Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators.

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1ß. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.

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.

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.

PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR.

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

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.

The LIFR Inhibitor EC359 Effectively Targets Type II Endometrial Cancer by Blocking LIF/LIFR Oncogenic Signaling.

Endometrial cancer (ECa) is the most common female gynecologic cancer. When comparing the two histological subtypes of endometrial cancer, Type II tumors are biologically more aggressive and have a worse prognosis than Type I tumors. Current treatments for Type II tumors are ineffective, and new targeted therapies are urgently needed. LIFR and its ligand, LIF, have been shown to play a critical role in the progression of multiple solid cancers and therapy resistance. The role of LIF/LIFR in the progression of Type II ECa, on the other hand, is unknown. We investigated the role of LIF/LIFR signaling in Type II ECa and tested the efficacy of EC359, a novel small-molecule LIFR inhibitor, against Type II ECa. The analysis of tumor databases has uncovered a correlation between diminished survival rates and increased expression of leukemia inhibitory factor (LIF), suggesting a potential connection between altered LIF expression and unfavorable overall survival in Type II ECa. The results obtained from cell viability and colony formation assays demonstrated a significant decrease in the growth of Type II ECa LIFR knockdown cells in comparison to vector control cells. Furthermore, in both primary and established Type II ECa cells, pharmacological inhibition of the LIF/LIFR axis with EC359 markedly decreased cell viability, long-term cell survival, and invasion, and promoted apoptosis. Additionally, EC359 treatment reduced the activation of pathways driven by LIF/LIFR, such as AKT, mTOR, and STAT3. Tumor progression was markedly inhibited by EC359 treatment in two different patient-derived xenograft models in vivo and patient-derived organoids ex vivo. Collectively, these results suggest LIFR inhibitor EC359 as a possible new small-molecule therapeutics for the management of Type II ECa.

The ILEI/LIFR complex induces EMT via the Akt and ERK pathways in renal interstitial fibrosis.

BACKGROUND: Chronic kidney disease (CKD) is characterized by high morbidity and mortality and is difficult to cure. Renal interstitial fibrosis (RIF) is a major determinant of, and commonly occurs within, CKD progression. Epithelial mesenchymal transition (EMT) has been identified as a crucial process in triggering renal interstitial fibrosis (RIF). Interleukin-like EMT inducer (ILEI) is an important promotor of EMT; this study aims to elucidate the mechanisms involved. METHODS: Male C57BL6/J mouse were randomly divided into 6 groups: sham (n = 10), sham with negative control (NC) shRNA (sham + NC, n = 10), sham with ILEI shRNA (sham + shILEI, n = 10), unilateral ureteral obstruction (UUO, n = 10), UUO with NC (UUO + NC, n = 10) and UUO with ILEI shRNA (UUO + shILEI, n = 10). Hematoxylin and eosin (H&E), Masson, and immunohistochemical (IHC) staining and western blotting (WB) were performed on murine kidney tissue to identify the function and mechanism of ILEI in RIF. In vitro, ILEI was overexpressed to induce EMT in HK2 cells and analyzed via transwell, WB, real-time PCR, and co-immunoprecipitation. Finally, tissue from 12 pediatric CKD patients (seven with RIF and five without RIF) were studied with H&E, Masson, and IHC staining. RESULTS: Our in vitro model revealed that ILEI facilitates RIF in the UUO model via the Akt and ERK pathways. Further experiments in vivo and in vitro revealed that ILEI promotes renal tubular EMT by binding and activating leukemia inhibitory factor receptor (LIFR), in which phosphorylation of Akt and ERK is involved. We further find markedly increased expression levels of ILEI and LIFR in kidneys from pediatric CKD patients with RIF. CONCLUSION: Our results indicate that ILEI may be a useful biomarker for renal fibrosis and a potential therapeutic target for modulating RIF.

Lidocaine Suppresses Gastric Cancer Development Through Circ_ANO5/miR-21-5p/LIFR Axis.

