ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) is associated with uncontrolled inflammatory responses. Loss of pulmonary angiotensin-converting enzyme 2 (ACE2) function has been associated with SARS-CoV-2 infection. The aberrant signalling and dysregulated inflammation characteristic of lung cancer have marked similarities with SARS-CoV-2 infection. Spearman's correlation analysis of The Cancer Genome Atlas (TCGA) datasets indicated an inverse correlation between ACE2 and IL6 in lung adenocarcinoma. qRT-PCR analysis revealed CoV-2-SRBD-mediated diminished ACE2 expression in lung cancer cells that was concomitant with increased IL6 expression. Western blot and qRT-PCR analysis suggested that treatment with methotrexate (MTx) dampened CoV-2-SRBD-mediated increase in JAK1/STAT3 phosphorylation, gp130, IL6, and folate-binding protein (FBP) expressions. MTx also rescued the diminished expression of ACE2 in CoV-2-SRBD transfected cells. As lung tissue injury in severely affected COVID-19 patients is characterised by aberrant inflammatory response, repurposing MTx as an effective therapy against critical regulators of inflammation in SARS-CoV-2 infection warrants investigation.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19 Drug Treatment , Glycyrrhizic Acid/therapeutic use , Immunosuppressive Agents/therapeutic use , Interleukin-6/biosynthesis , Methotrexate/therapeutic use , A549 Cells , Adenocarcinoma of Lung/pathology , Anti-Inflammatory Agents/therapeutic use , COVID-19/immunology , COVID-19/pathology , Cell Line, Tumor , Cytokine Receptor gp130/biosynthesis , Folate Receptor 2/biosynthesis , HMGB1 Protein/antagonists & inhibitors , HMGB1 Protein/metabolism , Humans , Interleukin-6/immunology , Janus Kinase 1/metabolism , Lung Neoplasms/pathology , Phosphorylation/drug effects , SARS-CoV-2/drug effects , STAT3 Transcription Factor/metabolism , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Chemoresistance is one of the barriers for the development of bladder cancer treatments. Previously, we showed that glycoprotein-130 (GP130) is overexpressed in chemoresistant bladder cancer cells and that knocking down GP130 expression reduced cell viability. In our current work, we showed that down-regulation of GP130 sensitized bladder cancer cells to cisplatin-based chemotherapy by activating DNA repair signaling. We performed immunohistochemistry and demonstrated a positive correlation between the levels of Ku70, an initiator of canonical non-homologous end joining repair (c-NHEJ) and suppressor of apoptosis, and GP130 in human bladder cancer specimens. GP130 knockdown by SC144, a small molecule inhibitor, in combination with cisplatin, increased the number of DNA lesions, specifically DNA double-stranded breaks, with a subsequent increase in apoptosis and reduced cell viability. Furthermore, GP130 inhibition attenuated Ku70 expression in bladder and breast cancer cells as well as in transformed kidney cells. In addition, we fabricated a novel polymer-lipid hybrid delivery system to facilitate GP130 siRNA delivery that had a similar efficiency when compared with Lipofectamine, but induced less toxicity.
Subject(s)
Apoptosis/drug effects , Cisplatin/pharmacology , Cytokine Receptor gp130/biosynthesis , DNA Repair , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Ku Autoantigen/metabolism , Neoplasm Proteins/metabolism , Signal Transduction/drug effects , Urinary Bladder Neoplasms/metabolism , Female , HEK293 Cells , Humans , MCF-7 Cells , Male , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathologyABSTRACT
The standard of age-related glomerulosclerosis is unclear. Both signal transducer and activator of transcription 3 (STAT3) and autophagy are involved in age-related kidney disease. Therefore, we aimed to explore the standard, as well as the potential mechanism(s). A total of 44 patients who underwent radical nephrectomy were enrolled. Pearson analysis was performed to investigate the parameters with ages. The patients were divided into the young- and aged-kidney groups. Kidney morphological changes were evaluated by histology staining, senescence was evaluated by senescence-associated-ß-galactosidase (SA-ß-gal) staining, and autophagosome was measured by transmission electron microscopy. Moreover, Western blot and/or immunohistochemistry were accomplished to assess the expression of p16, STAT3, and glycoprotein130 (GP130) and autophagy-related proteins. Furthermore, human glomerular mesangial cells were administrated with tocilizumab (TCZ) and/or IL-6, and then the above indexes were tested again. Sclerotic glomerular density and glomerular sclerosis rate were significantly higher in individuals more than 40 years old, and they were strongly correlated with ages. Moreover, the expression of p16, STAT3, GP130, and p62 was significantly increased, while LC3II and autophagosome were statistically decreased in the aged-kidney. Glomeruli were hardly to stain with SA-ß-gal. For the in vitro experiments, we observed that IL-6 significantly increased p16, STAT3, GP130, and p62, induced higher SA-ß-gal staining, while downregulated LC3II and autophagosome. Furthermore, TCZ statistically reversed the effects of IL-6 on the above expression of proteins. Glomerular sclerosis rate might be one standard for natural renal aging, and IL-6/STAT3-mediated autophagy may participate in the development of age-related glomerulosclerosis.
