Doxorubicin-Induced Cardiomyopathy in Children.

Doxorubicin-induced cardiotoxicity in childhood cancer survivors is a growing problem. The population of patients at risk for cardiovascular disease is steadily increasing, as five-year survival rates for all types of childhood cancers continue to improve. Doxorubicin affects the developing heart differently from the adult heart and in a subset of exposed patients, childhood exposure leads to late, irreversible cardiomyopathy. Notably, the prevalence of late-onset toxicity is increasing in parallel with improved survival. By the year 2020, it is estimated that there will be 500,000 childhood cancer survivors and over 50,000 of them will suffer from doxorubicin-induced cardiotoxicity. The majority of the research to-date, concentrated on childhood cancer survivors, has focused mostly on clinical outcomes through well-designed epidemiological and retrospective cohort studies. Preclinical studies have elucidated many of the cellular mechanisms that elicit acute toxicity in cardiomyocytes. However, more research is needed in the areas of early- and late-onset cardiotoxicity and more importantly improving the scientific understanding of how other cells present in the cardiac milieu are impacted by doxorubicin exposure. The overall goal of this review is to succinctly summarize the major clinical and preclinical studies focused on doxorubicin-induced cardiotoxicity. As the prevalence of patients affected by doxorubicin exposure continues to increase, it is imperative that the major gaps in existing research are identified and subsequently utilized to develop appropriate research priorities for the coming years. Well-designed preclinical research models will enhance our understanding of the pathophysiology of doxorubicin-induced cardiotoxicity and directly lead to better diagnosis, treatment, and prevention. © 2019 American Physiological Society. Compr Physiol 9:905-931, 2019.

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.

[Research progress of neuregulin 4 biological function].

Neuregulin 4 (NRG4) is a kind of protein containing epidermal growth factor (EGF)-like domains, mainly expressed and secreted by brown adipocytes. It specifically activates EGF receptor ErbB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) to stimulate cell proliferation, inhibit apoptosis and improve energy metabolism of cells. Increasing evidence has shown that NRG4 plays an important role in epithelial cell-related diseases, cardiovascular diseases, tumors and glycolipid metabolic diseases, and therefore it could be a potential therapeutic target of some diseases.

New adipokines.

Adipose tissue is now widely recognized as "an organ" able to synthesize and secrete hundred factors collectively called adipokines. These secreted molecules exert pleiotropic actions, notably on the regulation of glucose and lipid metabolism, inflammation, reproduction, or angiogenesis. Over the past two decades, a considerable amount of work was performed on the two "star" adipokines, leptin and adiponectin, particularly because of their involvement in energy metabolism. The present review is focused on the three most recently discovered adipokines that are clearly emerging as important actors in metabolism: apelin, fibroblast growth factor-21, and neuroregulin-4. Moreover, given a number of clinical and experimental data, these three adipokines represent promising targets in the context of metabolic disorders associated with obesity.

The brown fat secretome: metabolic functions beyond thermogenesis.

Brown fat is highly active in fuel oxidation and dissipates chemical energy through uncoupling protein (UCP)1-mediated heat production. Activation of brown fat leads to increased energy expenditure, reduced adiposity, and lower plasma glucose and lipid levels, thus contributing to better homeostasis. Uncoupled respiration and thermogenesis have been considered to be responsible for the metabolic benefits of brown adipose tissue. Recent studies have demonstrated that brown adipocytes also secrete factors that act locally and systemically to influence fuel and energy metabolism. This review discusses the evidence supporting a thermogenesis-independent role of brown fat, particularly through its release of secreted factors, and their implications in physiology and therapeutic development.

Cellular and pharmacological targets to induce coronary arteriogenesis.

The formation of collateral vessels (arteriogenesis) to sustain perfusion in ischemic tissue is native to the body and can compensate for coronary stenosis. However, arteriogenesis is a complex process and is dependent on many different factors. Although animal studies on collateral formation and stimulation show promising data, clinical trials have failed to replicate these results. Further research to the exact mechanisms is needed in order to develop a pharmalogical stimulant. This review gives an overview of recent data in the field of arteriogenesis.

