Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.     

Molecular investigations on the high-affinity nerve growth factor receptor.

Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

Characterization of the leukemia inhibitory factor receptor in the goldfish (Carassius auratus)

The cytokine leukemia inhibitory factor (LIF) and its receptor LIFR have been extensively characterized in mammals. LIF has been shown to mediate the proliferation, differentiation and activation of a number of cell types in various tissues. This paper reports on the identification of a novel LIFR isolated from goldfish (Carassius auratus) macrophages. Goldfish LIFR shares a 26% amino acid sequence identity with mammalian LIFR sequences; however it retains all of the conserved amino acid motifs that identify a functional LIFR such as the cytokine binding domains and the box-1 and box-2 motifs. The goldfish LIFR phylogenetically groups with the other identified LIFRs from human, mouse, rat and chicken, and it appears to be ancestral to the divergence of the oncostatin M receptor (OSMR). The tissue expression of goldfish LIFR is observed in the gill, kidney and brain as well as sorted goldfish macrophages which exhibit higher expression than monocytes and early progenitor cells.     

Analysis of the leukemia inhibitory factor receptor functional domains by chimeric receptors and cytokines

In contrast to other hematopoietic cytokine receptors, the leukemia inhibitory factor receptor (LIFR) possesses two cytokine binding modules (CBMs). Previous studies suggested that the NH(2)-terminal CBM and the Ig-like domain of the LIFR are most important for LIF binding and activity. Using the recently engineered designer cytokine IC7, which induces an active heterodimer of the LIFR and gp130 after binding to the IL-6R, and several receptor chimeras of the LIFR and the interleukin-6 receptor (IL-6R) carrying the CBM of the IL-6R in place of the COOH-terminal LIFR CBM, we could assign individual receptor subdomains to individual binding sites of the ligand. The NH(2)-terminal CBM and the Ig-like domain of the LIFR bind to ligand site III, whereas the COOH-terminal CBM contacts site I. Furthermore, we show that LIFR mutants carrying the IL-6R CBM instead of the COOH-terminal CBM can replace the IL-6R by acting as an alpha-receptor for IL-6. However, in situations where a signaling competent receptor is bound at IL-6 site I, ligand binding to site III is an absolute requirement for participation of the receptor in a signaling heterodimer with gp130; i.e., a functional receptor complex of IL-6 type cytokines cannot be assembled solely via site I and II as in the growth hormone receptor complex.     

N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.     

Stat3-dependent induction of interleukin-3 receptor expression in leukemia inhibitory factor-stimulated M1 mouse leukemia cells

M1 mouse leukemia cells differentiate to macrophages/monocytes by the stimulation of interleukin-6 (IL-6)/leukemia inhibitory factor (LIF). To identify new LIF-induced genes, we have performed representational difference analysis using M1 cells and cloned mouse interleukin-3 (IL-3) receptor beta subunit gene. The mRNA expression of both IL-3 receptor (IL-3R) alpha and beta subunits is upregulated after 1 h stimulation of LIF and remains to be elevated along the differentiation of M1 cells. This induction is almost completely suppressed in M1 cells expressing a dominant negative form of Stat3. Furthermore, we show that IL-3-induced Stat5 phosphorylation increases in LIF-stimulated M1 cells. These results suggest that Stat3 may play a role in the differentiation of myeloid cells by regulating IL-3R expression.     

Human leukemia inhibitory factor improves the viability of cultured ovine embryos.

