Leukemia Inhibitory Factor Receptor Research Articles

Characterization, Chromosomal Assignment, and Role of LIFR in Early Embryogenesis and Stem Cell Establishment of Rabbits

Leukemia inhibitory factor (LIF) is a multifunctional cytokine with an important role during early embryonic development, implantation, and as an inhibitor of murine embryonic stem cell differentiation. It exerts its effects by binding to the leukemia inhibitory factor receptor, a heterodimer of two transmembrane proteins, the specific leukemia inhibitory factor receptor subunit, and the common gp130. A partial cDNA clone coding for the membrane-bound form of the specific rabbit leukemia inhibitory factor receptor was isolated from the genital ridge of 13.5 days postcoitum fetus. Fluorescent in situ hybridization analysis revealed that the rabbit leukemia inhibitory factor receptor gene is located on chromosome OCU11p11.1. It has been shown that the membrane-bound rabbit leukemia inhibitory factor receptor mRNA is expressed during embryo implantation but not at earlier developmental stages. Rabbit embryonic stem cell-like line establishment is improved in the presence of LIF, and those cells express both leukemia inhibitory factor and its receptor. The withdrawal of leukemia inhibitory factor results the differentiation of embryonic stem cell-like cells to beating myocardial-like cells. Our findings suggest that the self-renewal mechanism is similar in mouse and rabbit embryonic stem cells, and expands our knowledge on the role of the LIF-LIFR signal pathway in early rabbit embryogenesis and rabbit embryonic stem cell establishment.

The role of CXCR4/SDF-1, CD117/SCF, and c-met/HGF chemokine signalling in the mobilization of progenitor cells and the parameters of the left ventricular function, remodelling, and myocardial perfusion following acute myocardial infarction

Chemokines and their receptors induce the qualitative and quantitative changes of the pool of bone marrow-derived and resident cardiac stem/progenitor cells, which in turn can modulate myocardial recovery after myocardial infarction (MI). Primary role of the chemokines, such as stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), hepatocyte growth factor (HGF) and leukaemia inhibitory factor (LIF) is the mobilization and homing of the bone marrow-derived stem/progenitor cells to the infarcted myocardium. In the setting of the acute MI there is an increased expression of SDF-1, HGF, LIF in the peri-infarct zone. The most important chemokine–chemokine receptor axis is SDF-1/CXCR4 which is significantly upregulated during acute MI. It corresponds with the significant mobilization of cells expressing cardiac and endothelial markers, which also express the chemokine receptors CXCR4, c-met, CD117 and leukemia inhibitory factor receptor. Specific population of small non-haematopoietic cells expressing the CXCR4 receptor was identified, which have the capability of cardiogenic differentiation. The mobilization of stem/progenitor cells expressing the receptors for chemoattractant cytokines is significantly correlated with the improvement of left ventricular ejection fraction (LVEF) and remodelling in 1-year follow-up after the acute MI. Patients with reduced LVEF after MI have also impaired mobilization of the stem/progenitor cells. The number of circulating cells is inversely correlated with the infarct area and positively with myocardial perfusion. Also the migratory response of the endothelial progenitor cells (EPCs) to the SDF-1 gradient is positively correlated with their capability to improve the perfusion and is one of the factors that seem to determine the outcome of the progenitor cell-based therapy after acute MI.

Cardiotrophin-1 Is an Osteoclast-Derived Stimulus of Bone Formation Required for Normal Bone Remodeling

Cardiotrophin (CT-1) signals through gp130 and the LIF receptor (LIFR) and plays a major role in cardiac, neurological, and liver biology. We report here that CT-1 is also expressed within bone in osteoclasts and that CT-1 is capable of increasing osteoblast activity and mineralization both in vitro and in vivo. Furthermore, CT-1 stimulated CAAT/enhancer-binding protein-delta (C/EBP delta) expression and runt-related transcription factor 2 (runx2) activation. In neonate CT-1(-/-) mice, we detected low bone mass associated with reduced osteoblasts and many large osteoclasts, but increased cartilage remnants within the bone, suggesting impaired resorption. Cultured bone marrow (BM) from CT-1(-/-) mice generated many oversized osteoclasts and mineralized poorly compared with wildtype BM. As the CT-1(-/-) mice aged, the reduced osteoblast surface (ObS/BS) was no longer detected, but impaired bone resorption continued resulting in an osteopetrotic phenotype in adult bone. CT-1 may now be classed as an essential osteoclast-derived stimulus of both bone formation and resorption.

