Exploratory assessment of the effect of systemic administration of soluble glycoprotein 130 on cognitive performance and chemokine levels in a mouse model of experimental traumatic brain injury.

Uncontrolled neuroinflammation mediates traumatic brain injury (TBI) pathology and impairs recovery. Interleukin-6 (IL-6), a pleiotropic inflammatory regulator, is associated with poor clinical TBI outcomes. IL-6 operates via classical-signaling through membrane-bound IL-6 receptor (IL-6R) and trans-signaling through soluble IL-6 receptor (s)IL-6R. IL-6 trans-signaling specifically contributes to neuropathology, making it a potential precision therapeutic TBI target. Soluble glycoprotein 130 (sgp130) prevents IL-6 trans-signaling, sparing classical signaling, thus is a possible treatment. Mice received either controlled cortical impact (CCI) (6.0 ± 0.2 m/s; 2 mm; 50-60ms) or sham procedures. Vehicle (VEH) or sgp130-Fc was subcutaneously administered to sham (VEH or 1 µg) and CCI (VEH, 0.25 µg or 1 µg) mice on days 1, 4, 7, 10 and 13 post-surgery to assess effects on cognition [Morris Water Maze (MWM)] and ipsilateral hemisphere IL-6 related biomarkers (day 21 post-surgery). CCI + sgp130-Fc groups (0.25 µg and 1 µg) were combined for analysis given similar behavior/biomarker outcomes. CCI + VEH mice had longer latencies and path lengths to the platform and increased peripheral zone time versus Sham + VEH and Sham + sgp130-Fc mice, suggesting injury-induced impairments in learning and anxiety. CCI + sgp130-Fc mice had shorter platform latencies and path lengths and had decreased peripheral zone time, indicating a therapeutic benefit of sgp130-Fc after injury on learning and anxiety. Interestingly, Sham + sgp130-Fc mice had shorter platform latencies, path lengths and peripheral zone times than Sham + VEH mice, suggesting a beneficial effect of sgp130-Fc, independent of injury. CCI + VEH mice had increased brain IL-6 and decreased sgp130 levels versus Sham + VEH and Sham + sgp130-Fc mice. There was no treatment effect on IL-6, sIL6-R or sgp130 in Sham + VEH versus Sham + sgp130-Fc mice. There was also no treatment effect on IL-6 in CCI + VEH versus CCI + sgp130-Fc mice. However, CCI + sgp130-Fc mice had increased sIL-6R and sgp130 versus CCI + VEH mice, demonstrating sgp130-Fc treatment effects on brain biomarkers. Inflammatory chemokines (MIP-1ß, IP-10, MIG) were increased in CCI + VEH mice versus Sham + VEH and Sham + sgp130-Fc mice. However, CCI + sgp130-Fc mice had decreased chemokine levels versus CCI + VEH mice. IL-6 positively correlated, while sgp130 negatively correlated, with chemokine levels. Overall, we found that systemic sgp130-Fc treatment after CCI improved learning, decreased anxiety and reduced CCI-induced brain chemokines. Future studies will explore sex-specific dosing and treatment mechanisms for sgp130-Fc therapy.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

Associations between serum estradiol and IL-6/sIL-6R/sgp130 complex in female patients with major depressive disorder.

