LIF is essential for ISC function and protects against radiation-induced gastrointestinal syndrome.

Leukemia inhibitory factor (LIF) is a cytokine essential for maintaining pluripotency of mouse embryonic stem cells. However, its role in adult intestinal stem cells (ISCs) is unclear. The adult intestinal epithelium has a high self-renewal rate driven by ISCs in crypts. Here, we find that LIF is present in the ISC niche in crypts and critical for the function of ISCs in maintaining the intestinal epithelial homeostasis and regeneration. Mechanistically, LIF maintains ß-catenin activity through the AKT/GSK3ß signaling to regulate ISC functions. LIF deficiency in mice impairs the renewal of the intestinal epithelium under the physiological condition. Further, LIF deficiency in mice impairs the regeneration of intestinal epithelium in response to radiation and shortens the lifespan of mice after high doses of radiation due to gastrointestinal (GI) syndrome, which can be rescued by administering recombinant LIF (rLIF). Importantly, LIF exhibits a radioprotective role in wild-type (WT) mice by protecting mice from lethal radiation-induced GI syndrome; administering rLIF promotes intestinal epithelial regeneration and prolongs survival in WT mice after radiation. These results reveal a previously unidentified and a crucial role of LIF in ensuring ISC function, promoting regeneration of the intestinal epithelium in response to radiation and protecting against radiation-induced GI syndrome.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

PURPOSE: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. METHODS: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. RESULTS: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. CONCLUSIONS: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.

Leukemia Inhibitory Factor Haplodeficiency Desynchronizes Glial Reactivity and Exacerbates Damage and Functional Deficits after a Concussive Brain Injury.

Reactions of both astrocytes and microglia to central nervous system injury can be beneficial or detrimental to recovery. To gain insights into the functional importance of gliosis, we developed a new model of adolescent closed-head injury (CHI) and interrogated the behavioral, physiological, and cellular outcomes after a concussive CHI in leukemia inhibitory factor (LIF) haplodeficient mice. These mice were chosen because LIF is important for astrocyte and microglial activation. Behaviorally, the LIF haplodeficient animals were equally impaired 4 h after the injury, but in the subsequent 2 weeks, the LIF haplodeficient mice acquired more severe motor and sensory deficits, compared with wild type mice. The prolonged accumulation of neurological impairment was accompanied by desynchronization of the gliotic response, increased cell death, axonal degeneration, diminished callosal compound action potential, and hypomyelination. Our results clearly show that LIF is an essential injury-induced cytokine that is required to prevent the propagation of secondary neurodegeneration.

α6ß1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

BACKGROUND: The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. RESULTS: Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and ß1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. ß1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of ß1-, α6- and αV-integrins.

K/B×N serum transfer arthritis is delayed and less severe in leukaemia inhibitory factor (LIF)-deficient mice.

This study is investigating the role of leukaemia inhibitory factor (LIF) in the development of inflammation and joint damage in the mouse K/B×N serum transfer arthritis model. LIF knock-out (LIF(-/-)) mice were generated by mating heterozygote females (LIF(+/-)) with heterozygote males. Arthritis was induced in 8-20-week-old LIF knock-out mice (LIF(-/-)) by intraperitoneal injection of pooled K/B×N sera (50 µl) on days 0 and 2. Clinical disease was scored daily for 6 days. Safranin-O and haematoxylin-stained sections were scored for synovitis, joint space exudate, cartilage degradation and bone damage. RNA was extracted from ankle joints and used to investigate gene expression levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1, LIF, LIF receptor, oncostatin M (OSM), OSM receptor, IL-6 and their common receptor subunit gp130 by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results show that wild-type mice developed severe clinically overt polyarthritis. In contrast, LIF(-/-) mice showed a more than 50% reduction in clinical arthritis severity. Significantly lower histological scores were observed in LIF(-/-) mice compared to wild-type disease controls. LIF(-/-) mice had histopathological scores that were similar to normal healthy mice. IL-6 subfamily cytokine and receptor subunit expression remained unchanged. The expression levels for IL-6 were reduced significantly in all the diseased mice, whether wild-type or LIF(-/-) mice (P < 0·001), compared to healthy wild-type mice. We conclude that LIF contributes to the development of disease in the K/B×N serum transfer model of arthritis. These results provide further evidence for the role of LIF in inflammation and cartilage bone resorption and provide impetus to test the effects of LIF blockade as a therapeutic strategy in rheumatoid arthritis.

Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity.

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/ß-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.

Endogenous leukemia inhibitory factor protects photoreceptor cells against light-induced degeneration.

PURPOSE: Expression of leukemia inhibitory factor (LIF) by a subset of Müller glia cells has recently been implicated in an endogenous survival response to photoreceptor injury in a model of inherited retinal degeneration. To investigate whether such a LIF-controlled survival pathway might be commonly induced upon photoreceptor injury independently of the nature of the toxic stimulus, we analyzed the role of LIF during light-induced retinal degeneration. METHODS: Lif(+/-) and Lif(-/-) mice were exposed to 15,000 lx of white light for 2 h. Retinal morphology and rhodopsin content were analyzed nine days after light exposure. Gene expression studies were done using real-time PCR. Protein levels were determined by western blotting using specific antibodies. RESULTS: A lack of LIF reduced survival of photoreceptor cells after light exposure. In the absence of LIF several genes encoding molecules involved in the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway were not activated after light exposure. Presence or absence of LIF did not affect AKT (also known as protein kinase B, PKB) signaling and had only a mild effect on extracellular regulated kinase (ERK) phosphorylation. Stress-induced glial fibrillary acidic protein (GFAP) induction was minimal in the absence of LIF. CONCLUSIONS: Our results suggest that increased retinal expression of LIF is a general response to photoreceptor injury. Independent of the nature of the toxic insult (gene mutation, light), LIF may activate an endogenous rescue pathway that protects viable photoreceptor cells, leading to an increased photoreceptor survival in the stressed retina. This defense system may depend on the Jak/STAT pathway and may involve endothelin 2 (EDN2) but not (or only minimally) AKT and ERK1,2 signaling.

Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells.

Embryonic stem (ES) cell pluripotency is regulated in part by transcription factor (TF) pathways that maintain self-renewal and inhibit differentiation. Stat3 and c-Myc TFs are essential for maintaining mouse ES cell self-renewal. c-Myc, together with Oct4, Sox2, and Klf4, is a reprogramming factor. While previous studies have investigated core transcriptional circuitry in ES cells, other TF pathways that promote ES cell pluripotency have yet to be investigated. Therefore, to further understand ES cell transcriptional networks, we used genome-wide chromatin immunoprecipitation and microarray analysis (ChIP-chip) to map Stat3 and c-Myc binding targets in ES cells. Our results show that Stat3 and c-Myc occupy a significant number of genes whose expression is highly enriched in ES cells. By comparing Stat3 and c-Myc target genes with gene expression data from undifferentiated ES cells and embryoid bodies (EBs), we found that Stat3 binds active and inactive genes in ES cells, while c-Myc binds predominantly active genes. Moreover, the transcriptional states of Stat3 and c-Myc targets are correlated with co-occupancy of pluripotency-related TFs, polycomb group proteins, and active and repressive histone modifications. We also provide evidence that Stat3 targets are differentially expressed in ES cells following removal of LIF, where culture of ES cells in the absence of LIF resulted in downregulation of Stat3 target genes enriched in ES cells, and upregulation of lineage specific Stat3 target genes. Altogether, we reveal transcriptional targets of two key pluripotency-related genes in ES cells--Stat3 and c-Myc, thus providing further insight into the ES cell transcriptional network.

Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia.