Multiple variants aggregate in the neuregulin signaling pathway in a subset of schizophrenia patients.

Despite the strongly held view that schizophrenia (SZ) shows substantial genetic heterogeneity, pathway heterogeneity, as seen in cancer where different pathways are affected in similar tumors, has not been explored. We explore this possibility in a case-only study of the neuregulin signaling pathway (NSP), which has been prominently implicated in SZ and for which there is detailed knowledge on the ligand- and receptor-processing steps through ß- and γ-secretase cleavage. We hypothesize that more than one damaging variants in the NSP genes might be necessary to cause disease, leading to an apparent clustering of such variants in only the few patients with affected NSP. We analyze linkage and next-generation sequencing results for the genes encoding components of the pathway, including NRG1, NRG3, ERBB4, ß-secretase and the γ-secretase complex. We find multiple independent examples of supporting evidence for this hypothesis: (i) increased linkage scores over NSP genes, (ii) multiple positive interlocus correlations of linkage scores across families suggesting each family is linked to either many or none of the genes, (iii) aggregation of predicted damaging variants in a subset of individuals and (iv) significant phenotypic differences of the subset of patients carrying such variants. Collectively, our data strongly support the hypothesis that the NSP is affected by multiple damaging variants in a subset of phenotypically distinct patients. On the basis of this, we propose a general model of pathway heterogeneity in SZ, which, in part, may explain its phenotypic variability and genetic complexity.

Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

BACKGROUND: Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. CONCLUSIONS/SIGNIFICANCE: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

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.

The role of neuregulin4 and HER4 in gastrointestinal malignant lymphoma.

The human epidermal growth factor (EGF) receptor (HER) family consists of four receptors that bind to ligands sharing an EGF-like motif. The HER family of receptor tyrosine kinases and their ligands (EGF family) are known to play a significant role in gastrointestinal cancer. In particular, the EGF receptor, HER1, is one of the main candidates for the molecular-targeted therapy of colon cancer, and HER2 is a candidate for the treatment of gastric cancer which overexpresses HER2. In contrast, the role of the HER and EGF families in malignant lymphoma has not been fully elucidated. In this study, we investigated the expression and function of the HER and EGF families in lymphoma cell lines and tumor samples. Reverse transcription polymerase chain reaction revealed that the ligands for HER1 were mainly expressed in gastric cancer and colon cancer cell lines, but not in lymphoma cell lines. On the other hand, the EGF family member, neuregulin (NRG) 4, was highly expressed in lymphoma cell lines. Immunohistochemical analyses of malignant lymphoma clinical samples revealed that NRG4 and HER4 were mainly expressed in mucosa-associated lymphoid tissue (MALT) and follicular lymphoma. Immunoprecipitation of Raji and Daudi cell lines revealed that recombinant NRG4 induced the tyrosine phosphorylation of HER4. Additionally, recombinant NRG4 activated the proliferation of lymphoma cell lines. These findings suggest that the NRG4-HER4 axis plays a major role in the proliferation of malignant lymphoma cells in the gastrointestinal tract.

Neuregulin signaling is dispensable for NMDA- and GABA(A)-receptor expression in the cerebellum in vivo.

