Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

PCBP1 regulates LIFR through FAM3C to maintain breast cancer stem cell self-renewal and invasiveness.

The poly(rC) binding protein 1 gene (PCBP1) encodes the heterogeneous nuclear ribonucleoprotein E1 (hnRNPE1), a nucleic acid-binding protein that plays a tumor-suppressive role in the mammary epithelium by regulating phenotypic plasticity and cell fate. Following the loss of PCBP1 function, the FAM3C gene (encoding the Interleukin-like EMT inducer, or "ILEI" protein) and the leukemia inhibitory factor receptor (LIFR) gene are upregulated. Interaction between FAM3C and LIFR in the extracellular space induces phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Overexpression and/or hyperactivity of STAT3 has been detected in 40% of breast cancer cases and is associated with a poor prognosis. Herein, we characterize feed-forward regulation of LIFR expression in response to FAM3C/LIFR/STAT3 signaling in mammary epithelial cells. We show that PCBP1 upregulates LIFR transcription through activity at the LIFR promoter, and that FAM3C participates in transcriptional regulation of LIFR. Additionally, our bioinformatic analysis reveals a signature of transcriptional regulation associated with FAM3C/LIFR interaction and identifies the TWIST1 transcription factor as a downstream effector that participates in the maintenance of LIFR expression. Finally, we characterize the effect of LIFR expression in cell-based experiments that demonstrate the promotion of invasion, migration, and self-renewal of breast cancer stem cells (BCSCs), consistent with previous studies linking LIFR expression to tumor initiation and metastasis in mammary epithelial cells.

OSMR deficiency aggravates pressure overload-induced cardiac hypertrophy by modulating macrophages and OSM/LIFR/STAT3 signalling.

BACKGROUND: Oncostatin M (OSM) is a secreted cytokine of the interleukin (IL)-6 family that induces biological effects by activating functional receptor complexes of the common signal transducing component glycoprotein 130 (gp130) and OSM receptor ß (OSMR) or leukaemia inhibitory factor receptor (LIFR), which are mainly involved in chronic inflammatory and cardiovascular diseases. The effect and underlying mechanism of OSM/OSMR/LIFR on the development of cardiac hypertrophy remains unclear. METHODS AND RESULTS: OSMR-knockout (OSMR-KO) mice were subjected to aortic banding (AB) surgery to establish a model of pressure overload-induced cardiac hypertrophy. Echocardiographic, histological, biochemical and immunological analyses of the myocardium and the adoptive transfer of bone marrow-derived macrophages (BMDMs) were conducted for in vivo studies. BMDMs were isolated and stimulated with lipopolysaccharide (LPS) for the in vitro study. OSMR deficiency aggravated cardiac hypertrophy, fibrotic remodelling and cardiac dysfunction after AB surgery in mice. Mechanistically, the loss of OSMR activated OSM/LIFR/STAT3 signalling and promoted a proresolving macrophage phenotype that exacerbated inflammation and impaired cardiac repair during remodelling. In addition, adoptive transfer of OSMR-KO BMDMs to WT mice after AB surgery resulted in a consistent hypertrophic phenotype. Moreover, knockdown of LIFR in myocardial tissue with Ad-shLIFR ameliorated the effects of OSMR deletion on the phenotype and STAT3 activation. CONCLUSIONS: OSMR deficiency aggravated pressure overload-induced cardiac hypertrophy by modulating macrophages and OSM/LIFR/STAT3 signalling, which provided evidence that OSMR might be an attractive target for treating pathological cardiac hypertrophy and heart failure.

Repositioning Mifepristone as a Leukaemia Inhibitory Factor Receptor Antagonist for the Treatment of Pancreatic Adenocarcinoma.

Pancreatic cancer is a leading cause of cancer mortality and is projected to become the second-most common cause of cancer mortality in the next decade. While gene-wide association studies and next generation sequencing analyses have identified molecular patterns and transcriptome profiles with prognostic relevance, therapeutic opportunities remain limited. Among the genes that are upregulated in pancreatic ductal adenocarcinomas (PDAC), the leukaemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, has emerged as potential therapeutic candidate. LIF is aberrantly secreted by tumour cells and promotes tumour progression in pancreatic and other solid tumours through aberrant activation of the LIF receptor (LIFR) and downstream signalling that involves the JAK1/STAT3 pathway. Since there are no LIFR antagonists available for clinical use, we developed an in silico strategy to identify potential LIFR antagonists and drug repositioning with regard to LIFR antagonists. The results of these studies allowed the identification of mifepristone, a progesterone/glucocorticoid antagonist, clinically used in medical abortion, as a potent LIFR antagonist. Computational studies revealed that mifepristone binding partially overlapped the LIFR binding site. LIF and LIFR are expressed by human PDAC tissues and PDAC cell lines, including MIA-PaCa-2 and PANC-1 cells. Exposure of these cell lines to mifepristone reverses cell proliferation, migration and epithelial mesenchymal transition induced by LIF in a concentration-dependent manner. Mifepristone inhibits LIFR signalling and reverses STAT3 phosphorylation induced by LIF. Together, these data support the repositioning of mifepristone as a potential therapeutic agent in the treatment of PDAC.

gp130 Cytokines Activate Novel Signaling Pathways and Alter Bone Dissemination in ER+ Breast Cancer Cells.

Breast cancer cells frequently home to the bone marrow, where they encounter signals that promote survival and quiescence or stimulate their proliferation. The interleukin-6 (IL-6) cytokines signal through the co-receptor glycoprotein130 (gp130) and are abundantly secreted within the bone microenvironment. Breast cancer cell expression of leukemia inhibitory factor (LIF) receptor (LIFR)/STAT3 signaling promotes tumor dormancy in the bone, but it is unclear which, if any of the cytokines that signal through LIFR, including LIF, oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), promote tumor dormancy and which signaling pathways are induced. We first confirmed that LIF, OSM, and CNTF and their receptor components were expressed across a panel of breast cancer cell lines, although expression was lower in estrogen receptor-negative (ER- ) bone metastatic clones compared with parental cell lines. In estrogen receptor-positive (ER+ ) cells, OSM robustly stimulated phosphorylation of known gp130 signaling targets STAT3, ERK, and AKT, while CNTF activated STAT3 signaling. In ER- breast cancer cells, OSM alone stimulated AKT and ERK signaling. Overexpression of OSM, but not CNTF, reduced dormancy gene expression and increased ER+ breast cancer bone dissemination. Reverse-phase protein array revealed distinct and overlapping pathways stimulated by OSM, LIF, and CNTF with known roles in breast cancer progression and metastasis. In breast cancer patients, downregulation of the cytokines or receptors was associated with reduced relapse-free survival, but OSM was significantly elevated in patients with invasive disease and distant metastasis. Together these data indicate that the gp130 cytokines induce multiple signaling cascades in breast cancer cells, with a potential pro-tumorigenic role for OSM and pro-dormancy role for CNTF. © 2021 American Society for Bone and Mineral Research (ASBMR).

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Immunization against leukemia inhibitory factor and its receptor suppresses tumor formation of breast cancer initiating cells in BALB/c mouse.

Immunotherapy is a promising approach for specific targeting of cancer cells. Leukemia inhibitory factor (LIF) regulates several features of cancers and cancer stem cells (CSCs) through binding to LIF receptor (LIFR). In this study, we investigated the consensus of LIF and LIFR immunization on the growth of mouse mammary tumors. For this purpose, mouse LIF and LIFR were designed as truncated proteins, expressed in E. coli and then injected to mice as individual and mixed antigens. The results showed the production of neutralizing antibodies and secretion of interferon-γ and interleukin-2 in response to immunization. In continue, the immunized mice were subjected for tumor formation challenge by inoculation of the breast CSCs derived from MC4-L2 cells. Development of the breast tumors was observed in all the control mice, while the tumors appeared in 75% of animals in the LIF group. LIFR injection, individually or in combination with LIF, strongly inhibited the tumor growth to only 25% of the mice. Moreover, a delay in tumor appearance was observed in the immunized mice compared to the controls. Immunostaining of the tumor sections confirmed the expression of LIF and LIFR. In conclusion, LIF and LIFR might be effective targets for immunotherapy of the tumors that express these proteins.

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling.

Metastasis remains a clinically unsolved issue in nasopharyngeal carcinoma. Here, we report that higher levels of cytoplasmic leukemia inhibitory factor (LIF) and LIF receptor are correlated with poorer metastasis/recurrence-free survival. Further, single nucleotide variations and signal peptide mutation of LIF are identified in NPC. Cytoplasmic LIF reprograms the invasive mode from collective to mesenchymal migration via acquisition of EMT and invadopodia-associated characteristics. Higher cytoplasmic LIF enhances cancer vascular dissemination and local invasion mechanistically through modulation of YAP1-FAK/PXN signaling. Immunohistochemical analyses of NPC biopsies reveal a positive correlation of cytoplasmic LIF expression with focal adhesion kinases. Pharmaceutical intervention with AZD0530 markedly reverses LIF-mediated cancer dissemination and local invasion through promotion of cytoplasmic accumulation of YAP1 and suppression of focal adhesion kinases. Given the significant role of LIF/YAP1-focal adhesion signaling in cancer dissemination, targeting of this pathway presents a promising opportunity to block metastasis.

Quantitative proteome analysis identifies MAP2K6 as potential regulator of LIFR-induced radioresistance in nasopharyngeal carcinoma cells.

Using Tandem Mass Tags (TMT) labeling and LC-MS/MS analysis of peptides from two cell lines (CNE2 and its radioresistant subclone CNE2-IR), we identified 754 proteins differentially expressed in CNE2-IR compared to CNE2. MAP2K6 was identified as a candidate radioresistance-related protein kinase. In vitro functional analysis revealed that over-expression of MAP2K6 significantly enhanced cell survival and colony formation following irradiation in NPC cells. Further, knockdown of MAP2K6 in radioresistant NPC cells led to decreased colony formation and increased apoptotic cells following irradiation. However, the effect of MAP2K6 in regulating the radioresistance in NPC cells did not seem to depend on p38/MAPK activity. Importantly, MAP2K6 might be required for leukemia inhibitory factor receptor (LIFR)-regulated radioresistance, as the expression levels of MAP2K6 affected LIFR/p70S6K signaling activation in NPC cells. Further, MAP2K6 kinase activity is required to activate LIFR/p70S6K signaling and to confer pro-survival effect on NPC cells. In conclusion, MAP2K6 might be an important regulator of LIFR-induced radioresistance in NPC.

A Zombie LIF Gene in Elephants Is Upregulated by TP53 to Induce Apoptosis in Response to DNA Damage.

Large-bodied organisms have more cells that can potentially turn cancerous than small-bodied organisms, imposing an increased risk of developing cancer. This expectation predicts a positive correlation between body size and cancer risk; however, there is no correlation between body size and cancer risk across species ("Peto's paradox"). Here, we show that elephants and their extinct relatives (proboscideans) may have resolved Peto's paradox in part through refunctionalizing a leukemia inhibitory factor pseudogene (LIF6) with pro-apoptotic functions. LIF6 is transcriptionally upregulated by TP53 in response to DNA damage and translocates to the mitochondria where it induces apoptosis. Phylogenetic analyses of living and extinct proboscidean LIF6 genes indicates that its TP53 response element evolved coincident with the evolution of large body sizes in the proboscidean stem lineage. These results suggest that refunctionalizing of a pro-apoptotic LIF pseudogene may have been permissive (although not sufficient) for the evolution of large body sizes in proboscideans.

Long non-coding RNA-CTD-2108O9.1 represses breast cancer metastasis by influencing leukemia inhibitory factor receptor.

Breast cancer (BC) is an aggressive malignant disease in women worldwide with a high tendency to metastasize. However, important biomarkers for BC metastasis remain largely undefined. In the present study, we identified that long non-coding RNA-CTD-2108O9.1 is downregulated in BC tissues and cells and acts as a metastatic inhibitor of BC. Mechanistic investigation determined that lncRNA-CTD-2108O9.1 represses metastasis by targeting leukemia inhibitory factor receptor (LIFR), which is designated as a metastasis suppressor in BC. Our study characterizes a significant tumor suppressor active in BC metastasis repression through the known metastasis inhibitor LIFR.

Mutations in the leukemia inhibitory factor receptor (LIFR) gene and Lifr deficiency cause urinary tract malformations.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. As CAKUT is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new CAKUT causing genes. Using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (LIFR) gene causing instability of the mRNA in a patient presenting with bilateral CAKUT and requiring kidney transplantation at one year of age. LIFR encodes a transmembrane receptor utilized by IL-6 family cytokines, mainly by the leukemia inhibitory factor (LIF). Mutational analysis of 121 further patients with severe CAKUT yielded two rare heterozygous LIFR missense variants predicted to be pathogenic in three unrelated patients. LIFR mutants showed decreased half-life and cell membrane localization resulting in reduced LIF-stimulated STAT3 phosphorylation. LIFR showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. Lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying LIFR variants. Additionally, a form of cryptorchidism was detected in all Lifr-/- mice and the patient carrying the LIFR frameshift mutation. Altogether, we demonstrate heterozygous novel or rare LIFR mutations in 3.3% of CAKUT patients, and provide evidence that Lifr deficiency and deactivating LIFR mutations cause highly similar anomalies of the urogenital tract in mice and humans.

Decreased epithelial progesterone receptor A at the window of receptivity is required for preparation of the endometrium for embryo attachment.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

Feedback Activation of Leukemia Inhibitory Factor Receptor Limits Response to Histone Deacetylase Inhibitors in Breast Cancer.

Histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits in subtypes of hematological malignancies. However, the efficacy of HDAC inhibitors in solid tumors remains uncertain. This study takes breast cancer as a model to understand mechanisms accounting for limited response of HDAC inhibitors in solid tumors and to seek combination solutions. We discover that feedback activation of leukemia inhibitory factor receptor (LIFR) signaling in breast cancer limits the response to HDAC inhibition. Mechanistically, HDAC inhibition increases histone acetylation at the LIFR gene promoter, which recruits bromodomain protein BRD4, upregulates LIFR expression, and activates JAK1-STAT3 signaling. Importantly, JAK1 or BRD4 inhibition sensitizes breast cancer to HDAC inhibitors, implicating combination inhibition of HDAC with JAK1 or BRD4 as potential therapies for breast cancer.

The Leukemia Inhibitory Factor Receptor Gene Is a Direct Target of RUNX1.

Activation of cytokine signaling via the leukemia inhibitory factor receptor (LIFR) plays an integral role in hematopoiesis, osteogenesis, and placental development, along with mediating neurotrophic mechanisms. However, the regulatory control of the LIFR gene has remained largely unexplored. Here, we characterize the LIFR gene as a novel target of the RUNX1 transcription factor. The RUNX1 transcription factor is an essential regulator of hematopoiesis and is a frequent target of point mutations and chromosomal alterations in leukemia. RUNX1 regulates hematopoiesis through its control of genes important for hematopoietic cell growth, proliferation, and differentiation, including a number of cytokines and cytokine receptors. LIFR is regulated by two alternate promoters: a placental-specific and a ubiquitously active general promoter. We show that both of these promoters are regulated by RUNX1. However, in myeloid cells LIFR expression is driven solely by the general LIFR promoter with our data indicating that the placental promoter is epigenetically silenced in these cells. While RUNX1 activates the LIFR general promoter, the oncogenic RUNX1-ETO fusion protein generated by the t(8;21) translocation commonly associated with acute myeloid leukemia represses promoter activity. The data presented here establish LIFR as a transcriptional target of RUNX1 and suggest that disruption of RUNX1 activity in myeloid cells may result in altered LIFR signaling in these cells.

Leukemia Inhibitory Factor (LIF) Inhibition during Mid-Gestation Impairs Trophoblast Invasion and Spiral Artery Remodelling during Pregnancy in Mice.

The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFRα) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFRα antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of α-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.

Lack of FSH support enhances LIF-STAT3 signaling in granulosa cells of atretic follicles in cattle.

Subordinate follicles (SFs) of bovine follicular waves undergo atresia due to declining FSH concentrations; however, the signalling mechanisms have not been fully deciphered. We used an FSH-induced co-dominance model to determine the effect of FSH on signalling pathways in granulosa cells of the second-largest follicles (SF in control cows and co-dominant follicle (co-DF2) in FSH-treated cows). The SF was smaller than DF in control cows while diameters of co-DF1 and co-DF2 in FSH-treated cows were similar. The presence of cleaved CASP3 protein confirmed that granulosa cells of SFs, but not of DFs and co-DFs, were apoptotic. To determine the effect of FSH on molecular characteristics of the second-largest follicles, we generated relative variables for the second largest follicle in each cow. For this, variables of SF or co-DF2 were divided by the variables of the largest follicle DF or co-DF1 in each cow. There was higher transcript abundance of MAPK1/3 and AKT1/2/3 but lower abundance of phosphorylated MAPK3/1 in SF than co-DF2 granulosa cells. Abundance of mRNA and phosphorylated protein of STAT3 was higher in granulosa cells of control SF than FSH-treated co-DF2. SF granulosa cells had higher levels of LIFR and IL6ST transcripts, the two receptors involved in STAT3 activation. Further, lower transcript abundance of interleukin 6 receptor (IL6R), another receptor involved in STAT3 activation, indicated that STAT3 activation in SF granulosa cells could be mainly due to leukemia inhibitory factor (LIF) signalling. These results indicate that atresia due to lack of FSH is associated with activated LIF-STAT3 signalling in SF granulosa cells, as FSH treatment reversed such activation.

Leukemia inhibitory factor (LIF).

Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. It utilises a receptor that consists of the LIF receptor ß and gp130 and this receptor complex is also used by ciliary neurotrophic growth factor (CNTF), oncostatin M, cardiotrophin1 (CT1) and cardiotrophin-like cytokine (CLC). Despite common signal transduction mechanisms (JAK/STAT, MAPK and PI3K) LIF can have paradoxically opposite effects in different cell types including stimulating or inhibiting each of cell proliferation, differentiation and survival. While LIF can act on a wide range of cell types, LIF knockout mice have revealed that many of these actions are not apparent during ordinary development and that they may be the result of induced LIF expression during tissue damage or injury. Nevertheless LIF does appear to have non-redundant actions in maternal receptivity to blastocyst implantation, placental formation and in the development of the nervous system. LIF has also found practical use in the maintenance of self-renewal and totipotency of embryonic stem cells and induced pluripotent stem cells.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.