Biomedical subjects

M X Sliwkowski

Publications and source records attributed to M X Sliwkowski.

At least 37 records · Page 2Linked to original sources

PubMed1998

Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis.

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.

Alanine↗

PubMed1998

Selection of heregulin variants having higher affinity for the ErbB3 receptor by monovalent phage display.

Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.

Amino Acid Sequence↗

PubMed1998

Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression.

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.

Animals↗

PubMed1997

Gamma-heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175.

A novel neuregulin isoform, termed gamma-HRG, was cloned and characterized from the human breast cancer cell line, MDA-MB-175. As observed with other neuregulins, gamma-HRG, is a product of alternative mRNA splicing of the neuregulin gene. Gamma-HRG contains the EGF-like and immunoglobulin-like domains that are commonly found in other family members, but lacks a transmembrane and cytoplasmic region. The new isoform possesses a unique N-terminal region that includes a hydrophobic domain that may function as a secretion signal. A purified recombinant version of gamma-HRG competes for binding to soluble ErbB3- and ErbB4-IgG fusion proteins with affinities similar to those observed for rHRGbeta1(177-244). Gamma-HRG has a wide distribution in mesenchymal or neuronal tissues but in contrast to other neuregulins, it is not present in breast, lung, liver and small intestine. Expression of gamma-HRG with its cognate receptors, ErbB3 and ErbB2 suggested that the growth of the MDA-MB-175 cell line might be a result of the autocrine stimulation of a growth factor signaling pathway. Treatment of MDA-MB-175 cells with an anti-ErbB2 monoclonal antibody that interferes with the ligand-dependent formation of ErbB2-ErbB3 heterodimer complexes shows a strong growth inhibitory effect on this cell line. Moreover, incubation with a receptor-IgG fusion protein that neutralizes secreted gamma-HRG, also inhibits cell growth. These data suggest that the secretion of gamma-HRG by MDA-MB-175 cells leads to the formation of a constitutively active receptor complex and stimulates the growth of these cells in an autocrine manner.

Alternative Splicing↗

PubMed1997

Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that binds and activates ErbB4.

We describe the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. We show that the EGF-like domain of NRG3 binds to the extracellular domain of ErbB4 in vitro. Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4.

Amino Acid Sequence↗

PubMed1997

Association of csk-homologous kinase (CHK) (formerly MATK) with HER-2/ErbB-2 in breast cancer cells.

Protein-tyrosine kinases, such as HER-2/ErbB-2, have been specifically linked to breast cancer. The Csk-homologous kinase (CHK), formerly MATK, is a tyrosine kinase that contains the Src homology 2 and 3 (SH2 and SH3) domains and demonstrates homology ( approximately 50%) to the Csk tyrosine kinase. Like Csk, CHK is able to phosphorylate and inactivate Src family kinases. In this report, we investigated whether CHK is expressed in breast cancer tissues and whether it participates in the ErbB-2 signaling pathway in T47D and MCF-7 breast cancer cell lines. Immunostaining of the CHK protein in breast tissues demonstrated that primary invasive ductal carcinomas, stage II (13 of 15 cases) and stage I (8 of 15 cases), expressed the CHK protein, while this protein was not detected in the adjacent normal tissues from the same patients. To study the role of CHK in the ErbB-2 signaling pathway, glutathione S-transferase fusion proteins containing the SH2 and SH3 domains of CHK were generated. CHK-SH2 and CHK-SH3-SH2, but not CHK-SH3 or CHK-NH2-SH3, precipitated the tyrosine-phosphorylated ErbB-2 upon stimulation with heregulin. EGF or interleukin-6 stimulation of T47D cells failed to induce CHK-SH2 association with ErbB-2, the EGF-receptor, or the interleukin-6 receptor. In vivo association of the tyrosine-phosphorylated ErbB-2 with CHK was observed in co-immunoprecipitation studies with anti-CHK antibodies. EGF-R, ErbB-3, and ErbB-4 were not detected in the CHK immunoprecipitates or in the precipitates of the GST-SH2 fusion proteins of CHK, suggesting that the association of CHK with ErbB-2 upon heregulin stimulation is receptor-specific (ErbB-2) and ligand-specific (heregulin). These results indicate that CHK might participate in signaling in breast cancer cells by associating, via its SH2 domain, with ErbB-2 following heregulin stimulation.

