Leukemia Inhibitory Factor Stimulation Research Articles

#1261 A small-molecule inhibitor of Bruton's tyrosine kinase reduces production of galactose-deficient IgA1 in IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is a chronic kidney disease characterized by glomerular immunodeposits that contain aberrantly glycosylated IgA1, i.e., IgA1 with some hinge-region O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1). These immunodeposits originate from circulating immune complexes that contain Gd-IgA1 bound by IgG autoantibodies. Consistent with this hypothesis, serum levels of Gd-IgA1 and IgG autoantibodies independently predict disease progression in IgAN patients. Biochemical studies using IgA1-secreting cell lines derived from peripheral blood of IgAN patients and healthy controls have revealed that Gd-IgA1 production is driven by altered biosynthetic pathways of IgA1 O-glycans. Specifically, cell lines from IgAN patients have altered expression and activity of several glycosyltransferases, C1GalT1 and ST6GalNAc2, and a C1GalT1-specific chaperone, C1GalT1C1. Genetic studies have shown that serum levels of Gd-IgA1 are genetically co-determined and genome-wide association studies (GWAS) identified genetic variations that impact the expression C1GALT1 and C1GALT1C1 genes. Moreover, macroscopic hematuria during an upper-respiratory-tract infection is common at the clinical onset of IgAN and during episodes of accentuated disease activity, indicating a connection between IgAN and mucosal inflammation. Notably, a pro-inflammatory cytokine interleukin-6 (IL-6), enhances production of Gd-IgA1 exclusively in the cell lines derived from IgAN patients. Leukemia inhibitory factor (LIF) has similar effects. LIF-induced effects are mediated by abnormal STAT1 signaling and pathways that include Lyn, a Src-family kinase. The corresponding gene, LYN, was recently identified in one of the 16 new IgAN-associated GWAS loci. Lyn, together with spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), is involved in B-cell-receptor (BCR)-signaling pathways that include activation and inhibitory signals. The aim of this study was to assess if Btk may be involved in Gd-IgA1 production in IgAN. Method IgA1-producing cell lines were cloned by limiting dilution of Epstein-Barr-virus-immortalized peripheral-blood B cells from patients with IgAN (n = 5) and healthy controls (n = 5). Cell signaling, induced by LIF or by BCR crosslinking with anti-IgA antibody, was assessed by flow cytometry or SDS-PAGE/immunoblotting using antibodies for specific phosphorylated sites of selected proteins. Data were normalized using antibodies for specific proteins. Total IgA and Gd-IgA1 were quantified in cell-culture medium using standard ELISA and lectin-ELISA, respectively. Gd-IgA1, normalized to IgA, was expressed in Units (U) of standard Gd-IgA1 protein. Results IgA1-producing cell lines from patients with IgAN and healthy controls secreted similar amounts of total IgA (17.2 ± 9.3 ug/ml vs. 14.6 ± 9.5 ug/ml), but the cells from IgAN patients secreted more Gd-IgA1 (24.2 ± 7.5 U vs. 15.5 ± 2.7 U; p < 0.05). Baseline phosphorylation of Btk (Y223), but not Syk (Y551), was higher in the cells from patients with IgAN than in those from healthy controls. We used an IgA1-secreting cell line from an IgAN patient to confirm that BCR crosslinking with F(ab’)2 anti-IgA antibody in IgA + CD19+ cells lines induced phosphorylation of Btk (Y223). Conversely, anti-IgM antibody, used as a negative control, did not activate Btk. LIF stimulation did not affect phosphorylation of Btk or Syk. A Btk inhibitor, PCI-32765 (ibrutinib), reduced Gd-IgA1 production in a dose-dependent fashion exclusively in the IgAN-derived cells. Notably, 1 uM PCI-32765 reduced Gd-IgA1 production to that for healthy controls (25.7 ± 1.2 U to 17.3 ± 1.8 U; p < 0.01). Conclusion Inhibition of Btk with a small-molecule inhibitor normalized Gd-IgA1 production in IgA1-secreting cells from patients with IgAN, revealing a previously unknown role of Btk in aberrant O-glycosylation of IgA1. Btk may thus represent a potential new target for disease-specific therapy of IgAN.

Leukemia inhibitory factor is involved in the pathogenesis of NSCLC through activation of the STAT3 signaling pathway.

