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At least 235 records · Page 13Linked to original sources
PubMed2003

Mutations in the immunoglobulin-like domain of gp190, the leukemia inhibitory factor (LIF) receptor, increase or decrease its affinity for LIF.

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.

Amino Acid Sequence↗
PubMed2003

Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex.

The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor alpha (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex.

Amino Acid Substitution↗
PubMed2003

Leukemia inhibitory factor (LIF), cardiotrophin-1, and oncostatin M share structural binding determinants in the immunoglobulin-like domain of LIF receptor.

Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.

Amino Acid Sequence↗
PubMed2003

Leukemia inhibitory factor receptor signaling negatively modulates nerve growth factor-induced neurite outgrowth in PC12 cells and sympathetic neurons.

Nerve growth factor (NGF) is required for the development of sympathetic neurons and subsets of sensory neurons. Our current knowledge on the molecular mechanisms underlying the biological functions of NGF is in part based on the studies with PC12 rat pheochromocytoma cells, which differentiate into sympathetic neuron-like cells upon NGF treatment. Here we report that the expression of leukemia inhibitory factor receptor (LIFR), one of the signaling molecules shared by several neuropoietic cytokines of the interleukin-6 family, is specifically up-regulated in PC12 cells following treatment with NGF. Attenuation of LIFR signaling through stable transfection of antisense- or dominant negative-LIFR constructs enhances NGF-induced neurite extension in PC12 cells. On the contrary, overexpression of LIFR retards the growth of neurites. More importantly, whereas NGF-induced Rac1 activity is enhanced in antisense-LIFR and dominant negative-LIFR expressing PC12 cells, it is reduced in LIFR expressing PC12 cells. Following combined treatment with NGF and ciliary neurotrophic factor, sympathetic neurons exhibit attenuated neurite growth and branching. On the other hand, in sympathetic neurons lacking LIFR, neurite growth and branching is enhanced when compared with wild type controls. Taken together, our findings demonstrate that LIFR expression can be specifically induced by NGF and, besides its known function in cell survival and phenotype development, activated LIFR signaling can exert negative regulatory effects on neurite extension and branching of sympathetic neurons.

Animals↗
PubMed2003

Affinity maturation of leukemia inhibitory factor and conversion to potent antagonists of signaling.

Leukemia inhibitory factor (LIF)-induced cell signaling occurs following sequential binding to the LIF receptor alpha-chain (LIFR), then to the gp130 co-receptor used by all members of the interleukin-6 family of cytokines. By monovalently displaying human LIF on the surface of M13 phage and randomizing clusters of residues in regions predicted to be important for human LIFR binding, we have identified mutations, which lead to significant increases in affinity for binding to LIFR. Six libraries were constructed in which regions of 4-6 amino acids were randomized then panned against LIFR. Mutations identified in three distinct clusters, residues 53-57, 102-103, and 150-155, gave rise to proteins with significantly increased affinity for binding to both human and mouse LIFR. Combining the mutations for each of these regions further increased the affinity, such that the best mutants bound to human LIFR with >1000-fold higher affinity than wild-type human LIF. NMR analysis indicated that the mutations did not alter the overall structure of the molecule relative to the native protein, although some local changes occurred in the vicinity of the substituted residues. Despite increases in LIFR binding affinity, these mutants did not show any increase in activity as agonists of LIF-induced proliferation of Ba/F3 cells expressing human LIFR and gp130 compared with wild-type LIF. Incorporation of two additional mutations (Q29A and G124R), which were found to abrogate cell signaling, led to the generation of highly potent antagonists of both human and murine LIF-induced bioactivity.

Animals↗
PubMed2005

Cross-talk among gp130 cytokines in adipocytes.