Subject(s)
Aging/metabolism , Autophagy , Glomerulosclerosis, Focal Segmental/metabolism , Interleukin-6/biosynthesis , STAT3 Transcription Factor/biosynthesis , Adult , Aged , Aged, 80 and over , Aging/pathology , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cytokine Receptor gp130/biosynthesis , Female , Gene Expression Regulation , Glomerulosclerosis, Focal Segmental/pathology , Humans , Male , Middle Aged , beta-Galactosidase/metabolismABSTRACT
Pulmonary arterial hypertension (PAH) is characterized by a progressive accumulation of pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterioles leading to the narrowing of the lumen, right heart failure, and death. Although most studies have supported the notion of a role for IL-6/glycoprotein 130 (gp130) signaling in PAH, it remains unclear how this signaling pathway determines the progression of the disease. Here, we identify ectopic upregulation of membrane-bound IL-6 receptor (IL6R) on PA-SMCs in PAH patients and in rodent models of pulmonary hypertension (PH) and demonstrate its key role for PA-SMC accumulation in vitro and in vivo. Using Sm22a-Cre Il6rfl/fl, which lack Il6r in SM22A-expressing cells, we found that these animals are protected against chronic hypoxia-induced PH with reduced PA-SMC accumulation, revealing the potent pro-survival potential of membrane-bound IL6R. Moreover, we determine that treatment with IL6R-specific antagonist reverses experimental PH in two rat models. This therapeutic strategy holds promise for future clinical studies in PAH.
Subject(s)
Cytokine Receptor gp130/biosynthesis , Familial Primary Pulmonary Hypertension/metabolism , Mice, Transgenic , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/metabolism , Up-Regulation , Vascular Remodeling , Animals , Arterioles/metabolism , Arterioles/pathology , Arterioles/physiopathology , Familial Primary Pulmonary Hypertension/genetics , Familial Primary Pulmonary Hypertension/pathology , Familial Primary Pulmonary Hypertension/physiopathology , Humans , Mice , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/pathology , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , Rats , Signal Transduction/geneticsABSTRACT
INTRODUCTION: The purpose of this study was to investigate the mechanical loading-induced changes in protein and mRNA expressions of interleukin-6 (IL-6) and its key signaling factors glycoprotein 130 (gp130), signal transducer and activator of transcription 3 (STAT3), and the Src homology phosphotyrosine phosphatase (SHP2) at the tension and compression sides of the teeth in mouse models. METHODS: A total of 55 C57B/6 mice (10 weeks old) were divided into 3 groups. Orthodontic force was applied in group A (experimental group, n = 30); the tooth movement device was placed without activation in group B (sham control group, n = 15), and group C (blank control group, n = 10). Tooth movement was induced by a nickel-titanium coil spring inserted between the maxillary left incisor and the first molar with a force of approximately 4 g. The animals were killed 12 days after the interventions; protein and mRNA expressions of IL-6, gp130, STAT3, and SHP2 in the periodontal tissues were observed with immunohistochemistry and in-situ hybridization, respectively. RESULTS: In contrast with the control groups, we observed enhanced expressions of IL-6, gp130, STAT3, and SHP2 protein and mRNA at the mesial and distal sides of the teeth with application of orthodontic forces in the experimental group. In contrast with the distal side, we observed enhanced expression of gp130 protein and mRNA at the mesial side in the experimental group. CONCLUSIONS: We observed enhanced expression of IL-6 and its key signaling factors gp130, STAT3, and SHP2 protein and mRNA at the tension and compression sides of the teeth with application of orthodontic forces. The mechanical loading applied for orthodontic tooth movement might induce changes in protein localization and mRNA expression patterns of IL-6 and its key signaling factors gp130, STAT3, and SHP2 at the tension and compression sides of the periodontal ligaments of the teeth in mouse models. The result might demonstrate the special role of IL-6 and its key signaling factors in the alveolar bone-modeling process.