Glycoproteins↗

PubMed1997

Heregulins and the ErbB-2/3/4 receptors in gliomas.

The activation of autocrine loops involving proto-oncogene related receptor tyrosine kinases has led to the analysis of a large number of growth factor systems in human glioma specimens and cell lines. The ErbB-2 system, also called HER-2 or neu, is analogous to the epidermal growth factor receptor system (EGF-R, ErbB-1). Neuregulins consist of a large family of proteins arising from alternative mRNA splicing of a single gene located at 8p22-p11. Activation of ErbB-2 by neuregulins occurs in heterodimeric complexes with ErbB-3 and ErbB-4. A panel of human glioma cell lines, which had previously been analyzed for ErbB-2 expression, was examined for ErbB-3 and ErbB-4 expression. Coordinate expression of ErbB-2, -3 or -4 was not observed in these cell lines. Despite the presence of a complete system capable of signaling in about half the cell lines, no constitutive activation of ErbB-2, -3 or -4 was observed, and autophosphorylation of ErbB-2 in response to heregulin was observed only in one cell line from the panel, NCE-G84. Moreover, the addition of recombinant heregulin or antibodies capable of disrupting ErbB-2/ErbB-3 complexes had no effect on cell proliferation. We conclude that the role of neuregulins and its receptors in the control of glioma cell proliferation may be limited or may be context dependent on in situ conditions which are lost in vitro. Alternatively, neuregulins may be involved in cell differentiation or survival in the central nervous system. Data supporting these conclusions are described in more detail herein.

Brain Neoplasms↗

PubMed1996

Insulin regulates heregulin binding and ErbB3 expression in rat hepatocytes.

The heregulin-ErbB system of ligands and receptors are newly described epidermal growth factor (EGF) and EGF receptor-related proteins that regulate growth, differentiation, and gene expression in numerous cell types. This study describes a receptor for heregulin beta-1 (HRGbeta1) on cultured rat hepatocytes and an inhibitory influence of insulin on HRGbeta1 binding. HRGbeta1 (30 nM) stimulated DNA synthesis 2-fold and was not augmented by insulin as is the case with EGF receptor ligands. A labeled peptide corresponding to the EGF domain of HRGbeta1 bound to a single population of 19,600 +/- 1,800 binding sites/cell with a Kd of 360 +/- 22 pM. Cross-linking experiments showed binding of HRGbeta1 to ErbB3 but not ErbB2 or ErbB4. HRGbeta1 induced phosphorylation of ErbB3 and decreased ErbB3 protein levels, suggesting that HRGbeta1 activates signaling through the ErbB3 receptor and influences receptor trafficking. Following plating, [125I]HRGbeta1 binding and ErbB3 protein levels increased 8- and 3-fold, respectively, over the first 12 h in culture. These increases required de novo protein synthesis and were inhibited with 50 nM insulin resulting in 3500 binding sites with a Kd of 265 pM. These data suggest that the heregulin-ErbB system can regulate liver functions and may be linked to the metabolic and nutritional status of the animal.

Animals↗

PubMed1996

Heregulin is rapidly translocated to the nucleus and its transport is correlated with c-myc induction in breast cancer cells.

Heregulins (neuregulins) are a family of proteins known to interact and activate the receptor tyrosine kinases ErbB2 in association with ErbB3 or ErbB4. Using immunofluorescence microscopy, electron microscopy autoradiography, and SDS-PAGE analysis of nuclear fractions, we show that the heregulin-beta1(1-244) isoform is rapidly internalized and translocated to the nucleus of SK-BR-3 breast cancer cells as an intact molecule. Heregulin-beta 1(1-244) treatment up-regulated expression of c-myc mRNA and protein, which was also observed to undergo its own translocation from the cytosol to the nucleus. c-myc thus appears to be a cellular target gene of HRGbeta 1(1-244), and its induction may be related to the nuclear translocation of heregulin.

Breast Neoplasms↗

PubMed1996

High-resolution solution structure of the EGF-like domain of heregulin-alpha.