The interleukin-6 (IL-6) family of cytokines is a family of structurally and functionally related proteins, including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These proteins are also known as gp130 cytokines because they all share gp130 as a common transducer protein within their functional receptor complexes. Several of these cytokines (LIF, OSM, CNTF, and CT-1) also utilize the LIF receptor (LIFR) as a component of their receptor complex. We have shown that all of these cytokines are capable of activating both the JAK/STAT and p42/44 mitogen-activated protein kinase signaling pathways in 3T3-L1 adipocytes. By performing a variety of preincubation studies and examining the ability of these cytokines to activate STATs, ERKs, and induce transcription of SOCS-3 mRNA, we have also examined the ability of gp130 cytokines to modulate the action of their family members. Our results indicate that a subset of gp130 cytokines, in particular CT-1, LIF, and OSM, has the ability to impair subsequent signaling activity initiated by gp130 cytokines. However, IL-6 and CNTF do not exhibit this cross-talk ability. Moreover, our results indicate that the cross-talk among gp130 cytokines is mediated by the ability of these cytokines to induce ligand-dependent degradation of the LIFR, in a proteasome-independent manner, which coincides with decreased levels of LIFR at the plasma membrane. In summary, our results demonstrate that an inhibitory cross-talk among specific gp130 cytokines in 3T3-L1 adipocytes occurs as a result of specific degradation of LIFR via a lysosome-mediated pathway.

Adipocytes↗
PubMed2000

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

3T3 Cells↗
PubMed2002

Cytotoxicity of paclitaxel or cisplatin on carcinoma cell lines is not inhibited by leukemia inhibitory factor (LIF).

We have established a reliable, reproducible and objective growth assay to measure whether leukemia inhibitory factor (LIF) was able to protect tumour-derived cell lines from toxic effects of the chemotherapy agents, cisplatin and paclitaxel. Using this assay, we demonstrated that LIF did not alter the cytotoxic action of these drugs, on a panel of seven cancer cell lines. This was not because of the inactivity of the LIF or because the cell lines did not express components of the LIF receptor. These findings suggest that the potential clinical use of LIF, as a neuroprotective agent, in conjunction with chemotherapy will not interfere with the anti-tumour treatment.

Antigens, CD↗
PubMed1993

Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix.

Leukemia Inhibitory Factor (LIF) interacts with two classes of high affinity binding sites on rat UMR cells cultured in monolayer. One class of binding sites was found to be localized in the extracellular matrix (ECM) after removal of cells from the culture dish. The interaction of LIF with ECM-localized binding sites is not dependent upon either glycosylation of LIF or the presence of extracellular glycosyaminoglycans. Chemical cross-linking studies demonstrate that LIF interacts with a 200-kD cell-associated protein and a 140-kD ECM-localized protein. A 140-kD protein could also be specifically precipitated from solubilised metabolically radiolabeled UMR ECM by antibodies directed against LIF by virtue of its ability to form a stable complex with unlabeled LIF. In addition, soluble LIF associated with this ECM-localized protein is biologically active in terms of inhibition of ES cell differentiation. The properties of ECM-localized 140-kD species are very similar to those of the secreted form of the LIF receptor suggesting that the ECM localization of LIF and LIF signal transduction may be closely coupled.

Alkaline Phosphatase↗
PubMed2004

Null leukemia inhibitory factor receptor (LIFR) mutations in Stuve-Wiedemann/Schwartz-Jampel type 2 syndrome.

Stuve-Wiedemann syndrome (SWS) is a severe autosomal recessive condition characterized by bowing of the long bones, with cortical thickening, flared metaphyses with coarsened trabecular pattern, camptodactyly, respiratory distress, feeding difficulties, and hyperthermic episodes responsible for early lethality. Clinical overlap with Schwartz-Jampel type 2 syndrome (SJS2) has suggested that SWS and SJS2 could be allelic disorders. Through studying a series of 19 families with SWS/SJS2, we have mapped the disease gene to chromosome 5p13.1 at locus D5S418 (Zmax=10.66 at theta =0) and have identified null mutations in the leukemia inhibitory factor receptor (LIFR or gp190 chain) gene. A total of 14 distinct mutations were identified in the 19 families. An identical frameshift insertion (653_654insT) was identified in families from the United Arab Emirates, suggesting a founder effect in that region. It is interesting that 12/14 mutations predicted premature termination of translation. Functional studies indicated that these mutations alter the stability of LIFR messenger RNA transcripts, resulting in the absence of the LIFR protein and in the impairment of the JAK/STAT3 signaling pathway in patient cells. We conclude, therefore, that SWS and SJS2 represent a single clinically and genetically homogeneous condition due to null mutations in the LIFR gene on chromosome 5p13.