Subject(s)
Interleukin-6/genetics , RNA, Messenger/biosynthesis , Tooth Movement Techniques , Animals , Cytokine Receptor gp130/biosynthesis , Cytokine Receptor gp130/genetics , Interleukin-6/biosynthesis , Male , Mice , Mice, Inbred C57BL , Protein Biosynthesis , Protein Tyrosine Phosphatase, Non-Receptor Type 11/biosynthesis , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , STAT3 Transcription Factor/biosynthesis , STAT3 Transcription Factor/geneticsABSTRACT
Interleukin (IL)-6 engages similar signaling mechanisms to leptin. Here, we find that central application of IL-6 in mice suppresses feeding and improves glucose tolerance. In contrast to leptin, whose action is attenuated in obesity, the ability of IL-6 to suppress feeding is enhanced in obese mice. IL-6 suppresses feeding in the absence of neuronal IL-6-receptor (IL-6R) expression in hypothalamic or all forebrain neurons of mice. Conversely, obese mice exhibit increased soluble IL-6R levels in the cerebrospinal fluid. Blocking IL-6 trans-signaling in the CNS abrogates the ability of IL-6 to suppress feeding. Furthermore, gp130 expression is enhanced in the paraventricular nucleus of the hypothalamus (PVH) of obese mice, and deletion of gp130 in the PVH attenuates the beneficial central IL-6 effects on metabolism. Collectively, these experiments indicate that IL-6 trans-signaling is enhanced in the CNS of obese mice, allowing IL-6 to exert its beneficial metabolic effects even under conditions of leptin resistance.
Subject(s)
Animal Nutritional Physiological Phenomena/genetics , Cytokine Receptor gp130/genetics , Interleukin-6/genetics , Obesity/genetics , Animals , Cytokine Receptor gp130/biosynthesis , Energy Metabolism/genetics , Glucose/metabolism , Humans , Hypothalamus/metabolism , Hypothalamus/pathology , Interleukin-6/metabolism , Mice , Mice, Obese , Neurons/metabolism , Neurons/pathology , Obesity/metabolism , Obesity/physiopathology , Prosencephalon/metabolism , Prosencephalon/pathologyABSTRACT
When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (â¼3000pgml-1) was injected (3dwk-1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.
Subject(s)
Achilles Tendon/metabolism , Extracellular Matrix/metabolism , Interleukin-6/pharmacology , Achilles Tendon/pathology , Animals , Collagen Type I/biosynthesis , Collagen Type I, alpha 1 Chain , Collagen Type III/biosynthesis , Cytokine Receptor gp130/biosynthesis , Extracellular Matrix/pathology , Gene Expression Regulation/drug effects , Male , Protein Inhibitors of Activated STAT/biosynthesis , Protein-Lysine 6-Oxidase/metabolism , Rats , Rats, Wistar , STAT3 Transcription Factor/biosynthesis , Suppressor of Cytokine Signaling 3 Protein/biosynthesis , Tissue Inhibitor of Metalloproteinase-1/biosynthesisABSTRACT
Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.
Subject(s)
DNA Methylation/genetics , Liver/metabolism , Macrophages/immunology , Malaria Vaccines/immunology , Malaria/immunology , Monocytes/immunology , Plasmodium chabaudi/immunology , Animals , Chemokine CX3CL1/biosynthesis , Chemokine CX3CL1/genetics , Cytokine Receptor gp130/biosynthesis , Cytokine Receptor gp130/genetics , Epigenesis, Genetic , Female , GATA2 Transcription Factor/biosynthesis , GATA2 Transcription Factor/genetics , Interleukin-6/genetics , Malaria/parasitology , Malaria/prevention & control , Mice , Mice, Inbred BALB C , Oligonucleotide Array Sequence Analysis , Parasitemia/immunology , Promoter Regions, Genetic/genetics , VaccinationABSTRACT
The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.
Subject(s)
Biomarkers , Congenital Disorders of Glycosylation/genetics , Cytokine Receptor gp130/genetics , Phosphotransferases (Phosphomutases)/genetics , Animals , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Cytokine Receptor gp130/biosynthesis , Disease Models, Animal , Fibroblasts/metabolism , Gene Expression Regulation , Genotype , Glycosylation , Humans , Mannose/genetics , Mannose/metabolism , Mice , MutationABSTRACT
Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers.