Leukemia inhibitory factor (LIF) is a tumor promoter in several cancer types. However, the role of LIF in non-small cell lung cancer (NSCLC) remains to be explored. The present study explored the hypothesis that LIF is important for NSCLC development by measuring LIF expression and its downstream signal transducer and activator of transcription 3 (STAT3) phosphorylation in tumor samples derived from patients with NSCLC. The association between LIF expression and clinical features was analyzed in two cancer subtypes. The effects of LIF on cell proliferation, migration and invasion were also evaluated in a NSCLC-derived cell line, A549. LIF mRNA and protein expression levels were significantly higher in tumor tissues compared with those in the corresponding adjacent and normal lung tissues. Regarding NSCLC subtypes, LIF expression was significantly higher in adenocarcinoma than in squamous cell carcinoma tissues. It was also found that phosphorylated-STAT3 levels were higher in tumor tissues compared with those in the corresponding adjacent and normal lung tissues, which was in agreement with the LIF expression levels in NSCLC tissues. Clinically, overexpression of LIF was positively correlated with aggressive tumor characteristics, including lymph node metastasis and advanced tumor stage. In A549 cells, LIF treatment enhanced cell proliferation, migration and invasion. LIF also increased STAT3 phosphorylation in A549 cells, and the STAT3 inhibitor Stattic decreased A549 cell migration and invasion following LIF stimulation. The present results demonstrate that LIF is overexpressed in NSCLC, and that LIF can promote NSCLC development through activation of the STAT3 signaling pathway. The present study indicates that LIF may serve as a potential prognostic marker for NSCLC.

Leukemia Inhibitory Factor Signaling Enhances Production of Galactose-Deficient IgA1 in IgA Nephropathy

Objectives: IgA nephropathy (IgAN) is thought to involve an autoimmune process wherein galactose-deficient IgA1 (Gd-IgA1), recognized as autoantigen by autoantibodies, forms pathogenic immune complexes. Mounting evidence has implicated abnormal activation of some protein-tyrosine kinases (PTKs) in IgAN. Furthermore, genome-wide association studies (GWAS) of IgAN provided insight into disease pathobiology and genetics. A GWAS locus on chromosome 22q12 contains genes encoding leukemia inhibitory factor (LIF) and oncostatin M, interleukin (IL)-6-related cytokines implicated in mucosal immunity and inflammation. We have previously shown that IL-6 mediates overproduction of Gd-IgA1 through aberrant STAT3 activation. Here, we show that LIF enhanced production of Gd-IgA1 in IgA1-secreting cells of patients with IgAN and provide initial analyses of LIF signaling. Methods: We characterized LIF signaling that is involved in the overproduction of Gd-IgA1, using IgA1-secreting cell lines derived from peripheral blood of patients with IgAN and healthy controls (HC). We used global PTK activity profiling, immunoblotting, lectin ELISA, and siRNA knock-down. Results: LIF stimulation did not significantly affect production of total IgA1 in IgA1-secreting cells from patients with IgAN or HC. However, LIF increased production of Gd-IgA1, but only in the cells from patients with IgAN. LIF stimulation enhanced phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN to a higher degree than in the cells from HC. siRNA knock-down of STAT1 blocked LIF-mediated overproduction of Gd-IgA1. Unexpectedly, this abnormal phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN was not mediated by JAK, but rather involved activation of Src-family PTKs (SFKs). Conclusion: Abnormal LIF/STAT1 signaling represents another pathway potentially leading to overproduction of Gd-IgA1 in IgAN, providing possible explanation for the phenotype associated with chromosome 22q12 GWAS locus. Abnormal LIF/STAT1 signaling and the associated SFKs may represent potential diagnostic and/or therapeutic targets in IgAN.

Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation

ObjectivesInsulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. MethodsTo explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. ResultsInterestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. ConclusionWe report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.

Leukemia inhibitory factor protects cholangiocarcinoma cells from drug-induced apoptosis via a PI3K/AKT-dependent Mcl-1 activation.

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation.Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis.In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.

Leukemia inhibitory factor increases glucose uptake in mouse skeletal muscle.

