The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo- and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-induced interactions of erbB3 with different erbB receptors. A dose-dependent inhibition of native erbB receptor dimerization by the erbB peptides has been observed in 32D cell lines transfected with different combinations of erbB receptors. The peptides effectively inhibited growth of two types of transformed cells overexpressing different erbB receptors, T6-17 and 32D, in standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell viability assays. The study identifies distinct loops within the membrane-proximal part of the subdomain IV as potential receptor-receptor interaction sites for the erbB receptors and demonstrates the possibility of disabling receptor activity by structure-based targeting of the dimerization interfaces. Molecular models for possible arrangement of the erbB1.EGF complex, consistent with the involvement of subdomain IV in inter-receptor interactions, are proposed. Small dimerization inhibitors described herein can be useful as probes to elucidate different erbB signaling pathways and may be developed as therapeutic agents.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HB(del/del)) and secretion-deficient (HB(uc/uc)) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HB(del/del) eyelids. Crosses between HB(del/del) mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HB(del/del) embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFalpha-null mice and in HB-EGF and TGFalpha double null mice revealed that HB-EGF and TGFalpha contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFalpha is required for leading edge extension in epithelial sheet migration during eyelid closure.

Overexpression of the erbB family of receptor tyrosine kinases has been implicated in a variety of tumors including breast, lung, prostate, and brain. Most solid tumors express one or more of these receptors, which can often be related to tumor aggressiveness and poor patient prognosis. CI-1033, a pan-erbB tyrosine kinase inhibitor, is a clinically promising agent that is active against all four members of the erbB receptor tyrosine kinase family. In vitro studies of human cancer cell lines indicate that CI-1033 results in prompt, potent, and sustained inhibition of tyrosine kinase activity. This inhibition is highly selective for erbB1 (epidermal growth factor receptor), erbB2, erbB3, and erbB4 without inhibiting tyrosine kinase activity of receptors such as platelet-derived growth factor receptor, fibroblast growth factor receptor, and insulin receptor, even at high concentrations. Treatment of athymic nude mice bearing xenografts of human A431 epidermoid carcinoma, H125 non-small cell lung carcinoma, and SF-767 glioblastoma results in highly significant suppression of tumor growth. The major toxicity in animals is diarrhea, which is more severe at higher doses. In animal models, all side effects are reversible on cessation of treatment. Thus, CI-1033, which is currently undergoing phase I clinical trials, holds significant potential for use in a broad range of solid tumors.

To delineate the role of matrix metalloproteinase 7 (MMP7) in mammary tumorigenesis, MMP7 was expressed in the normal murine mammary gland cell line, c57MG. MMP7 markedly enhanced the growth rate of the c57MG cells in three-dimensional culture and promoted tumor formation in vivo. Subsequent investigation showed that MMP7 (a) up-regulated ErbB4 receptor levels, (b) solubilized the ErbB4 receptor cognate ligand heparin-bound epidermal growth factor, and (c) mediated the proteolytic processing of ErbB4 to yield a soluble intracellular domain (ICD) that localized to the cytoplasm and the nucleus. Furthermore, overexpression of the ErbB4 ICD in the c57MG cell line recapitulated the proliferative effects of MMP7 in vitro and in vivo. These data indicate a novel mechanism for mammary epithelial cell transformation by MMP7.

Epilepsy is a common and refractory neurological disorder, but the neuronal regulatory mechanisms of epileptogenesis remain largely unclear. Activity-dependent transcription of genes for neurotrophins such as brain-derived neurotrophic factor (BDNF) has been shown to promote epileptogenesis; however, little is known about factors that may act as intrinsic, homeostatic or counterbalancing mechanisms. Using rodent models, here we show that limbic seizure activity upregulated NRG1-ErbB4 signaling and that epileptogenesis was inhibited by infusing NRG1 intracerebrally but exacerbated by neutralizing endogenous NRG1 with soluble ErbB4 extracellular domain, by inhibiting ErbB4 activation or by deleting the Erbb4 gene. Furthermore, specific depletion of ErbB4 in parvalbumin-expressing interneurons abolished NRG1-mediated inhibition of epileptogenesis and promoted kindling progression, resulting in increased spontaneous seizures and exuberant mossy fiber sprouting. In contrast, depleting ErbB4 in CaMKIIα-positive pyramidal neurons had no effect. Thus, NRG1-induced activation of ErbB4 in parvalbumin-expressing inhibitory interneurons may serve as a critical endogenous negative-feedback mechanism to suppress limbic epileptogenesis.