Subject(s)
Cytokine Receptor gp130/biosynthesis , Immunoglobulin Fc Fragments/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Cell Line , Cytokine Receptor gp130/genetics , Humans , Immunoglobulin Fc Fragments/genetics , Recombinant Fusion Proteins/geneticsABSTRACT
Gastric cancer shows the highest invasive and metastasis features, especially lymph metastasis, which is closely associated with poor prognosis of gastric cancer. Although there is evidence that interleukin-6 (IL-6) can promote gastric cancer progression, the underlying specific mechanisms and the mechanisms of gastric cancer lymphangiogenesis are largely unknown. In the present study, we explore whether IL-6 could promote the proliferation and invasion activity of gastric cancer cells, and whether IL-6 mediating VEGF-C production affected the lymphangiogenesis in gastric cancer cells. Our results revealed that IL-6 and its receptors (IL-6 and gp130) are broadly expressed in various gastric cancer cell lines including SGC-7901, MGC, MKN-28 and AGS. Exogenous IL-6 increased the ability of gastric cancer cell proliferation and invasion, which could be weakened by AG490. in addition, exogenous IL-6 promoted the VEGF-C production of gastric cancer cells and the lymphangiogenesis of HDLECs. As we expected, AG490 was able to reduce these effects. Western blot analysis showed that IL-6 increased JKA, STAT3, p-STAT3 and VEGF-C protein levels in the gastric cancer cells. However, the JKA, STAT3, p-STAT3 and VEGF-C protein expression levels were inhibited by AG490. Our data suggested that IL-6 mediates the singnal pathway of JAK-STAT3-VEGF-C promoting the growth, invasion and lymphangiogenesis in gastric cancer. Thus, IL-6 and its related signal pathways may be a promising target for treatment of gastric cancer growth and lymphangiogenesis.
Subject(s)
Interleukin-6/genetics , Lymphangiogenesis/genetics , STAT3 Transcription Factor/genetics , Stomach Neoplasms/genetics , Vascular Endothelial Growth Factor C/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cytokine Receptor gp130/biosynthesis , Cytokine Receptor gp130/genetics , Gene Expression Regulation, Neoplastic , Humans , Interleukin-6/biosynthesis , Janus Kinases/genetics , Lymphatic Metastasis , Neoplasm Invasiveness/genetics , STAT3 Transcription Factor/biosynthesis , Signal Transduction , Stomach Neoplasms/pathology , Vascular Endothelial Growth Factor C/biosynthesisABSTRACT
Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.
Subject(s)
Apolipoproteins/genetics , Ion Transport/genetics , Kidney Diseases/genetics , Lipoproteins, HDL/genetics , Mitogen-Activated Protein Kinases/physiology , Mutation, Missense , Potassium/metabolism , Amino Acid Substitution , Apolipoprotein L1 , Apolipoproteins/physiology , Black People/genetics , Cell Death , Cell Size , Cytokine Receptor gp130/biosynthesis , Cytokine Receptor gp130/genetics , Disease Progression , Enzyme Activation , Gene Frequency , Genetic Predisposition to Disease , HEK293 Cells , Humans , Kidney Diseases/ethnology , Lipoproteins, HDL/physiology , MAP Kinase Signaling System , Phosphorylation , Protein Processing, Post-Translational , Recombinant Fusion Proteins/metabolism , Risk , STAT3 Transcription Factor/metabolism , TransfectionABSTRACT
BACKGROUND: This study aimed to explore the responses to the interleukin-6 (IL-6)/soluble interleukin-6 receptor (sIL-6R) complex in peritumoral endothelial cells (PECs) and tumor endothelial cells (TECs), as well as determine the signaling pathways in the angiogenesis of hepatocellular carcinoma (HCC). METHODS: The expression of IL-6, IL-6R, gp130, CD68, HIF-1α, and microvessel density (MVD) were assessed with an orthotopic xenograft model in nude mice. ECs were incubated under hypoxic conditions to detect IL-6 and gp130. The proliferation of PECs and TECs in the presence of IL-6 and sIL-6R, as well as the expression of gp130, JAK2/STAT3, PI3K/AKT in endothelial cells were measured. RESULTS: Peritumoral IL-6, IL-6R, gp130, CD68, and HIF-1α expression, as