Corrigendum

Journal of NeurochemistryVolume 107, Issue 5 p. 1470-1470 Free Access Corrigendum This article corrects the following: Transregulation of leukemia inhibitor factor receptor expression and function by growth factors in neuroblastoma cells Martha D. Port, George S. Laszlo, Neil M. Nathanson, Volume 106Issue 4Journal of Neurochemistry pages: 1941-1951 First Published online: July 4, 2008 First published: 11 November 2008 https://doi.org/10.1111/j.1471-4159.2008.05635.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Port et al. (2008), the title should have read as follows: Transregulation of Leukemia Inhibitory Factor Receptor Expression and Function by Growth Factors in Neuroblastoma Cells Reference Port M. D., Laszlo G. S. and Nathanson N. M. (2008) Transregulation of leukemia inhibitor factor receptor expression and function by growth factors in neuroblastoma cells. J. Neurochem. 106, 1941– 1951. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume107, Issue5December 2008Pages 1470-1470 ReferencesRelatedInformation

Ciliary Neurotrophic Factor, Cardiotrophin-like Cytokine, and Neuropoietin Share a Conserved Binding Site on the Ciliary Neurotrophic Factor Receptor α Chain

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.

Heterokaryon-Based Reprogramming of Human B Lymphocytes for Pluripotency Requires Oct4 but Not Sox2

Differentiated cells can be reprogrammed through the formation of heterokaryons and hybrid cells when fused with embryonic stem (ES) cells. Here, we provide evidence that conversion of human B-lymphocytes towards a multipotent state is initiated much more rapidly than previously thought, occurring in transient heterokaryons before nuclear fusion and cell division. Interestingly, reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene profile, in which markers of human ES cells are expressed (hSSEA4, hFGF receptors and ligands), but markers that are specific to mouse ES cells are not (e.g., Bmp4 and LIF receptor). Using genetically engineered mouse ES cells, we demonstrate that successful reprogramming of human lymphocytes is independent of Sox2, a factor thought to be required for induced pluripotent stem (iPS) cells. In contrast, there is a distinct requirement for Oct4 in the establishment but not the maintenance of the reprogrammed state. Experimental heterokaryons, therefore, offer a powerful approach to trace the contribution of individual factors to the reprogramming of human somatic cells towards a multipotent state.

Human Naturally Occurring and Adaptive Regulatory T cells Secrete High Levels of Leukaemia Inhibitory Factor upon Activation

Leukaemia inhibitory factor (LIF) is a member of the IL-6 cytokine family which signals through cognate receptors and activates target genes involved in survival, apoptosis, proliferation, differentiation and suppression of differentiation in different cell types. Binding of LIF to the LIFRalpha/gp130 receptor complex has been shown to activate the Janus kinase-signal transducer and activator of transcription 3 pathway. Here we show that activation of naturally occurring and adaptive regulatory T cells leads to increased LIF expression which is abrogated by cyclic adenosine monophosphate. Furthermore, the LIF receptors gp130 and LIFRalpha are upregulated on the surface of activated T cells and signal transducer and activator of transcription 3 phosphorylation is increased. Interestingly, LIF was not required for suppressive function but rather appeared to have a stimulatory effect on T cells that served to modulate and counteract immunosuppression by regulatory T cells.

