Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.
ABSTRACT: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ?570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1? (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy.
Project description:NRG-1? (neuregulin-1?) serves multiple functions during embryonic heart development by signalling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1? induces cardiomyogenesis of mESCs (mouse embryonic stem cells) at the later stages of differen-tiation through ErbB4 receptor activation. In the present study we systematically examined NRG-1? induction of cardiac myocytes in mESCs and identified a novel time window, the first 48 h, for NRG-1?-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast with the later differentiation of mESCs in which NRG-1? induces cardiomyogenesis via the ErbB4 receptor, we found that knocking down ErbB3 or ErbB2 with siRNA during the early differentiation inhibited NRG-1?-induced cardiomyogenesis in mESCs. Microarray analysis of RNA expression at this early time point indicated that NRG-1? treatment in mESCs resulted in gene expression changes important to differentiation including up-regulation of components of PI3K (phosphoinositide 3-kinase), a known mediator of the NRG-1?/ErbB signalling pathway, as well as activation of CREB (cAMP-response-element-binding protein). Further study demonstrated that the NRG-1?-induced phosphorylation of CREB was required for cardiomyogenesis of mESCs. In summary, we report a previously unrecognized role for NRG-1?/ErbB3/CREB signalling at the pre-mesoderm stage for stem cell cardiac differentiation.
Project description:BACKGROUND: Evidence of genetic association between the NRG1 (Neuregulin-1) gene and schizophrenia is now well-documented. Furthermore, several recent reports suggest association between schizophrenia and single-nucleotide polymorphisms (SNPs) in ERBB4, one of the receptors for Neuregulin-1. In this study, we have extended the previously published associations by investigating the involvement of all eight genes from the ERBB and NRG families for association with schizophrenia. METHODS: Eight genes from the ERBB and NRG families were tested for association to schizophrenia using a collection of 396 cases and 1,342 blood bank controls ascertained from Aberdeen, UK. A total of 365 SNPs were tested. Association testing of both alleles and genotypes was carried out using the fast Fisher's Exact Test (FET). To understand better the nature of the associations, all pairs of SNPs separated by >or= 0.5 cM with at least nominal evidence of association (P < 0.10) were tested for evidence of pairwise interaction by logistic regression analysis. RESULTS: 42 out of 365 tested SNPs in the eight genes from the ERBB and NRG gene families were significantly associated with schizophrenia (P < 0.05). Associated SNPs were located in ERBB4 and NRG1, confirming earlier reports. However, novel associations were also seen in NRG2, NRG3 and EGFR. In pairwise interaction tests, clear evidence of gene-gene interaction was detected for NRG1-NRG2, NRG1-NRG3 and EGFR-NRG2, and suggestive evidence was also seen for ERBB4-NRG1, ERBB4-NRG2, ERBB4-NRG3 and ERBB4-ERBB2. Evidence of intragenic interaction was seen for SNPs in ERBB4. CONCLUSION: These new findings suggest that observed associations between NRG1 and schizophrenia may be mediated through functional interaction not just with ERBB4, but with other members of the NRG and ERBB families. There is evidence that genetic interaction among these loci may increase susceptibility to schizophrenia.
Project description:The beta isoform of Neuregulin-1 (NRG-1?), along with its receptors (ErbB2-4), is required for cardiac development. NRG-1?, as well as the ErbB2 and ErbB4 receptors, is also essential for maintenance of adult heart function. These observations have led to its evaluation as a therapeutic for heart failure. Animal studies and ongoing clinical trials have demonstrated beneficial effects of two forms of recombinant NRG-1? on cardiac function. In addition to the possible role for recombinant NRG-1?s as heart failure therapies, endogenous NRG-1?/ErbB signaling appears to play a role in restoring cardiac function after injury. The potential mechanisms by which NRG-1? may act as both a therapy and a mediator of reverse remodeling remain incompletely understood. In addition to direct effects on cardiac myocytes NRG-1? acts on the vasculature, interstitium, cardiac fibroblasts, and hematopoietic and immune cells, which, collectively, may contribute to NRG-1?'s role in maintaining cardiac structure and function, as well as mediating reverse remodeling.