well as MVD, gradually increased during tumor growth. Hypoxia could directly induce IL-6 expression, but not gp130 in PECs. The co-culture of IL-6/sIL-6R induced much higher PEC proliferation and gp130 expression, as well as the elevated phosphorylation of JAK2 and STAT3, however not the phosphorylation of PI3K and AKT. CONCLUSIONS: PECs exhibited higher proliferation in response to IL-6/sIL-6R co-treatment compared with TECs in HCC via the up-regulation of gp130 /JAK2/STAT3. PEC and its associated peritumoral angiogenesis microenvironment may be a potential novel target for anti-angiogenic treatment.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cytokine Receptor gp130/genetics , Interleukin-6/genetics , Janus Kinase 2/genetics , Liver Neoplasms/genetics , STAT3 Transcription Factor/genetics , Animals , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Coculture Techniques , Cytokine Receptor gp130/biosynthesis , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gene Expression Regulation, Neoplastic , Humans , Interleukin-6/biosynthesis , Janus Kinase 2/biosynthesis , Liver Neoplasms/pathology , Mice , Neoplasm Proteins/biosynthesis , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , STAT3 Transcription Factor/biosynthesis , Signal Transduction , Tumor Microenvironment/geneticsABSTRACT
INTRODUCTION: Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model. METHODS: Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression. RESULTS: Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo. CONCLUSION: Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.
Subject(s)
Cytokine Receptor gp130/biosynthesis , Hyperalgesia/metabolism , MicroRNAs/biosynthesis , STAT3 Transcription Factor/metabolism , Spinal Cord/metabolism , Stress, Psychological/metabolism , Animals , Gene Expression Regulation , Glial Fibrillary Acidic Protein/biosynthesis , Hyperalgesia/pathology , Janus Kinases/biosynthesis , Male , Rats , Rats, Wistar , Signal Transduction , Spinal Cord/pathology , Stress, Psychological/pathology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
PURPOSE: It is anticipated that the interleukin-6/glycoprotein 130 (IL-6/gp130) family of cytokines and Jak-STAT signaling may be amenable to therapeutic manipulation for retinal diseases. Müller cells, which exhibit morphologic and functional changes in prevalent retinal diseases, are implicated in their induction and action. METHODS: We characterized expression of endogenous IL-6/gp130 cytokines and Jak-STAT signaling after inducible Müller cell ablation in the neural retinas of adult mice. This resulted in photoreceptor apoptosis and reactive activation of surviving Müller cells. Analysis was performed by using a combination of quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry. Recombinant leukemia inhibitory factor (rLIF) was intravitreally injected in an attempt to inhibit photoreceptor degeneration following selective Müller cell ablation. RESULTS: Significant differential expression (both increases and decreases) of multiple IL-6/gp130 cytokines, such as LIF, oncostatin-M, and ciliary neurotrophic factor, occurred after Müller cell ablation, with concomitant increase in signal transducers and activators of transcription and extracellular kinases 1 and 2, particularly in surviving, activated Müller cells. Basic fibroblast growth factor was robustly increased in photoreceptors after selective Müller cell ablation. Multiple injections of rLIF failed to prevent photoreceptor degeneration. CONCLUSIONS: These results further characterize expression of IL-6/gp130 cytokines and Jak-STAT signaling in outer retinal disease, suggesting Müller cells are critical for their induction and action. Lack of rLIF-mediated neuroprotection contrasts with other retinal degenerations where Müller cell integrity remains intact or photoreceptor apoptosis occurs in a more rapid, synchronous manner. The presence of Müller cells may be critical for the functional benefits of rLIF and potentially other IL-6/gp130 cytokines.