Base Sequence↗
PubMed2001

Expression of leukemia inhibitory factor in human nerve following injury.

In animal models of peripheral nerve injury, leukemia inhibitory factor (LIF) is normally expressed at very low levels. Following nerve injury, its expression is rapidly increased in the nerve at the injury site and promotes both sensory and motor neuron survival. Once normal nerve function is restored, LIF expression returns to negligible levels. For this reason, LIF is considered to be a peripheral nerve trauma factor. We wished to determine whether LIF is also upregulated in human nerves following trauma and whether it is expressed in neuromas of varying age. Immunohistochemical staining for the presence of LIF was performed on injured and control human nerves from a number of subjects. Results demonstrate that LIF expression is increased in nerves within hours of injury and, in the case of neuroma formation, can persist for several years. LIF immunoreactivity was consistently found in Schwann cells, in peripheral nerve axons, and, at stages when an inflammatory response was present, also in neutrophils, mast cells, macrophages, and blood vessel walls. The level of staining within the connective tissue of injured nerves was elevated compared to control nerves, which may be due to the presence of LIF bound to the soluble secreted form of the LIF receptor. Whether the continued expression of LIF is unhealed injured nerves promotes the development of neuromas remains to be resolved.

Axons↗
PubMed1999

The proinflammatory and chondral activities of leukemia inhibitory factor in goat joints are partially a function of interleukin-1.

We wished to determine if the effects of injected recombinant human leukemia inhibitory factor (LIF) are a function of endogenous goat interleukin-1 (IL-1) production and, conversely, if the effects of injected recombinant human IL-1 are a function of endogenous LIF production in goat radiocarpal joints (RCJ). In preliminary experiments, murine LIF binding protein (MuLBP) and recombinant HuIL-1RA were found to independently attenuate the cartilage proteoglycan resorbing activity of goat synovial membrane-conditioned medium (GSMCM), implying activity against goat LIF and goat IL-1, respectively. The present study shows that the proinflammatory and chondral actions of rHuLIF in goat RCJ are partially attenuated by rHuIL-1RA. This implies that a small but important component of the in vivo activity of rHuLIF is a result of IL-1 production in the synovial joint. With the exception of proteoglycan synthesis, the absence of significant effects by MuLBP on the actions of rHuIL-1alpha in goat RCJ suggests that the proinflammatory and chondral effects of IL-1alpha in vivo are probably not mediated by LIF.

Animals↗
PubMed1995

Leukemia inhibitory factor binds to human breast cancer cells and stimulates their proliferation.

Leukemia inhibitory factor (LIF) is a cytokine that was originally described as a differentiation factor of a murine myeloid leukemia cell line and subsequently found to be an important mediator of embryonic development. Although extensively studied in the hematopoietic system, its effects on solid tumors are generally unknown. In the present study we investigated the role of LIF in human breast cancer cells. Using the reverse transcriptase-polymerase chain reaction, we found that the human breast carcinoma MCF-7 cell line expressed the message for both LIF receptor and its signal-transducing protein gp130, suggesting that these receptors might be biologically active. Binding studies with radiolabeled LIF demonstrated that MCF-7 cells interacted with this cytokine, and the ligand binding was specific and time, dose, and temperature dependent. In addition, a Scatchard analysis of the data revealed a single class of high-affinity (Kd 0.27 nM) receptors with a density of approximately 430 sites per cell. MCF-7 cells exposed to LIF internalized and degraded the ligand. LIF stimulated the growth of MCF-7 as well as other estrogen-dependent and independent breast cancer cell lines, but the effect on normal breast epithelial lines was less significant. Likewise, it stimulated colony formation by breast cancer cells obtained from five different breast cancer patients in a dose-dependent fashion. These results overall suggest that human breast tumor cells express functional LIF receptors that play a role in breast cancer cell proliferation.

Base Sequence↗
PubMed2006

s-SHIP associates with receptor complexes essential for pluripotent stem cell growth and survival.