Structural Organization of a Full-Length gp130/LIF-R Cytokine Receptor Transmembrane Complex

gp130 is a shared receptor for at least nine cytokines and can signal either as a homodimer or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here, we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Ralpha). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Ralpha hexameric complex, CNTF/CNTF-Ralpha heterodimerizes gp130 and LIF-R via noncooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic analysis of the full-length gp130/LIF-R/CNTF-Ralpha/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the "tall" class of gp130 family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

Transregulation of leukemia inhibitor factor receptor expression and function by growth factors in neuroblastoma cells

The cytokines that signal through the leukemia inhibitory factor (LIF) receptor are members of the neuropoietic cytokine family and have varied and numerous roles in the nervous system. In this report, we have determined the effects of growth factor stimulation on LIF receptor (LIFR) expression and signal transduction in the human neuroblastoma cell line NBFL. We show here that stimulation of NBFL cells with either epidermal growth factor or fibroblast growth factor decreases the level of LIFR in an extracellular signal-regulated kinase (Erk)1/2-dependent manner and that this down-regulation is due to an increase in the apparent rate of lysosomal LIFR degradation. Growth factor-induced decreases in LIFR level inhibit both LIF-stimulated phosphorylation of signal transducers and activators of transcription 3 and LIFR-mediated gene induction. We also show that Ser1044 of LIFR, which we have previously shown to be phosphorylated by Erk1/2, is required for the inhibitory effects of growth factors. Neurons are exposed to varying combinations and concentrations of growth factors and cytokines that influence their growth, development, differentiation, and repair in vivo. These findings demonstrate that LIFR expression and signaling in neuroblastoma cells can be regulated by growth factors that are potent activators of the mitogen-activated protein kinase pathway, and thus illustrate a fundamental mechanism that underlies crosstalk between receptor tyrosine kinase and neuropoietic cytokine signaling pathways.

Neuropoietin Attenuates Adipogenesis and Induces Insulin Resistance in Adipocytes

Recent findings have implicated gp130 receptor ligands, particularly ciliary neurotrophic factor (CNTF), as potential anti-obesity therapeutics. Neuropoietin (NP) is a recently discovered cytokine in the gp130 family that shares functional and structural features with CNTF and signals via the CNTF receptor tripartite complex comprised of CNTFRalpha, LIF receptor, and gp130. NP plays a role in the development of the nervous system, but the effects of NP on adipocytes have not been previously examined. Because CNTF exerts anti-obesogenic effects in adipocytes and NP shares the same receptor complex, we investigated the effects of NP on adipocyte development and insulin action. Using cultured 3T3-L1 adipocytes, we observed that NP has the ability to block adipogenesis in a dose- and time-dependent manner. We also observed that cultured adipocytes, as well as murine adipose tissue, are highly responsive to acute NP treatment. Rodents injected with NP had a substantial increase in STAT3 tyrosine phosphorylation and ERK 1 and 2 activation. We also observed the induction of SOCS-3 mRNA in 3T3-L1 adipocytes following NP treatment. Unlike CNTF, our studies have revealed that NP also substantially attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In addition, NP blocks insulin action in adipose tissue in vivo. These observations are supported by data demonstrating that NP impairs insulin signaling via decreased activation of both IRS-1 and Akt. In summary, we have observed that both adipocytes in vitro and in vivo are highly responsive to NP, and this cytokine has the ability to affect insulin signaling in fat cells. These novel observations suggest that NP, unlike CNTF, may not be a viable obesity therapeutic.

Regulation of cytokine signaling components in developing rat retina correlates with transient inhibition of rod differentiation by CNTF

Ciliary neurotrophic factor (CNTF) is known to inhibit the differentiation of rod photoreceptors from postmitotic precursor cells. During early postnatal development, photoreceptor precursors lose their responsiveness to CNTF. The underlying events causing this change in responsiveness are unknown. Moreover, whether rods express CNTF receptor alpha, a prerequisite for a direct response to the factor, is controversial. Since morphological studies have previously produced conflicting results, we have analyzed the expression of cytokine receptor components and potential ligands in the rat photoreceptor layer by real-time reverse transcription with the polymerase chain reaction after laser microdissection and by immunoblotting. Cytokine effects on rods were studied in explant cultures from newborn rat retina. CNTF receptor alpha (CNTFR alpha) and leukemia inhibitory factor receptor ss (LIFRss) were expressed in immature photoreceptors. Expression of the CNTF-specific alpha-subunit (but not of LIFRss) was downregulated specifically in the photoreceptor layer in parallel with the appearance of opsin-positive rods. The decrease of CNTFR alpha levels in explant cultures was closely correlated with the loss of precursor cell responsiveness to CNTF. Increasing the CNTF concentration in the culture medium led to prolonged CNTFR alpha expression and, concomitantly, to persistent inhibition of rod differentiation. Application of CNTF and LIF in vitro induced phosphorylation of STAT3. Inducibility of STAT3 activation by CNTF decreased with photoreceptor maturation, whereas the LIF effect persisted. Our results thus indicate that CNTF acts directly on photoreceptor precursors inhibiting their differentiation via activation of the JAK/STAT3 signal transduction pathway, and that this effect is temporally limited because of the downregulation of CNTFR alpha.