BACKGROUND: Lidocaine has been manifested to exert anti-tumor role in gastric cancer (GC) progression. However, the action mechanism by which Lidocaine functions in GC has not been fully elucidated. AIM: The study aimed to reveal the molecular mechanism of Lidocaine in GC progression. METHODS: Cell clonogenicity and viability were assessed by colony formation and methyl thiazolyl tetrazolium assays, respectively. Transwell assay was employed to detect cell migration and invasion. Flow cytometry was implemented to monitor cell apoptosis. Relative expression of circular RNA ANO5 (circ_ANO5), microRNA (miR)-21-5p and Leukemia inhibitory factor receptor (LIFR) was examined by quantitative reverse transcription-polymerase chain reaction. Western blot assay was performed to analyze the levels of LIFR and cell metastasis-related proteins. The target relationship between miR-21-5p and circ_ANO5 or LIFR was confirmed by dual-luciferase reporter assay. In addition, xenograft model was established to explore the role of Lidocaine in vivo. RESULTS: Lidocaine inhibited cell proliferation, migration and invasion, while promoted apoptosis of GC cells. Lidocaine upregulated circ_ANO5 and LIFR expression, but downregulated miR-21-5p expression in GC cells. Additionally, expression of circ_ANO5 and LIFR was decreased, while miR-21-5p expression was increased in GC cells. Circ_ANO5 depletion or miR-21-5p overexpression attenuated Lidocaine-induced anti-proliferative and anti-metastatic effects on GC cells. Circ_ANO5 could sponge miR-21-5p, and miR-21-5p targeted LIFR. Moreover, Lidocaine suppressed the tumor growth in vivo. CONCLUSION: Lidocaine might GC cell malignancy by modulating circ_ANO5/miR-21-5p/LIFR axis, highlighting a novel insight for GC treatment.

Cell signaling pathways in autosomal-dominant leukodystrophy (ADLD): the intriguing role of the astrocytes.

Autosomal-dominant leukodystrophy (ADLD) is a rare fatal neurodegenerative disorder with overexpression of the nuclear lamina component, Lamin B1 due to LMNB1 gene duplication or deletions upstream of the gene. The molecular mechanisms responsible for driving the onset and development of this pathology are not clear yet. Vacuolar demyelination seems to be one of the most significant histopathological observations of ADLD. Considering the role of oligodendrocytes, astrocytes, and leukemia inhibitory factor (LIF)-activated signaling pathways in the myelination processes, this work aims to analyze the specific alterations in different cell populations from patients with LMNB1 duplications and engineered cellular models overexpressing Lamin B1 protein. Our results point out, for the first time, that astrocytes may be pivotal in the evolution of the disease. Indeed, cells from ADLD patients and astrocytes overexpressing LMNB1 show severe ultrastructural nuclear alterations, not present in oligodendrocytes overexpressing LMNB1. Moreover, the accumulation of Lamin B1 in astrocytes induces a reduction in LIF and in LIF-Receptor (LIF-R) levels with a consequential decrease in LIF secretion. Therefore, in both our cellular models, Jak/Stat3 and PI3K/Akt axes, downstream of LIF/LIF-R, are downregulated. Significantly, the administration of exogenous LIF can partially reverse the toxic effects induced by Lamin B1 accumulation with differences between astrocytes and oligodendrocytes, highlighting that LMNB1 overexpression drastically affects astrocytic function reducing their fundamental support to oligodendrocytes in the myelination process. In addition, inflammation has also been investigated, showing an increased activation in ADLD patients' cells.