Members of the IL-6 family, IL-6 and ciliary neurotrophic factor (CNTF), have been shown to increase glucose uptake and fatty acid oxidation in skeletal muscle. However, the metabolic effects of another family member, leukemia inhibitory factor (LIF), are not well characterized. Effects of LIF on skeletal muscle glucose uptake and palmitate oxidation and signaling were investigated in ex vivo incubated mouse soleus and EDL muscles from muscle-specific AMPKα2 kinase-dead, muscle-specific SOCS3 knockout, and lean and high-fat-fed mice. Inhibitors were used to investigate involvement of specific signaling pathways. LIF increased muscle glucose uptake in dose (50-5,000 pM/l) and time-dependent manners with maximal effects at the 30-min time point. LIF increased Akt Ser(473) phosphorylation (P) in soleus and EDL, whereas AMPK Thr(172) P was unaffected. Incubation with parthenolide abolished LIF-induced glucose uptake and STAT3 Tyr(705) P, whereas incubation with LY-294002 and wortmannin suppressed both basal and LIF-induced glucose uptake and Akt Ser(473) P, indicating that JAK and PI 3-kinase signaling is required for LIF-stimulated glucose uptake. Incubation with rapamycin and AZD8055 indicated that mammalian target of rapamycin complex (mTORC)2, but not mTORC1, also is required for LIF-stimulated glucose uptake. In contrast to CNTF, LIF stimulation did not alter palmitate oxidation. LIF-stimulated glucose uptake was maintained in EDL from obese insulin-resistant mice, whereas soleus developed LIF resistance. Lack of SOCS3 and AMPKα2 did not affect LIF-stimulated glucose uptake. In conclusion, LIF acutely increased muscle glucose uptake by a mechanism potentially involving the PI 3-kinase/mTORC2/Akt pathway and is not impaired in EDL muscle from obese insulin-resistant mice.

Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells

Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells

Bone material properties in hypoparathyroidism

Bone material properties in hypoparathyroidism

Abstract 9396: A Genetic Fate-Mapping Study Showing That Leukemia Inhibitory Factor Stimulates Cardiac Stem Cell-Derived Cardiomyocyte Renewal After Myocardial Infarction

Introduction: Recent studies have contradicted the notion that the adult heart is a terminally differentiated organ. The source of cardiac regeneration and its relation with cardio-protective drugs remain controversial. Using a genetic fate-mapping model, we examined the rate of stem cell-derived cardiomyocyte renewal, focusing on the effect of leukemia inhibitory factor (LIF) on cardiac stem cells. Methods and Results: We generated MerCreMer-LacZ mice, in which more than 99.9% of the cardiomyocytes in the left ventricular field showed positive 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) staining just after tamoxifen injection. Thus, every Xgal-negative cardiomyocyte was derived from a stem or precursor cell after tamoxifen administration. During normal aging spanning 1 year, the number of Xgal-negative cardiomyocytes in the mice did not change significantly: 2 weeks, 9.8 ± 3.8 cells per section; 1 year, 9.8 ± 5.2 cells per section. In contrast, at 6 months after myocardial infarction (MI), the MI mice had more Xgal-negative cells (41.0 ± 9.3 cells per section) than the control mice (8.0 ± 2.6 cells per section; P < 0.001) did. We injected the LIF plasmid or phosphate-buffered saline (PBS) at the time of MI. At 1 month after MI, the MI + LIF group (118.6 ± 51.5 cells per section) had a greater number of Xgal-negative cells than the MI + PBS group (37.0 ± 5.5 cells per section; P < 0.05) did. Echocardiography showed significant recovery of functional shortening in the LIF-treated group only. To detect the source of LIF-induced cardiomyocyte renewal, we studied cardiac side population (SP) cells. SP cells were found to have LIF receptors, and the percentage of phosphorylated signal transducers and activators of transcription 3-positive and cyclin D1-positive SP cells increased after LIF stimulation in vitro. In addition, continuous 5-bromodeoxyuridine (BrdU) administration during the first week after MI resulted in 68.9% BrdU-positive SP cells in LIF-treated mice, but only 30.5% in PBS-treated mice, suggesting that LIF influenced SP cell proliferation. Conclusion: LIF may stimulate stem cell-derived cardiomyocyte regeneration in part by activating SP cells. Our findings should help provide a novel strategy for therapeutic cardiogenesis.