BACKGROUND: It has been hypothesized that the IL-6/sIL-6R/sgp130 complex, an inflammatory complex, plays a critical role in the pathogenesis of major depressive disorder (MDD). Estradiol (E2) is a sex steroid hormone involved in emotional regulation and MDD. This study aimed to investigate the relationship between E2 and IL-6/sIL-6R/sgp130 complex in patients with MDD. METHODS: Using enzyme-linked immunosorbent assay, the levels of IL-6, sIL-6Rα, and sgp130 were compared between 117 female patients with MDD and 122 healthy controls.The serum concentrations of E2 and other biomarkers were also measured. RESULTS: (1) The serum levels of IL-6 and sIL-6Rα in patients with MDD were significantly higher than those in the control group, while the serum levels of sgp130 and E2 were significantly lower (all P < 0.05). (2) Low levels of E2 were associated with high levels of IL-6 and low levels of sgp130 (all P < 0.01). (3) HAMD-24 score was positively correlated with the serum level of IL-6, but negatively correlated with the serum levels of sgp130 and E2(all P < 0.05). (4) IL-6 and sgp130 had certain prognostic values in MDD, and the combination of various indicators showed a significantly superior prognostic value. CONCLUSIONS: The IL6/sIL-6R/sgp130 complex in female patients with MDD was closely related to E2 level. In addition, IL-6 and sgp130 may be valuable serum biomarkers for the diagnosis and prognosis of MDD in women.

Dynamics of the Levels of Interleukin 6, Its Soluble Receptor, and Soluble Glycoprotein 130 in Patients with Chronic Heart Failure and Preserved or Reduced Ejection Fraction.

The features of IL-6 trans-signaling were studied in patients with heart failure with reduced (n=74) and preserved (n=31) ejection fraction (EF) during acute decompensation of HF (ADHF) and after 1 year. Patients with ADHF with reduced EF demonstrated higher levels of IL-6 and soluble glycoprotein 130 in comparison with those in patients with preserved EF: 10.18 (7.07; 16.14) pg/ml vs 6.35 (3.52; 11.00) pg/ml and 543.46 (455.37; 634.43) ng/ml vs 498.50 (408.16; 632.23) ng/ml, respectively. The levels of soluble IL-6 receptor little differed in these groups: 57.82 (47.55; 79.85) ng/ml vs 61.30 (44.97; 78.08) ng/ml. After 1 year, the levels of IL-6 in HF patients with reduced EF significantly decreased (5.36 (3.35; 8.35) pg/ml), while in patients with preserved EF, the decrease in this parameter was less pronounced (5.86 (4.05; 7.32) pg/ml), and the difference between groups disappeared. The levels of soluble glycoprotein 130 increased in both groups: 448.06 (357.74; 550.67) ng/ml vs 385.35 (344.29; 523.72) ng/ml. It should be noted that after 1 year (in stable patients), the levels of soluble IL-6 receptor increased in both groups: 65.75 (54.84; 75.39) ng/ml vs 70.81 (57.51; 82.25) ng/ml. Thus, despite the high levels of IL-6 in HF patients with reduced EF, the potential limiting IL-6 trans-signaling in these patients is higher than in patients with preserved EF.

Altered expression of leukemia inhibitory factor (LIF), LIFR, gp130, and IL11 in the embryo implantation site of rat model with prenatal androgen-induced polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that represents infertility in many reproductive-age women. Reduced implantation of blastocyst was proposed as an etiology for infertility in this syndrome. In this regard, many candidate genes such as leukemia inhibitory factor (LIF), LIF receptor (LIFR), glycoprotein 130 (gp130), and interleukin 11 (IL11) were proposed to be disrupted. Investigation of these genes is not ethically approved in pregnant women with PCOS. In this study, we aimed to compare the expression of LIF, LIFR, gp130, and IL11 before and during different gestational days in uterine tissues of prenatally-androgenized rat models of PCOS with control rats. The rat model of polycystic ovary syndrome was created by the injection of testosterone during prenatal life. RNA extraction and cDNA synthesis from uterine tissues were performed in both prenatal induced PCOS and control rats. Expression of LIF, LIFR, gp130, and IL11 genes was compared before pregnancy (GD0) and during pregnancy on GD0.5, GD4.5, GD5.5, and GD8.5 between two study groups (n = 6 each group) using SYBR Green real-time PCR. The expression of the LIF mRNAs significantly decreased on GD4.5, 5.5, and 8.5 in the PCOS rats compared to the controls (P-values: 0.0483, 0.0152, and 0.0043). Additionally, decreased expression of LIFR and gp130 was observed on GD0.5 to 8.5 in PCOS rats compared to controls (P-values: 0.022, 0.0480, 0.0043, 0.0022 for LIFR and 0.0189, 0.0022, 0.0087, 0.0022 for gp130). Moreover, IL-11 mRNA levels decreased in the PCOS group compared to their controls both before (P-value:0.0362) and during the gestational period (P-values:0.0085, 0.0043, 0.0389, 0.0087). Reduced expression of LIF, LIFR, gp130, and IL11 in the rats with PCOS indicates a possible disruption in the implantation and decidualization stages in this syndrome.