Neuregulin-1s (NRG-1s) are a family of growth and differentiation factors with multiple roles in the development and function in different organs including the nervous system. Among the proposed functions of NRG-1s in the nervous system is the regulation of genes encoding certain neurotransmitter receptors during synapse formation as well as of other aspects of synaptic function. Here, we have examined, in granule cells of the cerebellum in vivo, the role of NRGs in the induction of NMDA receptor (NMDA-R) and GABA(A) receptor (GABA(A)-R), which are thought to be induced by NRG-1 secreted by the synaptic inputs. To this end, we used the Cre/loxP system to genetically ablate the NRG receptors ErbB2 and ErbB4 selectively in these cells, thus eliminating all NRG-mediated signaling to them. Unlike previous reports using cultured granule cells to address the same question, we found that the developmental expression patterns of the mRNAs encoding the NR2C subunit of the NMDA-R and the beta2-subunit of the GABA(A)-R is normal in mice lacking the NRG receptors ErbB2 and ErbB4. Likewise, no alterations in cerebellar morphology nor in certain aspects of cerebellar wiring were resolved in these mutants. We conclude that NRG/ErbB signaling to the granule cells is dispensable for the normal development of their synaptic inputs.

Notch signalling coordinates tissue growth and wing fate specification in Drosophila.

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. Here, we report that these two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. We present evidence that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. Our data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium.

Neuregulins and neuronal plasticity: possible relevance in schizophrenia.

Polymorphisms in the Neuregulin 1 (NRG1) and ErbB4 receptor genes have been associated with schizophrenia in numerous cohort and family studies, and biochemical measurements from postmortem prefrontal cortex homogenates suggest that NRG/ErbB signalling is altered in schizophrenia. Moreover, recent work from our group, and from others, indicates that NRG/ErbB signalling has a role in regulating glutamatergic transmission--an intriguing finding given that glutamatergic hypofunction has been proposed to be involved in the pathogenesis underlying schizophrenia. Here we will provide a brief background of the complexity of the NRG/ErbB signalling system. We will then focus on how NRG1 reverses (depotentiates) long-term potentiation (LTP) at hippocampal Schaeffer collateral--CA1 glutamatergic synapses in the adult brain. Specifically, we found that NRG1 depotentiates LTP in an activity- and time-dependent manner. A role of endogenous NRG for regulating plasticity at hippocampal synapses is supported by experiments demonstrating that ErbB receptor antagonists completely block LTP depotentiation by brief theta-pulse stimuli, a subthreshold stimulus paradigm that reverses LTP in live animals. Preliminary results indicate that NRG1-mediated LTP depotentiation is NMDA receptor independent, and manifests as an internalization of GluR1-containing AMPA receptors. The importance of the NRG/ ErbB signalling pathway in regulating homeostasis at glutamatergic synapses, and its possible implications for schizophrenia, will be discussed.

Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing.

Cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3beta (GSK3beta) have been implicated in pathogenic processes associated with Alzheimer's disease because both kinases regulate tau hyperphosphorylation and enhance amyloid precursor protein (APP) processing leading to an increase in amyloid beta (Abeta) production. Here we show that young p25 overexpressing mice have enhanced cdk5 activity but reduced GSK3beta activity attributable to phosphorylation at the inhibitory GSK3beta-serine 9 (GSK3beta-S9) site. Phosphorylation at this site was mediated by enhanced activity of the neuregulin receptor complex, ErbB, and activation of the downstream phosphatidylinositol 3 kinase/Akt pathway. Young p25 mice had elevated Abeta peptide levels, but phospho-tau levels were decreased overall. Thus, cdk5 appears to play a dominant role in the regulation of amyloidogenic APP processing, whereas GSK3beta plays a dominant role in overall tau phosphorylation. In older mice, GSK3beta inhibitory phosphorylation at S9 was reduced relative to young mice. Abeta peptide levels were still elevated but phospho-tau levels were either unchanged or showed a trend to increase, suggesting that GSK3beta activity increases with aging. Inhibition of cdk5 by a specific inhibitor reduced cdk5 activity in p25 mice, leading to reduced Abeta production in both young and old mice. However, in young mice, cdk5 inhibition reversed GSK3beta inhibition, leading to an increase in overall tau phosphorylation. When cdk5 inhibitor was administered to very old, nontransgenic mice, inhibition of cdk5 reduced Abeta levels, and phospho-tau levels showed a trend to increase. Thus, cdk5 inhibitors may not be effective in targeting tau phosphorylation in the elderly.

Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells.

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

Neuregulins mediate calcium-induced glucose transport during muscle contraction.

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.

Multiple EGFR ligands participate in guiding migrating border cells.

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.

Phe45 of NRG2beta is critical for the affinity of NRG2beta for ErbB4 and for potent stimulation of ErbB4 signaling by NRG2beta*.

The Neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of growth factors. EGF family members regulate the signaling of ErbB family receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4. We have previously demonstrated that the EGF family hormone NRG2beta is a potent ErbB4 agonist, whereas NRG2alpha is a weak ErbB4 agonist. We have also previously demonstrated that Phe45 of NRG2beta regulates the potency of NRG2beta. Here, we address the hypotheses that Phe45 regulates the potency of NRG2beta by regulating the affinity of NRG2beta for ErbB4. We demonstrate that Phe45 of NRG2beta indeed regulates the affinity of NRG2beta for ErbB4. Furthermore, a hydrophobic or uncharged amino acid side chain at residue 45 contributes to NRG2beta binding to ErbB4. These data indicate that Phe45 of NRG2beta may regulate the affinity of NRG2beta for ErbB4 by interacting with hydrophobic amino acids in ErbB4.

Endogenous neuregulin restores radial glia in a (ferret) model of cortical dysplasia.

Radial glia are integral components of the developing neocortex. During corticogenesis, they form an important scaffold for neurons migrating into the cortical plate. Recent attention has focused on neuregulin (NRG1), acting through erbB receptors, in maintaining their morphology. We developed a model of developmental radial glial disruption by delivering an antimitotic [methylazoxy methanol (MAM)] to pregnant ferrets on embryonic day 24 (E24). We previously found that normal ferret cortex contains a soluble factor capable of realigning the disorganized radial glia back toward their normal morphology. Characterization of the reorganizing activity in normal cortex demonstrated that the probable factor mediating these responses was a 30-50 kDa protein. To test whether this endogenous soluble factor was NRG1, we used organotypic cultures of E24 MAM-treated ferret neocortex supplemented with the endogenous factor obtained from normal cortical implants, exogenous NRG1beta, antibodies that either blocked or stimulated erbB receptors, or a soluble erbB subtype that binds to available NRG1. We report that exogenous NRG1 or antibodies that stimulate erbB receptors dramatically improve the morphology of disrupted radial glia, whereas blockade of NRG1-erbB signaling prevents the radial glial repair. Our results suggest that NRG1 is an endogenous factor in ferret neocortex capable of repairing damaged radial glia and that it acts via one or more erbB receptors.

Development of an autocrine neuregulin signaling loop with malignant transformation of human breast epithelial cells.

Neuregulin (NRG) is a heparin-binding factor that activates members of the epidermal growth factor family of tyrosine kinase receptors including erbB2 that is overexpressed in more aggressive types of breast cancer. The exact role that NRG plays in breast cancer is complicated by the fact that NRG has been shown to have both proliferative and antiproliferative effects, depending on the breast cancer cell line used. Using an isogenic series of breast epithelial cell lines (MCF10A) ranging from benign to malignant, we found that the actions of NRG changed from antiproliferative to proliferative as the cells progress to cancer. This correlated with a progressive inability of NRG to down-regulate a group of proliferation genes identified previously using cDNA microarrays. As the cells progress to malignancy, they expressed higher levels of erbB2 and lower levels of erbB3 and secreted high levels of NRG into the culture media, resulting in high basal levels of erbB receptor phosphorylation. Disruption of this autocrine signaling loop by blocking ligand-induced receptor activation inhibited cancer cell proliferation. These results demonstrate that the transition of MCF10A cells from normal to premalignant to malignant correlates with the development of a constitutively active autocrine NRG signaling loop that promotes cell proliferation and suggest that disrupting this autocrine loop may provide an important therapeutic measure to control breast cancer cell growth.