Bone Disease from a Placental Hypoxia Pathway

Osteoclasts are cells of a hematopoetic lineage that resorb bone, and excessive activity of these cells can lead to low bone mass with an associated increased incidence of fractures. These cells are also implicated in disorders such as Paget's disease of the bone and multiple myeloma. Osteoclast differentiation requires the transcription factor AP-1, which is a heterodimer of a Jun family member and a Fos family member. Bozec et al . found that mice deficient for the Fos family member Fra-2, which is encoded by Fosl2 , exhibited increased osteoclast size (giant osteoclasts), number, and activity. The expression and abundance of the leukemia inhibitory factor (LIF) receptor (LIFR) were also decreased in the bones of Fosl2 knockout mice. Chromatin immunoprecipitation experiments and reporter gene assays showed that LIF was a direct target of Fra-2 and Jun. Although no bone phenotype was reported for LIF knockout mice, Bozec et al . found that these mice had osteoclasts that were larger and more numerous with increased resorptive activity, which led to decreased bone volume. Fetal liver-derived osteoclast progenitor cells deficient in either Fra-2 or LIF did not differentiate in culture and exhibited increased apoptosis compared with wild-type cells; however, differentiation into osteoclasts was rescued by the addition of LIF. In vivo, the expression of Bcl-2 , which encodes an antiapoptotic protein, was increased in the bones of Fra-2- or LIF-deficient animals. In vivo, osteoclasts were hypoxic and hypoxia-inducible factor-1α (HIF-1α) was abundant, and the mRNA for PHD2 (prolyl hydroxylase domain 2 protein, which contributes to HIF-1α destabilization) was decreased, suggesting that hypoxia may contribute to the giant osteoclast phenotype. In culture, the Fra-2- or LIF-deficient osteoclast progenitors exhibited decreased Bcl-2 expression and increased cell death at 21% oxygen, and increased Bcl-2 expression and less cell death at 3% oxygen. Thus, the loss of LIF signaling appears to alter the sensitivity of the cells to oxygen. Surprisingly, selective knockout of Fra-2 in the macrophage-osteoclast lineage did not show bone phenotypes in vivo, but isolated progenitor cells failed to differentiate in the absence of additional LIF. Instead, it appeared that the Fra-2 knockout mice had impaired placental function, because LIF and LIFR and Phd2 expression were decreased in the placenta, and HIF-1α and its targets were increased. Thus, proper bone oxygenation in utero requires a functional LIF signaling axis that required Fra-2, and a similar pathway in the bone progenitors was required for proper oxygen responsiveness. A. Bozec, L. Bakiri, A. Hoebertz, R. Eferl, A. F. Schilling, V. Komnenovic, H. Scheuch, M. Priemel, C. L. Stewart, M. Amling, E. F. Wagner, Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia. Nature 454 , 221-225 (2008). [PubMed]

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.

Systematic Comparison of Gene Expression between Murine Memory and Naive B Cells Demonstrates That Memory B Cells Have Unique Signaling Capabilities

Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Ag-specific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.