Embryonic stem (ES) cells are pluripotent cells that have the ability to either self-renew or differentiate into any cell type found in the mammalian body. The signaling pathways required for self-renewal of these cells are yet to be defined. Previously we identified a stem cell-specific isoform of the protein SH2 domain-containing 5'-inositol phosphatase (SHIP) that we call s-SHIP, which is expressed in both pluripotent ES cells and adult tissue-specific multipotent cells, such as hematopoietic stem cells (HSCs). s-SHIP lacks an SH2 domain but contains a 5'-inositol phosphatase domain and several protein-protein interaction domains that potentially enable its participation in many different signaling pathways. Here we show that s-SHIP associates with gp130, which forms a heterodimeric complex with the leukemia inhibitory factor receptor (LIFR). Signaling through LIFR and other receptors that heterodimerize with gp130 is critical for growth and survival of ES cells and HSCs. Our findings provide biochemical evidence that s-SHIP participates in signaling pathways important for the maintenance of pluripotent stem cell populations.

Animals↗
PubMed2001

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

Amino Acid Sequence↗
PubMed2003

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation.

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.

Animals↗
PubMed2004

Expression of leukemia inhibitory factor and its receptor is not altered in the decidua and chorionic villi of human anembryonic pregnancy.

BACKGROUND: Uterine expression of leukemia inhibitory factor (LIF) is absolutely essential for mouse, and critical for human, embryo implantation. However LIF is not required for post-implantation development of mouse embryo. The objective of this study was to examine the role of LIF system in post-implantation stage of human pregnancy. METHODS: Tissues from 25 patients with anembryonic pregnancy (AP; blighted ovum) and 25 matched patients with normal pregnancy (NP) were collected. LIF and its receptor beta (LIF-Rbeta) expression in the decidua and chorionic villi were analyzed by semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR), real-time quantitative PCR and immunohistochemical study. RESULTS: LIF mRNA levels were not different either between different tissues (decidua vs chorionic villi) or between different patients (NP vs AP). LIF-Rbeta mRNA levels were significantly higher in chorionic villi than in decidua but were not different between NP and AP. Immunohistochemical staining supported these findings and showed a predominate expression of LIF-Rbeta in the trophoblast cells. CONCLUSIONS: This study concluded that at early human post-implantation stage, LIF is produced from both decidua and chorionic villi and may exert its major action on trophoblasts. A baseline expression of LIF and LIF-Rbeta is probably needed for early pregnancy, but AP cannot be accounted for by the defective expression of either LIF or LIF-Rbeta in most circumstances.

Adult↗
PubMed1997

Epitope-function relationships of human leukemia inhibitory factor receptors using a novel set of anti-gp190 mAB.

The binding and functional properties of a set of six mAb directed against the human gp190 [leukemia inhibitory factor (LIF) receptor] signal transducing molecule were determined. Each of the antibodies reacted with a distinct epitope on gp190 expressed either by gp190-transfected Chinese hamster ovary cells or by the LIF receptor-positive choriocarcinoma JAR cell line. Two of the antibodies (1B4 and 6E6) had binding stoichiometries that were approximately 2-fold lower than those of other mAb (10B2, 12D9 and 7G7), suggesting either that gp190 is present as a pre-associated homodimer in the cell membrane or that part of gp190 is pre-associated with another component. Two mAb (1C7 and 1B4) were found to inhibit LIF binding on the two cell types studied. On JAR cells, this inhibition was, however, restricted to the high-affinity LIF component, suggesting different modes of LIF engagement with the low- and high-affinity receptor species. mAb 1C7 and 1B4 were also found to synergize for inhibiting LIF high-affinity binding. This synergy also extended to the inhibition of LIF- or oncostatin M (OSM)-induced proliferation of a Ba/F3 cell line co-transfected with human gp130 and gp190. However, this mAb combination inhibited LIF- but not OSM-induced haptoglobin secretion by HepG2 cells, suggesting that whereas haptoglobin secretion induced by LIF involves gp130/gp190 common LIF/OSM type I receptors, that induced by OSM mainly involves type II OSM receptors.

Animals↗