The zinc finger transcription factor gene Krox-20 is expressed in Schwann cells and is required for the myelination of peripheral nerves. We show that the regulation of Krox-20 expression in peripheral glial cells reveals three important steps in the development and differentiation of these cells. (i) Expression of Krox-20 in Schwann cells requires continuous neuronal signalling via direct axonal contact. Therefore Krox-20 appears to be a key component of the transduction cascade linking axonal signalling to myelination. (ii) Krox-20 inducibility is acquired by Schwann cells at the time that they are formed from their precursors. Diffusible factor(s) synthesised by the neural tube can mediate this transition and can be mimicked by NDFbeta or a combination of CNTF and bFGF. Furthermore, the neural tube activity is blocked by a hybrid protein containing the NDF-binding domain of the ErbB4 receptor, strongly implicating NDF in the physiological transition. (iii) In sensory ganglia, the microenvironment is capable of negatively regulating Krox-20, presumably by preventing the conversion of satellite glial cells toward a Schwann cell-like phenotype.

Proteases are now recognized as having an active role in a variety of processes aside from their recognized metabolic role in protein degradation. Within the ErbB system of ligands and receptors, proteases are known to be necessary for the generation of soluble ligands from transmembrane precursors and for the processing of the ErbB4 receptor, such that its intracellular domain is translocated to the nucleus. There are two protease activities involved in the events: proteases that cleave within the ectodomain of ligand (or receptor) and proteases that cleave the substrate within the transmembrane domain. The former are the ADAM proteases and the latter are the gamma-secretase complex and the rhomboid proteases. This review discusses the roles of each of these protease systems within the ErbB system.

Diminished left frontotemporal connectivity is among the most frequently reported findings in schizophrenia and there is evidence that altered neuronal myelination may in part account for this deficit. Several investigations have suggested that variations of the genes that encode the Neuregulin 1 (NRG1)-ErbB4 receptor complex are associated with schizophrenia illness. As NRG1--ErbB4 has been implicated in neuronal myelination, we investigated with diffusion tensor imaging (DTI) whether fractional anisotropy (FA)--a putative measure of neuronal myelination--is predicted by a risk haplotype of the ErbB4 gene. The effects of the ErbB4 genotype were investigated in healthy subjects (N=59; mean age: 22.6+/-1.8 years). We also measured reaction time (RT) during a selective attention/working memory paradigm (visual oddball). In the schizophrenia risk genotype group, we found lower FA in the temporal lobe white matter (WM) including frontotemporal fiber tracts, predominantly in the left hemisphere. RT was increased in the risk genotype group and correlated with FA in the affected brain region. As FA is considered to index structural integrity of WM, to which neuronal fiber myelination is contributing, our results suggest that variations of the ErbB4 genotype may confer risk for schizophrenia illness via its impact on left frontotemporal connectivity in human brain. Reliability and validity of the result is suggested by our observation that (1) the FA-genotype association was not only obtained in the entire sample but also in both the split halves and (2) a statistical relationship was found among RT, genotype and FA.

MicroRNAs (miRNAs) are a small class of non‑coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR‑193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR‑193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome‑wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3'UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer.

Metastatic spread is the single-most powerful predictor of poor outcome in Ewing sarcoma (ES). Therefore targeting pathways that drive metastasis has tremendous potential to reduce the burden of disease in ES. We previously showed that activation of the ERBB4 tyrosine kinase suppresses anoikis, or detachment-induced cell death, and induces chemoresistance in ES cell lines in vitro. We now show that ERBB4 is transcriptionally overexpressed in ES cell lines derived from chemoresistant or metastatic ES tumours. ERBB4 activates the PI3K-Akt cascade and focal adhesion kinase (FAK), and both pathways contribute to ERBB4-mediated activation of the Rac1 GTPase in vitro and in vivo. ERBB4 augments tumour invasion and metastasis in vivo, and these effects are blocked by ERBB4 knockdown. ERBB4 expression correlates significantly with reduced disease-free survival, and increased expression is observed in metastatic compared to primary patient-matched ES biopsies. Our findings identify a novel ERBB4-PI3K-Akt-FAK-Rac1 pathway associated with aggressive disease in ES. These results predict that therapeutic targeting of ERBB4, alone or in combination with cytotoxic agents, may suppress the metastatic phenotype in ES.