Osteoclasts are multinucleated haematopoietic cells that resorb bone. Increased osteoclast activity causes osteoporosis, a disorder resulting in a low bone mass and a high risk of fractures. Increased osteoclast size and numbers are also a hallmark of other disorders, such as Paget's disease and multiple myeloma. The protein c-Fos, a component of the AP-1 transcription factor complex, is essential for osteoclast differentiation. Here we show that the Fos-related protein Fra-2 controls osteoclast survival and size. The bones of Fra-2-deficient newborn mice have giant osteoclasts, and signalling through leukaemia inhibitory factor (LIF) and its receptor is impaired. Similarly, newborn animals lacking LIF have giant osteoclasts, and we show that LIF is a direct transcriptional target of Fra-2 and c-Jun. Moreover, bones deficient in Fra-2 and LIF are hypoxic and express increased levels of hypoxia-induced factor 1alpha (HIF1alpha) and Bcl-2. Overexpression of Bcl-2 is sufficient to induce giant osteoclasts in vivo, whereas Fra-2 and LIF affect HIF1alpha through transcriptional modulation of the HIF prolyl hydroxylase PHD2. This pathway is operative in the placenta, because specific inactivation of Fra-2 in the embryo alone does not cause hypoxia or the giant osteoclast phenotype. Thus placenta-induced hypoxia during embryogenesis leads to the formation of giant osteoclasts in young pups. These findings offer potential targets for the treatment of syndromes associated with increased osteoclastogenesis.

Leukemia inhibitory factor deficiency modulates the immune response and limits autoimmune demyelination: a new role for neurotrophic cytokines in neuroinflammation.

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.

Leukemia inhibitory factor signaling modulates both central nervous system demyelination and myelin repair.

Leukemia inhibitory factor (LIF) receptor signaling limits the severity of inflammatory demyelination in experimental autoimmune encephalomyelitis, a T-cell dependent animal model of multiple sclerosis (MS) [Butzkueven et al. (2002) Nat Med 8:613-619]. To identify whether LIF exerts direct effects within the central nervous system to limit demyelination, we have studied the influence of LIF upon the phenotype of mice challenged with cuprizone, a copper chelator, which produces a toxic oligodendrocytopathy. We find that exogenously administered LIF limits cuprizone-induced demyelination. Knockout mice deficient in LIF exhibit both potentiated demyelination and oligodendrocyte loss after cuprizone challenge, an effect that is ameliorated by exogenous LIF, arguing for a direct beneficial effect of endogenous LIF receptor signaling. Numbers of oligodendrocyte progenitor cells in cuprizone-challenged mice are not influenced by either exogenous LIF or LIF deficiency, arguing for effects directed to the differentiated oligodendrocyte. Studies on the influence of LIF upon remyelination after cuprizone challenge fail to reveal any significant effect of exogenous LIF. The LIF-knockout mice do, however, display impaired remyelination, although oligodendrocyte replenishment, previously identified to occur from the progenitor pool, is not significantly compromised. Thus endogenous LIF receptor signaling is not only protective of oligodendrocytes but can also enhance remyelination, and exogenous LIF has therapeutic potential in limiting the consequences of oligodendrocyte damage.

p53 regulates maternal reproduction through LIF.

Extensive studies have shown that p53 is important in tumour prevention. However, little is known about its normal physiological function. Here we show that p53 is important in reproduction, in a gender-specific manner. Significant decreases in embryonic implantation, pregnancy rate and litter size were observed in matings with p53-/- female mice but not with p53-/- male mice. The gene encoding leukaemia inhibitory factor (LIF), a cytokine critical for implantation, was identified as a p53-regulated gene that functions as the downstream mediator of this effect. p53 can regulate both basal and inducible transcription of LIF. Loss of p53 decreased both the level and function of LIF in uteri. Lower LIF levels were observed in the uteri of p53-/- mice than in those of p53+/+ mice, particularly at day 4 of pregnancy, when transiently induced high levels of LIF were crucial for embryonic implantation. This observation probably accounts for the impaired implantation of embryos in p53-/- female mice. Administration of LIF to pregnant p53-/- mice restored maternal reproduction by improving implantation. These results demonstrate a function for p53 in maternal reproduction through the regulation of LIF. Evidence is accumulating that p53 may have a similar function in humans.

Pinopodes are present in Lif null and Hoxa10 null mice.