miR-221-3p regulates hepatocellular carcinoma cell proliferation, migration and invasion via targeting LIFR.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common and fatal cancers in the world. This study aims to investigate the mechanism by which miR-221-3p regulates HCC cell proliferation, migration and invasion, so as to provide a new idea for targeted therapy towards HCC. MATERIALS AND METHODS: Expression quantification data including mature miRNA and mRNA were accessed from TCGA-LIHC dataset, and matched clinical information was obtained as well, which helped identify the miRNA of interest. Thereafter, effect of the miRNA on HCC cell biological functions was assessed with a series of in vitro experiments, such as qRT-PCR, MTT, wound healing assay and Transwell. To gain more insight into the mechanism of the miRNA in HCC, bioinformatics method was conducted to predict downstream target gene. The potential targeting relationship between the miRNA and the predicted mRNA was validated by dual-luciferase reporter assay. Western blot was performed to test protein expression. RESULTS: MiR-221-3p identified by differential expression analysis was found to be significantly elevated in HCC tissue. Overexpressing miR-221-3p noticeably enhanced HCC cell proliferative, migratory and invasive abilities. Leukemia inhibitory factor receptor (LIFR), confirmed as a downstream target of miR-221-3p in HCC by dual-luciferase reporter assay, was poorly expressed in HCC tissue and cells. Additionally, the expression of LIFR was decreased following the targeted binding between miR-221-3p and LIFR 3'-UTR, while increasing the expression of LIFR attenuated the promoting effect of miR-221-3p on HCC cells. CONCLUSION: MiR-221-3p is an oncogene in HCC cells, and it exerts its role in HCC cell viability and motility via targeting LIFR.

OSM/OSMR and Interleukin 6 Family Cytokines in Physiological and Pathological Condition.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and can bind two different receptors, Leukemia inhibitory factor receptor (LIFR) and Oncostatin M receptor (OSMR), through a complex containing the common glycoprotein 130 (gp130) subunit [...].

Association of novel stop-gained leukaemia inhibitory factor receptor gene (rs121912501) variant, leukaemia inhibitory factor and ovarian steroids with unexplained infertility among Pakistani women.

AIMS AND OBJECTIVES: Embryo implantation is a complex process that requires sequential steps at the interface of embryo interaction with decidual endometrium. Many women after experiencing multiple attempts of assisted reproductive techniques fail to get implantation because of instability of leukaemia inhibitory factor and leukaemia inhibitory factor receptor-signal transducer and activator of transcription factor 3 (LIF-LIFR STAT3) signalling cascade. Therefore, this study explores the association of ovarian steroids, LIF and LIFR stop-gained variant using the tetra primer amplification refractory mutation system-polymerase chain reaction (TARMS-PCR) with unexplained infertility (UEX-IF) among Pakistani women. MATERIALS AND METHODS: This is a case-control study, a total of 81 unexplained infertile women and 162 fertile controls (with age and BMI matched) were inducted. Serum estradiol, progesterone and LIF were determined using enzyme-linked immunosorbent assay (ELISA). T-ARMS-PCR was designed using Primer 1 software. Genomic DNA was extracted from peripheral blood and amplified using T-ARMS-PCR followed by sequencing for validation and comprehensive concordance. RESULTS: This study established differences in LIF levels (χ2  = 9.857, P < .05) between patients and controls as well as explored the decreased LIF significantly raised the risk of UEX-IF (OR = 2.316; 95% CI = 1.214, 4.416). Progesterone (P) was significantly associated with UEX-IF between fertile and infertile counterparts (χ2  = 20.347, P < .05). It was also observed that increased Progesterone reduced the risk of UEX-IF (OR = 0.306; 95% CI = 0.166, 0.567). A rapid and inexpensive method for genotyping novel LIFR gene polymorphism through T- ARMS-PCR was successfully developed. LIFR gene SNP (rs121912501) had significant association (χ2  = 200.681, P < .05) with UEX-IF. LIFR rs121912501 "TT" genotype (OR = 5.417; 95% CI = 1.868, 15.709) and "CT" genotype (OR = 3.104, 95% CI = 1.586,6.076) were at increased risk of infertility. CONCLUSION: UEX-IF can be caused by LIFR gene variation irrespective of increased P. It may open the doors for the discovery of new management plans for infertile women.

Hsa_circ_0001073 targets miR-626/LIFR axis to inhibit lung cancer progression.

