Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents.

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-alpha [corrected], like NRG1-beta [corrected], emerges as a narrow-specificity ligand, whereas NRG2-beta [corrected] is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.

Activity-dependent regulation of Neu differentiation factor/neuregulin expression in rat brain.

Neu differentiation factor (NDF/neuregulin) is widely expressed in the central and peripheral nervous systems, where it functions as a mediator of the interactions between nerve cells and Schwann, glia, oligodendrocyte, and muscle cells, to control cellular proliferation, differentiation, and migration. NDF binds to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Here we demonstrate that NDF and its ErbB-4 receptor are highly reactive to changes in ambient neuronal activity in the rodent brain in a region-selective manner. Generation of epileptic seizures by using kainic acid, a potent glutamate analog, elevated levels of NDF transcripts in limbic cortical areas, hippocampus, and amygdala. Concomitantly, ErbB-4 mRNA was increased with a similar spatial distribution, but transcription of the other NDF receptor, ErbB-3, did not change. A more moderate stimulation, forced locomotion, was accompanied by an increase in NDF transcripts and protein in the hippocampus and in the motor cortex. Similar changes were found with ErbB-4, but not ErbB-3. Last, a pathway-specific tetanic stimulation of the perforant path, which produced long-term potentiation, was followed by induction of NDF expression in the ipsilateral dentate gyrus and CA3 area of the hippocampus. Taken together, these results indicate that NDF is regulated by physiological activity and may play a role in neural plasticity.

Up-regulation of Tie gene expression by leukemia inhibitory factor and steel factor in CD34+ cells from human umbilical cord blood.

Tie, a new receptor tyrosine kinase, is expressed in vascular endothelial and hematopoietic cells. To determine whether Tie might be involved in early hematopoiesis, we asked whether the Tie gene is expressed in normal human hematopoietic stem/progenitor cells and if the expression of this gene could be regulated. Using a single-cell reverse-transcriptase polymerase chain reaction (RT-PCR) assay to study expression of the Tie gene in the subset of human umbilical cord blood (UCB) CD34+++ primitive stem/ progenitor cells with extensive replating capacity, we demonstrated at the single isolated cell level that Tie was expressed in these cells. The expression of Tie gene in CD34+ cells was at a low level but was enhanced up to two- to fourfold by steel factor (SLF) or leukemia inhibitory factor (LIF), two cytokines that regulate production of stem/progenitor cells, as assessed using competitive PCR and semi-quantitative RT-PCR assays. The fold increases were observed as early as 2 h for SLF and 4 h for LIF and remained elevated for 24 h. These results demonstrate modulation of gene regulation in the rare populations of CD34+ cells and suggest the possibility that Tie may play a role in the proliferation and differentiation of immature hematopoietic cells.

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

Neu differentiation factor/neuregulin isoforms activate distinct receptor combinations.

The multiple isoforms of Neu differentiation factor (NDF/neuregulin) induce a pleiotropic cellular response that is isoform-specific and cell type-dependent. The molecular basis of this heterogeneity was addressed by comparing the two major groups of isoforms, alpha and beta. Both groups bind to the catalytically impaired receptor tyrosine kinase ErbB-3, whose mitogenic stimulation by NDF requires transactivation by other ErbB proteins, either ErbB-1 or ErbB-2. By expressing each pair of receptors in interleukin 3-dependent myeloid cells, we found that both isoforms induced mitogenic signals in cells co-expressing the combination of ErbB-3 with ErbB-2. However, only the beta isoform stimulated cells that expressed both ErbB-3 and ErbB-1, and neither isoform was active on cells expressing ErbB-3 alone. Both isoforms bind to all ErbB-3-expressing cells, albeit with different affinities, but the co-stimulatory mitogenic effect is correlated with the ability of each auxiliary receptor to transphosphorylate ErbB-3. These results imply that NDF isoforms differ in their ability to induce receptor heterodimers; whereas both types of isoforms signal through ErbB-3/ErbB-2 heterodimers, only beta isoforms are able to stabilize ErbB-3/ErbB-1 heterodimers.

NDF induces expression of a novel 46 kD protein in estrogen receptor positive breast cancer cells.

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-beta 2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BR-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-beta-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2-6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells.

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3.

An immunological approach reveals biological differences between the two NDF/heregulin receptors, ErbB-3 and ErbB-4.

The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

Purification and characterization of two related but distinct metalloproteinase inhibitors secreted by bovine aortic endothelial cells.

Two metalloproteinase inhibitors were purified from serum-free medium conditioned by bovine aortic endothelial cells. One of these inhibitors, with a molecular weight of 30,000-34,000 (reduced) is identified as tissue inhibitor of metalloproteinases; the second inhibitor has a molecular weight of 27,500 (reduced) and 20,400 (unreduced), is not recognized by an antiserum against bovine tissue inhibitor of metalloproteinases, appears unglycosylated, and has 51% identity with tissue inhibitor of metalloproteinases by NH2-terminal amino acid sequence analysis. This inhibitor has antiproteinase activities similar to those of tissue inhibitor of metalloproteinases, with inhibition of classical collagenase, type IV collagenase, and gelatinases but not trypsin, plasmin, or bacterial collagenase. Other properties shared with tissue inhibitor of metalloproteinases include trypsin sensitivity, acid and heat resistance, and inactivation by reduction-alkylation. The presence of these inhibitors in endothelial cells suggests that they may play important roles in protecting the integrity of the vascular basement membrane.

In vivo activation of neutrophil function in hamsters by recombinant human granulocyte colony-stimulating factor.

The in vivo effect of Escherichia coli-derived recombinant human granulocyte colony-stimulating factor on neutrophil function was studied in golden Syrian hamsters. Significant increases in superoxide generation and specific binding of N-formylmethionyl-leucyl-phenylalanine were observed in neutrophils isolated 4 h following a single subcutaneous injection of the factor (30 micrograms/kg). However, phagocytotic activity was not significantly stimulated in hamsters treated with the factor. Recombinant human granulocyte colony-stimulating factor hastened the recovery of peripheral neutrophil counts in animals made leukopenic by prior treatment with cyclophosphamide. Beginning several hours after infection, resistance to lethal infection following intraperitoneal injection of Staphylococcus aureus was increased when neutropenic animals were treated daily with the factor. This protective effect was associated with increased peritoneal neutrophil counts and a decreased incidence of positive peritoneal bacterial cultures at 24 h after the start of treatment. These results suggest that recombinant human granulocyte colony-stimulating factor may be a useful adjunct in the treatment of bacterial infections in neutropenic patients.

Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo.

A fragment of plasmid NAH7 from Pseudomonas putida PpG7 has been cloned and expressed in Escherichia coli HB101. Growth of the recombinant Escherichia coli in nutrient medium results in the formation of indigo. The production of this dye is increased in the presence of tryptophan or indole. Several bacteria that oxidize aromatic hydrocarbons to cis-dihydrodiols also oxidize indole to indigo. The results suggest that indigo formation is due to the combined activities of tryptophanase and naphthalene dioxygenase.