Gene expression in cultured endometrium from women with different outcomes following IVF

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Expression of short‐form oncostatin M receptor as a decoy receptor in lung adenocarcinomas

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines, and binds to the OSM receptor (OSMR) to inhibit cancer growth. Four forms of OSMR have been identified: leukemia inhibitory factor receptor (LIFR), OSMR beta, short-form OSMR (OSMRs) and soluble OSMR (sOSMR). In this study, we examined the type and expression of OSMR in lung adenocarcinomas (LADCs). Expression of OSMR was determined by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, immunohistochemistry and confocal immunofluorescent microscopy (CIM). Our results showed that, among the four forms of OSMR, OSMRs was mainly expressed in LADC, and expression level of OSMRs correlated with patient survival. CIM revealed that OSMRs was localized on the cell membrane of LADC cell lines in vitro. OSMRs acts as a decoy receptor by reducing the inhibitory effect of OSM on cell growth. Decrease in OSMRs expression by siRNA increased cell sensitivity to OSM, and ectopic expression of OSMRs reduced cell sensitivity to OSM. These results suggest that expression of OSMRs, which operates as a decoy receptor for OSM, is correlated with disease progression and adverse prognosis in patients with LADC.

Progesterone and Interferon Tau Regulation of LIFR and IL6ST in the Peri-implantation Ovine Uterus.

Leukemia inhibitory factor (LIF) plays a key role in blastocyst implantation and development and also regulates uterine receptivity and conceptus (embryo and associated extraembryonic membranes) implantation in many species. In sheep, LIF is expressed by uterine epithelia throughout the estrous cycle and early pregnancy, and LIF is hypothesized to act on the trophectoderm of the rapidly expanding blastocyst. In fact, human LIF improves the implantation rate of ovine embryos. However, little information is available on expression of LIF receptor (LIFR) and co-receptor interleukin 6 signal transducer (IL6ST; also known as gp130), which is a signal transducer shared by many cytokines, including IL6 and LIF. These studies determined effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on LIFR and IL6ST expression in the ovine uterus. In Study One, ewes (n=5/day/status) were hysterectomized on either Day 10, 12, 14 or 16 of the estrous cycle or Day 10, 12, 14, 16, 18 or 20 of pregnancy. In both cyclic and pregnant ewes, endometrial LIFR mRNA levels increased after Day 10, but the increase was greater in pregnant than cyclic ewes (day x status, P<0.01). IL6ST mRNA levels were higher in pregnant than cyclic ewes (status, P<0.04), and increased 2.8-fold from Days 10 to 20 of pregnancy (linear, P<0.05). LIFR mRNA and protein were localized to the endometrial luminal (LE) and superficial glands (sGE), whereas IL6ST mRNA and protein were localized primarily in uterine glands. Both LIFR and IL6ST were expressed in conceptus trophectoderm. In Study Two, Day 5 cyclic ewes were ovariectomized and fitted with uterine catheters. Ewes (n=5/treatment) received daily injections of either 50 mg P4 or P4 with the anti-progestin ZK 136,317 from Days 5 to 16 and intrauterine injections of either control proteins (CX) or recombinant ovine IFNT from Days 11 to 16. All ewes were hysterectomized on Day 17. Endometrial LIFR mRNA was not affected by P4 (P4+CX vs P4+ZK+CX, P>0.10). However, IFNT infusion increased LIFR mRNA levels by 1.7-fold (P4+CX vs P4+IFN, P<0.04), but not in P4+ZK-treated ewes. IL6ST mRNA was increased 2.6-fold by P4 (P4+CX vs P4+ZK+CX, P<0.03), and IFNT infusion increased (P<0.05) IL6ST mRNA abundance by 1.5- and 2.6-fold, respectively, in P4- as well as P4+ZK-treated ewes. Study Three used a model of accelerated blastocyst development in which bred ewes were treated daily with vehicle as a control or 25 mg P4 beginning on Day 1.5 post-mating. In ewes treated to Day 9, endometrial LIFR mRNA abundance was increased 2-fold by P4 (P<0.03), while IL6ST mRNA was unaffected (P>0.10). In ewes treated to Day 12, LIFR mRNA was increased 1.6-fold by P4 (P<0.05), while IL6ST was not affected (P>0.10). Treatment of ewes with the anti-progestin RU486 from Days 9 to 12 reduced endometrial LIFR mRNA levels by 2.5-fold (P<0.005) and IL6ST mRNA by 2.8-fold (P<0.02) as compared to P4-treated ewes. These results indicate that LIFR and IL6ST are both stimulated by IFNT and regulated by P4 in a complex stage- and cell-specific manner. Coexpression of LIFR and IL6ST was observed only in the upper glands of the endometrium and conceptus trophectoderm. Thus, uterine LIF may have actions on both the uterine glands and trophectoderm in order to mediate endometrial receptivity and conceptus growth and development in sheep. (Supported by NIH Grant 5 R01 HD32534)