OBJECTIVE: To assess pinopode formation in Lif null and Hoxa10 null mice with infertility secondary to failed implantation. DESIGN: Controlled animal experiment. SETTING: Animal research and laboratory facility. ANIMAL(S): Lif null, Hoxa10 null, and ICR mice and Sprague-Dawley rats. INTERVENTION(S): Endometrial tissue was collected during the peri-implantation period and after ovariectomy. MAIN OUTCOME MEASURE(S): Endometrial epithelial tissue was examined under scanning-electron microscopy and assigned a score depending on the number of pinopodes present. RESULT(S): Pinopode scores in ICR, Lif null, and Hoxa10 null mice were comparable throughout the peri-implantation period, rising on day 3.5 of pregnancy and remaining elevated through to day 7.5, suggesting that pinopodes are not a good indicator of receptivity in mice. In contrast, pinopode scores in rats clearly demarcated the window of receptivity, appearing on day 4 of pregnancy and declining sharply on day 6. Pinopode scores were low in E(2)-treated ovariectomized mice, but unexpectedly, pinopode scores in vehicle-injected ovariectomized ICR mice were markedly elevated. CONCLUSION(S): Lif null and Hoxa10 null mice, in which implantation is impaired, have a similar number of pinopodes to fertile ICR mice. Pinopodes do not define a window of implantation in mice.

Altered neuronal responses and regulation of neurotrophic proteins in the medial septum following fimbria-fornix transection in CNTF- and leukaemia inhibitory factor-deficient mice.

Degeneration of axotomized GABAergic septohippocampal neurones has been shown to be enhanced in ciliary neurotrophic factor (CNTF)-deficient mice following fimbria-fornix transection (FFT), indicating a neuroprotective function of endogenous CNTF. Paradoxically, however, the cholinergic population of septohippocampal neurones was more resistant to axotomy in these mutants. As leukaemia inhibitory factor (LIF) has been identified as a potential neuroprotective factor for the cholinergic medial septum (MS) neurones, FFT-induced responses were compared in CNTF(-/-), LIF(-/-) and CNTF/LIF double knockout mice. In CNTF(-/-) mice, FFT-induced cholinergic degeneration was confirmed to be attenuated as compared with wildtype mice. The expression of both LIF and LIF receptor beta was increased in the MS providing a possible explanation for the enhanced neuronal resistance to FFT in these animals. However, ablation of the LIF gene also produced paradoxical effects; following FFT in LIF(-/-) mice no loss of GABAergic or cholinergic MS neurones was detectable during the first postlesional week, suggesting that other efficient neuroprotective mechanisms are activated in these animals. In fact, enhanced activation of astrocytes, a source of neurotrophic proteins, was indicated by increased up-regulation of glial fibrillary acidic protein and vimentin expression. In addition, mRNA levels for neurotrophin signalling components (e.g. nerve growth factor, p75(NTR)) were differentially regulated. The positive effect on axotomized cholinergic neurones seen in CNTF(-/-) and LIF(-/-) mice as well as the increased up-regulation of astrogliose markers was abolished in CNTF/LIF double knockout animals. Our results indicate that endogenous CNTF and LIF are involved in the regulation of neuronal survival following central nervous system lesion and are integrated into a network of neurotrophic signals that mutually influence their expression and function.

Leukemia inhibitory factor restores the hypertrophic response to increased loading in the LIF(-/-) mouse.

Cytokines and growth factors are thought to contribute to skeletal muscle hypertrophy. Leukemia inhibitory factor (LIF), a cytokine, enhances skeletal muscle regeneration; however the role of LIF in skeletal muscle hypertrophy remains uncertain. We examined the hypertrophic ability of the plantaris and soleus muscles in wild-type mice (WT) and LIF knock-out mice [LIF(-/-)] in response to increased mechanical load. Using the functional overload model to induce increases in mechanical load on the plantaris and soleus muscle, WT mice demonstrated increases in plantaris and soleus mass after 7, 21, and 42 days of loading. However, the LIF(-/-) mice had no significant increases in plantaris muscle mass at any time point, while the soleus muscle exhibited a delayed hypertrophic response. Systemic delivery of LIF to the LIF(-/-) mice returned the hypertrophic response to the same levels as the WT mice after 21 days of functional overload. These data demonstrate for the first time that LIF expression in loaded skeletal muscle is critical for the development of skeletal muscle hypertrophy in the functional overload model.