The solution structure of the 63-residue heregulin-alpha (HRG-alpha) epidermal growth factor (EGF)-like domain, corresponding to residues 177-239 of HRG-alpha, has been determined to high resolution using data from two-dimensional and three-dimensional homo- and heteronuclear NMR spectroscopy. The structure is based on a total of 887 internuclear distance and dihedral restraints derived from data obtained using unlabeled and uniformly 15N-labeled protein samples, at pH 4.5, 20 degrees C. A total of 20 structures were calculated using a hybrid distance geometry-simulated annealing approach with the program DGII, followed by restrained molecular dynamics using the program DISCOVER. The average maximum violations are 0.12 +/- 0.01 angstroms and 1.4 +/- 0.3 degrees for distance and dihedral restraints, respectively. The backbone (N,C(alpha),C) atomic rms distribution about the mean coordinates for residues 3-23 and 31-49 is 0.29 +/- 0/07 angstroms. The N-and C-terminal residues (1-2 and 50-63) and 24-30 are disordered. Comparison of the HRG-alpha EGF-like domain structure with the previously determined structure of human EGF [Hommel et al. (1992) J. Mol. Biol. 227, 271-282] reveals a high degree of structural similarity; excluding the N-terminal region (residues 1-13), the disordered phi-loop region (residues 24-30) that contains a three-residue insertion in HRG-alpha relative to hEGF, and the disordered C-terminal region (residues 50-63), the C(alpha) alignment between the HRG-alpha and hEGF minimized mean structures has a rms difference of approximately 1 angstrom. In HRG-alpha the N-terminal residues 2-6 form a well-defined beta strand rather than being disordered as found for hEGF. This structural difference correlates with functional data which suggest that the N-terminal region of the HRG-alpha EGF-like domain is responsible for the observed receptor specificity differences between HRG-alpha and EGF.

Amino Acid Sequence↗

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↗

PubMed1996

Identification of Gas6 as a growth factor for human Schwann cells.

Schwann cells are one of the principal components of the peripheral nervous system. They play a crucial role in nerve regeneration and can be used clinically in the repair of injured nerves. We have established serum-free, defined culture conditions that rapidly expand adult human Schwann cells without fibroblast growth. We find that Gas6, a ligand for the Axl and Rse/Tyro3 receptor protein tyrosine kinase family, stimulates human Schwann cell growth, increasing both cell number and thymidine incorporation. Gas6 has synergistic effects with the other known human Schwann cell mitogens, heregulin/glial growth factor and forskolin. Addition of Gas6 causes phosphorylation of Axl and Rse/Tyro3 simultaneously and results in ERK-2 activation. A combination of Gas6 with heregulin and forskolin, on a defined background, supports maximal Schwann cell proliferation, while preserving the typical Schwann cell morphology and expression of the Schwann cell markers S-100, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor. Gas6 mRNA is present in both spinal motor neurons and large neurons of the dorsal root ganglia, and neural injury has been reported to upregulate Rse/Axl in the schwann cell. This is the first demonstration of a potentially important biological role for the human Gas6/Rse-Axl system.

Animals↗

PubMed1996

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Animals↗

PubMed1996

Establishment of Schwann cell lines from normal adult and embryonic rat dorsal root ganglia.

Schwann cells, an important component of the peripheral nervous system, interact with neurons to mutually support growth and replication in the embryo and survival and differentiated function in the adult. The ability of adult Schwann cells to re-enter the cell cycle after nerve injury is crucial to their role in nerve repair. This ability suggests that it should be possible to obtain non-transformed, cell lines which maintain the characteristics of proliferating adult Schwann cells in vivo, as well as obtaining Schwann cells from rapidly dividing embryonic tissues. One approach to obtaining normal functionally differentiated cell lines has been to start primary cultures in serum-free medium containing growth factors and attachment proteins specifically selected to favor the replication of the cell type of interest. By culturing dispersed dorsal root ganglia on laminin, in serum-free medium with hormones and growth factors, we repeatedly generate homogenous Schwann cell cultures which yield normal Schwann cell lines from the dorsal root ganglia (DRG) of both embryonic and adult rats. These cells maintain the phenotype of Schwann cells as determined by morphology and staining for GFAP, S100, p75 NGF receptor, laminin, and MAG production in co-culture with DRG neurons.