Subject(s)
Cytokine Receptor gp130/genetics , Ependymoglial Cells/metabolism , Gene Expression Regulation , RNA/genetics , Retinal Degeneration/genetics , STAT3 Transcription Factor/genetics , Animals , Apoptosis , Blotting, Western , Cytokine Receptor gp130/biosynthesis , Disease Models, Animal , Ependymoglial Cells/pathology , Immunohistochemistry , Mice , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , STAT3 Transcription Factor/biosynthesis , Signal TransductionABSTRACT
Obesity is a known risk factor for breast cancer. We have recently identified that adipokine leptin regulates the expression of a proto-oncogenic enzyme sphingosine kinase 1 (SK1). Signal transducer and activator of transcription 3 (STAT3) has been linked to breast cancer progression and here we investigate the mechanism of leptin-induced STAT3 activation in ER-negative breast cancer. Gene and protein expression in human primary and secondary breast cancer tissues was analysed using quantitative real-time polymerase chain reaction (qRT-PCR) assay and immunofluorescence. Leptin-induced signalling was analysed in human ER-negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Gene expression and receptor signalling was modified using small interfering RNA and neutralising antibodies. In human ER-negative breast tumours and lymph node metastases, the expression of leptin receptor significantly correlated with SK1. In ER-negative breast cancer cells, SK1 knockdown led to a significant reduction in leptin-induced STAT3 phosphorylation. Knockdown of another known activator of STAT3 signalling, gp130 also resulted in a significant decrease in leptin-induced STAT3 phosphorylation. ELISA assay showed that leptin produces a significant amount of IL-6 in an SK1-dependent manner. IL-6 neutralising antibodies significantly reduced p-STAT3. Immunofluorescent staining of human primary and secondary breast tumours showed significant correlation between SK1 and IL-6 (P < 0.001), SK1 and p-STAT3 (P < 0.01) and IL-6 and p-STAT3 (P < 0.01). Our findings demonstrate that leptin-induced STAT3 is partially cross activated through SK1-mediated IL6 secretion and gp130 activation. Positive correlations in human tissues suggest the potential significance of this pathway in ER-negative breast cancer.
Subject(s)
Breast Neoplasms/genetics , Cytokine Receptor gp130/biosynthesis , Interleukin-6/biosynthesis , Leptin/genetics , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , STAT3 Transcription Factor/biosynthesis , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Interleukin-6/genetics , Lymphatic Metastasis , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Receptors, Estrogen/genetics , Receptors, Leptin/genetics , STAT3 Transcription Factor/genetics , Signal Transduction , Transcriptional Activation/geneticsABSTRACT
UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), the most common tumor of AIDS patients worldwide. A key characteristic of KS tumors is extremely high levels of vascular slits and extravasated red blood cells, making neoangiogenesis a key component of the tumor. The main KS tumor cell is the spindle cell, a cell of endothelial origin that maintains KSHV predominantly in the latent state. In cultured endothelial cells, latent KSHV infection induces angiogenic phenotypes, including longer-term stabilization of capillary-like tube formation in Matrigel, a basement membrane matrix. The present studies show that KSHV infection of endothelial cells strongly downregulates transforming growth factor ß2 (TGF-ß2). This downregulation allows the stabilization of capillary-like tube formation during latent infection, as the addition of exogenous TGF-ß2 inhibits the KSHV-induced stability of these structures. While two KSHV microRNAs are sufficient to downregulate TGF-ß2 in endothelial cells, they are not required during KSHV infection. However, activation of the gp130 cell surface receptor is both necessary and sufficient for downregulation of TGF-ß2 in KSHV-infected cells. IMPORTANCE: Kaposi's sarcoma is a highly vascularized, endothelial cell-based tumor supporting large amounts of angiogenesis. There is evidence that KSHV, the etiologic agent of KS, induces aberrant angiogenesis. For example, KSHV induces stabilization of capillary-like tube formation in cultured endothelial cells. A clearer understanding of how KSHV regulates angiogenesis could provide potential therapeutic targets for KS. We found that KSHV downregulates TGF-ß2, a cytokine related to TGF-ß1 that is known to inhibit angiogenesis. The downregulation of this inhibitor promotes the stability of capillary-like tube formation insofar as adding back TGF-ß2 to infected cells blocks KSHV-induced long-term tubule stability. Therefore, KSHV downregulation of TGF-ß2 may increase aberrant vascularization in KS tumors through increased capillary formation and thereby aid in KS tumor promotion.