Reposition of the anti-inflammatory drug diacerein in an in-vivo colorectal cancer model.

Excessive interleukin (IL)-6 production is a driver for malignancy and drug resistance in colorectal cancer (CRC). Our study investigated a seven-week post-treatment with the anti-inflammatory drug, Diacerein (Diac), alone or in combination with 5-fluorouracil (5-FU), using a 1,2-dimethylhydrazine (DMH) rat model of CRC. Diac alone and 5-FU+Diac reduced serum levels of carcino-embryonic antigen (CEA), while all regimens decreased serum levels of colon cancer-specific antigen (CCSA), a more specific CRC biomarker. Additionally, Diac, 5-FU and their combination suppressed colonic content/gene expression of IL-6, its downstream oncogene, Kirsten rat sarcoma viral oncogene homolog (K-Ras), and consequently Notch intracellular domain and nuclear factor-kappa B (NF-κB) p65. In turn, NF-κB downstream factors, viz., matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), c-Myc, and B-cell lymphoma-2 (Bcl-2) were also downregulated, while E-cadherin was elevated. Additionally, the drugs reduced the immunoreactivity of CD31 to prove their anti-angiogenic effect, while the TUNEL assay confirmed the apoptotic effect. The apoptotic effect was confirmed by transferase dUTP nick-end labeling assay. Moreover, these drugs inhibited colon content of p-Akt, ß-catenin, and cyclin D1 immunoreactivity. The drugs also activated the tumor suppressor glycogen synthase kinase 3- ß (GSK3-ß) and upregulated the expression of the Nur77 gene, which represents the second arm of IL-6 signaling. However, only 5-FU upregulated miR-200a, another K-Ras downstream factor. The in-vitro cytotoxic and migration/invasion assays verified the molecular trajectories. Accordingly, we evaluated the antineoplastic effect of Diac alone and its possible chemosensitization effect when added to 5-FU. This combination may target critical oncogenic pathways, including the IL-6/K-Ras/Notch/NF-κB p65 axis, p-Akt/GSK3-ß/ß-catenin/cyclin D-1 hub, and Nur77.

Localization of interleukin-6 signaling complex in epithelialized apical lesions of endodontic origin.

OBJECTIVES: We aimed to determine the immunolocalization patterns of the interleukin (IL)-6 signaling complex in epithelialized and non-epithelialized apical lesions of endodontic origin (ALEOs). MATERIALS AND METHODS: Epithelialized (n = 8) and non-epithelialized (n = 7) ALEOs were obtained from teeth with indication of extraction in patients with clinical diagnosis of apical periodontitis. All tissues were subjected to routine processing for histopathologic examination and primary antibodies for IL-6, IL-6 receptor (R), and glycoprotein (gp)-130 were used for immunohistochemistry and double immunofluorescence co-localization. RESULTS: IL-6, IL-6R, and gp-130 were immunolocalized in endothelial cells and mononuclear leukocytes in a diffuse pattern within the connective tissue of epithelialized and non-epithelialized ALEOs. In the epithelialized lesions, two different patterns were identified: IL-6 signaling complex was localized within the proliferating epithelium in a diffuse intracellular pattern and in a cell membrane localization pattern within the mature epithelial lining, showing a decreased intensity towards the surface layers. CONCLUSIONS: IL-6, IL-6R, and gp-130 localized to mononuclear inflammatory cells, vascular endothelial cells, and immature proliferating epithelia in a diffuse pattern and in mature lining epithelia in a localized cell membrane pattern, supporting a role for epithelial proliferation during cyst formation. Additional cell membrane co-localization of IL-6 receptor complex suggests classic signaling involvement in addition to trans-signaling.