Embryos were collected from ewes on Day 6 after estrus (Day 0 = estrus), placed in M2 culture medium, and assigned to 1 of 4 treatment groups. Some embryos were transferred to recipient ewes on Day 6 of their estrous cycle either in pairs (group 1) or singularly (group 2) within 3 h of collection. The remaining embryos were individually cultured for 48 h in an atmosphere of 5% CO2 in humidified air in either synthetic oviduct fluid (SOF) medium (group 3) or SOF containing 1,000 U/ml of recombinant human leukemia inhibitory factor (hLIF) (SOF + hLIF: group 4). These embryos were then transferred to recipient ewes on Day 8 of their estrous cycle. The addition of hLIF to culture medium significantly improved the development of the embryos compared with control embryos prior to transfer (blastocysts hatching from the zona pellucida: group 3 = 16% vs. group 4 = 64%, p less than 0.05; those degenerative: group 3 = 27% vs. group 4 = 9%, p less than 0.05) and the subsequent pregnancy rates of the recipient ewes, receiving a single embryo, at Day 70 of pregnancy (group 3 = 16% vs. group 4 = 50%, p less than 0.05). The pregnancy rate of ewes given embryos cultured for 48 h in SOF + hLIF prior to transfer (50%; group 4) was similar to the group 2 ewes receiving a single embryo soon after collection (52%), but the pregnancy rate for both groups was significantly lower than that for the group 1 ewes receiving two embryos soon after collection (89%: 53% twins, 36% singles; p less than 0.05).     

Leukemia inhibitory factor, leukemia inhibitory factor receptor, and glycoprotein 130 in rhesus monkey uterus during menstrual cycle and early pregnancy

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.     

Birth control vaccine targeting leukemia inhibitory factor

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin‐6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF‐receptor (LIF‐R) that span the regions involved in ligand‐receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF‐R‐mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long‐lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF‐R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF‐R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF‐R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF‐R is also important for implantation and pregnancy in women. Mol. Reprod. Dev. 79:97–106, 2012. © 2011 Wiley Periodicals, Inc.     

Production of a monoclonal antibody directed against the high-affinity nerve growth factor receptor.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.     

Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways

Liver development is regulated by soluble factors as well as cell-cell contacts. We previously reported that oncostatin M (OSM) induced hepatic maturation in a primary culture of embryonic day 14 liver cells. While OSM expression in the liver starts in mid gestation and decreases in postnatal stages, hepatocyte growth factor (HGF) is mainly expressed in the liver in the first few days after birth. In this study, we compared the effect of OSM and HGF on the differentiation of fetal hepatic cells in vitro. Like OSM, HGF in the presence of dexamethasone induced expression of glucose-6-phosphatase, tyrosine amino transferase and carbamoyl-phosphate synthase, and accumulation of glycogen in fetal hepatic cells, although to a lesser extent than OSM. Interestingly, while both OSM and HGF up-regulated production of albumin, secretion of albumin occurred only in response to OSM. In addition, although hepatic maturation induced by OSM depends on STAT3, HGF failed to activate STAT3 and HGF-induced differentiation was independent of STAT3. These results indicate that OSM and HGF induce hepatic maturation through different signaling pathways.     

Kinetic analyses of the binding of leukemia inhibitory factor to receptor on cells and membranes and in detergent solution.

The equilibrium and kinetic properties of leukemia inhibitory factor (LIF) binding to a range of cell types have been compared. When binding was examined at 4 degrees C, the majority of cells were found to express a single class of high affinity LIF receptor (KD = 20-100 pM; ka = 2-8 x 10(8) min-1 M-1; kd = 0.0004-0.0011 min-1). In contrast, certain activated macrophage populations expressed apparently independent classes of high and low affinity LIF receptor. The low affinity receptors differed from the high affinity receptors in terms of the dissociation rate of the receptor-ligand complex (KD = 1-2 nM; ka = 3-7 x 10(8) min-1 M-1; kd = 0.30-0.67 min-1). At 37 degrees C, the interaction of LIF with its high affinity receptor was more complicated, since occupied LIF receptors were internalized more rapidly than unoccupied receptors, internalized LIF was hydrolyzed and released from the cell, and new receptors were synthesized and expressed on the cell surface. Interestingly, when membranes were prepared from cells that expressed only high affinity receptors, both high and low affinity receptors were detected, while after detergent solubilization of membranes only low affinity receptors were apparent. These results are discussed in terms of a structural model for the LIF receptor in which interaction of a low affinity binding subunit and a second nonbinding subunit is required for the generation of the high affinity receptor.