Reconstitution of the response to leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in hepatoma cells

Ciliary neurotrophic factor (CNTF) has been described as a neuro-active cytokine that shares functional similarities with the leukemia inhibitory factor (LIF). We demonstrate here that, like LIF, CNTF stimulates expression of acute phase plasma proteins in rat H-35 hepatoma cells. Transfection of the LIF receptor into Hep3B hepatoma cells reconstituted LIF and oncostatin M regulation of acute phase plasma protein genes. Co-expression of the LIF receptor and the CNTF receptor, but not expression of either subunit alone, generated CNTF responsiveness in Hep3B cells, suggesting cooperativity of these receptor subunits. Evidence is presented for direct interaction of the LIF receptor with the intracellular signal transduction machinery.     

Modulation of integrin alpha-v-beta-1 expression on human tumor cells by leukemia inhibitory factor (LIF) and oncostatin M (OSM)

Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytotic cells of the myeloid lineage. Natural killer (NK) cells are spontaneously cytotoxic large granular lymphocytes that are involved in immunosurveillance against cancer and infections. Their activity is modulated by hormones of the hypothalamic-pituitary-adrenal axis. We wished to determine whether two human corticostatins/defensins, HP-1 and HP-4, are able to change in vitro the spontaneous NK activity of human peripheral blood mononuclear cells (PBMC) and the responses either to the stimulatory cytokines immune interferon (IFN-gamma) or interleukin 2 (IL-2) and to the inhibitory hormone cortisol. NK cell activity was measured in a 4-h direct cytotoxicity assay with K562 cells as a target. HP-1 and HP-4 (10 (-8) -10 (-9) M) significantly inhibited the spontaneous and cytokine-inducible NK activity, and increased the cortisol-dependent inhibition. Radioimmunoassay of HPLC purified fractions obtained from sonicated NK cells showed HP-1 in the two cell preparations examined. We also evaluated the effects of HP-1 and HP-4 (10 (-8) M -10(-9) M) upo IFN-gamma and interleukin 6 (IL-6) production by PBMC stimulated with phytohemagglutinin (PHA) or concanavalin A (ConA). IFN-gamma was titrated with the biological assay using WISH cells as indicators and vescicular stomatitis virus (VSV) as the challenge virus. IL-6 was measured using an enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that corticostatins/defensins are novel modulators of lymphocyte functions in vitro. Their immunodepressing properties could add complexity and plasticity to hypothalamic-pituitary-adrenal-cytokine circuits in vivo.     

Effects of leukemia inhibitory factor and oncostatin M on bone mineral formed in in vitro rat bone-marrow stromal cell culture: physicochemical aspects

Leukemia inhibitory factor (LIF) and oncostatin M (OSM), two pleiotropic cytokines involved in bone remodeling, have both anabolic and catabolic activities. This study analyzed the effects of LIF and OSM on the physicochemical characteristics of mineral phases formed in a rat bone-marrow stromal cell culture model. Stromal cells were cultured for three weeks in the presence of 10(-8) M dexamethasone, 50 microgram/mL ascorbic acid and 10 mM Na beta-glycerophosphate with or without 10 ng/ml LIF or OSM. Subsequently, the physicochemical characteristics of the mineralization nodules formed were analyzed by energy dispersive X ray microanalysis (EDX) and Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy. EDX and FT-IR spectroscopy revealed the influence of LIF and OSM on the physicochemical characteristics of mineral phases. FT-Raman spectroscopy showed modifications of the main vibrational modes of the organic matrix. These alterations induced by growth factors could help define new strategies for the prevention and treatment of skeletal disorders.     

Cloning and characterization of a specific receptor for mouse oncostatin M

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.     

Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc(2)155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.     

Upmodulation of alpha v beta 1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: correlation with increased cell adhesion on fibronectin

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 1.5-2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. alpha v beta 1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.     

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.     

Neural precursor differentiation into astrocytes requires signaling through the leukemia inhibitory factor receptor

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.     

The mannose 6-phosphate/insulin-like growth factor II receptor is a nanomolar affinity receptor for glycosylated human leukemia inhibitory factor

Comparison of the binding properties of non-glycosylated, glycosylated human leukemia inhibitory factor (LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor beta subunit showed that most of the low affinity (nanomolar) receptors expressed by a variety of cell lines are not due to gp190. These receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not Escherichia coli-derived LIF or CHO LIF treated with endoglycosidase F. CHO LIF binding to these receptors is neither affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically inhibited by low concentrations of mannose 6-phosphate (Man-6-P) (IC50 = 40 microM), suggesting that they could be related to Man-6-P receptors. The identity of this LIF binding component with the Man-6-P/insulin-like growth factor-II receptor (Man-6-P/IGFII-R) was supported by several findings. (i) It has a molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa); (ii) the complex of LIF cross-linked to this receptor is immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii) this antibody inhibits LIF and IGFII binding to the receptor with comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of Man-6-P/IGFII-R in LIF processing and biological activity is discussed.     