Estrogen downregulates gp130 expression in HUVECs by regulating ADAM10 and ADAM17 via the estrogen receptor.

Glycoprotein130 (gp130) is an important signal transducer in the interleukin-6 (IL-6) trans-signaling pathway, which plays a crucial role in chronic inflammation in atherosclerosis. Studies suggest that estrogen can inhibit IL-6/gp130 signaling and reduce the risk of coronary artery disease, but the precise mechanism is unclear. The aim of this study was to investigate whether and how estrogen regulates gp130 in human umbilical vein endothelial cells (HUVECs). HUVECs were first treated with IL-6 and soluble IL-6 receptor (sIL-6R) to induce inflammation, then treated with estradiol. We then measured the expression of gp130, a disintegrin and metalloproteinase 10 (ADAM10) and 17 (ADAM17) by RT-PCR and western blot. Levels of soluble gp130 (sgp130) in the culture supernatant were measured by ELISA. We found that IL-6 and sIL-6R increased expression of gp130 protein and decreased levels of sgp130 protein, without affecting gp130 mRNA levels. Estradiol treatment reversed these effects in a concentration- and time-dependent manner. These effects were regulated by ADAM10 and ADAM17 via an estrogen receptor α/ß-dependent mechanism. These results shed further light on the mechanism underlying the clinical effects of estrogen therapy in atherosclerosis and coronary artery disease.

Activating the interleukin-6-Gp130-STAT3 pathway ameliorates ventricular electrical stability in myocardial infarction rats by modulating neurotransmitters in the paraventricular nucleus.

BACKGROUND: Malignant ventricular arrhythmia (VA) is the most common cause of death associated with acute myocardial infarction (MI). Recent studies have revealed direct involvement of the paraventricular nucleus (PVN) in the occurrence of VA. However, the underlying mechanisms remain incompletely understood. In this study, we investigated changes in the interleukin-6 (IL-6)-glycoprotein 130-signal transducer and activator of transcription 3 (STAT3) pathway in the PVN during acute MI and the effects of this pathway on ventricular stability. METHODS: Rats were divided into a control group, a MI group, a PVN-injected anti-IL-6 antibody group and a PVN-injected SC144 group to observe how IL-6 and its downstream glycoprotein 130-STAT3 pathway in the PVN affect ventricular stability. The left anterior descending coronary artery was ligated to induce MI. After that, an anti-IL-6 antibody and SC144 were injected into the PVNs of rats. All data are expressed as the mean ± SE and were analysed by ANOVA with a post hoc LSD test. p < 0.05 was considered to indicate statistical significance. RESULTS: After MI, the concentration of the inflammatory factor IL-6 increased, and its downstream glycoprotein 130-STAT3 pathway was activated in the PVN. After injection of MI rat PVNs with the anti-IL-6 antibody or glycoprotein 130 inhibitor (SC144), glutamate levels increased and γ-aminobutyric acid (GABA) levels decreased in the PVN. Plasma norepinephrine concentrations also increased after treatment, which increased the vulnerability to VA. CONCLUSIONS: In summary, IL-6 in the PVN exerts a protective effect in MI rats, and the glycoprotein 130-STAT3 pathway plays a key role in this process. We anticipate that our findings will provide new ideas for the prevention and treatment of arrhythmia after MI.

Adipose Tumor Microenvironment.