ErbB4, a member of the epidermal growth factor receptor family, plays a role in normal breast and breast cancer development by regulating mammary epithelial cell proliferation, survival and differentiation. In this study, we show that WWP1, a C2-WW-HECT type E3 ubiquitin ligase, binds, ubiquitinates and destructs ErbB4-CYT1, but much less efficiently for CYT2, isoforms (both JMa and JMb). The protein-protein interaction occurs primarily between the first and third WW domains of WWP1 and the second PY motif of ErbB4. Knockdown of WWP1 by two different small interfering RNAs increases the endogenous ErbB4 protein levels in both MCF7 and T47D breast cancer cell lines. In addition, overexpression of the wild type, but not the catalytic inactive WWP1, dramatically decreases the endogenous ErbB4 protein levels in MCF7. Importantly, we found that WWP1 negatively regulates the heregulin-beta1-stimulated ErbB4 activity as measured by the serum response element report assay and the BRCA1 mRNA expression. After a systematic screening of all WWP1 family members by small interfering RNA, we found that AIP4/Itch and HECW1/NEDL1 also negatively regulate the ErbB4 protein expression in T47D. Interestingly, the protein expression levels of both WWP1 and ErbB4 are higher in estrogen receptor-alpha-positive than in estrogen receptor-alpha-negative breast cancer cell lines. These data suggest that WWP1 and its family members suppress the ErbB4 expression and function in breast cancer.

Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special 'niche' microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood-testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF-RET-GFRA1 signaling, FGF2-MAP2K1 signaling, CXCL12-CXCR4 signaling, CCL9-CCR1 signaling, FSH-nociceptin/OPRL1, retinoic acid/FSH-NRG/ERBB4, and AR/RB-ARID4A/ARID4B) are also addressed.

Neuregulin 1 (Nrg-1) isoforms have been shown to influence the emergence and growth of oligodendrocytes, the CNS myelin-forming cells. We have investigated how Nrg-1 signaling of ErbB receptors specifically controls the early stages of oligodendrocyte generation from multipotential neural precursors (NPs). We show here that embryonic striatal NPs express multiple Nrg-1 transcripts and proteins as well as their specific receptors, ErbB2 and ErbB4, but not ErbB3. The major isoform synthesized by striatal NPs is a transmembrane type III isoform called cysteine-rich domain Nrg-1. To examine the biological effect of Nrg-1, we added soluble ErbB3 (sErbB3) to growing neurospheres. This inhibitor of Nrg-1 bioactivity decreased mitosis of NPs and increased their apoptosis, resulting in a significant reduction in neurosphere size and number. When NPs were induced to migrate and differentiate by adhesion of neurospheres to the substratum, the level of type III isoforms detected by RT-PCR and Western blot decreased in parallel with a reduction in Nrg-1 fluorescence intensity in differentiating astrocytes, neurons, and oligodendrocytes. Pretreatment of growing neurospheres with sErbB3 induced a threefold increase in the proportion of oligodendrocytes generated from NPs migrating out of the neurosphere. This effect was not observed with an unrelated soluble receptor. Addition of sErbB3 during NP growth and differentiation enhanced oligodendrocyte maturation as shown by expression of galactocerebroside and myelin basic protein. We propose that both type III Nrg-1 signaling and soluble ErbB receptors modulate oligodendrocyte development from NPs.

Epiregulin (EPR) is a recently described member of the epidermal growth factor (EGF) family of peptide growth factors. The ever expanding size of the EGF family has made distinguishing the activities of these hormones paramount. We show here that EPR activates two members of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and ErbB4. Therefore by these criteria, EPR is qualitatively similar to another EGF family hormone, betacellulin (BTC). Yet, here we also demonstrate quantitative differences between EPR and BTC. EPR stimulates higher levels of EGFR phosphorylation than does BTC, whereas BTC stimulates higher levels of ErbB4 phosphorylation than does EPR. Moreover, the EPR and BTC dose response curves show that although EGFR is more sensitive to EPR than is ErbB4, ErbB4 is more sensitive to BTC than is EGFR. Finally, ErbB2, which is not activated by EPR when expressed on its own, increases the sensitivity of ErbB4 for activation by EPR. Therefore, these results establish that EPR exhibits novel activities and modes of regulation, which may have significant implications for EPR function in vivo.

OBJECTIVE

To evaluate the antitumor activity and toxicity of two doses of CI-1033 in patients with platinum-refractory or recurrent ovarian cancer, and to determine baseline expression of epidermal growth factor receptor in tumor cells.

METHODS

This phase II, open-label clinical trial evaluated CI-1033 in patients with ovarian cancer who failed prior platinum-based therapy. Two oral doses of CI-1033 were evaluated--a 50-mg and a 200--mg oral dose administered daily for 21 days in a 28-day cycle. Patients were evaluated for tumor response and toxicity; in addition, archival baseline tumor samples were analyzed by immunohistochemistry for erbB1 to erbB4 status.

RESULTS

One hundred five eligible patients were treated. Baseline demographic characteristics were balanced in this heavily pretreated patient population. The median number of prior chemotherapy regimens received was four. The most commonly encountered drug-related adverse events for both dose arms were gastrointestinal (diarrhea, nausea, stomatitis) toxicity, asthenia, and rash. No responses were observed. Stable disease was confirmed in 34% and 26% of patients in the 200-mg and 50-mg arms, respectively, and 1-year survival rates were 38.5% and 37.7%, respectively. Baseline erbB3 and erbB4 revealed the highest frequencies of expression, while erbB2 was the lowest.

CONCLUSIONS

CI-1033 did not show activity in unscreened patients with advanced ovarian cancer. At 50 mg/d, CI-1033 had a more favorable adverse events profile than at 200 mg/d. erbB3 and erbB4 receptors showed the highest expression in tumor samples while erbB2 revealed the least. There appears to be no association between baseline erbB expression and disease stability.

Neuregulin-1 (NRG-1) regulates developmental neuronal survival and synaptogenesis, astrocytic differentiation, and microglial activation. Given these NRG-1 actions, we hypothesized that the synaptic loss, gliosis, inflammation, and neuronal death occurring in Alzheimer disease (AD) is associated with altered expression of NRG-1 and its receptors (the erbB2, erbB3, and erbB4 membrane tyrosine kinases). We examined the expression and distribution of NRG-1 and the erbB kinases in the hippocampus of AD patients and cognitively normal controls and in transgenic mice that coexpress AD-associated mutations of the beta amyloid precursor protein (APP(K670N,M671L)) and presenilin-1 (PS1(M146L)). In the hippocampi of both control humans and wild type mice, NRG-1 and the 3 erbB receptors are expressed in distinct cellular compartments of hippocampal neurons. All 4 molecules are associated with neuronal cell bodies, but only NRG-1, erbB2, and erbB4 are present in synapse-rich regions. In AD and in the doubly transgenic mouse, erbB4 is expressed by reactive astrocytes and microglia surrounding neuritic plaques. In AD brains, microglia and, to a lesser extent, dystrophic neurites, also upregulate NRG-1 in neuritic plaques, suggesting that autocrine and/or paracrine interactions regulate NRG-1 action within these lesions. NRG-1 and erbB4, as well as erbB2, are similarly associated with neuritic plaques in the doubly transgenic mice. Thus, in AD the hippocampal distribution of NRG-1 and erbB4 is altered. The similarities between the alterations in the expression of NRG-1 and its receptors in human AD and in APP(K670N;M671L)/PS1(M146L) mutant mice suggests that this animal model may be very informative in deciphering the potential role of these molecules in AD.

Neuregulins have been shown to play an important role in the development of the central nervous system, but their function in adult tissues is still unclear. We investigated the expression of the neuregulin receptors erbB3 and erbB4 in the adult rat brain by in situ hybridization histochemistry. Areas with considerable expression of erbB4 receptor mRNA include cortex, amygdala, hippocampus, medial habenula, reticular thalamic nucleus, several hypothalamic nuclei, subthalamic nucleus, substantia nigra pars compacta, and ventral tegmental area. Immunostaining for tyrosine hydroxylase and dopamine depletion by 6-hydroxydopamine indicate that erbB4 is expressed in dopamine neurons in the latter two nuclei. Substantial erbB4 expression is also present in clusters of cells along the ventral and medial border of the striatum/nucleus accumbens and in the subependymal zone along the lateral and olfactory ventricles (rostral migratory stream), suggesting a role for neuregulins in adult cell proliferation. In contrast, erbB3 mRNA is mostly expressed in white matter throughout the brain and in the ependyma of the ventral half of the third ventricle (tanycytes). These results demonstrate that expression of erbB3 and erbB4 receptors is widespread in the adult rat brain and suggest a function for neuregulins into adulthood.

