Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Neuregulin expression in solid tumors: prognostic value and predictive role to anti-HER3 therapies.

BACKGROUND: Neuregulins (NRG) are a family of epidermal growth factor ligands which act through binding to HER3 and HER4 receptors. NRGs are widely expressed in solid tumors. Their prognostic significance or their role as predictors of benefit from anti-HER3 therapy is not known. RESULTS: Of 29 included studies, 7 studies reported the association between NRG and outcome. NRG was most commonly expressed in breast, prostate, colon and bladder cancers. NRG expression was not associated with either OS or PFS (HR: 3.47, 95% CI 0.78-15.47, p = 0.10 and HR: 1.64, 95% CI 0.94-2.86, p = 0.08, respectively). In 4 placebo controlled trials of anti-HER3 therapy, the addition of anti-HER3 antibodies to control therapy in unselected patients was not associated with improved PFS (HR: 0.88, 95% CI 0.75-1.04. p = 0.14). However, in patients with high NRG expression, there was significantly delayed progression (HR: 0.35, 95% CI 0.23-0.52, p < 0.001). Anti-HER3 antibodies were associated with increased risk of diarrhea, nausea and rash. METHODS: A search of electronically available databases identified studies exploring clinical outcomes based on NRG expression, as well as placebo-controlled trials of HER3-directed therapy reporting results based on NRG expression status. Data were combined in a meta-analysis using generic inverse variance and random effects modeling for studies reporting the hazard ratio (HR) for overall (OS) or progression-free survival (PFS). Mantel-Haenszel random-effect modeling was used for odds ratio (OR) for 3-year and 5-year OS and PFS. CONCLUSIONS: NRG expression is not associated with either OS or PFS, but is a predictor of benefit from anti-HER3 antibodies.

PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila.

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.

Expression of neuregulin 4 splice variants in normal human tissues and prostate cancer and their effects on cell motility.

The neuregulin 4 gene encodes at least five different variants (designated A1, A2, B1, B2 and B3) produced as a result of alternative splicing. We have determined their sites of expression in normal human adult tissues using isoform-specific antibodies. Their expression is cell type specific and differs in subcellular location suggesting that they may have varied functions in these contexts. We have shown in a panel of prostate cancers that each form is present to differing degrees, and that principal component analysis indicates that there are three patterns of expression. Some isoforms were positively correlated with high prostate-specific antigen levels and others were inversely associated with Gleason score. Synthetic, refolded A forms promoted lamellipodia and filopodia formation in cells expressing the ErbB4 (CTa) receptor and stimulated cell motility in wound healing assays. The data suggest that the different forms have varied sites of expression and function, and this includes effects on cell architecture and motility.

The complete family of epidermal growth factor receptors and their ligands are co-ordinately expressed in breast cancer.

The levels of expression of the four receptors and eleven ligands composing the epidermal growth factor family were measured using immunohistochemical staining in one hundred cases of breast cancer. All of the family were expressed to some degree in some cases; however, individual cases showed a very wide range of expression of the family from essentially none to all the factors at high levels. The highest aggregate level of expression of a receptor was HER2 followed by HER1, then HER3, then HER4. The ligands (including two splice variants of the NRG1 and NRG2 genes) broadly fell into three groups, those with the highest aggregate expression were Epigen, Epiregulin, Neuregulin 1alpha, Neuregulin 2alpha, Neuregulin 2beta, Neuregulin 4 and TGFalpha, moderate expression was seen with EGF, Neuregulin 1beta and Neuregulin 3, and relatively low levels of expression were seen of HB-EGF, Betacellulin and Amphiregulin. Statistical analysis using Spearman's Rank Correlation showed a positive correlation of expression between each of the factors. Analysing the data using the Cox Proportional Hazards model showed that, in this dataset, the most powerful predictors of relapse free interval and overall survival were the combined measurement of only Epigen and Neuregulin 4.

Expression of ErbB receptors and their cognate ligands in gastric and colon cancer cell lines.

