Neuregulin-4 is associated with plasma glucose and increased risk of type 2 diabetes mellitus.

BACKGROUND: Neuregulin-4 is a cytokine with many functions and is primarily produced by fat tissue. AIM OF THE STUDY: The aim of the study was to observe the relationship between serum neuregulin-4 levels and diabetes regulation in type 2 diabetes mellitus (T2DM), and to compare neuregulin-4 levels of diabetic subjects with those in healthy controls. METHODS: Patients with T2DM were included to the study. Healthy subjects were enrolled as controls. Subjects with T2DM with glycated haemoglobin (HbA1c) <7% were classed as well controlled and those with HbA1c >7% were classed as poorly controlled. Neuregulin-4 levels of the study and control groups were compared. RESULTS: The neuregulin-4 levels of the poorly controlled T2DM, well-controlled T2DM and control groups were significantly different (p = 0.005). Neuregulin-4 was significantly correlated with fasting plasma glucose (r = 0.247, p = 0.002) but not with HbA1c. In a regression analysis model, 0.1 point elevation in neuregulin-4 levels increased the rate of existence of T2DM 4.4-fold (odds ratio 4.4, 95% confidence interval 1.26-15.1; p = 0.02). CONCLUSION: Neuregulin-4 is significantly increased in patients with T2DM compared with control subjects, which means that it could be a marker of T2DM. Since neuregulin-4 was correlated with fasting glucose, we suggest that elevated neuregulin-4 could predict poor control in T2DM for short periods when HbA1c is not useful.  Moreover, one unit elevation in neuregulin-4 (0.1 ng/ml) increases the rate of existence of T2DM 4.4-fold, independently from other variables.

[Extracellular matrix of cerebral tumors with different invasiveness]. / Különbözo invazivitású agydaganatok extracelluláris mátrixának expressziója.

OBJECTIVES: Ineffective surgical and radiotherapy of glioblastoma is mainly due to its intensive infiltrating behavior. Contrarily, brain metastases of anaplastic carcinomas are well-circumscribed intracerebral lesions that can be easily exstirpated in most cases. The molecules of the extracellular matrix (ECM) play a pivotal role in the peritumoral infiltration. In this study the mRNA expression of the ECM components was investigated in two types of intracerebral malignoma with different invasion activity. Our aim was to identify the ECM molecules that are responsible for the different intensity of peritumoral infiltration of tumors from different origin. METHODS: The mRNA expression of twenty-three ECM molecules was determined by quantitative reverse transcriptase polymerase chain reaction. Four pieces of glioblastoma and four pieces of intracerebral lung adenocarcinoma metastasis from neurosurgical operation were investigated. Immunohistochemical investigations were performed in case of five molecules. RESULTS: The mRNA expression of nine molecules (brevican, neurocan, neuroglycan-C, syndecan-1,2,4, tenascin-C, versican and matrix-metalloproteinase-[MMP]2) differed significantly by comparison of the two tumor types. By immunohistochemistry, neurocan, syndecan, versican and MMP-2 showed alteration in staining intensity according to the mRNA expression, while MMP-9 showed higher staining intensity in the metastatic tumor. CONCLUSIONS: The identified molecules can play an important role in the different infiltration activity of tumors from different origin. Thus these ECM-components could serve as targets for anti-invasion therapy in the future.

Characterization of the cell membrane-associated products of the Neuregulin 4 gene.

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Dynamic expression of Erbb pathway members during early mammary gland morphogenesis.

Similar to other epithelial appendages, mammary anlagen progress from stratified epithelium through placode and bud stages. Embryonic mammary morphogenesis is elicited by a combination of local cell migration, adhesion changes and proliferation, and these same developmental processes impact breast cancer etiology. The Erbb signaling network plays important roles in postnatal mammary gland morphogenesis and carcinogenesis. Neuregulin3 (Nrg3), an Erbb family ligand, has recently been shown to be involved in the specification of mammary glands in mice. To further examine the possible involvement of other Erbb family members and their ligands in early mammary morphogenesis, we have characterized their expression patterns during this process. We used whole mount in situ hybridization to analyze the expression patterns of these genes at stages prior to and during mammary placode formation. Immunohistochemistry was used to examine expression patterns at later bud stages. The Neuregulin ligands, Nrg1, Nrg2, Nrg3, Nrg4 and the receptors, Erbb1, Erbb2, Erbb3, Erbb4, were expressed either at stages prior to morphological appearance of the mammary placode or from the time that the placode is first morphologically distinct through to later bud stages. The expression patterns presented here suggest that multiple members of this signaling network are potential mediators of early mammary morphogenesis.

ErbB/HER ligands in human breast cancer, and relationships with their receptors, the bio-pathological features and prognosis.

