Leukemia Inhibitory Factor Secretion Research Articles

Polarized secretion of Leukemia Inhibitory Factor

BackgroundThe direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor (LIF) belongs to the interleukin-6 (IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1β is known to promote IL-6 secretion from the stimulated membrane (apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1β stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney (MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF.ResultsWhen Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1β increased LIF production. After treating the apical surface with IL-1β, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones.ConclusionThe site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.

Inhibition of Myoblast Differentiation by Tumor Necrosis Factor α Is Mediated by c-Jun N-terminal Kinase 1 and Leukemia Inhibitory Factor

The proinflammatory cytokine, TNFalpha plays a major role in muscle wasting occurring in chronic diseases and muscular dystrophies. Among its other functions, TNFalpha perturbs muscle regeneration by preventing satellite cell differentiation. In the present study, the role of c-Jun N-terminal kinase (JNK), a mediator of TNFalpha, was investigated in differentiating myoblast cell lines. Addition of TNFalpha to C2 myoblasts induced immediate and delayed phases of JNK activity. The delayed phase is associated with myoblast proliferation. Inhibition of JNK activity prevented proliferation and restored differentiation to TNFalpha-treated myoblasts. Studies with cell lines expressing MyoD:ER chimera and lacking JNK1 or JNK2 genes indicate that JNK1 activity mediates the effects of TNFalpha on myoblast proliferation and differentiation. TNFalpha does not induce proliferation or inhibit differentiation of JNK1-null myoblasts. However, differentiation of JNK1-null myoblasts is inhibited when they are grown in conditioned medium derived from cell lines affected by TNFalpha. We investigated the induced synthesis of several candidate growth factors and cytokines following treatment with TNFalpha. Expression of IL-6 and leukemia inhibitory factor (LIF) was induced by TNFalpha in wild-type and JNK2-null myoblasts. However, LIF expression was not induced by TNFalpha in JNK1-null myoblasts. Addition of LIF to the growth medium of JNK1-null myoblasts prevented their differentiation. Moreover, LIF-neutralizing antibodies added to the medium of C2 myoblasts prevented inhibition of differentiation mediated by TNFalpha. Hence, TNFalpha promotes myoblast proliferation through JNK1 and prevents myoblast differentiation through JNK1-mediated secretion of LIF.

Inhibition of STAT3 Expression and Signaling in Resveratrol-Differentiated Medulloblastoma Cells

In this study, the potential influence of resveratrol (3,5,4′-trihydroxy-trans-stilbene) in signal transducer and activator of transcription 3 (STAT3) signaling of medulloblastoma cells was evaluated by checking the status of STAT3 signaling and its downstream gene expression in two medulloblastoma cell lines (UW228-2 and UW228-3) with and without resveratrol treatment. The results revealed that resveratrol induced neuronal differentiation of medulloblastoma cells. Signal transducer and activator of transcription 3 expression and phosphorylation were detected in normally cultured UW228-2 and UW228-3 cells that were apparently attenuated after resveratrol treatment. The expression of STAT3 downstream genes, survivin, cyclin D1, Cox-2, and c-Myc, was suppressed but Bcl-2 was enhanced by resveratrol. Meanwhile, the production and secretion of leukemia inhibitory factor, a STAT3 activator, became active in resveratrol-treated cells. To further ascertain the significance of STAT3 signaling for medulloblastoma cells, AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 cells. Phosphorylation of STAT3 was inhibited by AG490 accompanied with growth suppression, differentiation-like changes, and down-regulation of survivin, cyclin D1, Cox-2, and c-Myc. Our data thus suggest the importance of STAT3 signaling in maintenance and survival of medulloblastoma cells. This signaling may be the major target of resveratrol. Enhanced leukemia inhibitory factor and Bcl-2 expressions in resveratrol-treated cells might reflect a compensatory response to the loss of STAT3 function.