Animals↗

PubMed1995

HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

Animals↗

PubMed1995

Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2.

The ability of sensory axons to stimulate Schwann cell proliferation by contact was established in the 1970s. Although the mitogen responsible for this proliferation has been localized to the axon surface and biochemically characterized, it has yet to be identified. Recently a family of proteins known as heregulins (HRGs) has been isolated, characterized, and shown to interact with a number of class 1 receptor tyrosine kinases, including the erbB2, erbB3, and erbB4 gene products. These factors include glial growth factor, a Schwann cell mitogen. We have tested the effects of antibodies against components of this system (HRG beta 1 and p185erbB2) in cocultures of rat sensory neurons and human (or rat) Schwann cells to elucidate the role of these proteins in axon-induced Schwann cell proliferation. 2C4, a monoclonal antibody specific for the human p185erbB2 receptor tyrosine kinase, bound to the surface of human Schwann cells and reduced human Schwann cell incorporation of [3H]thymidine by > 90% compared with untreated controls in this coculture system. This antibody had no effect on rat Schwann cell incorporation of [3H]thymidine under similar conditions. A polyclonal antibody raised against HRG beta 1 reduced human and rat Schwann cell incorporation of [3H]thymidine by nearly 80% and up to 49%, respectively, relative to controls. These results imply that a HRG, or a HRG-like molecule, is a component of the axonal mitogen. This mitogen is presented to Schwann cells by axons and induces proliferation through an interaction that involves p185erbB2 on Schwann cells.

Animals↗

PubMed1995

Regulation of the acetylcholine receptor epsilon subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation.

Structural specialization of the postsynaptic skeletal muscle membrane is in part mediated by the motor neuron-induced transcriptional regulation of synaptic muscle nuclei. ARIA, a factor that stimulates production of acetylcholine receptors (AChRs), is a candidate signaling molecule for such regulation. Here we examine the transynaptic inducing potential of this polypeptide factor. ARIA immunoreactivity is detectable at synaptic sites in vivo. In vitro, recombinant heregulin beta 1 (rHRG beta 1), the human homolog of ARIA, induces expression of the AChR epsilon gene, the subunit most sensitive to synaptic input. The inducing property of rHRG beta 1 is demonstrated most dramatically in primary muscle cultures from transgenic mice bearing an epsilon promoter-nuclear lacZ reporter transgene. Transient transfection experiments using the Sol 8 muscle cell line indicate that sequences that confer responsiveness to ARIA are located within a 150 bp epsilon subunit promoter region and are E box-independent. These results suggest that ARIA performs a vital role by directing spatially restricted gene expression at the neuromuscular junction.

Animals↗

PubMed1995

The influence of heregulins on human Schwann cell proliferation.

The use of Schwann cell (SC) autotransplantation to influence neural repair in humans is dependent upon identifying mitogens that will effectively expand human Schwann cells (SCs) in culture. The recent purification and molecular cloning of glial growth factor (GGF), a potent mitogen for rat Schwann cells, has led to the recognition that a family of proteins (GGF/HRG/NDF/ARIA) are alternatively spliced products of a single gene. The heregulins (HRGs) have been characterized with respect to their influence on human breast cancer cell lines; here we examined whether the HRGs have mitogenic activity for human SCs. Using DNA synthesis assays and serial passaging of cells in culture, we demonstrate that HRG is an effective mitogen for human SCs and that, in the presence of agents that elevate cAMP, it is possible to expand these cells over multiple passages without overwhelming fibroblast contamination. One putative target for this family of proteins is p185erbB2, and EGF-like receptor tyrosine kinase that is encoded by the erbB2 protooncogene. In this report we also demonstrate that the erbB2/3/4 messages as well as the erbB2/3 receptor proteins are present within cultured human SCs. The addition of HRG to human SCs results in tyrosine phosphorylation of a 185 kDa protein. In the presence of stimulatory concentrations of HRG, a blocking monoclonal antibody (2C4) to p185erbB2 is capable of significantly inhibiting phosphorylation of a 185 kDa protein as well as the subsequent incorporation of 3H-thymidine within the human SC. These latter results implicate an important role for p185erbB2 in mediating the mitogenic response of human SCs to HRGs.

Adenylyl Cyclases↗