Cardiotrophin-like Cytokine Factor Research Articles

Detection of CLCF1 protein expression by flow cytometry

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.

Reduced serum CLCF1 levels in hyperthyroidism patients and T3-treated mice.

Characteristic symptoms of hyperthyroidism include weight loss, heart palpitation and sweating. Thyroid hormones (TH) can stimulate thermogenesis through central and peripheral mechanisms. Previous studies have shown an association between dysfunction of Cardiotrophin-like cytokine factor 1 (CLCF1) and cold-induced sweating syndrome (CISS), with recent research also indicating a link between CLCF1 and brown adipose tissue (BAT) thermogenesis. However, it remains unclear whether CLCF1 and TH have synergistic or antagonistic effects on thermogenesis. This study aims to investigate the influence of thyroid hormone on circulating CLCF1 levels in humans and explore the potential role of thyroid hormone in regulating energy metabolism by modulating CLCF1 in mice. By recruiting hyperthyroid patients and healthy subjects, we observed significantly lower serum CLCF1 levels in hyperthyroid patients compared to healthy subjects, with serum CLCF1 levels independently associated with hyperthyroidism after adjusting for potential confounders. Tissue analysis from mice treated with T3 revealed a decrease in Clcf1 expression in BAT and iWAT of C57BL/6 mice. These findings suggest that TH may play a role in regulating Clcf1 expression in thermogenic adipose tissue and impacting thermogenesis.

Abstract 1414: HBM9014, a first-in-class fully human anti-LIFR antibody with excellent preclinical efficacy and safety profile

Abstract Background: Leukemia inhibitory factor (LIF) and cardiotrophin like cytokine factor 1 (CLCF1) are multi-functional cytokines from IL-6 family. They are reported to be highly expressed in a variety of human cancers such as pancreatic carcinoma, cholangiocarcinoma, NSCLC, GBM, head and neck cancer etc. Their expression is associated with poor prognosis and survival. LIFR is the key component of LIF/CLCF1 receptors. Antibody targeting LIFR has the advantage to block both LIF and CLCF1 pathways thus inhibiting tumor growth more effectively than products to block single cytokines. Methods: Fully human Anti-LIFR antibodies were generated from H2L2 Harbour Mice® transgenic platform. In vitro function of the antibodies was tested for their abilities to block LIF/CLCF1 induced downstream signaling pathway and tumor spheroid proliferation. In vivo efficacy was tested in multiple tumor models as monotherapy and in combination with cisplatin. DRF toxicokinetics was evaluated on NHP with a 4-week repeat-dose toxicity study. Results: HBM9014, a fully human anti-LIFR antibody generated by H2L2 Harbour Mice®, specifically binds LIFR thus blocks the interaction between LIF/CLCF1 and their respective receptors, inhibits downstream STAT3 phosphorylation, and reduces exogenous and endogenous LIF induced tumor spheroid proliferation in vitro. Furthermore, HBM9014 shows potent anti-tumor activity in multiple tumor models in vivo. In a GLP NHP 4-week repeat-dose toxicity study, HBM9014 is well tolerated up to 150 mg/kg with no discernable drug-related toxicity. Conclusions: HBM9014 specifically binds to the key component of LIF and CLCF1 receptor, LIFR, thus blocks both LIF and CLCF1 signaling pathways. HBM9014 shows significant cell proliferation inhibition alone or in combo with small molecule inhibitors in 3D culture of multiple cancer cell lines. It also shows significant in vivo antitumor efficacy as monotherapy, and enhanced efficacy in combination with Cisplatin in multiple mouse models. Importantly, despite the benefits of double blocking, HMB9014 has an excellent safety profile. Taken together, HMB9014 is a promising drug candidate for advanced cancer with favorable efficacy and safety profiles that warrant clinical validation. Citation Format: Jue Zeng, Dongxu Zhang, Louis Liu, Lingbing Zhang. HBM9014, a first-in-class fully human anti-LIFR antibody with excellent preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1414.

CLCF1 inhibits energy expenditure via suppressing brown fat thermogenesis.

Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis which plays an important role in thermogenesis and energy metabolism. However, the regulatory factors that inhibit BAT activity remain largely unknown. Here, cardiotrophin-like cytokine factor 1 (CLCF1) is identified as a negative regulator of thermogenesis in BAT. Adenovirus-mediated overexpression of CLCF1 in BAT greatly impairs the thermogenic capacity of BAT and reduces the metabolic rate. Consistently, BAT-specific ablation of CLCF1 enhances the BAT function and energy expenditure under both thermoneutral and cold conditions. Mechanistically, adenylate cyclase 3 (ADCY3) is identified as a downstream target of CLCF1 to mediate its role in regulating thermogenesis. Furthermore, CLCF1 is identified to negatively regulate the PERK-ATF4 signaling axis to modulate the transcriptional activity of ADCY3, which activates the PKA substrate phosphorylation. Moreover, CLCF1 deletion in BAT protects the mice against diet-induced obesity by promoting BAT activation and further attenuating impaired glucose and lipid metabolism. Therefore, our results reveal the essential role of CLCF1 in regulating BAT thermogenesis and suggest that inhibiting CLCF1 signaling might be a potential therapeutic strategy for improving obesity-related metabolic disorders.

CLCF1 signaling restrains thermogenesis and disrupts metabolic homeostasis by inhibiting mitochondrial biogenesis in brown adipocytes

Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we found that cardiotrophin-like cytokine factor 1 (CLCF1) signaling led to loss of brown fat identity, which impaired thermogenic capacity. CLCF1 levels decreased during thermogenic stimulation but were considerably increased in obesity. Adipocyte-specific CLCF1 transgenic (CLCF1-ATG) mice showed impaired energy expenditure and severe cold intolerance. Elevated CLCF1 triggered whitening of brown adipose tissue by suppressing mitochondrial biogenesis. Mechanistically, CLCF1 bound and activated ciliary neurotrophic factor receptor (CNTFR) and augmented signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 transcriptionally inhibited both peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α and 1β, which thereafter restrained mitochondrial biogenesis in adipocytes. Inhibition of CNTFR or STAT3 could diminish the inhibitory effects of CLCF1 on mitochondrial biogenesis and thermogenesis. As a result, CLCF1-TG mice were predisposed to develop metabolic dysfunction even without external metabolic stress. Our findings revealed a brake signal on nonshivering thermogenesis and suggested that targeting this pathway could be used to restore brown fat activity and systemic metabolic homeostasis in obesity.

Identification of Lncrna-Mrna Networks in Hepg2 Cells upon ATP7B Knockout and Copper Accumulation.

Hepatolenticular degeneration (HLD) is an inherited disorder caused by the mutation in the adenosine triphosphatase copper transporting β gene (ATP7B). W aimed to explore the genetic changes in HLD using bioinformatics analysis. The study was conducted in Nepal, in 2019. The GSE107323 dataset was downloaded and the differentially expressed lncRNAs (DElncRNAs) as well as differentially expressed genes (DEGs) induced by ATP7B knockout (KO) and copper toxicity were clustered using Mfuzz clustering analysis. LncRNAs and genes with high coexpression (correlation coefficient > 0.9) and pathways involving the DEGs were used to construct the lncRNA-gene-pathway network. ATP7B KO and ATP7B KO + copper induced 51 overlapping DEGs and 687 overlapping DElncRNAs, respectively. Mfuzz analysis identified four clusters, including two clusters of consistently upregulated and downregulated DEGs/DElncRNAs. The lncRNA-gene-pathway network consisted of 13 DElncRNAs, 10 DEGs, and two pathways, including "hsa04630: Jak-STAT signaling pathway" and "hsa04920: Adipocytokine signaling pathway". Eight downregulated genes, including erythropoietin (EPO), insulin receptor substrate 1 (IRS1), and PPARG coactivator 1 alpha (PPARGC1A), and two upregulated genes (cardiotrophin-like cytokine factor 1 and cyclin D3) were involved in the two pathways. These genes were targeted by multiple lncRNAs, including PCAT6 and MALAT1. Collectively, the differentially expressed lncRNA-mRNA axes play crucial roles in HLD pathogenesis through mediating cell proliferation and inflammation. Moreover, the EPO, IRS1, or PPARGC1A genes were potent therapeutic targets for HLD.

Structural insights into the assembly of gp130 family cytokine signaling complexes

The interleukin-6 (IL-6) family cytokines signal through gp130 receptor homodimerization or heterodimerization with a second signaling receptor and play crucial roles in various cellular processes. We determined cryo-electron microscopy structures of five signaling complexes of this family, containing full receptor ectodomains bound to their respective ligands ciliary neurotrophic factor, cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor, IL-27, and IL-6. Our structures collectively reveal similarities and differences in the assembly of these complexes. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a~30angstrom range but with distinct distances and orientations. We also reveal how CLCF1 engages its secretion chaperone cytokine receptor-like factor 1.Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina.

Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in the light-damaged retina and induced proliferation of rod precursor cells in the undamaged retina, but not Müller glia. While rod precursor cell proliferation was previously shown to be Insulin-like growth factor 1 receptor (IGF-1R)-dependent, co-injection of IGF-1 with CLCF1/CRLF1 failed to induce further proliferation of either Müller glia or rod precursor cells. Together, these findings demonstrate that CNTFR ligands have a neuroprotective effect and are required for induction of Müller glia proliferation in the light-damaged zebrafish retina.

Uncovering the promiscuous activity of IL-6 proteins: A multi-dimensional analysis of phylogeny, classification and residue conservation.

The IL‐6 family of cytokines, known for their pleiotropic behavior, share binding to the gp130 receptor for signal transduction with the necessity to bind other receptors. Leukemia inhibitory factor receptor is triggered by the IL‐6 family proteins: leukemia inhibitory factor (LIF), oncostatin‐M (OSM), cardiotrophin‐1 (CT‐1), ciliary neurotrophic factor (CNTF), and cardiotrophin‐like cytokine factor 1 (CLCF1). Besides the conserved binding sites to the receptor, not much is known in terms of the diversity and characteristics of these proteins in different organisms. Herein, we describe the sequence analysis of LIF, OSM, and CT‐1 from several organisms, and m17, a LIF ortholog found in fishes, regarding its phylogenetics, intrinsic properties, and the impact of conserved residues on structural features. Sequences were identified in seven classes of vertebrates, showing high conservation values in binding site III, but protein‐dependent results on binding site II. GRAVY, isoelectric point, and molecular weight parameters were relevant to differentiate classes in each protein and to enable, for the first time and with high fidelity, the prediction of both organism class and protein type just using machine learning approaches. OSM sequences from primates showed an increased BC loop when compared to the remaining mammals, which could influence binding to OSM receptor and tune signaling pathways. Overall, this study highlights the potential of sequence diversity analysis to understand IL‐6 cytokine family evolution, showing the conservation of function‐related motifs and evolution of class and protein‐dependent characteristics. Our results could impact future medical treatment of disorders associated with imbalances in these cytokines.

Intrahepatic paracrine signaling by cardiotrophin-like cytokine factor 1 ameliorates diet-induced NASH in mice.

The mammalian liver harbors heterogeneous cell types that communicate via local paracrine signaling. Recent studies have delineated the transcriptomic landscape of the liver in NASH that provides insights into liver cell heterogeneity, intercellular crosstalk, and disease-associated reprogramming. However, the nature of intrahepatic signaling and its role in NASH progression remain obscure. Here, we performed transcriptomic analyses and identified cardiotrophin-like cytokine factor 1 (CLCF1), a member of the IL-6 family cytokines, as a cholangiocyte-derived paracrine factor that was elevated in the liver from diet-induced NASH mice and patients with NASH. Adenovirus-associated virus-mediated overexpression of CLCF1 in the liver ameliorated NASH pathologies in two diet-induced NASH models in mice, illustrating that CLCF1 induction may serve an adaptive and protective role during NASH pathogenesis. Unexpectedly, messenger RNA and protein levels of leukemia inhibitory factor receptor (LIFR), a subunit of the receptor complex for CLCF1, were markedly downregulated in NASH liver. Hepatocyte-specific inactivation of LIFR accelerated NASH progression in mice, supporting an important role of intrahepatic cytokine signaling in maintaining tissue homeostasis under metabolic stress conditions. Together, this study sheds light on the molecular nature of intrahepatic paracrine signaling during NASH pathogenesis and uncovers potential targets for therapeutic intervention.

Abstract 3185: Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms

Abstract Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remain underexploited. Previously, we validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. From our genetically engineered mouse and our syngeneic modelling of LUAD we have observed that eCNTFR seems to work via two mechanisms, firstly in a tumor intrinsic fashion where it may modulate altered guanosine triphosphate loading, as we see differential effectiveness of eCNTFR-Fc, depending on the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate. Alternatively, eCNTFR also effects the tumor microenvironment and alters the composition of the tumor immune infiltrate, where using single cell genomics we see vast changes to the myeloid compartment, and alteration of the CAF populations, which we have previously shown to be the primary location of CLCF1 expression in mouse LUAD. To further enhance the efficacy of eCNTFR-Fc we have identified potential targets for combination therapy from both the autonomous and non-cell autonomous mechanisms of eCNTFR-Fc. We conclude that eCNTFR-Fc acts through two distinct mechanisms to be a highly effective treatment for Kras mutant LUAD, and combining eCNTER with targets, identified by a rational combination approach, could further enhance its therapeutic potential. Citation Format: Kieren D. Marini, Emma C. Champion, Jennifer R. Cochrane, E. Alejandro Sweet-Cordero. Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3185.