Isoflavones regulate secretion of leukemia inhibitory factor and transforming growth factor β and expression of glycodelin in human endometrial epithelial cells

Isoflavones have attracted much attention due to their association with health benefits; however, comprehensive understanding of the beneficial impacts of isoflavones on uterine biology at the molecular level remains unexplored. In the present study, our data showed that isoflavones aglycones AglyMax, genistein, and equol, but not daidzein, within the range of plasma concentration, displayed bioavailability in regulating the secretion of leukemia inhibitory factor (LIF) and transforming growth factor beta (TGF-beta) in Ishikawa cells, which was blocked by an estrogen receptor antagonist ICI 182 780, mitogen-activated protein kinase kinase (MEK)1/2 inhibitor PD98059, and p38 mitogen-activated protein kinase inhibitor SB203580. We also found that AglyMax and genistein increased in cyclic AMP release and the expression of glycodelin protein in Ishikawa cells assayed using western blot and immunochemical staining. The MEK1/2 inhibitor PD98059 and the protein kinase A inhibitor H89, but not SB203580, attenuated this glycoprotein expression. Moreover, isoflavone aglycones AglyMax stimulated LIF, and TGF-beta secretion, and glycodelin expression in separate primary endometrial epithelial cells in the follicular phase or luteal phase from healthy subject donors. Overall, our findings suggest that isoflavones may alter the uterine expression of estrogen-responsive genes.

Inflammatory responses induced by interleukin-17 family members in human colonic subepithelial myofibroblasts

We investigated the potential role of interleukin (IL)-17 family members (IL-17A to IL-17F) in the induction of inflammatory responses in human colonic subepithelial myofibroblasts (SEMFs). The expression of the inflammatory cytokines IL-6, IL-8, leukemia inhibitory factor (LIF), and matrix metalloproteinases (MMP)-1 and MMP-3 were evaluated by enzyme-linked immunosorbent assay and Northern blotting. Activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-17A and IL-17F significantly enhanced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. The effects of IL-17A were relatively stronger than those induced by IL-17F. The effects of IL-17B, IL-17C, IL-17D, and IL-17E were modest as compared with those induced by IL-17A and IL-17F. Both IL-17A and IL-17F augmented IL-1beta-induced secretion of IL-6, IL-8, LIF, MMP-1, and MMP-3. A similar augmentation was also observed in tumor necrosis factor (TNF)-alpha-induced cytokine and MMP secretion. IL-17A and IL-17F rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38 MAPKs, and c-Jun-NH(2)-terminal kinase (JNK) as early as 15 min after stimulation. Inhibitors for ERK (PD98059 and U0216) and p38 MAPK (SB203580) significantly reduced the IL-17F-induced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. Among IL-17 family members, IL-17A and IL-17F strongly stimulate human colonic SEMFs, inducing inflammatory responses.

Isoflavones regulate secretion of leukemia inhibitory factor and transforming growth factor β and expression of glycodelin in human endometrial epithelial cells

Isoflavones have attracted much attention due to their association with health benefits. However, comprehensive understanding of the beneficial impacts of isoflavones on uterine biology at the molecular level remains unexplored. Therefore, we tested the hypothesis that isoflavones, including isoflavones aglycones AglyMax-70, genistein, daidzein and equol within the range of the plasma concentration displays disparate bioavailability in regulating the secretion of Leukaemia inhibitory factor (LIF), transforming growth factor β (TGF-β), and expression of glycodelin in Ishikawa cells and in normal human endometrial epithelial cells. Prospective, in vitro experimental study. Human endometrial epithelial Ishikawa cells obtained from the Human Science Research Resources Bank (Osaka, Japan), the normal human endometrial epithelial cells were isolated from endometrium tissues. We used epithelial cells in culture after the first passage. We tested the isoflavones aglycones AglyMax-70, genistein, daidzein and equol within the range of the plasma concentration displays disparate bioavailability in regulating the secretion of LIF, TGF-β, and expression of glycodelin in Ishikawa cells and in normal human endometrial epithelial cells. In the present study, isoflavones aglycones AglyMax-70, genistein, and equol, but not daidzein, within the range of plasma concentration displays bioavailability in regulating secretion of LIF and TGF-β in Ishikawa cells, which was blocked by an estrogen receptor antagonist ICI 182 780, MEK1/2 inhibitor PD98059, and p38 MAP kinase inhibitor SB203580. We also found that AglyMax-70 and genistein increased in cyclic AMP release and expression of glycodelin protein in Ishikawa cells assayed by using Western blot and immunochemistrical staining. The MEK1/2 inhibitor PD98059 and PKA inhibitor H89, but not SB203580, attenuated this glycoprotein expression. Moreover, isoflavones aglycones AglyMax stimulated LIF, TGF-β secretion and glycodelin expression in separated primary endometrial epithelial cells at the follicular phase or luteal phase from health subject donors. Overall, the present in vitro studies demonstrated that isoflavones aglycones elevated the secretion of LIF and TGF-β and induced expression of glycodelin in Ishikawa cells and isolated human primary endometrial epithelial cells. Thus soy isoflavones aglycones acting as a dietary supplement may have benefit for uterine biology and early pregnancy.

