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At least 19 recordsLinked to original sources
PubMed1997

Intra-tumoral application of a heregulin-exotoxin-a fusion protein causes rapid tumor regression without adverse systemic or local effects.

Tumor toxins are recombinant, bifunctional proteins which comprise a tumor-cell-specific recognition domain and an enzymatic toxin domain. We have evaluated the in vivo effects of a tumor toxin that specifically recognizes the erbB-3 and erbB-4 receptors (HRG beta 1-ETA). High doses of HRG beta 1-ETA administered systemically (intracardially or intraperitoneally) caused acute liver necrosis and were lethal. The same dose of tumor toxins applied subcutaneously had no detectable histopathological effects. The anti-tumor activity of HRG beta 1-ETA was tested in nude mice with xenografts of a human breast tumor, MAXF1162. The MAXF1162 tumor grew rapidly upon s.c. implantation. Intra-tumoral application of HRG beta 1-ETA (7 times 5 micrograms over a period of 21 days) induced complete regression of tumors. At the time the treatment was terminated, no tumor cells were detectable microscopically. Evaluation of the liver of treated animals revealed no significant toxicity in the effective dose range. These experiments indicate that tumor toxins can become valuable for local tumor treatment and for reduction of tumor burden.

Animals↗
PubMed1996

Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells.

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

Animals↗
PubMed1995

ErbB3 and ErbB2/neu mediate the effect of heregulin on acetylcholine receptor gene expression in muscle: differential expression at the endplate.

Motor neurons modulate acetylcholine receptor (AChR) gene expression in skeletal muscle by two signalling pathways: the transmitter-evoked depolarization of muscle membrane inhibits AChR gene transcription throughout the myofibre presumably via activation of a serine/threonine kinase, while the transcription rates of AChR genes in the synaptic region are increased by nerve-derived trophic factors including AChR-inducing activity (ARIA). To gain further insight into these interactions we characterized the receptor for heregulin (HRG)/ARIA in muscle. We showed that HRG increases AChR alpha-subunit mRNA levels via tyrosine phosphorylation of ErbB3 and ErbB2/neu in myotubes. The protein tyrosine phosphatase inhibitor, pervanadate, potentiated the responses to HRG that were in turn blocked by the tyrosine kinase inhibitor erstatin, indicating the relevance of tyrosine phosphorylation to these events. The effects of HRG were inhibited by enhanced cellular serine/threonine phosphorylation which has been implicated in the repression of AChR genes by electrical activity. Immunocytochemical analysis of adult rat muscle revealed that while ErbB2/neu is present throughout the entire surface of the myofibre membrane, ErbB3 expression is exclusively restricted to the endplate suggesting its involvement in synapse-specific transcription of AChR genes by HRG/ARIA.

Animals↗
PubMed1995

Human Schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion.

Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts or HASCs (which contain both SCs and fibroblasts). Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRG beta 1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 microM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P0 (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies.

Adult↗
PubMed1996

Analysis of heregulin-induced ErbB2 phosphorylation with a high-throughput Kinase receptor activation enzyme-linked immunosorbant assay.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

Animals↗
PubMed1994

Isolation of a heregulin-like growth factor secreted by estrogen receptor-negative MDA-MB-231 human breast cancer cells that stimulates estrogen receptor-positive cells.

A 52 kDa peptide growth factor secreted by the estrogen receptor (ER)-negative human breast cancer cell line MDA-MB-231 was purified to homogeneity. It induced transient membrane ruffling, lamellipodia formation, cell motility and proliferation exclusively of ER-positive human breast cancer cells. Partial sequencing revealed a high homology to the protein family of heregulins. However, the obtained amino acid sequences of the new factor were not completely identical to any of the members of the heregulin family. This finding together with the observation that the successful purification protocol was significantly different from that used to isolate other members of the heregulin family indicate the isolation of a novel heregulin-like proliferation factor for ER-positive human breast cancer cells.

Amino Acid Sequence↗
PubMed1995

Selective regulation of steroid receptor expression in MCF-7 breast cancer cells by a novel member of the heregulin family.

A 52 kDa heregulin secreted by estrogen receptor (ER)-negative human breast cancer cells induced rapid growth of ER-positive MCF-7 breast cancer cells with a stimulatory effect observed at 10(-11)M. This heregulin down-regulated the message for ER in MCF-7 cells within 24 hours after stimulation. Similarly the ER protein was down-regulated within 24 to 48 hours after stimulation of cells. However, this down-regulation occurred without activation of the ER, since the progesterone receptor (PR) level of cells stimulated with the 52 kDa heregulin did not increase over the time period measured. As a control, estradiol down-regulated and activated ER as shown by a pronounced increase in PR content of MCF-7 cells. This finding indicates an important role of this heregulin in the down-regulation of ER in estrogen-dependent human breast cancer cells.

