Naili Liu

Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors.

We show that beta forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage.

Structural and functional aspects of the multiplicity of Neu differentiation factors.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships

The Neu/HER‐2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast‐derived Neu, the ovarian protein did not display covalent cross‐linking to radiolabeled NDF, and was devoid of ligand‐induced association with phosphatidylinositol 3′‐kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(−9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin‐like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF‐induced phosphorylation and covalent cross‐linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.

Binding of Neu Differentiation Factor with the Extracellular Domain of Her2 and Her3

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDFβ2 was shown to bind sHer3 with a dissociation constant of 26 ± 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDFβ2 for sHer3 was observed upon addition of sHer2 to the NDFβ2-sHer3 mixture. Binding of NDFβ2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

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‐β2 (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‐BP‐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‐β‐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.

Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.