Leukemia Inhibitory Factor (LIF): Marrow-Derived Human Mesenchymal Stem Cell (huMSC) Secretion and Potential Impact on Umbilical Cord Blood (UCB) CD133+ Hematopoietic Stem Cells (HSC) Differentiation.

In vitro expansion of UCB using cytokines has been pursued to overcome the limited stem cell content of a single UCB graft. We have previously demonstrated that a feeder layer of huMSC inhibits UCB HSC differentiation during short-term cytokine-driven expansion in vitro. The protein LIF has been shown to inhibit the differentiation of neurosphere stem cells during 3 week culture over a monolayer of murine stromal cells (C. Shih, et al., 2001). We sought to investigate the hypothesis that LIF secreted by bone marrow (BM) derived huMSCs is involved in inhibition of UCB HSC differentiation during short-term cytokine-driven expansion in vitro. BM derived huMSCs (third passage) were cultured at 2x10^6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72 hours and analyzed for LIF secretion levels by ELISA (Quantikine). LIF secreted by huMSC was noted at all four time points, with peak secretion at 48 h (mean 52.1±3.3 pg/ml) (n=3). UCB was obtained according to institutional guidelines after normal full-term deliveries, collected into bags with citrate dextrose (Allegiance), and MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi) and surface stained for LIF receptor (LIF-R). Surface expression of LIF-R on gated CD133+ cells was 2.61%. Total LIF-R expression in isolated CD133+ cells was further confirmed by Western blot (n=3) using anti-LIF-R antibody (Chemicon). Isolated UCB CD133+ were plated in 24 well plates at 3.3x10^3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B (Gibco). UCB CD133+ were culture-expanded for 96h with or without the addition of recombinant human LIF (Chemicon) (10ng/ml) in a combination of cytokines including: IL-3 (20 ng/mL), IL-6 (20 ng/mL), Flt-3L (100 ng/mL) (R&D), SCF (100 ng/mL), G-CSF (20 ng/mL), and EPO (0.1 U/mL) (Amgen Inc.). At 0, 48, and 96 h cell counts were obtained and flow analysis was performed including surface staining for: CD133, CD34, CD38, HLA-DR, CD33, CD71, and CD41B (Becton Dickinson). At 48 hours, higher cell counts in cultures without LIF were noted 6.3x10^4 ± 0.9/ml, compared with cultures with LIF 4.4x10^4 ± 0.8/ml. However, at 96 h cell counts equalized when comparing cultures with or without LIF at 7.510x10^4 ± 0.9/ml, and 7.8x10^4±0.9/ml respectively (n=6). Surface expression of differentiation markers on gated CD133+ cells did not differ when comparing cultures (n=3). In summary, we observed LIF secretion by MSC peaks at 48 h at a concentration 3 logs lower than that previously used to inhibit stem cell differentiation (10 ng/ml). Although LIF-R is expressed on CD133+ HSC, no difference in cell expansion nor surface phenotype of UCB 133+ cells was observed at early time points (96h) during expansion in cytokines with or without the addition of LIF. Taken together, huMSC mediated inhibition of cytokine-driven UCB HSC differentiation is not attributable to LIF secretion alone and may require direct cell contact between UCB CD133 HSC and huMSC. Studies are ongoing to determine whether LIF may augment huMSC-based UCB CD133 expansion.

Effects of IFN-β, leptin and simvastatin on LIF secretion by T lymphocytes of MS patients and healthy controls

In multiple sclerosis (MS), oligodendrocyte injury is believed to be caused by an aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that inflammatory responses in the central nervous system are not exclusively detrimental, but may also exert protective effects. Such protective effects are potentially mediated by the local secretion of neurotrophic factors by immune cells. We previously reported that T cells and monocytes in vitro and in inflammatory MS lesions produce leukaemia inhibitory factor (LIF), a member of the neuropoietic family of neurotrophins. In the present study, we report a reduced LIF production by CD4 + T cells of relapsing remitting MS patients as compared to healthy controls. Furthermore, immunomodulatory agents such as leptin, IFN-β and simvastatin were studied for their potential to alter LIF and secretion of other cytokines by T cells and monocytes of relapsing remitting MS patients and healthy controls. Low doses of simvastatin, but not IFN-β or leptin enhanced LIF secretion by CD4 + T cells of RR-MS patients. We further demonstrated that LIF did not influence viability, proliferation and cytokine secretion of T cells. Together these data provide new information on the regulation of LIF secretion by immune cells. Further insights into the complex regulation of neurotrophic factors such as LIF may prove useful for treatment of MS.