Epidermal growth factor (EGF) is a member of a structurally related family containing heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGFalpha) that exerts neurotrophic activity on midbrain dopaminergic neurons. To examine neurotrophic abnormality in Parkinson's disease (PD), we measured the protein content of EGF, TGFalpha, and HB-EGF in post-mortem brains of patients with Parkinson's disease and age-matched control subjects. Protein levels of EGF and tyrosine hydroxylase were decreased in the prefrontal cortex and the striatum of patients. In contrast, HB-EGF and TGFalpha levels were not significantly altered in either region. The expression of EGF receptors (ErbB1 and ErbB2, but not ErbB3 or ErbB4) was down-regulated significantly in the same forebrain regions. The same phenomenon was mimicked in rats by dopaminergic lesions induced by nigral 6-hydroxydopamine infusion. EGF and ErbB1 levels in the striatum of the PD model were markedly reduced on the lesioned side, compared with the control hemisphere. Subchronic supplement of EGF in the striatum of the PD model locally prevented the dopaminergic neurodegeration as measured by tyrosine hydroxylase immunoreactivity. These findings suggest that the neurotrophic activity of EGF is maintained by afferent signals of midbrain dopaminergic neurons and is impaired in patients with Parkinson's disease.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a multifunction protein containing an N-terminal C2 domain, four tandem WW domains for substrate binding, and a C-terminal catalytic HECT domain for ubiquitin transferring. WWP1 has been suggested to function as the E3 ligase for several PY motif-containing proteins, such as Smad2, KLF5, p63, ErbB4/HER4, RUNX2, JunB, RNF11, SPG20, and Gag, as well as several non-PY motif containing proteins, such as TβR1, Smad4, KLF2, and EPS15. WWP1 regulates a variety of cellular biological processes including protein trafficking and degradation, signaling, transcription, and viral budding. WWP1 has been implicated in several diseases, such as cancers, infectious diseases, neurological diseases, and aging. In this review article, we extensively summarize the current knowledge of WWP1 with special emphasis on the roles and action of mechanism of WWP1 in signaling and human diseases.

OBJECTIVE

The purpose of this research was to quantitatively analyze tumor-specific overexpression of all ErbB receptors and ErbB4 isoforms in transitional cell carcinoma (TCC) of the bladder.

METHODS

A real-time reverse transcription-PCR protocol was set up to simultaneously quantitate the mRNA levels of all four of the ErbB receptors and ErbB4 isoforms. Exon-intron structure of the ErbB4 gene was determined for ErbB4 isoform analysis. The assay was validated by analyzing: (a) defined ErbB cDNAs; (b) cell lines transfected with defined ErbB cDNAs; and (c) cancer cell lines with ErbB status controlled by Western blotting. ErbB mRNA expression was quantitated from 29 clinical samples representing TCC, interstitial cystitis, or histologically normal bladder. Cutoff expression levels predicting neoplasia at 95% probability were determined. ErbB expression and amplification was analyzed by immunohistochemistry and chromogenic in situ hybridization.

RESULTS

Experiments with control cDNAs and cell lines demonstrated that the assay was both specific and sensitive, and that ErbB mRNA levels closely correlated with protein levels in cancer cell lines. Determination of cutoff expression levels indicated tumor-specific overexpression of ErbB2, ErbB3, and specific ErbB4 isoforms in a subset of TCC patients. Significant overexpression of ErbB mRNAs was also detected in cases without amplification of the respective gene or when the protein product was not localized at the cell membrane.

CONCLUSIONS

Bladder cancer patients with tumor-specific overexpression of ErbB receptors or their isoforms were identified. Real-time reverse transcription-PCR could be used for ErbB receptor status quantitation to produce prognostic and predictive information for cancer therapy.