Reduction in miR‐141 is Induced by Leukemia Inhibitory Factor and Inhibits Proliferation in Choriocarcinoma Cell Line JEG‐3

Starting from the peri-implantation period, leukemia inhibitory factor (LIF) is a major regulator of trophoblast functions. Micro-RNAs (miRNA) are short non-coding RNA sequences, which regulate expression of genes at post-transcriptional level. The influence of LIF on miRNA expression in trophoblastic cells has not yet been analyzed and was focus of this investigation. JEG-3 choriocarcinoma cells have been stimulated with LIF for 1, 2, 4, 6, and 24 hr. The expression of miR-9, miR-141, miR-21, miR-93, and let-7 g has been analyzed by real-time PCR. Subsequently, miR-141 has been silenced and over-expressed to test its role in the proliferation of JEG-3 cells after 24 and 48 hr. MiR-141 has been significantly downregulated by more than 50% after LIF stimulation, while miR-21 and miR-93 expression has been significantly upregulated. Silencing of miR-141 completely inhibited the proliferation of JEG-3 cells, while over-expression had no effect. LIF regulates expression of miRNA in trophoblastic cells, which may be responsible for several functional effects induced by LIF.

STAT3/Pim-1 signaling pathway plays a crucial role in endothelial differentiation of cardiac resident Sca-1+ cells both in vitro and in vivo

Cardiac stem cells potentially differentiate into cardiac cells, including cardiomyocytes and endothelial cells (ECs). Previously we demonstrated that STAT3 activation by IL-6 family cytokines, such as leukemia inhibitory factor (LIF), induces the endothelial differentiation of cardiac Sca-1+ cells. In this study, we addressed molecular mechanisms for EC differentiation of Sca-1+ cells. First, DNA array experiments were performed to search for the molecules induced by LIF. Among 134 genes that LIF upregulated by more than 4 fold, we focused on Pim-1 gene transcript, because Pim-1 is associated with the differentiation of some cell lineages. Real time RT-PCR analyses confirmed that LIF stimulation upregulated Pim-1 expression. Adenoviral transfection of dominant negative (dn) STAT3 inhibited LIF-mediated induction of Pim-1, while the overexpression of constitutively active STAT3 upregulated Pim-1 expression, suggesting that STAT3 activation is necessary and sufficient for Pim-1 induction. Moreover, in STAT3-deficient Sca-1+ cells, LIF failed to induce Pim-1 expression and EC differentiation. Importantly, the overexpression of dnPim-1 abrogated the induction of EC markers, indicating Pim kinase activity is indispensable for STAT3-mediated EC differentiation in vitro. Finally, Sca-1+ cells labeled with LacZ were transplanted into post-infarct myocardium and the transdifferentiation was estimated. The overexpression of wild-type STAT3 by adenovirus vector significantly promoted EC differentiation, while STAT3 gene ablation reduced the frequency of differentiating cells in post-infarct myocardium. Furthermore, transplanted Sca-1+ cells overexpressing dnPim-1 showed the reduced frequency of EC differentiation and capillary density. Collectively, Pim-1 kinase is upregulated by STAT3 activation in cardiac Sca-1+ cells and plays a pivotal role in EC differentiation both in vitro and in vivo.

Stimulation of IL-6 Cytokines in Fibroblasts by Toll-like Receptors 2

The osteotropic interleukin-6 (IL-6) types of cytokines IL-6, IL-11, and leukemia inhibitory factor (LIF), but not oncostatin M, are expressed by human gingival fibroblasts, and their expressions are regulated by IL-1 and tumor necrosis factor-α (TNF-α). In the present study, we investigated whether signaling through Toll-like receptor 2 (TLR2) can affect the expression of these cytokines in human gingival fibroblasts. Lipopolysac-charide (LPS) from P. gingivalis was found to stimulate IL-6 and LIF mRNA and protein, but not IL-11 or OSM mRNA. Using two synthetic ligands acting specifically at TLR2 and siRNA knockdown of TLR2, we demonstrated the important role of TLR2 in the stimulation of IL-6 and LIF in gingival fibroblasts. Analysis of these data suggests that signaling through the innate immune system controls the expression of osteotropic cytokines in human gingival fibroblasts.