Distinct roles for leukemia inhibitory factor receptor alpha-chain and gp130 in cell type-specific signal transduction

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR alpha-chain (LIFRalpha) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRalpha and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRalpha or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRalpha receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRalpha cytoplasmic domains may differ depending upon the tissue and cellular response initiated.     

Leukemia inhibitory factor and oncostatin M influence the mineral phases formed in a murine heterotopic calcification model: a Fourier transform-infrared microspectroscopic study

The study of bone mineralization processes is of considerable interest in understanding bone diseases and developing new therapies for skeletal disorders, particularly since bone homeostasis requires numerous cell types and a large cytokine network. Cell culture models of mineralization have often been used to study the cellular mechanisms of mineralization, but few data have been reported concerning the influence of extracellular matrix components and cytokines on the physicochemical properties of mineral. The purpose of this study was to analyze the effects of two cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), involved in bone metabolism on the physicochemical properties of bone mineral formed in a murine in vivo mineralization model. Murine bone marrow cells implanted under the kidney capsule in the presence or absence of cytokines led to heterotopic ossicle formation. A scanning electron microscopic microprobe revealed that heterotopic calcification had a lower (approximately 20%) Ca/P ratio after cytokine treatment as compared with the control without cytokine. Transmission electron microscopic analysis of cytokine-treated ossicles showed numerous areas with low mineral density, whereas electron diffraction pattern revealed an apatitic phase. These areas were not observed in the absence of cytokine. Moreover, Fourier transform-infrared microspectroscopy showed at the molecular level that the presence of either cytokine induced many microscopic areas in which short-range order organization, such as incorporation of carbonate and crystallinity/maturity of ossicle mineral, were modified. LIF and OSM influenced mineral phase formation in the present model and may thus be key protagonists in bone mineral development and skeletal diseases.     

Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit 1

Although neurotrophins have traditionally been regarded as neuronal survival factors, recent work has suggested a role for these factors in synaptic plasticity. In particular, brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission in hippocampal neurons through trkB receptor stimulation and postsynaptic phosphorylation mechanisms. Activation of trkB also modulates hippocampal long-term potentiation, in which postsynaptic N-methyl-D-aspartate glutamate receptors play a key role. However, the final common pathway through which BDNF increases postsynaptic responsiveness is unknown. We now report that BDNF, within 5 min of exposure, elicits a dose-dependent increase in phosphorylation of the N-methyl-D-aspartate receptor subunit 1. This acute effect occurred in hippocampal synaptoneurosomes, which contain pre- and postsynaptic elements, and in isolated hippocampal postsynaptic densities. Nerve growth factor, in contrast, caused no enhancement of phosphorylation. These results suggest a potential mechanism for trophin-induced potentiation of synaptic transmission.     

Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes

The expression of the proinflammatory cytokine leukemia inhibitory factor (LIF) has been reported in the cartilage and synovium of rheumatoid arthritis (RA) patients. Here, we show that high levels of LIF were constitutively produced by cultures of synovium pieces. Low levels of LIF were produced spontaneously by isolated synoviocytes, but interleukin (IL)-1 beta caused a fourfold enhancement of this secretion. The anti-inflammatory cytokine IL-4 reduced the production of LIF by synovium pieces by 75%, as observed earlier with IL-6, IL-1 beta and tumor necrosis factor (TNF)-alpha. IL-4 had a direct effect since it inhibited LIF production by unstimulated and IL-1 beta- or TNF-alpha-stimulated synoviocytes. Conversely, IL-4 enhanced the production of IL-6, which shares with LIF biological activities and receptor components. The inhibitory effect of IL-4 was dose dependent and was reversed using a blocking anti-IL-4 receptor antibody. Similar inhibitory action of IL-4 on LIF production was observed on synovium pieces from patients with osteoarthritis and on normal synoviocytes. IL-10, another anti-inflammatory cytokine acting on monocytes, had no effect on LIF production by either synovium pieces or isolated synoviocytes. Thus, the production of LIF by synovium tissue was inhibited by IL-4 through both a direct effect on synoviocytes and an indirect effect by inhibition of the production of LIF-inducing cytokines.     

Brain-derived neurotrophic factor (BDNF) modulates inhibitory, but not excitatory, transmission in the CA1 region of the hippocampus

Brain-derived neurotrophic factor (BDNF) modulates inhibitory, but not excitatory, transmission in the CA1 region of the hippocampus. J. Neurophysiol. 80: 3383-3386, 1998. Brain-derived neurotrophic factor (BDNF) has been reported to have rapid effects on synaptic transmission in the hippocampus. We report here that bath application of BDNF causes a small but significant decrease in stimulus-evoked inhibitory postsynaptic currents (IPSCs) on CA1 pyramidal cells, which is prevented by the tyrosine kinase inhibitor lavendustin A. BDNF causes a decrease in the 1/CV2 of the IPSC, and also reduces paired-pulse depression of the IPSC, suggesting a presynaptic site of action. In contrast, BDNF did not have a detectable effect on field excitatory postsynaptic potentials measured in stratum radiatum. We conclude that BDNF has a selective depressant action on inhibitory transmission in the hippocampus, due at least in part to a presynaptic mechanism.