Characterization of a CLCF1 conditional knock-out mice model

Abstract The Cardiotrophin-like Cytokine Factor 1 (CLCF1) is a cytokine of the IL6 family with important pro-neurotrophic and immuno-modulating functions. However, the mechanisms behind CLCF1 activities and CLCF1 properties in pathological models remain poorly understood. This is partly due to a lack of tools for the study of CLCF1 functions in vivo. Indeed, the complete knock-out of CLCF1 in mice is lethal at P1: underdeveloped motor neurons of the face and jaw prevent the pups from suckling. To overcome this obstacle, we obtained a CLCF1 conditional knock-out mice model generated using the CRISPR-Cas9 technology. We then bred these mice with Vav-Cre mice to induce CLCF1 deletion in hematopoietic cells. We are now conducting immuno-phenotyping experiments of the main immune populations in those animals. Considering the activities of CLCF1 that were previously shown in vitro or in vivo using overexpression models, we hypothesize that the knock-out of CLCF1 in immune cells will lead to decreased numbers of B and myeloid cells. This new CLCF1 conditional knock-out mice model will be a potent tool to confirm and further study the activities, mechanisms, and pathological roles of CLCF1 in vivo. Supported by grants from CIHR (Canadian Institutes of Health Research)

CLCF1 is up-regulated in renal ischemia reperfusion injury and may associate with FOXO3.

BackgroundIschemia-reperfusion injury (IRI) is one of the most important risk factors for acute kidney injury. In kidney transplantation, renal IRI can induce delayed graft function (DGF). However, the mechanisms that link IRI to DGF remain unclear. This study aimed to find molecular markers of renal IRI which are also associated with DGF.MethodsA previously constructed database of differentially expressed genes in a murine IRI model was compared with a published DGF database. The expression of cardiotrophin-like cytokine factor 1 (CLCF1) was detected using immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (qPCR) assays. Serum CLCF1 was measured using an enzyme-linked immunosorbent assay (ELISA), and serum creatinine (Cr) was tested to evaluate kidney function.ResultsBy comparing the IRI database and the DGF database, we identified 107 differentially expressed genes, including 79 upregulated and 28 downregulated genes. CLCF1 was one of the upregulated genes found in the 2 databases. The levels of CLCF1 in IRI-treated kidney tissues and serum CLCF1 were upregulated compared to sham-operated mice. CLCF1 belongs to the interleukin-6 (IL-6) family, and the forkhead box O3 (FOXO3) gene plays a key role in regulating IL-6 expression. We observed that FOXO3 knockout induced an increase in serum CLCF1 levels in sham-operated mice. However, FOXO3 knockout failed to increase CLCF1 levels in IRI-treated mice.ConclusionsCLCF1 is upregulated in renal IRI and may be regulated by FOXO3. Our data indicated that CLCF1 might be a potential biomarker linking renal IRI to DGF in kidney transplantation.

Bromodomain-containing protein 4 activates cardiotrophin-like cytokine factor 1, an unfavorable prognostic biomarker, and promotes glioblastoma in vitro

BackgroundGlioblastoma (GBM) is one of the most common and malignant brain tumors. Cardiotrophin-like cytokine factor 1 (CLCF1) is a member of the IL-6 superfamily. However, the clinical significance, potential role, and molecular mechanism of CLCF1 in GBM remain obscure. Here, the expression and prognostic significance of CLCF1 was investigated in GBM.MethodsThe Cancer Genome Atlas (TCGA) GBM and Chinese Glioma Genome Atlas (CGGA) datasets were downloaded and analyzed by using Gene Expression Profiling Interactive Analysis (GEPIA). Next, 3 shRNAs targeting CLCF1 were designed, and silencing efficiency was examined with real-time polymerase chain reaction (PCR). Cell Counting Kit 8 (CCK-8), flow cytometry, transwell, and wound healing assays were used to study the function of CLCF1 in glioma cells.ResultsWe found increased expression of CLCF1 as an unfavorable prognostic marker in GBM. Functionally, down-regulation of CLCF1 significantly reduced cell proliferation, induced cell apoptosis and cell cycle G2 phase arrest, and weakened the migration and invasion of GBM cells. Downstream pathway analysis was conducted, and potential targets in cytokine receptors, extracellular matrix (ECM) receptors, apoptosis, and the cell cycle were uncovered. Finally, transcriptional regulators were analyzed, and bromodomain-containing protein 4 (BRD4) was found to activate CLCF1 in GBM.ConclusionsCLCF1, transcriptionally activated by BRD4, promotes glioma and serves as an unfavorable marker in GBM.

