Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.     

Regulation of neuregulin/ErbB signaling by contractile activity in skeletal muscle

Putative roles of neuregulin (NRG) and theErbB receptors in skeletal muscle biology include myogenesis, AChreceptor expression, and glucose transport. To date, however, thephysiological regulation of NRG/ErbB signaling has not been examined.We tested the hypothesis that contractile activity in vivo inducesNRG/ErbB activation. Rat hindlimb muscle contraction was elicited witha single bout of electrical stimulation (RX) or treadmill running (EX).Western blot and immunofluorescence confirmed the expression ofmultiple NRG isoforms and the ErbB2, ErbB3, and ErbB4 receptors inadult skeletal muscle. Both RX and EX significantly increasedphosphorylation of all NRG receptors. Furthermore, contraction induceda shift in the expression profile of NRG, consistent with proteolytic processing of a transmembrane isoform. Thus two distinct modes ofexercise activated processing of NRG with concomitant stimulation ofErbB2, ErbB3, and ErbB4 signaling in vivo. To our knowledge, this isthe first demonstration of physiological regulation of NRG/ErbBsignaling in any organ and implicates this pathway in the metabolic andproliferative responses of skeletal muscle to exercise.

     

The role of neuregulin/ErbB2/ErbB4 signaling in the heart with special focus on effects on cardiomyocyte proliferation

The signaling complex consisting of the growth factor neuregulin-1 (NRG1) and its tyrosine kinase receptors ErbB2 and ErbB4 has a critical role in cardiac development and homeostasis of the structure and function of the adult heart. Recent research results suggest that targeting this signaling complex may provide a viable strategy for treating heart failure. Clinical trials are currently evaluating the effectiveness and safety of intravenous administration of recombinant NRG1 formulations in heart failure patients. Endogenous as well as administered NRG1 has multiple possible activities in the adult heart, but how these are related is unknown. It has recently been demonstrated that NRG1 administration can stimulate proliferation of cardiomyocytes, which may contribute to repair failing hearts. This review summarizes the current knowledge of how NRG1 and its receptors control cardiac physiology and biology, with special emphasis on its role in cardiomyocyte proliferation during myocardial growth and regeneration.     

ErbB3/HER3 does not homodimerize upon neuregulin binding at the cell surface

To understand signaling by the neuregulin (NRG) receptor ErbB3/HER3, it is important to know whether ErbB3 forms homodimers upon ligand binding. Previous biophysical studies suggest that the ErbB3 extracellular region remains monomeric when bound to NRG. We used a chimeric receptor approach to address this question in living cells, fusing the extracellular region of ErbB3 to the kinase-active intracellular domain of ErbB1. The ErbB3/ErbB1 chimera responded to NRG only if ErbB2 was co-expressed in the same cells, whereas an ErbB4/ErbB1 chimera responded without ErbB2. We, therefore, suggest that ErbB3 is an obligate heterodimerization partner because of its inability to homodimerize.     

Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons

Neuregulin-1 (NRG1) plays an important role in neural development, synapse formation, and synaptic plasticity by activating ErbB receptor tyrosine kinases. Although ligand-induced endocytosis has been shown to be important for many receptor tyrosine kinases, whether NRG1 signaling depends on ErbB endocytosis remains controversial. Here, we provide evidence that ErbB4, a prominent ErbB protein in the brain, becomes internalized in NRG1-stimulated neurons. The induced ErbB4 endocytosis requires its kinase activity. Remarkably, inhibition of ErbB endocytosis attenuates NRG1-induced activation of Erk and Akt in neurons. These observations indicate a role of ErbB endocytosis in NRG1 signaling in neurons.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.     

Inter-conversion of neuregulin2 full and partial agonists for ErbB4

The EGF family hormone NRG2β potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2α 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 NRG2β 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 NRG2α 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 NRG2α L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2α/K45F. Collectively, these data indicate that Gln43 of NRG2β is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.     