Astrocytes Relay the Message

Oligodendrocytes are the glial cells that form the insulated membrane sheath called myelin on axons of the central nervous system. Electrical activity of the neurons increases myelination by stimulating differentiation of oligodendrocyte progenitor cells. This is mediated by adenosine, presumably formed from the adenosine triphosphate (ATP) released during neuronal electrical activity. Ishibashi et al. established a coculture system that allowed them to ask whether the neuron also provided instructive signals to mature oligodendrocytes, which may have important implications for alterations in myelination that occur during adulthood. Preparations of predominantly oligodendrocytes precursors (with a small percentage of astrocytes) were cocultured with dorsal root ganglion (DRG) neurons until the oligodendrocytes differentiated; then the neurons were stimulated or agonists were applied, and the percent of myelinated axons was determined. In response to stimulation of the neurons, a higher percentage of axons were myelinated than in cocultures that were not stimulated or in which action potentials were blocked. Application of the nonhydrolyzable ATP analog 2MeSATP or the P2X receptor agonist α-β methyl ATP also stimulated the increase in myelination in the absence of electrical activity, and apyrase, which hydrolyzes ATP, prevented the increase in myelination in response to electrical activity. The cytokine leukemia inhibitory factor (LIF) had been previously implicated in stimulating myelination, and Ishibashi et al. found that secretion of LIF was stimulated by electrical activity and that activity blocking antibodies against LIF prevented 2MeATP from increasing myelination. Examination of the cocultures indicated that LIF was produced by the small population of astrocytes and that the increase in transcription and secretion induced by electrical activity was blocked by apyrase. In cocultures with purified oligodendrocytes lacking any astrocytes, 2MeATP did not enhance myelination, confirming that the astrocytes were responsible for the ATP-stimulated increase in myelination. Addition of purified astrocytes, but not astrocytes from LIF-knockout mice, to the pure oligodendrocyte-DRG neuron cocultures restored the increase in myelination in response to 2MeATP. Thus, astrocytes appear to serve a key role in converting the neuronal electrical signal (mediated by released ATP) into a cytokine (LIF) signal that stimulates mature oligodendrocytes to form myelin (see Spiegel and Peles for commentary). T. Ishibashi, K. A. Dakin, B. Stevens, P. R. Lee, S. V. Kozlov, C. L. Stewart, R. D. Fields, Astrocytes promote myelination in response to electrical impulses. Neuron 49 , 823-832 (2006). [PubMed] I. Spiegel, E. Peles, A new player in CNS myelination. Neuron 49 , 777-778 (2006). [PubMed]

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.

Leukemia inhibitory factor is produced by myelin‐reactive T cells from multiple sclerosis patients and protects against tumor necrosis factor‐α‐induced oligodendrocyte apoptosis

In multiple sclerosis (MS), damage to oligodendrocytes is believed to be caused by an aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that these T cells are not exclusively detrimental but might also exert protective effects. We report for the first time that myelin-reactive T-cell clones from eight MS patients (6/19) and five healthy controls (4/11) produce leukemia inhibitory factor (LIF), a member of the neuropoietic family of neurotrophins. In addition, T-cell clones specific for tetanus toxoid, CD4(+) and CD8(+) T cells, and monocytes, but not B cells, secreted LIF. LIF-producing T lymphocytes and macrophages were also identified immunohistochemically in both active and chronic-active MS lesions. We further demonstrated dose-dependent protective effects of LIF on tumor necrosis factor-alpha-induced apoptosis of oligodendrocytes. In conclusion, our data demonstrate that peripheral and CNS-infiltrating T cells from MS patients produce LIF, a protective factor for oligodendrocytes. This study emphasizes that secretion of LIF may contribute to the neuroprotective effects of autoreactive T cells.