Subject(s)
Endothelial Cells/physiology , Endothelial Cells/virology , Herpesvirus 8, Human/physiology , Host-Pathogen Interactions , Neovascularization, Pathologic , Transforming Growth Factor beta2/antagonists & inhibitors , Cell Line , Cytokine Receptor gp130/biosynthesis , HumansABSTRACT
Cells have various receptors on their surface for responding to extracellular signals that involve intercellular communication. Although the mechanism of signal transduction by such wild-type receptors has been studied intensively, there has been minimal effort in investigating whether such receptors could signal when unnaturally coupled. In this study, we investigated whether unnatural receptor pairs comprising interleukin-2 (IL-2) and interleukin-6 (IL-6) receptor subunits could transduce a signal through forced dimerization. We replaced the extracellular domain of IL-2R and IL-6R signaling subunits (IL-2Rß, IL-2Rγ, and gp130) with the FK506-binding protein (FKBP) or the FKBP12-rapamycin binding (FRB) domain, the protein pair known to be heterodimerized by rapamycin. When expressed in a hematopoietic cell line, unnatural heterodimers (IL-2Rß-gp130 and IL-2Rγ-gp130 pairs) successfully transduced a signal. While the IL-2Rγ-gp130 pair maximally mimicked gp130 signaling, the IL-2Rß-gp130 pair gave weaker gp130 signaling and no significant induction of IL-2Rß signaling, indicating a high potential of the IL-2Rγ chain in terms of activating the coupled partners. This is the first report demonstrating that heterodimeric combinations of IL-2R and IL-6R subunits are functional for signaling. Further extension of this approach may attain a creative design of artificial receptor pairs that have distinct signaling properties when compared with natural receptors.
Subject(s)
Cytokine Receptor gp130/chemistry , Dimerization , Interleukin-2 Receptor beta Subunit/chemistry , Protein Subunits/chemistry , Cell Line , Cytokine Receptor gp130/biosynthesis , Cytokine Receptor gp130/genetics , Gene Expression Regulation , Humans , Interleukin-2 Receptor beta Subunit/genetics , Protein Subunits/biosynthesis , Protein Subunits/genetics , Signal Transduction , Sirolimus/chemistry , Tacrolimus Binding Protein 1A/chemistry , Tacrolimus Binding Protein 1A/geneticsABSTRACT
Glycoprotein gp130 is involved in the intracellular transduction of signals from receptors ofinterleukin-6--related cytokines. The linkage between Il6st gene encoding gp130 and predisposition to excessive freezing (catalepsy) in mice was shown. The aim of present study was to investigate the Il6st mRNA concentration, the level and the rate of glycosilation of gp130 in five brain structures in catalepsy-resistant AKR/J mice strain and in catalepsy-prone CBA/LacJ, AKR.CBA-D13Mit76 with the CBA-derived Il6st gene variant in the AKR/J genome, and ASC created by selection of back-crosses between CBA and AKR strains on catalepsy. Highest concentrations of the nonglycosilated and the glycosilated gp130 protein levels were detected in the midbrain. High levels of Il6st mRNA were discovered in the midbrain, the striatum and the hypothalamus in all mouse strains. The level of Il6st mRNA in the striatum of AKR.CBA-D13Mit76 mice was significantly higher compared with AKR/J. An association between hereditary catalepsy and Il6st expression in the striatum in mice was suggested.
Subject(s)
Brain/metabolism , Catalepsy/metabolism , Cytokine Receptor gp130/biosynthesis , Freezing Reaction, Cataleptic , Gene Expression Regulation , Nerve Tissue Proteins/biosynthesis , RNA, Messenger/biosynthesis , Animals , Brain/pathology , Catalepsy/genetics , Catalepsy/pathology , Genetic Predisposition to Disease , Mice , Species SpecificityABSTRACT
Unexplained infertility affects 25% of infertile couples. Cytokines and growth factors have been suggested to play an important role in the initial process of successful implantation in humans and failures in their production may be a cause of unexplained infertility. Leukemia inhibitory factor (LIF) and Interleukin-6 (IL-6) have demonstrated their importance in implantation in both animal and human studies. Lower expression of LIF is found in proliferative phase and maximal expression is found in secretory phase of the menstrual cycle. Lower expression of LIF is also found in secretory phase endometrium in patients with infertility. However, studies investigating whether the levels of LIF in proliferative phase are associated with multiple implantation failures (MIFs) are limited. 30 Endometrial biopsies in proliferative phase from unexplained infertile women with MIF with normal hormone levels were collected. The expression of LIF, IL-6 and its receptor gp130 were measured by immunohistochemistry and western blotting. Moderate expression of LIF in the proliferative phase and high expression of LIF in the secretory phase were found in fertile women. However, lower expression of LIF was found in unexplained infertile women with MIF compared to fertile women. There was no difference in endometrial IL-6 and gp130 expression between unexplained infertile women with MIF and fertile women. LIF expression is independent of the process of embryo and dependent partially on the maternal sex hormone levels. Our data suggest that the initial lower expression of LIF in proliferative phase may be one of the causes for multiple failure of implantation.