Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells.

Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-beta2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Specific inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14-16 h after HRG, when the cells were entering S-phase, was without effect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/SDI1) gene expression was rapidly induced by HRG, but significant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These findings demonstrate that MEK activation is critical to HRG-induced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.

Granulosa cell tumors express erbB4 and are sensitive to the cytotoxic action of heregulin-beta2/PE40.

The molecular genetic events involved in the etiology of human granulosa cell (GC) tumors, which represent approximately 7% of all malignant ovarian neoplasms, are unknown. Amplification and/or overexpression of the ERBB genes are a feature of many cancer types, and overexpression of erbB2 correlates with poor prognosis in epithelial ovarian cancer. In the present study, we used immunohistochemistry to determine the level and frequency of expression of different erbB receptors in GC tumors. Ten of 12 tumors expressed erbB4 at moderate to high levels in >50% of cancer cells, whereas erbB2 (6 of 12) and erbB3 (2 of 12) were expressed less frequently. Western blot experiments showed that the only available GC tumor cell line, COV434, also expressed erbB receptors. Heregulin (HRG)-beta2, a ligand for erbB3 and erbB4 receptors, stimulated tyrosine phosphorylation of the erbB receptors, which was accompanied by activation of Erk1 and Erk2, two mitogen-activated protein kinases with a functional role in mitogenesis. Importantly, HRG increased cell proliferation in COV434 cells, and treatment with HRG/PE40, a ligand toxin shown previously to be cytotoxic against human breast cancer cells overexpressing erbB receptors, led to a dramatic and irreversible decrease in cell number. These results indicate that erbB receptor signaling pathways may be critical in the control of GC tumor cell proliferation and that HRG/PE40 is a potential therapeutic agent for the treatment of GC tumors.

Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3.

Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Co-expression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library.

An immunoglobulin phage display library constructed from a tumour-associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c-erbB-2. Sixteen independent clones were confirmed as positive binders based on ELISA analysis of soluble Fabs. Nucleotide sequencing demonstrated that the V(H) region of 12 clones belonged to four different V gene families, and the clones demonstrated varying degrees of somatic mutation compared with germ-line sequences. Fab fragments were examined for cross-reactivity by ELISA and shown to be negative against a panel of irrelevant self and non-self antigens, including bovine serum albumin (BSA), mouse immunoglobulin, tetanus toxoid, heregulin-PE40-FLAG and insulin. Reactivity of Fabs in vitro was verified by immunocytochemistry, which showed binding to the c-erbB-2 over-expressing breast cancer cell line SKBR3 but not to the low-expressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 x 10(7) kappa light chain and gamma heavy chain clones), when derived from an individual whose colorectal tumour over-expressed c-erbB-2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti-c-erbB-2 Fabs recovered from this library suggests that they may have resulted from a humoral immune response in the individual, and that in vivo antibody responses to tumour-associated antigens may be exploited in vitro for the production of tumour-specific recombinant antibodies.

Heregulin (HRG)-induced mitogenic signaling and cytotoxic activity of a HRG/PE40 ligand toxin in human breast cancer cells.

The heregulins (HRGs) are a family of growth factors that bind direction to erbB3 and erbB4 and induce tyrosine phosphorylation of erbB2 via receptor heterodimerization. Since erbB2, erbB3, and erbB4 (erbB2-4) are often overexpressed in human breast cancer cells, we produced recombinant HRGs and a HRG-based ligand toxin to investigate the signaling events triggered by HRGs and the ability of these ligands to specifically target such cells. Recombinant HRG beta 2 stimulated the tyrosine phosphorylation of erbB2-4 in ZR-75-1 human breast cancer cells. This was accompanied by the tyrosine phosphorylation of Shc and the formation of complexes between Shc and the adapter protein Grb2. Complexes were also detected between Shc and erbB2-4. However, GRb2 was detected in erbB2 and erbB4 but not erbB3 immunoprecipitates. Thus, these receptors exhibit mechanistic differences in their coupling to Ras signaling, and HRG beta 2 administration triggers multiple inputs into the Ras signaling pathway, involving receptor-Grb2, receptor-Shc, and Shc-Grb2 complexes. HRG beta 2 addition also stimulated the association of erbB3 with phosphatidylinositol-3-kinase. In accordance with the activation of key mitogenic signaling pathways, HRG beta 2 stimulated the proliferation of MCF-7 and T-47D human breast cancer cells. Moreover, when tested for the ability to stimulate cell cycle re-entry of T-47D cells arrested under serum-free conditions, HRG beta 2 was more effective than insulin, previously the most potent mitogen identified using this system. Finally, a HRG beta 2 PE40 ligand toxin was constructed and found to exhibit cytotoxic activity against human breast cancer cells overexpressing erbB3 alone or in combination with erbB4 and/or erbB2.

A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), is expressed in rat brain.

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells.

Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [125I]oGH molecules at high affinity (Kd = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes (Kd = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865-1872). The expressed receptor also binds ovine placental lactogen (oPL, 205,000 binding sites per cell) with high affinity (Kd = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis (M(r) 148,000 and M(r) 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M(r) 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species (M(r) 133,000 and M(r) 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c-fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148-1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265-274).

The sheep growth hormone receptor: molecular cloning and ontogeny of mRNA expression in the liver.

Two overlapping cDNA clones encoding the sheep growth hormone (GH) receptor were isolated from a sheep liver cDNA library. The translated amino acid sequence predicts a polypeptide precursor of 634 amino acids with a calculated molecular weight of 70,799. The mature GH receptor comprises an extracellular domain of 242 amino acids, a hydrophobic transmembrane region of 24 amino acids, and a cytoplasmic domain of 350 amino acids. The nucleotide and translated amino acid sequences display extensive similarity with sequences established for GH receptors from a number of other mammalian species. A prominent transcript of 4.5 kb and a minor transcript of 1.9 kb are detected following Northern blot hybridization of poly(A)+ RNA isolated from sheep liver. The onset of GH receptor mRNA expression in the liver is developmentally regulated: GH receptor transcripts are first detected by Northern blot hybridization in liver taken from a term (145 days of gestation) fetus and reach maximum levels within one week following birth. Ribonuclease protection assays reveal heterogeneity within the 5' untranslated region of GH receptor mRNA transcripts detected in liver and a number of other tissues. At least one transcript appears to be expressed in a liver-specific fashion, supporting a role for alternative RNA splicing in the tissue-specific regulation of sheep GH receptor expression.