of EGR1 coupled with low levels of NAB2 can result in high, unrestrained EGR1 transcriptional activity in human prostate cancers.

Expression profiling reveals hepsin overexpression in prostate cancer.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in men. Despite this fact, many of the genetic changes that coincide with prostate cancer progression remain enigmatic. We have addressed this problem by characterizing the expression profiles of several benign and malignant human prostate samples, and we have identified several genes that are differentially expressed between benign and malignant glands. One gene that was overexpressed encodes the serine protease hepsin. We used an independent sample set to confirm that hepsin is overexpressed in prostate tumors, and in situ hybridization demonstrates that hepsin is specifically overexpressed in the carcinoma cells themselves. These facts, together with the molecular properties of hepsin, make it an ideal target for prostate cancer therapy.

Identification of genes induced in peripheral nerve after injury. Expression profiling and novel gene discovery.

Peripheral nerve injury results in axonal degeneration and in phenotypic changes of the surrounding Schwann cells, whose presence is critical for nerve regeneration. To identify genes induced after nerve injury in Schwann cells, we developed a strategy that included differential screening of a subtractive library enriched for cDNAs expressed in injured nerve, sequence analysis, and expression profiling. By using real time quantitative reverse transcriptase-polymerase chain reaction, we found that injury-induced genes could be categorized into four temporal expression patterns. Among the clones we identified were a number that were homologous only to expressed sequence tags in the data base. These were stratified based on their expression profile, presence of identifiable sequence motifs, homologies to other proteins, and evolutionary conservation. We chose one representative gene, nin283, to analyze in detail. The nin283 gene encodes a 227-residue protein containing both a zinc finger and a RING finger motif. nin283 is highly expressed in the central nervous system, particularly in the developing cortical plate in embryos. It is also expressed in peripheral ganglia and is induced by nerve growth factor in PC12 cells. Subcellular localization analysis demonstrated that Nin283 is located in the endosome/lysosome compartment, suggesting that it may participate in ubiquitin-mediated protein modification.

The transcriptional corepressor NAB2 blocks Egr-1-mediated growth factor activation and angiogenesis.

Effective tissue repair results from a rapid, temporally orchestrated series of events. At the site of local tissue injury, the production of many growth factors and cytokines is, in part, stimulated by the early growth response transcription factors such as Egr-1. Egr-1 protein binds to a family of corepressor proteins called NAB which function to block or limit Egr-1 trans-activation of cognate target genes. NAB2 blocks Egr-1 activation of the tissue factor (TF) promoter, Egr-1 stimulated production of PDGF-AB, HGF, TGFbeta(1), and VEGF and the endogenous expression of PDGF-AB and TGFbeta(1). Expression of a wild-type NAB2 but not a dominant negative NAB2 mutant abrogates Egr-1 driven TF promoter activity and tubule formation in an in vitro model of angiogenesis. These findings may have importance in any tissue that is subject to scarring after acute or chronic injury.

The transcription factor early growth-response factor 1 modulates tumor necrosis factor-alpha, immunoglobulin E, and airway responsiveness in mice.

Early growth-response factor 1 (Egr-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important in inflammation, cell growth, apoptosis, and the pathogenesis of disease. In vitro studies suggest that Egr-1 is capable of regulating the expression of tumor necrosis factor-alpha (TNF-alpha) and other genes involved in airway inflammation and reactivity following allergen stimulation. On the basis of these data, we hypothesized that in the absence of Egr-1, the TNF-alpha response and subsequent downstream inflammatory events that usually follow allergen challenge would be diminished. To test our hypothesis Egr-1 knock-out (KO) mice were examined in an ovalbumin (OVA)-induced model of airway inflammation and reactivity, and compared with identically treated wild-type (WT) control mice. In response to OVA sensitization and airway challenge, KO mice had diminished TNF-alpha mRNA and protein in the lungs and mast cells compared with WT mice. Interestingly, the KO mice had elevated IgE levels at baseline and after allergen challenge compared with WT mice. Furthermore, the airways of KO mice were hyporesponsive to methacholine challenge at baseline and after allergen challenge. These data indicate that Egr-1 modulates TNF-alpha, IgE, and airway responsiveness in mice.

Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early growth response gene and NF-AT family members.

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the resulting apoptosis of CD95-bearing cells is a critical component of peripheral T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of the expression of Fas ligand has been attributed to a number of factors, including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a direct contribution of NF-AT is controversial. The present study confirms a role for Egr factors and indicates that NF-AT is essential for optimal expression of murine Fas ligand through a direct interaction with an NF-AT consensus element. The role of these factors was further defined by studying the differential expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2, but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated the lack of Egr3 in Th1 cells. Differential usage of these transcription factors by Th1 and Th2 cells suggests a potential mechanism underlying the differential expression of Fas ligand by distinct T cell lineages.

Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells.

PURPOSE: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice. METHODS: The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography. RESULTS: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed. CONCLUSIONS: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.

c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.

Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis.

In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1(-/-)) and heterozygous (Egr-1(+/-)) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1(+/-) cells (22.8%; p < 0.0001) when compared with Egr-1(-/-) cells (3.5%). Radiation elevated p53 protein in Egr-1(+/-) cells in 3-6 h. However, in Egr-1(-/-) cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1(+/-) cells but not in Egr-1(-/-) cells. Interestingly, transient overexpression of EGR-1 in p53(-/-) MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53(+/+) MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed in Egr-1(-/-) MEF cells before and after radiation when compared with the levels observed in Egr-1(+/-) cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1(-/-) cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1(-/-) MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53.

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin.

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure. Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR(alpha)1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR(&agr;) receptors, as evidenced by the predominant expression of GFR(&agr;)2 in these neurons after ganglion formation.

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha2 (GFRalpha2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFRalpha2 mRNA and virtually all GFRalpha2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFRalpha2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.

EGR1 target genes in prostate carcinoma cells identified by microarray analysis.

The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-beta1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57(KIP2)). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators.