During mammalian hindbrain development, sensory axons grow along highly stereotyped routes within the cranial mesenchyme to reach their appropriate entry points into the neuroepithelium. Thus, trigeminal ganglion axons always project to rhombomere (r)2, whilst facial/acoustic ganglia axons always project to r4. Axons are never observed to enter the mesenchyme adjacent to r3, raising the possibility that r3 mesenchyme contains an axon growth-inhibitory activity. Conversely, in mice which lack the erbB4 receptor (normally expressed in r3), trigeminal and facial/acoustic ganglia axons misproject into r3 mesenchyme, suggesting that the putative axon barrier is absent. To investigate this hypothesis, we have developed an in vitro model in which dissociated wild-type embryonic trigeminal ganglion neurons are cultured on longitudinal cryosections of embryonic mouse head. We observed that on wild-type embryonic day 10 (E10) cryosections, neurites generally failed to grow into r3 mesenchyme from the adjacent r2 or r4 mesenchyme. This barrier was removed if cryosections were pretreated with chondroitinase or were washed with excess chondroitin 6-sulphate or hypertonic saline. By contrast, when trigeminal neurons were seeded onto cryosections of E10 erbB4 -/- embryo heads their neurites readily entered mutant r3 mesenchyme. Immunohistochemical analysis demonstrated chondroitin-sulphated proteoglycans throughout the cranial mesenchyme in both wild-type and erbB4 -/- embryos. We propose that trigeminal axons are excluded from wild-type r3 mesenchyme by a growth-inhibitory activity which associates with chondroitin-sulphated proteoglycans and that the synthesis of this activity may rely on signals transduced by erbB receptors.

The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use (125)I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.

BACKGROUND

Previous studies have revealed altered expression of epidermal growth factor receptor (EGFR)-family members and their endogenous inhibitor leucine-rich and immunoglobulin-like domains 1 (LRIG1) in renal cell carcinoma (RCC). In this study, we analyzed the gene expression levels of EGFR-family members and LRIG1, and their possible associations with clinical parameters in various types of RCC.

METHODS

Gene expression levels of EGFR-family members and LRIG1 were analyzed in 104 RCC samples, including 81 clear cell RCC (ccRCC), 15 papillary RCC (pRCC), and 7 chromophobe RCC (chRCC) by quantitative real-time RT-PCR. Associations between gene expression levels and clinical data, including tumor grade, stage, and patient survival were statistically assessed.

RESULTS

Compared to kidney cortex, EGFR was up-regulated in ccRCC and pRCC, LRIG1 and ERBB2 were down-regulated in ccRCC, and ERBB4 was strongly down-regulated in all RCC types. ERBB3 expression did not differ between RCC types or between RCC and the kidney cortex. The expression of the analyzed genes did not correlate with patient outcome.

CONCLUSIONS

This study revealed that the previously described up-regulation of EGFR and down-regulation of ERBB4 occurred in all analyzed RCC types, whereas down-regulation of ERBB2 and LRIG1 was only present in ccRCC. These observations illustrate the need to evaluate the different RCC types individually when analyzing molecules of interest and potential biological markers.

Transitional epithelium of the urinary bladder can be damaged during, for example, catheterization, overstretching due to obstructed voiding, or partial resection. The subsequent repair process can be stimulated by specific proteins such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha). However, little is known about the role of EGF-like growth factors and their respective receptors in human urothelial repair. In this study, we examined the effects of EGF, TGFalpha, amphiregulin and heregulin-alpha (HRGalpha) on proliferation, wound closure, and the expression of their receptors c-erbB1-c-erbB4 in primary cultures of human urothelial cells in vitro. Under conditions representing intact urothelium, all EGF-like growth factors except HRGalpha induced proliferation. TGFalpha induced proliferation up to four times. Amphiregulin increased expression of c-erbB1. Treatment with either TGFalpha or amphiregulin resulted in higher c-erbB1 activation and c-erbB3 levels. None of the growth factors affected the constitutive expression of c-erbB2 and c-erbB4. In the repair model, both EGF and TGFalpha stimulated the wound closure most strongly. This was mainly achieved by increased cellular migration. Receptor expression was not affected by the addition of exogenous growth factor. The role of c-erbB2 in wound healing was further investigated with the use of antisense DNA. Wound closure could be delayed up to 50% by antisense c-erbB2 but not by mismatched or sense oligonucleotides. Excessive production (e.g. in bladder tumors) or application of EGF, TGFalpha or amphiregulin, but not HRGalpha may lead to either hyperplasia or a faster repair of damaged urothelium in vivo. These effects seem to be mediated not only via c-erbB1 but also via c-erbB2. Our results suggest that modified members of the EGF-EGFR family are potential targets for future therapies for bladder wound healing and malignancy.

Although arachidonic acid has been demonstrated to stimulate a wide variety of cellular functions, the responsible mechanisms remain poorly defined. We now report that arachidonic acid stimulated the activity of class Ia phosphatidylinositol 3-kinase (PI3K) in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. Pretreatment of endothelial cells with AG-1478, an inhibitor of the ErbB receptor family, resulted in the suppression of PI3K activation by arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to stimulate PI3K activity in neutrophils was suppressed by indomethacin and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and lipoxygenases, respectively, but not by 17-octadecynoic acid, an inhibitor of omega-hydroxylation of arachidonic acid by cytochrome P450 monooxygenases. Consistent with this, the activity of PI3K in neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid. Arachidonic acid also transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473. Although PI3K was not required for the activation of the mitogen-activated protein kinases, ERK1, ERK2, and p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted via PI3K to stimulate the respiratory burst. These results not only define a novel mechanism through which some of the actions of arachidonic acid are mediated but also demonstrate that, in addition to ErbB1 (epidermal growth factor receptor), ErbB4 can also be transactivated by a non-epidermal growth factor-like ligand.

OBJECTIVE

ErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma.

METHODS

A tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar.

RESULTS

While ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro.

CONCLUSIONS

These results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer.

The EGF family hormone NRG2beta potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2alpha splicing isoform has lower affinity for ErbB4, does not potently stimulate ErbB4 phosphorylation, and fails to stimulate ErbB4 coupling. Here we investigate these differences. The NRG2beta Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. This failure to stimulate ErbB4 coupling is not due to differential ligand purity, glycosylation, or stability. The NRG2alpha K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. Thus, this failure to stimulate ErbB4 coupling is not due to inadequate affinity for ErbB4. In contrast, the NRG2alpha L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2alpha/K45F. Collectively, these data indicate that Gln43 of NRG2beta is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction.

The erbB4 gene encodes one of the four members of the mammalian ErbB family of transmembrane tyrosine kinases. The ErbB4 protein plays a role as a receptor for the neuregulins, a large group of structurally related molecules and a few other epidermal growth factor (EGF)-related polypeptides, such as heparin-binding EGF, betacellulin and epiregulin. The importance of this receptor tyrosine kinase in development has been demonstrated by the generation of mice with a targeted inactivation of the erbB4 gene. Such mice die by embryonic day eleven due to defective trabeculation in the heart, precluding analysis of phenotypes at later stages in development and in the adult. Now, using two unique genetic approaches our laboratories succeeded in overcoming this obstacle. In the first approach, the heart defects of ErbB4 null mutant mice were rescued by transgenic expression of an ErbB4 cDNA under a cardiac-specific myosin promoter. This allowed the generation of ErbB4 mutants that develop into adulthood and are fertile. In the second approach, the role of ErbB4 during mammary gland development was specifically addressed by Cre-mediated deletion of both erbB4 alleles within the mammary epithelium. Below we discuss the progress made studying these genetic models in understanding the physiological roles of ErbB4 with a focus on the mammary gland and the nervous system.

Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types, including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24-25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels was significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNI. TH protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson's disease.

The ErbB family of tyrosine kinase receptors mediates a variety of cellular responses to Neuregulin (NRG) 1; however, the intracellular signaling pathways downstream of ErbB4 and their functional outcomes remained to be elucidated. Here we show that NRG1 stimulated the proliferation of human HeLa cells expressing ErbB4, where the phosphorylation relay of extracellular signal-regulated kinase, a mitogen-activated protein kinase (MAPK), and serum response factor (SRF), a transcription factor, was induced, and the c-fos transcription was activated. By contrast, these all were attenuated in cells transfected with an ErbB4 mutant substituting the green fluorescence protein for the intracellular domain. We also demonstrated that a MAPK kinase inhibitor suppressed the NRG1-stimulated SRF phosphorylation, c-fos expression, and cell proliferation. Thus, the current study may unravel an ErbB4-mediated signaling pathway that is responsible for the NRG1-induced c-fos gene expression through the MAPK cascade-dependent SRF phosphorylation and thereby cell proliferation.

BACKGROUND

Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture.

RESULTS

In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3epsilon and zeta in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins.

CONCLUSIONS

Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances.

The neuregulin/ErbB system is a growth factor/receptor cascade that has been proven to be essential in the development of the heart and the sympathetic nervous system. However, the basis of the specificity of ligand-receptor recognition remains to be elucidated. In this study, the structures of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes were modeled based on the available structures of the homologous proteins. The binding free energies of NRG-1beta to ErbB3 and ErbB4 were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) computational method. In addition, computational alanine-scanning mutagenesis was performed in the binding site of NRG-1beta and the difference in the binding free energies between NRG-1beta mutants and the receptors was calculated. The results specify the contribution of each residue at the interaction interfaces to the binding affinity of NRG-1beta with ErbB3 and ErbB4, identifying several important interaction residue pairs that are in agreement with previously acquired experimental data. This indicates that the presented structural models of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes are reliable and could be used to guide future studies, such as performing desirable mutations on NRG-1beta to increase the binding affinity and selectivity to the receptor and discovering new therapeutic agents for the treatment of heart failure.

ErbB4 is a member of the ErbB family of receptor tyrosine kinases. Because of a paucity of appropriate pharmacologic tools, little is known about ErbB4 functions in vivo. In response to this need, we hypothesized that a recombinant form of the extracellular domain of ErbB4 would antagonize ligand-induced receptor tyrosine phosphorylation and subsequent downstream signaling and could be used to probe ErbB4 function. Indeed, we show here that one such ErbB4 protein, secErbB4-26/549, is a potent inhibitor of ligand-induced ErbB4 tyrosine phosphorylation and of ligand-induced ErbB4 coupling to biological responses. Furthermore, we demonstrate that secErbB4-26/549 antagonizes ligand-induced ErbB4 signaling by acting as a ligand sink. Thus, secErbB4-26/549 is suitable for elucidating the effects of ErbB4 ligand-induced ErbB signaling in a variety of biological contexts.

Although schizophrenia is generally considered to occur as a consequence of multiple genes that interact with one another, very few methods have been developed to model epistasis. Phenotype definition has also been a major challenge for research on the genetics of schizophrenia. In this report, we use novel statistical techniques to address the high dimensionality of genomic data, and we apply a refinement in phenotype definition by basing it on the occurrence of brain changes during the early course of the illness, as measured by repeated magnetic resonance scans (i.e., an 'intermediate phenotype.') The method combines a machine-learning algorithm, the ensemble method using stochastic gradient boosting, with traditional general linear model statistics. We began with 14 genes that are relevant to schizophrenia, based on association studies or their role in neurodevelopment, and then used statistical techniques to reduce them to five genes and 17 single nucleotide polymorphisms (SNPs) that had a significant statistical interaction: five for PDE4B, four for RELN, four for ERBB4, three for DISC1 and one for NRG1. Five of the SNPs involved in these interactions replicate previous research in that, these five SNPs have previously been identified as schizophrenia vulnerability markers or implicate cognitive processes relevant to schizophrenia. This ability to replicate previous work suggests that our method has potential for detecting a meaningful epistatic relationship among the genes that influence brain abnormalities in schizophrenia.

The neuregulin-1 (NRG-1)/ErbB system has emerged as a cardioprotective system that becomes activated during myocardial stress, most convincingly shown in response to cardiotoxic chemotherapy. Direct evidence of increased ventricular ErbB receptor activity in heart failure unrelated to cardiotoxic drugs is, however, limited. We investigated changes in NRG-1 expression, ErbB receptor phosphorylation and downstream activation of intracellular ErbB targets during rapid pacing and progressive ventricular dysfunction in the dog. Heart failure was induced in dogs by 7 weeks of rapid pacing. Ventricular function was assessed by echocardiography. Messenger RNA expression was investigated in ventricular biopsies using quantitative PCR. Activation of NRG-1/ErbB signaling and of downstream targets was investigated using immunoprecipitation and/or Western blotting. Over the course of 7 weeks of pacing and ventricular dilatation, ventricular levels of NRG-1, but not of other ErbB4 ligands, and of ADAM19, a protease promoting NRG-1 release, progressively increased. In parallel, levels of activated ErbB2 and ErbB4, phosphorylated at tyrosine residues 877/1248 and 1284 respectively, became progressively higher. Similarly, levels of total and phosphorylated PI-3 kinase increased. Surprisingly, however, and in contrast with activation of downstream targets of ErbB receptors in normal hearts, Akt and ERK1/2, remained inactivated. This study shows that ventricular ErbB2 and ErbB4 receptors become activated during the development of pacing-induced heart failure, but that downstream signaling is, at least partly, abrogated. Abrogation of cardioprotective signaling after ErbB activation is an unanticipated phenomenon in the progression of heart failure with possibly major pathophysiological significance. The underlying mechanisms should be further elucidated.

OBJECTIVE

Heregulins (HRGs) regulate the proliferation and differentiation of various cell types. However, very little is known about their function in the gastrointestinal tract. The aim of this study was to investigate the role of HRGs on gastrointestinal cells.

METHODS

We examined the expression of erbB receptors and HRG-alpha in human gastric cancer cell lines, rat gastrointestinal epithelial cells, and human gastric fibroblasts by Western blot analysis or reverse-transcription polymerase chain reaction. Receptor phosphorylation and heterodimerization induced by HRG-alpha were detected by Western blot analysis. We also evaluated the in vitro effects of HRG-alpha on cell proliferation and restitution.

RESULTS

Cancer cell lines and rat epithelial cells expressed erbB2 and erbB3, but protein expression of erbB4 was not detected. HRG-alpha was detected only in gastric fibroblasts. HRG-alpha activated tyrosine phosphorylation of epidermal growth factor receptor (EGFR), erbB2, and erbB3 and induced not only erbB3/erbB2 but also erbB3/EGFR and erbB2/EGFR heterodimer formation in MKN-28 cancer cells. Simultaneous cultivation of MKN-28 cells with gastric fibroblasts resulted in tyrosine phosphorylation of erbB3 in MKN-28 cells. HRG-alpha also stimulated proliferation of MKN-28 cells and gastric epithelial cells.

CONCLUSIONS

The data suggest that HRG-alpha may affect epithelial cell proliferation through mesenchymal-epithelial interaction in the gastric mucosa.

Excessive mucus production has been linked to many of the pathologic features of respiratory diseases, including obstruction of the airways, decline in lung function, increased rates of mortality, and increased infections. The mucins, MUC5AC and MUC5B, contribute to the viscoelastic properties of mucus, and are found at elevated levels in the airways of individuals with chronic respiratory diseases. The T helper type 2 cell cytokine, IL-13, is known to regulate MUC5AC expression in goblet cells of the airways, although much less is known about the regulation of MUC5B expression. In a study to further understand the mediators of MUC5AC and MUC5B expression, neuregulin (NRG) 1beta1 was identified as novel regulator of goblet cell formation in primary cultures of human bronchial epithelial cells (HBECs). NRG1beta1 increased expression of MUCAC and MUC5B proteins in a time- and dose-dependent fashion in HBEC cultures. NRG1beta1-induced expression of MU5AC and MUC5B was shown to involve v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) and ErbB3 receptors, but not ErbB4 receptors. Treatment of HBECs with inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, and phosphatidylinositol 3-kinase indicated that these kinases were involved in NRG1beta1-induced MUC5AC and MUC5B expression. Additionally, NRG1beta1 was shown to induce the phosphorylation of the ErbB2 receptor, AKT, and extracellular signal-regulated kinase 1/2. NRG1beta1 protein was found increased in the airways of antigen-challenged mice, together with increases in MUC5AC and MUC5B message. Together, these data indicate that NRG1beta1 is a novel mediator of MUC5AC and MUC5B expression in HBECs, and may represent a novel therapeutic target for mucus hypersecretion in respiratory diseases.

Top-down attention is crucial for meaningful behaviors and impaired in various mental disorders. However, its underpinning regulatory mechanisms are poorly understood. We demonstrate that the hippocampal-prefrontal synchrony associates with levels of top-down attention. Both attention and synchrony are reduced in mutant mice of ErbB4, a receptor of neuregulin-1. We used chemical genetic and optogenetic approaches to inactivate ErbB4 kinase and ErbB4+ interneurons, respectively, both of which reduce gamma-aminobutyric acid (GABA) activity. Such inhibitions in the hippocampus impair both hippocampal-prefrontal synchrony and top-down attention, whereas those in the prefrontal cortex alter attention, but not synchrony. These observations identify a role of ErbB4-dependent GABA activity in the hippocampus in synchronizing the hippocampal-prefrontal pathway and demonstrate that acute, dynamic ErbB4 signaling is required to command top-down attention. Because both neuregulin-1 and ErbB4 are susceptibility genes of schizophrenia and major depression, our study contributes to a better understanding of these disorders. VIDEO ABSTRACT.

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.

We previously identified synaptic cell adhesion molecule 1 (SynCAM1) as a component of a genetic network involved in the hypothalamic control of female puberty. Although it is well established that SynCAM1 is a synaptic adhesion molecule, its contribution to hypothalamic function is unknown. Here we show that, in addition to the expected neuronal localization illustrated by its presence in GnRH neurons, SynCAM1 is expressed in hypothalamic astrocytes. Cell adhesion assays indicated that SynCAM is recognized by both GnRH neurons and astrocytes as an adhesive partner and promotes cell-cell adhesiveness via homophilic, extracellular domain-mediated interactions. Alternative splicing of the SynCAM1 primary mRNA transcript yields four mRNAs encoding membrane-spanning SynCAM1 isoforms. Variants 1 and 4 are predicted to be both N and O glycosylated. Hypothalamic astrocytes and GnRH-producing GT1-7 cells express mainly isoform 4 mRNA, and sequential N- and O-deglycosylation of proteins extracted from these cells yields progressively smaller SynCAM1 species, indicating that isoform 4 is the predominant SynCAM1 variant expressed in astrocytes and GT1-7 cells. Neither cell type expresses the products of two other SynCAM genes (SynCAM2 and SynCAM3), suggesting that SynCAM-mediated astrocyte-astrocyte and astrocyte-GnRH neuron adhesiveness is mostly mediated by SynCAM1 homophilic interactions. When erbB4 receptor function is disrupted in astrocytes, via transgenic expression of a dominant-negative erbB4 receptor form, SynCAM1-mediated adhesiveness is severely compromised. Conversely, SynCAM1 adhesive behavior is rapidly, but transiently, enhanced in astrocytes by ligand-dependent activation of erbB4 receptors, suggesting that erbB4-mediated events affecting SynCAM1 function contribute to regulate astrocyte adhesive communication.

Employing the transgenic BALB-neuT mouse tumor model, we explored the in vivo biologic relevance of immunocompetent epitopes shared among the four ErbB receptors. The outcome of neu-mediated tumorigenesis was compared following vaccination with isogeneic normal rat ErbB2/Neu (LTR-Neu) or xenogeneic human ErbB receptors (LTR-EGFR, LTR-ErbB2, LTR-ErbB3 and LTR-ErbB4), each recombinantly expressed in an NIH3T3 murine cell background. Vaccination using rat LTR-Neu at the stage of atypical hyperplasia potently inhibited neu-mediated mammary tumorigenesis. Moreover, all human ErbB receptors specifically interfered with tumor development in BALB-neuT mice. Relative increase in tumor-free survival and reduction in tumor incidence corresponded to structural similarity shared with the etiologic neu oncogene, as rat orthologue LTR-Neu proved most effective followed by the human homologue LTR-ErbB2 and the other three human ErbB receptors. Vaccination resulted in high titer specific serum antibodies, whose tumor-inhibitory effect correlated with cross-reactivity to purified rat Neu extracellular domain in vitro. Furthermore, a T cell response specific for peptide epitopes of rat Neu was elicited in spleen cells of mice immunized with LTR-Neu and was remotely detectable for discrete peptides upon vaccination with LTR-ErbB2 and LTR-EGFR. The most pronounced tumor inhibition by LTR-Neu vaccination was associated with leukocyte infiltrate and tumor necrosis in vivo, while immune sera specifically induced cytotoxicity and apoptosis of BALB-neuT tumor cells in vitro. Our findings indicated that targeted inhibition of neu oncogene-mediated mammary carcinogenesis is conditional upon the immunization schedule and discrete immunogenic epitopes shared to a variable extent by different ErbB receptors.

The Cancer Genome Atlas (TCGA) sequencing analysis of head and neck squamous cell carcinoma (HNSCC) recently reported on gene fusions, however, few human papillomavirus (HPV) positive samples were included, and the functional relevance of identified fusions was not explored. We therefore performed an independent analysis of gene fusions in HPV-positive oropharyngeal SCC (OPSCC). RNA sequencing was performed on 47 HPV-positive OPSCC primary tumors and 25 normal mucosal samples from cancer unaffected controls on an Illumina TruSeq platform. MapSplice2 was used for alignment and identification of fusion candidates. Putative fusions with less than five spanning reads, detected in normal tissues, or that mapped to the same gene were filtered out. Selected fusions were validated by RT-PCR and Sanger sequencing. Within 47 HPV-positive OPSCC tumors, 282 gene fusions were identified. Most fusions (85.1%) occurred in a single tumor, and the remaining fusions recurred in 2-16 tumors. Gene fusions were associated with significant up regulation of 16 genes (including EGFR and ERBB4) and down regulation of four genes (PTPRT, ZNF750, DLG2, SLCO5A1). Expression of these genes followed similar patterns of up regulation and down regulation in tumors without these fusions compared to normal tissue. Five of six gene fusions selected for validation were confirmed through RT-PCR and sequencing. This integrative analysis provides a method of prioritizing functionally relevant gene fusions that may be expanded to other tumor types. These results demonstrate that gene fusions may be one mechanism by which functionally relevant genes are altered in HPV-positive OPSCC.

Schizophrenia (SZ) is primarily an adult psychiatric disorder in which disturbances caused by susceptibility genes and environmental insults during early neurodevelopment initiate neurophysiological changes over a long time course, culminating in the onset of full-blown disease nearly two decades later. Aberrant postnatal brain maturation is an essential mechanism underlying the disease. Currently, symptoms of SZ are treated with anti-psychotic medications that have variable efficacy and severe side effects. There has been much interest in the prodromal phase and the possibility of preventing SZ by interfering with the aberrant postnatal brain maturation associated with this disorder. Thus, it is crucial to understand the mechanisms that underlie the long-term progression to full disease manifestation to identify the best targets and approaches towards this goal. We believe that studies of certain SZ genetic susceptibility factors with neurodevelopmental implications will be key tools in this task. Accumulating evidence suggests that neuregulin-1 (NRG1) and disrupted-in-schizophrenia-1 (DISC1) are probably functionally convergent and play key roles in brain development. We provide an update on the role of these emerging concepts in understanding the complex time course of SZ from early neurodevelopmental disturbances to later onset and suggest ways of testing these in the future.