Project description:AIMS: A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity. METHODS AND RESULTS: Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3'UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed 'decoy' genes that had tandem complementary sequences for miR-146a in the 3'UTR of a luciferase gene. When miR-146a 'decoy' genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced. CONCLUSION: These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.
Project description:Cardiac microvascular endothelial cells (CMECs) are important angiogenic components and are injured rapidly after cardiac ischaemia and anoxia. Cardioprotective effects of Qiliqiangxin (QL), a traditional Chinese medicine, have been displayed recently. This study aims to investigate whether QL could protect CMECs against anoxic injury and to explore related signalling mechanisms. CMECs were successfully cultured from Sprague-Dawley rats and exposed to anoxia for 12 hrs in the absence and presence of QL. Cell migration assay and capillary-like tube formation assay on Matrigel were performed, and cell apoptosis was determined by TUNEL assay and caspase-3 activity. Neuregulin-1 (NRG-1) siRNA and LY294002 were administrated to block NRG-1/ErbB and PI3K/Akt signalling, respectively. As a result, anoxia inhibited cell migration, capillary-like tube formation and angiogenesis, and increased cell apoptosis. QL significantly reversed these anoxia-induced injuries and up-regulated expressions of NRG-1, phospho-ErbB2, phospho-ErbB4, phospho-Akt, phospho-mammalian target of rapamycin (mTOR), hypoxia-inducible factor-1? (HIF-1?) and vascular endothelial growth factor (VEGF) in CMECs, while NRG-1 knockdown abolished the protective effects of QL with suppressed NRG-1, phospho-ErbB2, phospho-ErbB4, phospho-Akt, phospho-mTOR, HIF-1? and VEGF expressions. Similarly, LY294002 interrupted the beneficial effects of QL with down-regulated phospho-Akt, phospho-mTOR, HIF-1? and VEGF expressions. However, it had no impact on NRG-1/ErbB signalling. Our data indicated that QL could attenuate anoxia-induced injuries in CMECs via NRG-1/ErbB signalling which was most probably dependent on PI3K/Akt/mTOR pathway.
Project description:Understanding the mechanisms of neurodegeneration is crucial for development of therapies to treat neurological disorders. S100 proteins are extensively expressed in the injured brain but S100's role and signalling in neural cells remain elusive. We recently demonstrated that the S100A4 protein protects neurons in brain injury and designed S100A4-derived peptides mimicking its beneficial effects. Here we show that neuroprotection by S100A4 involves the growth factor family receptor ErbB4 and its ligand Neuregulin 1 (NRG), key regulators of neuronal plasticity and implicated in multiple brain pathologies. The neuroprotective effect of S100A4 depends on ErbB4 expression and the ErbB4 signalling partners ErbB2/Akt, and is reduced by functional blockade of NRG/ErbB4 in cell models of neurodegeneration. We also detect binding of S100A4 with ErbB1 (EGFR) and ErbB3. S100A4-derived peptides interact with, and signal through ErbB, are neuroprotective in primary and immortalized dopaminergic neurons, and do not affect cell proliferation/motility - features which make them promising as potential neuroprotectants. Our data suggest that the S100-ErbB axis may be an important mechanism regulating neuronal survival and plasticity.