Neu differentiation factor (NDF), a member of the epidermal growth factor (EGF)-related peptide family, activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. In a similar fashion, EGF receptor (EGFR) agonists induce heterodimers of EGFR and ErbB2. In this paper, we show that the ErbB2-overexpressing breast tumor cells SKBR3, AU565, and MDA-MB453 are growth inhibited by NDF. Cells with elevated levels of ErbB2 but little or no NDF receptors (SKOV3 and MDA-MB361) or cells with low levels of ErbB2 (T47D and MCF7) are not growth inhibited. None of the EGFR agonists tested (EGF, beta-cellulin, or heparin-binding EGF) inhibited growth of ErbB2-overexpressing cells. These results suggest that formation of an ErbB2/NDF receptor heterodimer, but not of an ErbB2/EGFR heterodimer, promotes growth inhibition. In addition, NDF caused a down-regulation of ErbB2 but not of ErbB3. The mechanism underlying the inhibitory effect was examined further in SKBR3 cells, which are 95% growth inhibited by NDF. A G2-M arrest is seen 24 h after NDF treatment, and increased apoptosis is detectable from day 2 onward. The results demonstrate for the first time that NDF induces apoptosis of tumor cells overexpressing ErbB2.

BACKGROUND

Neuregulins are a family of peptide growth factors that promote cell growth and viability. The potential role of neuregulin-erbB signaling in hypertrophic growth and later failure in the adult heart in vivo is not known.

RESULTS

We used ribonuclease protection assays to quantify mRNA levels of neuregulin, erbB2, and erbB4 in left ventricular (LV) tissue and myocytes of normal rats and rats with aortic stenosis with pressure-overload hypertrophy 6 and 22 weeks after banding. At both stages of hypertrophy, Northern blot analyses of mRNA from LV myocytes showed upregulation of atrial natriuretic peptide, a molecular marker of hypertrophy (P<0.05). LV tissue neuregulin message levels were similar in animals with aortic stenosis compared with controls (P=NS) and were not detectable in myocytes. LV erbB2 and erbB4 message levels in LV tissue and myocytes were maintained during early compensatory hypertrophy in 6-week aortic stenosis animals compared with age-matched controls; in contrast, erbB2 and erbB4 message levels were depressed in 22-week aortic stenosis animals at the stage of early failure (both P<0.01 vs age-matched controls). Immunoblotting of erbB2 and erbB4 also showed normal protein levels in 6-week aortic stenosis animals compared with controls; however, erbB2 and erbB4 protein levels were depressed in 22-week aortic stenosis animals (48% decrease in erbB2, P<0.05, and 43% decrease in erbB4, P<0.01) relative to age-matched controls.

CONCLUSIONS

The neuregulin receptors erbB2 and erbB4 are downregulated at both the message and protein levels at the stage of early failure in animals with chronic hypertrophy secondary to aortic stenosis. These data suggest a role for disabled erbB receptor signaling in the transition from compensatory hypertrophy to failure.

During the last decade, several novel members of the Epidermal Growth Factor family of peptide growth factors have been identified. Most prominent among these are the Neuregulins or Heregulins. To date, four different Neuregulin genes have been identified (Neuregulin1-4) and several different splicing isoforms have been identified for at least two of these genes (Neuregulin1 and Neuregulin2). While Neuregulin1 isoforms have been extensively studied, comparatively little is known about Neuregulin3, Neuregulin4, or the Neuregulin2 isoforms. Indeed, there has been no systematic comparison of the activities of these molecules. Here we demonstrate that Neuregulin2alpha and Neuregulin2beta stimulate ErbB3 tyrosine phosphorylation and coupling to biological responses. In contrast, Neuregulin3 and Neuregulin4 fail to activate ErbB3 signaling. Furthermore, Neuregulin2beta, but not Neuregulin2alpha, stimulates ErbB4 tyrosine phosphorylation and coupling to biological responses. Finally, both Neuregulin3 and Neuregulin4 stimulate modest amounts of ErbB4 tyrosine phosphorylation. However, whereas Neuregulin3 stimulates a modest amount of ErbB4 coupling to biological responses, Neuregulin4 fails to stimulate ErbB4 coupling to biological responses. This suggests that there are qualitative as well as quantitative differences in ErbB family receptor activation by Neuregulin isoforms.