BACKGROUND: ErbB receptors and their cognate ligands are implicated in cancer progression. Their expression in gastrointestinal cancer, however, has not been systemically studied. MATERIALS AND METHODS: The expression of four ErbB receptors and a panel of ErbB ligands were determined by reverse transcription-PCR in two gastric (TMK1, MKN-45) and two colon (SW1116, HT-29) cancer cell lines. Cell proliferation was measured by MTT assay while gene knockdown was achieved by RNA interference. RESULTS: ErbB1, ErbB2 and ErbB3 receptors and five known or putative ErbB ligands, namely, epiregulin, epidermal growth factor (EGF), heparin-binding EGF, transforming growth factor alpha (TGFalpha) and neuroglycan-C were expressed in all four cell lines. Knockdown of neuroglycan-C, however, did not affect cell proliferation. CONCLUSION: This study profiles the expression of ErbB receptors and their cognate ligands in gastric and colon cancer cells. These findings might lay the basis for the development of ErbB pathway-directed therapeutics for gastrointestinal cancer.

[Effects of recombined neuregulin on healthy Macaca mulatta hearts].

To explore the effects of recombined Neuregulin on the heart of healthy Macaca mulatta, 10 healthy adult Macaca mulatta were randomly divided into two groups and were injected with the same doses of recombined Neuregulin and normal saline, respectively. At the same time, related indices were detected by 2-dimensional echocardiography and M-mode echocardiography. All indices were compared between the two groups and among different phases. Recombined Neuregulin had effects on LVEDD (left ventricular end-diastolic diameter), LVEDV (left ventricular end-diastolic volume), LVESV (left ventricular end-systolic volume) and SV (Stroke volume), and the effects changed with time. However, no significant changes were seen on EF (Ejection fraction) and FS (Fractional shortening). In conclusion, recombined Neuregulin has effects on the left ventricular volume of healthy Macaca mulatta, but no significant effect on cardiac contractility.

Neuregulin expression modulates clinical response to trastuzumab in patients with metastatic breast cancer.

PURPOSE: Human epidermal growth factor receptor 2 (HER-2) overexpression has been associated with the genesis and progression of a subset of breast cancers. The function of HER-2 may be upregulated by overexpression or by the availability of neuregulins (NRGs), a group of transmembrane growth factors. Transmembrane NRGs strongly activated HER-2 and cell proliferation in breast cancer cells that did not overexpress HER-2, and treatment with trastuzumab prevented the proliferative action of transmembrane NRG. This raised the relevant clinical question of whether patients considered as HER-2 negative, but expressing transmembrane NRG, may benefit from treatment with trastuzumab. PATIENTS AND METHODS: MCF7 cells expressing transmembrane NRG (MCF7-NRG2c) were injected into mice, and their sensitivity to trastuzumab was assessed. A retrospective study of 124 patients with early-stage or metastatic breast cancer was conducted. Expression of transmembrane NRG was evaluated by immunohistochemistry. In 11 patients, Western blot for NRGs was also carried out. Statistics were performed to analyze possible correlations between NRG expression and response to trastuzumab-based therapies, event-free survival, and overall survival (OS). RESULTS: Trastuzumab inhibited tumor growth in mice injected with MCF7-NRG2c cells. Transmembrane NRG was frequently expressed in breast cancer patients. Overexpression of transmembrane NRG significantly correlated with a longer event-free survival and OS in patients with low or normal HER-2 expression who were treated with trastuzumab-based therapies but not in patients with HER-2 overexpression. CONCLUSION: We suggest that the spectrum of patients who may benefit from trastuzumab-based therapies may be widened to include patients with metastatic breast cancer without HER-2 amplification but who express transmembrane NRGs.

Identification and characterization of novel spliced variants of neuregulin 4 in prostate cancer.

PURPOSE: The neuregulin (NRG) 1, 2, and 3 genes undergo extensive alternative mRNA splicing, which results in variants that show structural and functional diversity. The aims of this study were to establish whether the fourth member of this family, NRG4, is expressed in prostate cancer, if it is alternatively spliced and whether any functional differences between the variants could be observed. EXPERIMENTAL DESIGN: The expression of NRG4 was determined using immunohistochemical staining of 40 cases of primary prostate cancer. Bioinformatic analysis and reverse transcription-PCR (RT-PCR) using NRG4 isotype-specific primers on a panel of normal and prostate cancer cell lines were used to identify alternatively spliced NRG4 variants. Expression of these variants was determined using isotype-specific antibodies. Transfection into Cos-7 cells of two of these green fluorescent protein-tagged variants allowed analysis of their subcellular location. Four of the variants were chemically synthesized and tested for their ability to activate the ErbB4 receptor. RESULTS: NRG4 was variably expressed in the cytoplasm in the majority of prostate cancer cases, and in a subset of cases in the membrane, high levels were associated with advanced disease stage. Four novel NRG4 splice variants (NRGA2, NRG4 B1-3) were characterized, where each seemed to have a different subcellular location and were also expressed in the cytoplasm of the prostate tumors. NRG4 B3 was also present in endothelial cells. In transfected cells, the A type variant (NRG4 A1) was localized to the membrane, whereas the B type variant (NRG4 B1), which lacks the predicted transmembrane region, had an intracellular localization. Only the variants with an intact epidermal growth factor-like domain activated ErbB4 signaling. CONCLUSION: NRG4 overexpression is associated with advanced-stage prostate cancer. The alternative splice variants may have different roles in cell signaling, some acting as classic receptor ligands and some with as-yet unknown functions.