Serum levels of plasminogen activator urokinase receptor and cardiotrophin-like cytokine factor 1 in patients with nephrotic syndrome.

41 patients with chronic glomerulonephritis (CGN) were included in our study. 17 patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of plasminogen activator urokinase receptor (uPAR) and cardiotrophin-like cytokine factor 1 (CLCF-1)were measured by ELISA. The serum levels of uPAR were higher in FSGS patients before treatment than in patients with other morphological forms (MCD, IgA nephropathy, MN, and MPGN). The levels of uPAR in serum did not correlate with daily proteinuria, serum creatinine/eGFR, arterial hypertension, the number of sclerosed glomeruli, or tubulointerstitial fibrosis. No correlations were found between the levels of CLCF-1 in serum and creatinine levels/glomerular filtration rate, the percentage of sclerosed glomeruli, or the severity of tubulointerstitial fibrosis. There were no significant differences between the histological variants of nephritis. However, we found correlations between CLCF-1 levels and proteinuria and lipid levels. The data indicate an increase in the serum uPAR levels of FSGS before treatment. CLCF-1 levels in serum do not depend on histological forms of CGN, kidney function, or immunosuppressive treatment, but they correlate with proteinuria and serum lipids in patients with NS.

CRLF1 and CLCF1 in Development, Health and Disease

Cytokines and their receptors have a vital function in regulating various processes such as immune function, inflammation, haematopoiesis, cell growth and differentiation. The interaction between a cytokine and its specific receptor triggers intracellular signalling cascades that lead to altered gene expression in the target cell and consequent changes in its proliferation, differentiation, or activation. In this review, we highlight the role of the soluble type I cytokine receptor CRLF1 (cytokine receptor-like factor-1) and the Interleukin (IL)-6 cytokine CLCF1 (cardiotrophin-like cytokine factor 1) during development in physiological and pathological conditions with particular emphasis on Crisponi/cold-induced sweating syndrome (CS/CISS) and discuss new insights, challenges and possibilities arising from recent studies.

Cardiotrophin Like Cytokine Factor 1 (CLCF1) alleviates bone loss in osteoporosis mouse models by suppressing osteoclast differentiation through activating interferon signaling and repressing the nuclear factor-κB signaling pathway.

A growing body of evidence suggests that immune factors that regulate osteoclast differentiation and bone resorption might be promising therapeutic agents for the treatment of osteoporosis. The expression of CLCF1, an immune cell-derived molecule, has been reported to be reduced in patients with postmenopausal osteoporosis. This suggests that it may be involved in bone remodeling. Thus, we explored the functional role of CLCF1 in osteoclastogenesis and bone loss associated with osteoporosis. Surprisingly, the administration of recombinant CLCF1 repressed excessive bone loss in ovariectomized mice and prevented RANKL-induced bone loss in calvarial mouse model. Likewise, the addition of recombinant CLCF1 to RANKL-stimulated monocytes resulted in a significant suppression in the number of differentiated osteoclasts with small resorption areas being observed on dentine slices in vitro. At the same dosage, CLCF1 did not exhibit any detectable negative effects on the differentiation of osteoblasts. Mechanistically, the inhibition of osteoclast differentiation by the CLCF1 treatment appears to be related to the activation of interferon signaling (IFN) and the suppression of the NF-κB signaling pathway. Interestingly, the expression of the main components of IFN-signaling namely, STAT1 and IRF1, was detected in macrophages as early as 1h after stimulation with CLCF1. Consistent with these results, the blockade of STAT1 in macrophages abolished the inhibitory effect of CLCF1 on osteoclast differentiation in vitro. These collective findings point to a novel immunoregulatory function of CLCF1 in bone remodeling and highlight it as a potentially useful therapeutic agent for the treatment of osteoporosis.

Analysis of the polarization states of the alveolar macrophages in chronic obstructive pulmonary disease samples based on miRNA-mRNA network signatures.