SHP2 mediates gp130-dependent cardiomyocyte hypertrophy via negative regulation of skeletal alpha-actin gene

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal α-actin (α-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in α-SKA gene expression. Thus, α-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of α-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in α-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes α-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate α-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2WT), dominant-negative SHP2 (SHP2C/S), or β-galactosidase (β-gal), on α-SKA gene expression. LIF did not upregulate α-SKA mRNA in cardiomyocytes overexpressing either β-gal or SHP2WT. In cardiomyocytes overexpressing SHP2C/S, LIF induced upregulation of α-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2C/S, but not in myocytes overexpressing β-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates α-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.

N-Glycans modulate the activation of gp130 in mouse embryonic neural precursor cells

gp130 is a ubiquitously expressed glycoprotein and signal transducer of interleukin 6 family of cytokines. It has been reported that gp130 has 11 potential N-glycosylation sites in the extracellular domain, and nine of them are actually N-glycosylated. However, the structure and functional role of the carbohydrate chains carried by gp130 are totally unknown. In this study, we examined the functional role of N-glycans of gp130 in mouse neuroepithelial cells. In neuroepithelial cells treated with tunicamycin, an N-glycosylation inhibitor, unglycosylated form of gp130 was detected. The unglycosylated gp130 was not phosphorylated in response to leukemia inhibitory factor stimulation. Although the unglycosylated gp130 was found to be expressed on the cell surface, it could not form a heterodimer with leukemia inhibitory factor receptor. These results suggest that N-glycans are required for the activation, but not for the localization, of gp130 in neuroepithelial cells.

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.

Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates their signaling pathways. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by v-src and Jak2, using a phospho-specific antibody against STAP-2 phosphorylated at Tyr250. Mutational analyses revealed that Tyr250 was involved in the STAT3-enhancing activity of STAP-2. We further found that leukemia inhibitory factor (LIF) stimulated STAP-2 Tyr250 phosphorylation in 293T and Hep3B cells. Moreover, endogenous STAP-2 was phosphorylated at Tyr250 following LIF stimulation of murine M1 cell line. Taken together, our findings demonstrate that endogenous STAP-2 is phosphorylated at Tyr250 and that this phosphorylation is involved in its function.

Differential stimulation‐induced receptor localization in lipid rafts for interleukin‐6 family cytokines signaling through the gp130/leukemia inhibitory factor receptor complex

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are cytokines which signal through receptor complexes that include the receptor subunits glycoprotein 130 (gp130) and the LIF receptor (LIFR), but CNTF also requires the non-signal transducing CNTF receptor (CNTFR) for binding. We show here that in IMR-32 neuronal cells endogenously expressing the receptor subunits for LIF and CNTF, CNTFR, but not gp130 or LIFR, is found in detergent-resistant lipid rafts. In addition, stimulation of these cells with CNTF resulted in a rapid translocation of a portion of gp130 and LIFR into detergent-resistant lipid rafts while an equivalent stimulation with LIF did not. Disruption of lipid rafts by cholesterol depletion of cell membranes blocked the CNTF-induced translocation of LIFR and gp130. Interestingly, while cholesterol-depletion did not inhibit signal transducer and activator of transcription 3 phosphorylation by either CNTF or LIF stimulation, it strongly inhibited both CNTF- and LIF-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 and Akt. LIF and CNTF generally appear to have redundant effects in cells responsive to both cytokines. Intriguingly, the data presented here suggest a possible mechanism whereby CNTF or other cytokines that signal through CNTFR could generate signals distinct from those elicited by cytokines such as LIF which utilize a LIFR/gp130 heterodimer, via association with or exclusion from lipid rafts.