Project description:Investigation of peripheral gene expression patterns of transcripts within the NRG-ErbB signaling pathway, other than neuregulin-1 (NRG1), among patients with schizophrenia and more specifically treatment-resistant schizophrenia (TRS) is limited. The present study built on our previous work demonstrating elevated levels of NRG1 EGF?, EGF?, and type I(Ig2) containing transcripts in TRS by investigating 11 NRG-ErbB signaling pathway mRNA transcripts (NRG2, ErbB1, ErbB2, ErbB3, ErbB4, PIK3CD, PIK3R3, AKT1, mTOR, P70S6K, eIF4EBP1) in whole blood of TRS patients (N?=?71) and healthy controls (N?=?57). We also examined the effect of clozapine exposure on transcript levels using cultured peripheral blood mononuclear cells (PBMCs) from 15 healthy individuals. Five transcripts (ErbB3, PIK3CD, AKT1, P70S6K, eIF4EBP1) were significantly elevated in TRS patients compared to healthy controls but only expression of P70S6K (Pcorrected?=?0.018), a protein kinase linked to protein synthesis, cell growth, and cell proliferation, survived correction for multiple testing using the Benjamini-Hochberg method. Investigation of clinical factors revealed that ErbB2, PIK3CD, PIK3R3, AKT1, mTOR, and P70S6K expression were negatively correlated with duration of illness. However, no transcript was associated with chlorpromazine equivalent dose or clozapine plasma levels, the latter supported by our in vitro PBMC clozapine exposure experiment. Taken together with previously published NRG1 results, our findings suggest an overall upregulation of transcripts within the NRG-ErbB signaling pathway among individuals with schizophrenia some of which attenuate over duration of illness. Follow-up studies are needed to determine if the observed peripheral upregulation of transcripts within the NRG-ErbB signaling pathway are specific to TRS or are a general blood-based marker of schizophrenia.
Project description:Alterations in gamma-frequency oscillations are implicated in psychiatric disorders, and polymorphisms in NRG-1 and ERBB4, genes encoding Neuregulin-1 (NRG-1) and one of its receptors, designated ErbB4, are associated with schizophrenia. Here we show that NRG-1 selectively increases the power of kainate-induced, but not carbachol-induced, gamma oscillations in acute hippocampal slices. NRG-1beta is more effective than NRG-1alpha, a splice variant with lower affinity for ErbB receptors, and neither isoform affects the network activity without prior induction of gamma oscillations. NRG-1beta dramatically increases gamma oscillation power in hippocampal slices from both rats (2062 +/- 496%) and mice (710 +/- 299%). These effects of NRG-1beta are blocked by PD158780, a pan-specific antagonist of ErbB receptors, and are mediated specifically via ErbB4 receptors, because mice harboring a targeted mutation of ErbB4 do not respond to NRG-1. Moreover, we demonstrate that 50% of gamma-amino butyric acidergic parvalbumin (PV)-positive interneurons, which heavily contribute to the generation of gamma oscillations, express ErbB4 receptors. Importantly, both the number of PV-immunoreactive interneurons (-31%) and the power of kainate-induced gamma oscillations (-60%) are reduced in ErbB4 knockout mice. This study provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in persons with schizophrenia.
Project description:The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal ? oscillations--a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal ? oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases ? oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for ? oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1-induced increase in ? oscillation power. This unique link between D4R and ErbB4 signaling on ? oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and ? oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.
Project description:ErbB4 is a member of the ErbB family of receptor tyrosine kinases. This family includes ErbB2 (HER2/Neu), a validated therapeutic target in breast cancer. Several studies indicate that ErbB4 functions as a tumor suppressor in breast cancer, whereas others indicate that ErbB4 functions as an oncogene. Here the authors explore the context in which ErbB4 functions as an oncogene. Silencing expression of either ErbB2 or ErbB4 in breast tumor cell lines results in reduced stimulation of anchorage independence and cell motility by the ErbB4 agonist neuregulin 2?. ErbB2 tyrosine kinase activity, but not ErbB4 tyrosine kinase activity, is required for neuregulin 2? to stimulate cell proliferation. Moreover, sites of ErbB4 tyrosine phosphorylation, but not sites of ErbB2 tyrosine phosphorylation, are required for neuregulin 2? to couple to cell proliferation. These data suggest that targeting ErbB2 expression or tyrosine kinase activity may be effective in treating ErbB4-dependent breast tumors, even those tumors that lack ErbB2 overexpression.