BackgroundMultiple gene expression studies have been performed to investigate the biomarkers of chronic obstructive pulmonary disease (COPD). However, few studies have related COPD to macrophage cells.MethodsThe gene expression levels of clinical samples of COPD smokers (COPD; n=6), healthy smokers (Smoke; n=11), and never smokers (Never; n=4) were downloaded from the Gene Expression Omnibus (GEO) repository of GSE124180. The expression levels of messenger RNAs (mRNAs) and microRNAs (miRNAs) in macrophage cells of M0 (n=7), M1 (n=7), and M2 (n=7) were downloaded from the GEO repository of GSE46903 and GSE51307. Differentially expressed (DE) mRNAs (DEmRNAs) were identified by edgeR and GEO2R, with an adjusted P value <0.05 and |log2fold change (FC)| ≥1 chosen as the cut-off threshold. The potential target genes of miRNA were identified using miRanda (v3.3a) and TargetScan (v6.0) with default settings. Gene Ontology (GO) and Reactome pathway analyses were performed.ResultsThe composition of macrophages was quite different between COPD, Never, and Smoke samples. The proportion of M1 cells was lower than that of M0 and M2 cells in Smokers and COPD samples. Most of the genes specifically up-regulated in M1 are related to inflammation/immunity. The expression levels of miR-30a-5p, miR-200c-3p, miR-20b-5p, miR-199b-5p, and miR-301b-3p in M1 macrophages were all lower than that of M0. Their expression levels in M2 macrophages compared with M1 varied, with higher expression in miR-30a-5p, miR-20b-5p, and lower expression in miR-200c-3p, and miR-301b-3p. The mRNAs of the fms related receptor tyrosine kinase 1 (FLT1), cardiotrophin like cytokine factor 1 (CLCF1), phosphodiesterase 4D (PDE4D), coagulation factor III, and tissue factor (F3) were dysregulated in COPD and macrophage cells.ConclusionsThe present study mined the miRNA-mRNA signature which might play an essential role in COPD and macrophage polarization.

Influences of the IL-6 cytokine family on bone structure and function

The IL-6 family of cytokines comprises a large group of cytokines that all act via the formation of a signaling complex that includes the glycoprotein 130 (gp130) receptor. Despite this, many of these cytokines have unique roles that regulate the activity of bone forming osteoblasts, bone resorbing osteoclasts, bone-resident osteocytes, and cartilage cells (chondrocytes). These include specific functions in craniofacial development, longitudinal bone growth, and the maintenance of trabecular and cortical bone structure, and have been implicated in musculoskeletal pathologies such as craniosynostosis, osteoporosis, rheumatoid arthritis, osteoarthritis, and heterotopic ossifications. This review will work systematically through each member of this family and provide an overview and an update on the expression patterns and functions of each of these cytokines in the skeleton, as well as their negative feedback pathways, particularly suppressor of cytokine signaling 3 (SOCS3). The specific cytokines described are interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), cardiotrophin-like cytokine factor 1 (CLCF1), neuropoietin, humanin and interleukin 27 (IL-27).

MO210SERUM LEVELS OF PLASMINOGEN ACTIVATOR UROKINASE RECEPTOR AND CARDIOTROPHIN-LIKE CYTOKINE FACTOR 1IN PATIENTS WITH NEPHROTIC SYNDROME

Abstract Background and Aims The pathogenesis of primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) remains unknown to date. Some circulating permeability factors are discussed. This work assessed molecule candidates for permeability in serum samples of patients with nephrotic syndrome (NS). Method Forty-one patients with chronic glomerulonephritis (CGN) were included in our study. Seventeen patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of uPAR and CLCF-1 were measured by ELISA. Results The serum levels of plasminogen activator urokinase receptor (uPAR) were higher in FSGS patients before treatment than in patients with other morphological forms (MCD, IgA nephropathy, MN and MPGN). The levels of uPAR in serum did not correlate with daily proteinuria, serum creatinine/eGFR, arterial hypertension, the number of sclerosed glomeruli or tubulointerstitial fibrosis. No correlations were found between the levels of cardiotrophin-like cytokine factor 1 (CLCF-1) in serum and creatinine levels/glomerular filtration rate, the percentage of sclerosed glomeruli or the severity of tubulointerstitial fibrosis. There were no significant differences between the histological variants of nephritis. However, we found correlations between CLCF-1 levels and proteinuria (Rs = 0. 397, p = 0.015) and triglycerides levels (Rs = 0. 475, p = 0.003). Conclusion The data indicate an increase in the serum uPAR levels of FSGS before treatment. CLCF-1 levels in serum do not depend on histological forms of CGN, kidney function or immunosuppressive treatment, but they correlate with proteinuria and serum lipids in patients with NS.