BACKGROUND: The aim of this study is to provide an expression profile of ErbB/HER ligands in breast cancer. We analysed the relationships with their receptors, the bio-pathological features and prognosis. PATIENTS AND METHODS: Epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), amphiregulin (AREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG) and neuregulins1-4 (NRG1-4) were quantified in 363 tumours by real-time reverse transcription-polymerase chain reaction using TaqMan probes. RESULTS: Ligands were detected in 80%-96% of the cases, except NRG3 (42%) and EREG (45.5%). At least one ligand was expressed in 304 cases (cut-off: upper quartile). Almost all combinations of receptor and ligand co-expressions were observed, but TGFalpha is preferentially expressed in tumours co-expressing EGFR/HER3, NRG3 in those co-expressing EGFR/HER4, AREG and EREG in those co-expressing HER2/HER4. EGF and AREG were associated with estradiol receptors, small tumour size, low histoprognostic grading, high HER4 levels. TGFalpha, HB-EGF and NRG2 were negatively related to these parameters. In Cox univariate analyses, EGF was a prognostic factor. CONCLUSION: Our study demonstrates that (i) ErbB/HER ligands, including BTC and EREG, are expressed in most breast cancers; and (ii) TGFalpha, HB-EGF and NRG2 high expressions are related to the biological aggressiveness of the tumours.

Co-expression of neuregulins 1, 2, 3 and 4 in human breast cancer.

We have produced antibodies to the NRG2-alpha, NRG2-beta, NRG3 and NRG4 proteins and used these, and previously described antibodies to NRG1-alpha and NRG1-beta, to detect expression of each ligand by immunocytochemical staining in a series of 45 breast cancers. Each protein was expressed in a proportion of cases. Statistical analysis suggested that expression of one factor was associated with a high probability that other members of the family were co-expressed. NRG2-alpha expression was associated with node positivity (p-value = 0.005). The mRNAs for NRG1, 2, 3 and 4 were found in established breast cancer cell lines and NRG1, 2 and 3 mRNAs were detected in primary breast cancers. Expression of NRG4 mRNA was shown by in situ hybridization in sections from primary breast cancers. This data demonstrates that each member of the NRG family of ligands is expressed in breast cancer and suggests that they may be involved in regulating cell behaviour.

Expression of neuregulin and activation of erbB receptors in vestibular schwannomas: possible autocrine loop stimulation.

HYPOTHESIS: We sought to determine whether vestibular schwannomas are capable of producing and responding to the glial growth factor neuregulin. BACKGROUND: Neuregulin is a neuronally derived trophic factor that interacts with erbB2 and erbB3 receptors on Schwann cells and is required for normal Schwann cell proliferation, survival, and development. Vestibular schwannomas grow several millimeters or even centimeters away from adjacent axons, suggesting that vestibular schwannomas do not depend critically on axons for their proliferation or survival. This raises the possibility that vestibular schwannomas themselves produce and respond to trophic factors in an autocrine fashion. METHODS: Pathologic specimens from eight patients undergoing microsurgical removal of vestibular schwannomas and one patient undergoing vestibular nerve section were immunostained with anti-neuregulin, anti-erbB2, anti-erbB3, and anti-phosphorylated-erbB2 antibodies. Three patients had received previous gamma knife radiation therapy and two patients had neurofibromatosis Type 2. RESULTS: The Scarpa ganglion neurons express neuregulin, and normal vestibular Schwann cells express erbB2 and erbB3. Vestibular schwannomas from all eight patients demonstrated neuregulin, erbB2, and erbB3 immunoreactivity. In addition, all vestibular schwannomas demonstrated immunoreactivity to anti-phosphorylated-erbB2 antibody that only recognizes erbB2 when it is phosphorylated, or activated. CONCLUSION: These results demonstrate that vestibular schwannomas express neuregulin and its receptors, erbB2 and erbB3. Because erbB2 exists in an activated state, as evidenced by phosphorylated-erbB2 immunoreactivity, it likely responds to the locally produced neuregulin. This suggests the possibility that vestibular schwannomas produce and respond to neuregulin in an autocrine fashion.

ErbB signaling regulates lineage determination of developing pancreatic islet cells in embryonic organ culture.

The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of beta-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.

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 found in cultured-sciatic nerve conditioned medium causes neuronal differentiation of PC12 cells.

The present work deals with the search and identification of the molecule or combination of molecules, present in a medium conditioned by cultured rat-sciatic nerves (CM), able to cause neuronal differentiation of PC12 cells. The molecular mass range of the active fraction, as well as the thermostability and heparin affinity of the active component found in previous work, all characteristics shared with neuregulin (NRG) family members, led us to search for a NRG protein in the CM. Nerves were previously cultured for 8 days and the CM collected every 24 h, the following 3 days. The CM was concentrated (30,000 NMWL) and fractionated by quaternary ammonium chromatography and Cibacron blue affinity chromatography. The most active fraction B1.2 was further characterized by heparin affinity chromatography, size exclusion HPLC, Western blotting and immunoprecipitation. Results reveal abundance of NRG mRNA in the cultured nerves, presence of a 54 kDa NRG protein in the CM that increases along fractionation, and progressive diminution of fraction B1.2 differentiation activity on PC12 cells by gradual removal of the NRG protein by immunoprecipitation. The abundance of Schwann cells and the lack of axons in the cultured nerves suggest Schwann cells as the main NRG source, to which fibroblasts and perineurial cells might contribute.