L2/100 – Increased secretion of leukaemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signalling and airway contractility

L2/100 – Increased secretion of leukaemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signalling and airway contractility

Secretory role for human uterodomes (pinopods): secretion of LIF

The differentiation of human endometrial epithelium is a dynamic event, which occurs throughout the menstrual cycle in preparation for pregnancy. The appearance of uterodomes (pinopods) in this regard was first introduced in rodents with an established pinocytotic function, whereas little evidence was available in humans in this context. This study was undertaken to identify the potential physiological roles of uterodomes in the implantation process. To address this, endometrial biopsies from early, mid- and late luteal phases of the menstrual cycle of 23 fertile female patients with regular menses were used. Scanning and transmission electron microscopies (SEM and TEM) as well as immunofluorescence and immunogold TEM were performed to study the morphological changes and the expression pattern of leukaemia inhibitory factor (LIF) at uterodomes. Our results illustrated a high level of LIF expression in the human uterodomes, which was colocalized with the well-known biochemical markers of exocytosis, including syntaxin-1, 25-kDa synaptosomal protein (SNAP-25) and vesicle-associated membrane protein-2 (VAMP-2). Our morphological and immunocytochemical findings illustrated a secretory function for human uterodomes for the first time. In conclusion, this novel function for uterodomes provides an important clue in detection of their physiological function(s) during the process of the plasma membrane transformation.

Human Endometrial Leukemia Inhibitory Factor and Interleukin-6: Control of Secretion by Transforming Growth Factor-β-Related Members

Objective(s): The implantation process is closely linked to the fundamental question of the tolerance of the maternal immune system. The main objective of this study was to investigate whether different members of the transforming growth factor-β (TGF-β) superfamily could intervene in the first steps of embryo implantation by modulating the secretion of proimplantatory leukemia inhibitory factor (LIF) and in the tolerance of the fetal graft by regulating proinflammatory interleukin (IL)-6 secretion by human endometrial epithelium (EEC) in vitro. Methods: EEC were isolated from biopsies collected from 16 informed and consenting fertile women and were cultured for 72 h. Cytokine measurements (LIF and IL-6) were realized by ELISA. Results: TGF-β₁ (from 10^–12 to 10^–8M), -β₂, -β₃ and activin A (10^–10 and 10^–8M) increased LIF secretion by EEC cultures. Inhibin B (10^–10 and 10^–8M) did not stimulate LIF production by human EEC. Contrastingly, TGF-β₁ (from 10^–12 to 10^–8M), -β₂, -β₃ and activin A (10^–10 and 10^–8M) reduced IL-6 release by the same cells. Activin A at 10^–8 M also significantly reduced the stimulating effect of IL-1β (10^–9M) which is known to stimulate LIF production by EEC. Only the highest concentration of inhibin B (10^–8M) reduced IL-6 secretion by EEC, but did not modulate IL-1β-induced stimulation of IL-6 secretion. Conclusion(s): Besides their role in the control of the process of implantation and in the induction of embryonic mesoderm, different members of the TGF-β superfamily may also contribute in the reproductive process by enhancing endometrial proimplantatory LIF secretion and reducing proinflammatory IL-6 release by EEC.

Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the approximately 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, alpha-crystallin B, alpha-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.

Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium.

The elucidation of the molecular mechanisms by which the embryo contributes to its implantation is an area of extensive research. The main objective of this study was to investigate the pattern of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) secretion by human endometrial epithelium, and their regulation by human chorionic gonadotropin (hCG) and other growth factors present at the embryonic-endometrial interface. Endometrial epithelial cells (EEC) were isolated from biopsies collected at both proliferative and secretory phases of fertile women. HCG (1-50 IU/ml) increased LIF secretion by EEC cultures derived from follicular phase (up to 285+/-75%) or from secretory phase (up to 212+/-16%). In contrast, hCG reduced IL-6 secretion by EEC in both phases. The hCG/LH receptor gene was transcribed by EEC as evidenced by RT-PCR. Insulin-like growth factors 1 and 2 increased LIF secretion by EEC. Transforming growth factor beta1 stimulated LIF and reduced IL-6 secretion. Through hCG, the blastocyst may be involved in the control of its implantation (via an increase of proimplantatory LIF) and tolerance (via an inhibition of proinflammatory IL-6). Other growth factors present at the embryonic-endometrial interface are also involved in the control of LIF and IL-6 endometrial secretion.