Breast Neoplasms↗
PubMed1996

The EGF receptor binding of recombinant heregulinbeta1/EGF hybrids is blocked by heregulin residue glutamate 195.

Defined sequences from the EGF-like domain of human heregulin-beta1 (HRGbeta1) were recombined with a synthetic gene for human epidermal growth factor (hEGF) in an attempt to locate receptor-specific determinants within the HRGbeta1 molecule that blocks its inappropriate association with the EGF receptor (EGFR). Receptor competition assays detected only minor changes in relative EGFR affinity for those hybrids containing up to 12 N-terminal HRGbeta1 residues. However, extending the N-terminal substitution to include 20 HRGbeta1 residues resulted in a 100-fold drop in relative EGFR binding. Both interruption of the major beta-sheet structure of hEGF by insertion of a three amino acid loop present in HRGbeta1 and replacement of nearly the entire C-terminal hEGF subdomain with segments of HRGbeta1 sequence resulted in a 5-fold decreased EGFR affinity. The results presented here demonstrate that while a substantial portion of the hEGF and HRGbeta1 protein sequences were nearly interchangeable with regard to EGFR binding, the introduction of HRGbeta1 residue Glu195 effected a major decrease in EGFR binding.

Amino Acid Sequence↗
PubMed1995

Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle.

Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.

Aging↗
PubMed1996

Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon switch.

During the development of the mammalian neuromuscular junction, acetylcholine receptors (AChRs) become localized to the postsynaptic muscle membrane. As this process nears completion, the fetal form of the receptor, containing a gamma subunit (composition alpha 2 beta gamma delta) is gradually replaced by an epsilon subunit-containing adult form (alpha 2 beta epsilon delta). To understand how this transition is controlled, we compared the expression and regulation of the AChR gamma and epsilon subunits in developing, adult, and cultured muscles. Immunostaining with subunit-specific antibodies showed that replacement of gamma subunit- by epsilon subunit-containing AChRs occurs largely during the first postnatal week in fast-twitch muscles, and occurs homogeneously throughout individual endplates. In the slow-twitch soleus, however, this transition is delayed, and in the multiply innervated slow fibers of extraocular muscle, gamma subunit expression persists into adulthood. The transcriptional bases of the AChR subunit transition, and of these intermuscular variations, were demonstrated in mice bearing transgenes containing promoter elements from the AChR gamma and epsilon subunit genes, each coupled to a nuclear-localized beta-galactosidase (nlacZ) reporter. We show that transgene expression is stimulated by the nerve-derived inducer of AChR expression, ARIA, in myotubes cultured from gamma-nlacZ as well as epsilon-nlacZ mice. However, the expression of gamma-nlacZ, but not epsilon-nlacZ, is increased by treatment of myotubes with TTX, and the ARIA sensitivity of gamma-nlacZ is dependent on the electrical state of the myotube. Thus, the promoters of the gamma and epsilon subunit genes may integrate ARIA- and activity-dependent signals in different ways to generate their complementary patterns of expression.

Animals↗
PubMed1997

Heregulin degradation in the absence of rapid receptor-mediated internalization.

Heregulin receptors are unable to mediate the rapid internalization of bound ligand as demonstrated in cells transfected with chimeric or wild-type ErbB-2, -3, or -4 receptors (Baulida et al., 1996, J. Biol. Chem. 271, 5251-5257; Pinkas-Kramanski et al., 1996, EMBO J. 15, 2452-2467). This observation is now extended to include mammary carcinoma cell lines (SK-BR-3 and MDA-543) which express endogenous ErbB-2 and ErbB-3 receptors. Also, the fate of receptor-bound heregulin is examined. While receptor-bound heregulin is not rapidly internalized, the ligand is subject to a slow process of inactivation and degradation, which requires heregulin incubation at 37 degrees C with cells that express heregulin receptors. The degradation of heregulin is blocked to a significant extent by chloroquine, an inhibitor of endosome fusion with lysosomes, indicating that heregulin is slowly internalized and degraded. However, this process is not sufficiently rapid to produce ligand-dependent down-regulation of heregulin receptors.

3T3 Cells↗
PubMed1997

Schwann cells express NDF and SMDF/n-ARIA mRNAs, secrete neuregulin, and show constitutive activation of erbB3 receptors: evidence for a neuregulin autocrine loop.