Multiple promoter elements required for leukemia inhibitory factor‐stimulated M2 muscarinic acetylcholine receptor promoter activity

Treatment of neuronal cells with leukemia inhibitory factor (LIF) results in increased M(2) muscarinic acetylcholine receptor promoter activity. We demonstrate here that multiple promoter elements mediate LIF stimulation of M(2) gene transcription. We identify a LIF inducible element (LIE) in the M(2) promoter with high homology to a cytokine-inducible ACTG-containing sequence in the vasoactive intestinal peptide promoter. Mutagenesis of both a STAT (signal transducers and activators of transcription) element and the LIE in the M(2) promoter is required to attenuate stimulation of M(2) promoter activity by LIF completely. Mobility shift assays indicate that a LIF-stimulated complex binds to a 70 base pair M(2) promoter fragment. Furthermore, a STAT element within this fragment can bind to LIF-stimulated nuclear STAT1 homodimers in vitro. Mutagenesis experiments show that cytokine-stimulated activation of M(2) promoter activity requires tyrosine residues on glycoprotein 130 (gp130) that are also required for both STAT1 and STAT3 activation. Dominant negative STAT1 or STAT3 can block LIF-stimulated M(2) promoter activity. Real-time RT-PCR analysis indicates that LIF-stimulated induction of M(2) mRNA is partially dependent on protein synthesis. These results show that regulation of M(2) gene transcription in neuronal cells by LIF occurs through a complex novel mechanism that is dependent on LIE, STAT and de novo protein synthesis.

1141635071 Suppressor of Cytokine Signalling 3 (SOCS3) controls trophoblast invasion and proliferation

Introduction: Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular mediator of trophoblast invasion. In other cells, Suppressor Of Cytokine Signalling 3 (SOCS3) is known to be a major antagonist of STAT3 activation. For auto‐regulation and to avoid excessive signals, SOCS3 gene expression is partly induced by STAT3.Methods: The human choriocarcinoma cell line JEG‐3 was used for all experiments. STAT3 expression and STAT3 tyrosine (705) phosphorylation were analyzed by polyacrylamid gele electrophoresis followed by Western blotting. Interleukin‐6 (IL‐6) or Leukemia Inhibitory Factor (LIF) were used for stimulation. SOCS3 expression was silenced by RNA interference. For this approach in a first step, small interfering RNA (siRNA) was used. Subsequently, Jeg‐3 cells were transfected with genes for the respective, self‐designed small hairpin RNA (shRNA), neomycin resistance and green fluorescent protein. Application of neomycin and cell sorting by green fluorescence were used for elimination of non‐transfected cells. Non‐genomic siRNA and shRNA were respectively used for controls. Colorimetric proliferation assays were performed.Results: In IL‐6 stimulated cells, SOCS3 knockdown leads to increased STAT3 tyrosine phosphorylation. After LIF stimulation, proliferation in SOCS3 knockdown cells is significantly higher than in controls. STAT3 expression is not affected by SOCS3 knockdown.Conclusion: SOCS3 plays a major role in intracellular regulation of trophoblast proliferation and invasiveness. The balance of STAT3 and SOCS3 may tune the behavior of invading trophoblast cells.

Leukemia Inhibitory Factor Induces Endothelial Differentiation in Cardiac Stem Cells

The importance of interleukin 6 (IL-6)-related cytokines in cardiac homeostasis has been studied extensively; however, little is known about their biological significance in cardiac stem cells. Here we describe that leukemia inhibitory factor (LIF), a member of IL-6-related cytokines, activated STAT3 and ERK1/2 in cardiac Sca-1+ stem cells. LIF stimulation resulted in the induction of endothelial cell-specific genes, including VE-cadherin, Flk-1, and CD31, whereas neither smooth muscle nor cardiac muscle marker genes such as GATA4, GATA6, Nkx-2.5, and calponin were up-regulated. Immunocytochemical examination showed that about 25% of total cells were positively stained with anti-CD31 antibody 14 days after LIF stimulation. Immunofluorescent microscopic analyses identified the Sca-1+ cells that were also positively stained with anti-von Willebrand factor antibody, indicating the differentiating process of Sca-1+ cells into the endothelial cells. IL-6, which did not activate STAT3 and ERK1/2, failed to induce the differentiation of cardiac stem cells into the endothelial cells. In cardiac stem cells, the transduction with dominant negative STAT3 abrogated the LIF-induced endothelial differentiation. And the inhibition of ERK1/2 with the MEK1/2 inhibitor U0126 also prevented the differentiation of Sca-1+ cells into endothelial cells. Thus, both STAT3 and ERK1/2 are required for LIF-mediated endothelial differentiation in cardiac stem cells. Collectively, it is proposed that LIF regulates the commitment of cardiac stem cells into the endothelial cell lineage, contributing to neovascularization in the process of tissue remodeling and/or regeneration.