Activation of ErbB2 by overexpression or by transmembrane neuregulin results in differential signaling and sensitivity to herceptin.

The ligands of the epidermal growth factor family and their receptors, the ErbB proteins, have been linked to the development of different types of cancer. Particular attention has focused on ErbB2, whose activation may occur by receptor overexpression or by ligand-induced oligomerization with other ErbB receptors. Whether these two modes of ErbB2 activation cause the same biological responses is unknown. Here, we uncovered important differences in the signaling, proliferation rates, and the response to anti-ErbB2 antibodies when comparing MCF7 cells expressing the ligand neuregulin, to MCF7 cells overexpressing ErbB2. Expression of neuregulin caused higher proliferation than ErbB2 overexpression. Transmembrane neuregulin expression was accompanied by constitutive activation of ErbB2, ErbB3, and ErbB4 receptors. ErbB2 overexpression caused tyrosine phosphorylation of ErbB2, whereas ErbB3 and ErbB4 were only slightly tyrosine phosphorylated. Autocrine transmembrane neuregulin also caused constitutive activation of several signaling pathways, such as the Erk1/2, Erk5, and Akt routes, which have been linked to breast cancer cell proliferation. Interestingly, expression of neuregulin increased p21 levels and this was required for the proliferation of MCF7 cells. Treatment with the anti-ErbB2 receptor antibody Herceptin had an inhibitory effect on proliferation only in cells expressing neuregulin but not on cells overexpressing ErbB2, and its inhibitory activity was accompanied by a decrease in p21. These results suggest that Herceptin may also be of help in the treatment of tumors in which neuregulin feeds the tumoral tissue.

Dynamic EGFR-Ras signalling in Drosophila leg development.

In Drosophila, as in many other animals, EGFR-Ras signalling has multiple developmental roles from oogenesis to differentiation. In leg development, in particular, it has been described to be responsible for the establishment of distal leg fates in a graded manner. Here, we investigate the patterns of expression of activators of EGFR-Ras signalling, as well as some of the effectors, in order to better understand the patterning of the distal leg, and to investigate further roles of this signalling pathway. These patterns, together with genetic data obtained by different mutant conditions for EGFR-Ras members and transgene expression, suggest two rounds of signalling in leg development. Early, the EGFR ligand Vein is the main player in distal leg patterning, possibly supported later by another ligand activated by Rhomboid. Later, in a second wave of signalling when all the proximal-distal leg fates have been specified, domains of EGFR/Ras activation appear inside each leg segment to regulate Notch-mediated joint development, and also some organs such as tendons and sensory organs. This second wave relies on a ligand activated by Rhomboid.

ErbB-4 and neuregulin expression in the adult mouse olfactory bulb after peripheral denervation.

ErbB-4 is expressed by the periglomerular and the mitral/tufted cells of the adult mouse olfactory bulb (OB) and in the present work we tested whether this expression is regulated by the olfactory nerve input to the OB. Reversible zinc sulphate lesions of the olfactory mucosa were made in adult mice and the deafferented OB analysed by immunohistochemistry, Western blotting and semiquantitative RT-PCR. Following deafferentation, the expression of erbB-4, erbB-2 and neuregulin-1 (NRG-1) mRNAs in the OB was altered. At early stages (7-14 days) after lesion the levels of expression of olfactory marker protein (OMP), tyrosine hydroxylase (TH), erbB-4 and NRG-1 mRNAs were decreased, whilst expression of erbB-2 increased and that of NRG-2 was not significantly altered. We observed at least two distinct time courses for these expression changes. The lowest amounts of mRNA for erbB-4 and NRG-1 were observed at day 7 after lesion, whilst mRNAs for TH and OMP were lowest at day 14. At day 28 after the lesion, when olfactory receptor neuron axons had reinnervated the olfactory bulb, the expression levels of OMP, TH, erbB-2, erbB-4 and NRG-1 were identical to control values. These results indicate that the expression of erbB-4 mRNA and protein in periglomerular and mitral cells is controlled by peripheral olfactory innervation. The tight correlation in NRG-1 and erbB-4 expression levels also suggests a possible functional link that deserves further exploration.