Circular RNAs (circRNAs) are associated with lung cancer progression. However, it is unclear whether and how circRNA hsa_circ_0001073 (circ_0001073) are involved in lung cancer progression. circ_0001073, microRNA (miR)-626, and leukemia inhibitory factor receptor (LIFR) abundances were determined via quantitative reverse transcription polymerase chain reaction or western blot. Cell viability, invasion, and apoptosis were analyzed by cell counting kit-8, transwell analysis and flow cytometry, respectively. The target correlation was tested by dual-luciferase reporter analysis or RNA immunoprecipitation. Results showed that circ_0001073 abundance was down-regulated in lung cancer cells. circ_0001073 constrained cell viability and invasion and facilitated apoptosis in lung cancer cells. miR-626 was targeted via circ_0001073, and circ_0001073 inhibited lung cancer progression via reducing miR-626 expression. LIFR was targeted via miR-626, and miR-626 knockdown constrained cell viability and invasion and facilitated apoptosis in lung cancer cells via up-regulating LIFR. circ_0001073 increased LIFR expression via miR-626 in lung cancer cells. In conclusion, circ_0001073 represses lung cancer progression via miR-626/LIFR axis, indicating the potential value of circ_0001073 in lung cancer treatment.

Concomitant Activation of OSM and LIF Receptor by a Dual-Specific hlOSM Variant Confers Cardioprotection after Myocardial Infarction in Mice.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.

The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are closely related members of the interleukin-6 (IL-6) cytokine family. Both cytokines share a common origin and structure, and both interact through a specific region, termed binding site III, to activate a dimeric receptor complex formed by glycoprotein 130 (gp130) and LIF receptor (LIFR) in humans. However, only OSM activates the OSM receptor (OSMR)-gp130 complex. The molecular features that enable OSM to specifically activate the OSMR are currently unknown. To define specific sequence motifs within OSM that are critical for initiating signaling via OSMR, here we generated chimeric OSM-LIF cytokines and performed alanine-scanning experiments. Replacement of the OSM AB loop within OSM's binding site III with that of LIF abrogated OSMR activation, measured as STAT3 phosphorylation at Tyr-705, but did not compromise LIFR activation. Correspondingly, substitution of the AB loop and D-helix in LIF with their OSM counterparts was sufficient for OSMR activation. The alanine-scanning experiments revealed that residues Tyr-34, Gln-38, Gly-39, and Leu-45 (in the AB loop) and Pro-153 (in the D-helix) had specific roles in activating OSMR but not LIFR signaling, whereas Leu-40 and Cys-49 (in the AB loop), and Phe-160 and Lys-163 (in the D-helix) were required for activation of both receptors. Because most of the key amino acid residues identified here are conserved between LIF and OSM, we concluded that comparatively minor differences in a few amino acid residues within binding site III account for the differential biological effects of OSM and LIF.

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.

High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays.

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

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.

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.

Effects of Leukaemia Inhibitory Factor Receptor on the Early Stage of Osteogenic Differentiation of Human Bone Marrow Mesenchymal Cells.

Leukaemia inhibitory factor (LIF) has a wide variety of biological activities. While recent studies have focused on the role of LIF in osteoblast differentiation, the exact role of LIFR during the early stage of osteogenic differentiation remains unclear. We observed that LIFR expression gradually decreased during the early stage of osteogenic differentiation of hMSCs. To evaluate how LIFR regulates osteogenic differentiation in greater depth, we transfected hMSCs with LIFR overexpression and siRNA lentiviral plasmids. Cells were divided into four groups: a negative overexpression control group, a LIFR overexpression group, a negative siRNA control group, and a LIFR siRNA group. On different days (0, 3, and 6) of the osteogenic differentiation of hMSCs, alkaline phosphatase (ALP) activity was assayed with an ALP staining and activity assay kit. Cells were harvested to assess the mRNA and protein expression of LIF, LIFR, and osteogenesis-related factors (ALP; RUNX2; osteonectin) by qRT-PCR and western blot analyses, respectively. In addition, culture supernatants were tested for the LIF content by ELISA. Our results showed that overexpression of LIFR significantly suppressed the osteoblast differentiation of hMSCs. In contrast, LIFR siRNA markedly improved this osteoblast differentiation as determined by ALP staining and activity measurements. Moreover, RUNX2, ALP, and ONN expression was also significantly changed by altering LIFR expression. We further analysed the expression of LIF and LIFR, revealing consistent LIF and LIFR trends during the osteogenic differentiation of hMSCs. Together, these results suggested that LIFR may be a novel negative regulator during the early stage of hMSC osteogenic differentiation.