Expression,localization, and regulation of the neuregulin receptor ErbB3 in mouse heart

Neuregulins (NRG) belong to the EGF family of growth factors, which are ligands of the ErbB receptors. Their expression in the adult heart is essential, especially when the heart is submitted to cardiotoxic stress such as that produced by anthracyclines. It is considered that ErbB4 is the only NRG receptor expressed by the adult heart. Upon binding, ErbB4 may dimerize with ErbB2 to generate signals inside cells. However, here we show the presence of ErbB3 in the mouse heart from birth to adulthood by Western blotting and real‐time RT‐PCR. The expression level of ErbB3 mRNA was lower than that of ErbB2 or ErbB4, but was more stable throughout postnatal development. In isolated heart myocytes, ErbB3 localized to the Z‐lines similarly to ErbB1. Perfusion of isolated hearts with NRG‐1β induced phosphorylation of ErbB3, as well as ErbB2 and ErbB4. In adult mice, both ErbB2 and ErbB3, but not ErbB1 or ErbB4, were rapidly down‐regulated upon the induction of heart hypertrophy. In conclusion, our results demonstrate that ErbB3, in addition to ErbB4, is a receptor for neuregulin‐1β in the adult mouse heart. J. Cell. Physiol. 226: 450–455, 2011. © 2010 Wiley‐Liss, Inc.     

An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.     

Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4

Mouse embryos with a loss-of-function mutation in the gene encoding the receptor tyrosine kinase ErbB4 exhibit misprojections of cranial sensory ganglion afferent axons. Here we analyse ErbB4-deficient mice, and find that morphological differences between wild-type and mutant cranial ganglia correlate with aberrant migration of a subpopulation of hindbrain-derived cranial neural crest cells within the paraxial mesenchyme environment. In transplantation experiments using new grafting techniques in cultured mouse embryos, we determine that this phenotype is non-cell-autonomous: wild-type and mutant neural crest cells both migrate in a pattern consistent with the host environment, deviating from their normal pathway only when transplanted into mutant embryos. ErbB4 signalling events within the hindbrain therefore provide patterning information to cranial paraxial mesenchyme that is essential for the proper migration of neural crest cells.     

Expression of the neuregulin receptor ErbB4 in the brain of the rhesus monkey (Macaca mulatta)

We demonstrated recently that frontal cortical expression of the Neuregulin (NRG) receptor ErbB4 is restricted to interneurons in rodents, macaques, and humans. However, little is known about protein expression patterns in other areas of the brain. In situ hybridization studies have shown high ErbB4 mRNA levels in various subcortical areas, suggesting that ErbB4 is also expressed in cell types other than cortical interneurons. Here, using highly-specific monoclonal antibodies, we provide the first extensive report of ErbB4 protein expression throughout the cerebrum of primates. We show that ErbB4 immunoreactivity is high in association cortices, intermediate in sensory cortices, and relatively low in motor cortices. The overall immunoreactivity in the hippocampal formation is intermediate, but is high in a subset of interneurons. We detected the highest overall immunoreactivity in distinct locations of the ventral hypothalamus, medial habenula, intercalated nuclei of the amygdala and structures of the ventral forebrain, such as the islands of Calleja, olfactory tubercle and ventral pallidum, and medium expression in the reticular thalamic nucleus. While this pattern is generally consistent with ErbB4 mRNA expression data, further investigations are needed to identify the exact cellular and subcellular sources of mRNA and protein expression in these areas. In contrast to in situ hybridization in rodents, we detected only low levels of ErbB4-immunoreactivity in mesencephalic dopaminergic nuclei but a diffuse pattern of immunofluorescence that was medium in the dorsal striatum and high in the ventral forebrain, suggesting that most ErbB4 protein in dopaminergic neurons could be transported to axons. We conclude that the NRG-ErbB4 signaling pathway can potentially influence many functional systems throughout the brain of primates, and suggest that major sites of action are areas of the "corticolimbic" network. This interpretation is functionally consistent with the genetic association of NRG1 and ERBB4 with schizophrenia.     

Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses

Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.     

Association of PSD-95 with ErbB4 facilitates neuregulin signaling in cerebellar granule neurons in culture

The growth factor neuregulin 1 (NRG) selectively induces an increase in the gamma-aminobutyric acid (GABA)(A) receptor beta2 subunit protein in rat cerebellar granule neurons in culture. We previously demonstrated that NRG acts by triggering ErbB4 receptor phosphorylation and subsequent signaling through the mitogen-activated kinase (MAPK), phosphatidyl inositol-3 kinase (PI-3K) and cyclin-dependent kinase 5 (cdk5) pathways. In this report we show that the scaffolding protein, PSD-95, plays a key role in mediating the effects of NRG and that reducing its level attenuates the NRG-induced increase in beta2 subunit expression. PSD-95 appears to facilitate the effects of NRG through its association with ErbB4, an interaction that is augmented by NRG-activated cdk signaling. Inhibition of cdk activity with roscovitine attenuates the association of PSD-95 with ErbB4. The effects of cdk5 are not blocked by U0126, an inhibitor of MAPK signaling, indicating that cdk5 functions independently of cross-talk with this pathway. These findings raise the possibility that NRG-induced activation of cdk5 works in part by recruiting PSD-95, a protein involved in regulating synaptic plasticity, to associate with ErbB4. This interaction may be a positive feedback loop that augments NRG signaling and its downstream effects on GABA(A) receptor beta2 subunit expression.     

Effects of ectoparasite infestation during pregnancy on physiological stress and reproductive output in a rodent-flea system

Biotic and abiotic stressors impose various fitness costs on individuals across a variety of taxa. In vertebrates, these stressors typically trigger complex neuroendocrine responses that stimulate glucocorticoid (GC) secretion from the adrenal cortex. Short-term elevation of GCs can be adaptive as it shifts energy toward physiological processes that cope with acute stressors; however, chronic increases in GC levels could have detrimental effects on fitness. Parasitism can be considered an important biotic stressor in nature and a possible cause of reproductive failure that could substantially affect an individual’s fitness. Thus, we aimed to test the effects of parasitism and maternal stress, as measured by GCs, during pregnancy and the relationship between these variables and measures of reproductive output using a rodent-flea system. Female Egyptian spiny mice (Acomys cahirinus) were randomly assigned to flea (Parapulex chephrenis) infested or uninfested treatments before and during pregnancy. The offspring of these females were flea-free. Feces were collected at five time points during the experiment to determine maternal fecal glucocorticoid metabolite (FGCM) concentrations. Overall, infested females had lower FGCM levels during gestation but higher FGCM levels post-parturition and larger mass changes than uninfested females. Additionally, models related to pup quality and quantity often included some measure of maternal investment or body condition moderating relationships between infestation and stress. This suggests that flea parasitism or high GC levels alone might not significantly impact host reproduction but rather females can experience different effects depending on their level of investment, which could be limited by body condition and/or the number of pups present in a litter.     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.     

Neuregulin／Erb信号转导通路在神经发育中的作用

Neuregulins(NRG)是一在结构上相类似的多肽家族,它们由四个不同的基因编码。NRG有三种异构体（NRG1、2和3）。这些异构体在及脑内有高表达,包括发育中的脑和成熟的脑。这些异构体中,对NRG1研究最为广泛。NRG1对神经系统的发育有多种重要的调节作用。它能促进胶质细胞的生化和分化;也能调节小脑、颗粒细胞沿胶质细胞的迁移。在突触形成过程中,NRG1可诱导以下三种受体的表达：神经肌肉接头和中枢 神经系统内的乙酰胆碱受体的表达;小脑胶质细胞中NMDA受体的NR2C亚单位的表达;小脑胶质细胞中GABAA受体的表达。近年来的研究表明,NRG受体多分布于突触后膜致密区,提示NRG可能对突触的可塑性有重要的作用。本文综述了NRG的研究进展,特别对其功能及其信号转导机制有较多的讨论。