Neuregulins and erbB receptor expression in adult human oligodendrocytes.

We have previously reported that neonatal rat oligodendrocytes (OLGs) express and secrete neuregulins (NRGs) (Raabe et al., 1997). This laboratory has also shown that NRGs stimulate the differentiation of neonatal rat OLGs and that these cells express the erbB receptors for NRGs (Raabe et al., 1997). In this study, we have characterized NRG expression in adult human OLG cultures isolated from the temporal lobe resection of intractable epilepsy patients. Using immunocytochemistry and Western blotting, we find that adult human OLGs contain both the alpha and beta isoforms of NRGs. In addition, Western blots show that the adult human OLGs secrete both isoforms as N-glycosylated molecules. These cells also express all four erbB receptor subtypes, and possibly an activated erbB receptor. The observation that these cells synthesize and secrete their own NRGs, and possibly express a tyrosine-phosphorylated erbB receptor, is consistent with autocrine and/or paracrine signaling. Amplification of this signaling may provide a useful mechanism to stimulate differentiation of adult human OLGs in demyelinating disease.

Neuregulin induces the expression of mesodermal genes in the ectoderm of Xenopus laevis.

The primary patterning event in early vertebrate development is the formation of mesoderm and subsequent induction of the neural tube by the mesoderm. Some of the transforming growth factor (TGF)-beta family (Activin, Vg1) and the fibroblast growth factor (FGF) family molecules have been implicated for their roles in mesoderm induction. Here we show first the evidence that neuregulin, an epidermal growth factor (EGF)-like growth factor known for its role in neural and muscle differentiation, participates in mesoderm induction. Neuregulin could induce the ectopic expression of mesoderm specific gene Xbra in animal cap explants reared to the midgastrula stage, when animal caps dissected from late blastula were cultured with Neuregulin at a low concentration (10 ng/ml). In situ hybridization study showed that alpha-cardiac actin was expressed in animal caps that were treated with Neuregulin overnight. Skeletal and cardiac muscle specific genes such as MyoD family genes (myoD, MRF4, myf5) and SL1 as well as NCAM, a pan neural marker, were also ectopically expressed by treatment with Neuregulin. However, the expression of NCAM is presumed to be a secondary result of the initial mesoderm induction by Neuregulin. The temporal expression pattern of neuregulin during the early developmental stages was analyzed by RT-PCR in order to determine if neuregulin is expressed at the time of mesoderm induction. It has been found that the neuregulin transcript was already detected from the 16-cell stage (stage 5) and continued to be expressed till the tailbud stage (stage 25), the latest embryonic stage analyzed in this study. Considering that the mesoderm is induced at early blastula before the start of zygotic transcription, maternal neuregulin is expressed at the right time to participate in mesoderm induction. These data strongly suggest that neuregulin plays an important role in mesoderm induction.

Neuregulin and erbB receptor expression in normal and diseased human white matter.

Human white matter from non-neurologic cases, multiple sclerosis (MS) and other neurologic diseases (OND, inflammatory and non-inflammatory), was subjected to immunocytochemistry and Western blotting for expression of the neuregulin, glial growth factor-2 (GGF2), and its receptors, erbB2, erbB3 and erbB4. GGF2 has previously been shown to have mitogenic effects upon oligodendrocytes in vitro and an enhancing effect upon remyelination in animals with autoimmune demyelination. In all types of human white matter examined, expression of the ligand GGF2 and its three receptors was consistently found on oligodendrocytes, with higher levels being seen in cases of MS. Expression was also seen, albeit at lower levels, on astrocytes and microglial cells, the latter most commonly in MS and OND. In human lymph node tissue, some lymphocytes were positive for erbB2, erbB3 and erbB4. Western blots confirmed the presence of all three receptors in normal, MS and OND white matter. GGF2 and erbB receptor expression did not correlate with areas of remyelination and reactivity occurred throughout the tissue, with some increase in intensity at the edge of MS lesions. Examination of precursor oligodendrocyte immunoreactivity (with anti-PDGF-Ralpha and NG2), revealed widespread expression throughout both normal and diseased white matter. The presence of GGF2 and its receptors on oligodendrocytes and lymphocytes render this cell type a candidate for functional signaling via this pathway, perhaps in relationship to myelinating activity.