Cultured Schwann cells secreted low levels (30 pg/ml/1.5 x 10(6) cells) of a 45-kDa neuregulin protein and showed constitutive activation of a neuregulin receptor, Erb-B3, suggesting the existence of an autocrine loop involving neuregulins in Schwann cells. RT-PCR analyses indicated that Schwann cells and fibroblasts in culture produced SMDF/n-ARIA and NDF but not GGF neuregulin messages. Schwann cell and fibroblast neuregulin messages encoded both beta and alpha domains; Schwann cell transcripts encoded only transmembrane neuregulin forms while fibroblast messages encoded transmembrane and secreted forms. SMDF/n-ARIA and NDF messages were also expressed in early postnatal rat sciatic nerve, suggesting a role for neuregulins in peripheral nerve development. An anti-neuregulin antibody inhibited the mitogenic response of Schwann cells to cultured neurons and to extracts of cultured neurons or embryonic brain, consistent with the accepted paracrine role of neuregulins on Schwann cells. Surprisingly, the same antibody inhibited Schwann cell proliferation stimulated by several unrelated mitogens including bFGF, HGF, and TGF-beta1. These data implicate both paracrine and autocrine pathways involving neuregulin form(s) in Schwann cell mitogenic responses.

Animals↗
PubMed1997

Chick muscle expresses various ARIA isoforms: regulation during development, denervation, and regeneration.

Acetylcholine receptor inducing activity (ARIA) is a glycoprotein released from the motor neuron to stimulate the synthesis of acetylcholine receptors (AChRs) on the postsynaptic muscle fiber. Transcripts encoding ARIA were detected not only in brain but also in muscle, and immunohistochemical staining showed that muscle-derived ARIA was restricted to the neuromuscular junctions. RT-PCR analysis revealed three biological active isoforms of ARIA in chick muscle, namely ARIA beta 1, ARIA alpha 2, and ARIA beta 2 that were classified based on their variation in the carboxylterminus of the EGF-like domain. The expression of these ARIA isoforms in muscle change during development denervation, and nerve regeneration. ARIA beta 1, ARIA alpha 2, and ARIA beta 2 were expressed in embryonic and young chick muscles, while ARIA beta 1 was the major isoform expressed in adult chicken. The embryonic-like expression of ARIA alpha 2 and ARIA beta 2 was induced after nerve injury in adult chicken. However, the prominent expression of ARIA beta 1 in adult-like profile was restored after nerve regeneration. A splicing variation in the region between Ig-like and EGF-like domains of ARIA was also revealed; a zero-amino acid insertion (ARIASP0), a 17-amino acid insertion (ARIASP17), or a 34-amino acid insertion (ARIASP34) were identified. Unlike ARIASP0, the expression of ARIASP17 and ARIASP34 was found in muscle and sciatic nerve only. The expression of ARIASP0, ARIASP17, and ARIASP34 in chick muscle remained unchanged during development and after nerve injury. Moreover, the specific expression of these ARIA isoforms in cultured myotubes was not affected by drug treatments or by coculturing with neurons. Our findings provide strong evidence that muscle ARIA may play an important role in the formation of neuromuscular junctions.

Aging↗
PubMed1997

Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence.

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.

Alkaline Phosphatase↗
PubMed1994

Use of capillary electrophoresis-electrospray ionization mass spectrometry in the analysis of synthetic peptides.

We have constructed a capillary electrophoresis (CE) system with UV detection and have successfully interfaced it to an electrospray ionization mass spectrometry (ES-MS) system. A synthesized fragment of heregulin-beta (212-226) was thought to be a single component by re-injection into an HPLC system, but results from CE-UV-ES-MS indicated that a dehydration product was present in the desired peptide sample. A synthetic heregulin-alpha (177-241) was isolated by preparative HPLC, but re-injection on an analytical system indicated a tailing peak. CE-UV-ES-MS indicated a mixture whose two major components were of the same nominal molecular mass (within experimental error), suggesting the presence of an isomer or a deamidation product. The results show that CE-UV-ES-MS can be used as an orthogonal analytical technique to solve practical problems encountered in peptide synthesis laboratories.

Amino Acid Sequence↗
PubMed1993

ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family.

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human here-gulin, ligands for the receptor tyrosine kinase encoded by the neu (c-erbB2, HER2) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

Animals↗
PubMed1995

Truncated form of pro-acetylcholine receptor-inducing activity (ARIA) induces AChR alpha-subunit but not AChE transcripts in cultured chick myotubes.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR). We used reverse transcription-polymerase chain reaction (RT-PCR) to obtain a partial pro-ARIA cDNA clone from methonine-1 to serine-358 including the full functional sequence of ARIA. Northern blot analysis of mRNAs from the embryonic chick brain and muscle showed a transcript with a size of approximately 7.5 kb. The cloned cDNA was subcloned into an eukaryotic expression vector and stably transfected into human embryonic kidney 293 cells. The conditioned medium of the transfected cells was found to increase the level of transcript encoding for the alpha-subunit of AChR by approximately 4.4-fold, but not for acetylcholinesterase (AChE), in the cultured chick myotubes.

Acetylcholinesterase↗