The antiprogestin Org 31710 inhibits human blastocyst-endometrial interactions in vitro

To investigate in the effect of a new anti-progestin compound, Org 31710, on human blastocyst attachment to cultured endometrial epithelial cells. Experimental in vitro study. Endometrial cell cultures were prepared from endometrial biopsies obtained five to seven days after occurrence of the endogenous LH-peak (J.Reprod. Fertil. 2000; 120, 337–350). The anti-progestin Org 31710 was added to the culture medium in the test group at a concentration of 1μM, whereas the control cultures were cultured without presence of anti-progestin. Human donated frozen/thawed or surplus embryos were cultured to the expanded or hatched blastocyst stage in either Blast Assist II (Medi-Cult, Denmark), or in S2 (Vitrolife Ltd. Gothenburg, Sweden), before they were applied to the endometrial cultures and co-cultured for 96 hours. Secretion of glycodelin and Leukemia Inhibitory Factor (LIF) into the culture medium was measured by RIA and ELISA techniques. Endometrial cell cultures were examined by immunohistochemistry for expression of progesterone receptors, Interleukin-1 receptor type-1 and Integrin subunit β3. Blastocysts attached to the surface of endometrial cell cultures and endometrial pinopode formation were identified and studied by scanning electron microscopy (SEM). For statistical evaluation of attachment rates, Fishers exact test was used, whereas the van Elteren's stratified version of Wilcoxon's two-sample test was used for evaluation of pinopode formation and secretion of glycodelin and LIF into the culture medium. 11 out of 16 human hatched blastocysts attached to control cultures, whereas none of 10 hatched blastocysts attached when Org 31710 was added to the culture medium (p=0.0007). Immunohistochemical studies did not demonstrate significant differences between the groups. Biochemical analyses displayed a trend towards higher glycodelin secretions (p=0.08) and SEM a tendency towards less pinopode formation in the Org 31710 group, but the results did not reach statistical significance. The presence of swollen microvilli, precursors of endometrial pinopodes, was significantly reduced on cultures with Org 31710 (p=0.03). The percentage of endometrial cells displaying pinopode formation at attachment sites varied considerably. The study presents a model for human blastocyst-endometrial interactions responding to a new antiprogestin compound. The morphological and biochemical changes in the antiprogestin group are compatible with anti-implantation properties. However, the exact mechanism behind the anti-attachment properties of Org 31710 on endometrial cells (in culture) and blastocysts is yet to be elucidated.

Expression of leukemia inhibitory factor (LIF) and its receptor gp190 in human liver and in cultured human liver myofibroblasts. Cloning of new isoforms of LIF mRNA

BackgroundThe cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver.ResultsLIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4.ConclusionsWe show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation.

Leptin regulates the expression of leukemia inhibitory factor by human endometrial cells

Leptin regulates the expression of leukemia inhibitory factor by human endometrial cells

Fill a Gab(1) in Cardiac Hypertrophy Signaling

Cardiac hypertrophy represents one of the most central issues in heart failure, involving many aspects of changes in cardiac myocyte from cellular morphology to gene expression and contractile function.1,2 Although the process is at the beginning compensatory in response to hemodynamic load or myocardial injury, cardiac hypertrophy is often followed by pathological remodeling in myocardium and a transition to overt heart failure.3 A large number of extracellular ligands, their cognate receptors and corresponding intracellular signal transduction pathways have been implicated in the hypertrophic process from both in vitro and in vivo studies.4–13 Among them, cardiotrophin-1 (CT-1) was discovered as a potent activator of cardiac hypertrophy14 that shares extensive sequence and functional similarities with leukemia inhibitory factor (LIF) as members of interleukin-6 (IL-6)–related cytokine family.15 LIF/CT-1 bind to gp130/LIF cytokine receptor β heterodimer and activate downstream signaling pathways, including members of signal transducer and activator of transcription (STATs), mitogen-activated protein (MAP) kinases such as extracellular signal–regulated kinases (ERK1/2), and phosphatidylinositol 3-kinase (PI3K)/Akt.15,16 Studies have demonstrated that both LIF and CT-1 promote myocyte hypertrophy, survival, and a unique pattern of embryonic/fetal gene induction.17–22 LIF and CT-1 expression and gp130 signaling also correlated with mechanical stress in myocytes in culture23 and human and experimental models of heart failure.24–30 More significantly, targeted inactivation of gp130 in ventricular myocytes via Cre-loxP–mediated tissue-specific knockout resulted in rapid chamber dilation and a significant increase in myocyte apoptosis upon pressure overload.31 All these findings underscore the potential function of gp130 signaling in …