Leukemia Inhibitory Factor Regulates Trophoblast Giant Cell Differentiation via Janus Kinase 1-Signal Transducer and Activator of Transcription 3-Suppressor of Cytokine Signaling 3 Pathway

Suppressor of cytokine signaling 3 (SOCS3) inhibits leukemia-inhibitory factor (LIF) signaling and acts as a negative regulator. Deletion of SOCS3 causes embryonic lethality because of placental failure, and genetic reduction of LIF or the LIF receptor (LIFR) in SOCS3-deficient mice rescues placental defects and embryonic lethality; this indicates that SOCS3 is an essential inhibitor of LIFR signaling. However, the downstream signaling molecule that acts as a link between the LIFR and SOCS3 has not been identified. In this study we explored the downstream signaling of LIFR. The administration of LIF to SOCS3-heterozygous pregnant mice promotes trophoblast giant cell differentiation and accelerates placental failure in SOCS3-deficient mice. SOCS3-deficient trophoblast stem cells show enhanced and prolonged signal transducer and activator of transcription 3 (Stat3) activation by LIF stimulation. Further, in the trophoblasts of SOCS3-deficient placenta and differentiating cells from the choriocarcinoma-derived cell line Rcho-1 cells, constitutive activation of Stat3 is observed. The forced expression of SOCS3, dominant-negative Stat3, and dominant-negative Janus kinase 1 (JAK1) in Rcho-1 cells significantly suppressed the trophoblast giant cell differentiation of these cells. In addition, the number of trophoblast giant cells is significantly reduced concomitant with an increased number of precursor trophoblasts in JAK1-deficient placentas. Finally, JAK1 deficiency rescues placental defects and embryonic lethality in SOCS3-deficient mice. These results indicate that the LIFR signaling is finely coordinated by JAK1, Stat3, and SOCS3 and regulates trophoblast giant cell differentiation. In addition, these data establish that LIFR-JAK1-Stat3-SOCS3 signaling is an essential pathway for the regulation of trophoblast giant cell differentiation.

Neuroinflammation facilitates LIF entry into brain: role of TNF

Leukemia inhibitory factor (LIF) is a proinflammatory cytokine mediating a variety of central nervous system (CNS) responses to inflammatory stimuli. During lipopolysaccharide (LPS)-induced inflammation, blood concentrations of LIF increase, correlating with lethality of sepsis. Circulating LIF crosses the blood-brain barrier (BBB) by a saturable transport system. Here we determine how this transport system is regulated in neuroinflammation. Using transport assays that quantify the influx rate and volume of distribution of LIF in mice, we show that LPS facilitated the permeation of LIF from the blood to the brain without compromising the paracellular permeability of the BBB as determined by coadministration of fluorescein. Concurrently, gp130 (shared by the interleukin-6 family of cytokines), but not gp190 (the specific receptor for LIF) or cilliary neutrophic factor (CNTF-Ralpha, a unique receptor for cilliary neurotrophic factor that also uses gp130 and gp190), showed increased levels of mRNA and protein expression in cerebral microvessels from the LPS-treated mice. The upregulation of gp130 by LPS was at least partially mediated by vascular tumor necrosis factor receptor (TNFR)1 and TNFR2. This was shown by elevated TNFR1 and TNFR2 mRNA and protein in cerebral microvessels after LPS and by the absence of the LPS effect on gp130 in knockout mice lacking these receptors. The results show that neuroinflammation by LPS induces endothelial signaling and enhances cytokine transport across the BBB.

Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.