The term "adipose tissue" represents a multicellular and multifunctional organ involved in lipid storage, in hormone and temperature regulation, and in the protection of bones and vital organs from impact-based damage. Emerging evidence now suggests a more malignant role of adipose tissue in promoting cancer onset and progression via the release of secreted factors such as interleukin-6 (IL6) and extracellular vesicles (EVs). These adipose-source factors subsequently affect various aspects of tumorigenesis and/or cancer progression by either directly enhancing the tumor cell oncogenic phenotype or indirectly by the stimulating adjacent normal cells to adopt a more pro-cancer phenotype. Due to the recent growing interest in the role of IL6 and EVs released by adipose tissue in cancer promotion and progression, we are focusing on the protumorigenic impact of fat tissue via IL6 and EV secretion.

Interaction between epidermal growth factor receptor and interleukin-6 receptor in NSCLC progression.

Even though the interaction between epithelial growth factor receptor (EGFR) and interleukin-6 receptor (IL-6R) has been found in many tumors, there is a lack of relevant in-depth study of lung cancer. The following study investigates the interaction of EGFR and IL-6R in lung cancer. In the current study, EGFR, IL-6, and glycoprotein 130 (GP130) were highly expressed in non-small cell lung cancer (NSCLC) tissue samples and were associated with clinicopathological features and poor prognosis of patients with NSCLC. Furthermore, the effect of EGF and IL-6 on biological behavior of lung cancer cells (cell proliferation, invasion, cycle, and apoptosis) and the expression of EGFR, GP130, p-protein kinase B (p-AKT), and p-p44/42 mitogen-activated protein kinase (p-p44/42 MAPK) was significantly stronger compared with other treatment groups (all P < 0.05). These results suggest that EGFR and IL-6R have synergistic effects on NSCLC progression. This could help to solve the problem of EGFR inhibitors in the treatment of lung cancer resistance and improve the efficacy of current treatment for lung cancer through a combination of EGFR and IL-6R signaling pathways.

Interaction of estrogen receptor ß5 and interleukin 6 receptor in the progression of non-small cell lung cancer.

Numerous studies have shown that the estrogen receptor beta (ERß) and interleukin 6 receptor (IL-6R) had interaction in many tumors, including lung cancer. Previous studies found that ERß5 exhibits a different biological function compared with the other subtypes of ERß. Therefore, this study mainly explores the interaction between ERß5 and IL-6R in the progression of lung cancer. We found that the expression of ERß5, IL-6 and glycoprotein 130 (GP130) were significantly increased (P < 0.001) and the 5-year survival rate with the co-expression of ERß5 and GP130 is significantly lower (P = 0.0315) in non-small cell lung cancer (NSCLC) patients. The cell proliferation, invasion, and cell cycle were markedly increased, and the cell apoptotic was markedly inhibited with the concurrent action of ERß5 and IL-6 in A549 cells (P < 0.05). In addition, the expression of ERß5, GP130, p-AKT, and p-44/42 MAPK was also significantly increased in A549 cells (P < 0.05). These results indicate that ERß5 and GP130 can synergistically promote the progression of NSCLC and maybe combined as an independent prognostic factor in patients. In addition, these results also provide a theoretical basis for the combined targeting therapy of ERß5 and GP130 in NSCLC.

Bazedoxifene exhibits growth suppressive activity by targeting interleukin-6/glycoprotein 130/signal transducer and activator of transcription 3 signaling in hepatocellular carcinoma.

The interleukin (IL)-6/glycoprotein (GP)130/signal transducer and activator of transcription (STAT)3 pathway is emerging as a target for the treatment of hepatocellular carcinoma. IL-6 binds to IL-6R, forming a binary complex, which further combines with GP130 to transduce extracellular signaling by activating STAT3. Therefore, blocking the interaction between IL-6 and GP130 may inhibit the IL-6/GP130/STAT3 signaling pathway and its biological effects. It has been reported that bazedoxifene acetate (BAZ), a selective estrogen receptor modulator approved by the US Food and Drug Administration, could inhibit IL-6/GP130 protein-protein interactions. Western blot, immunofluorescence staining, wound healing and colony formation assays were used to detect the effect of BAZ on liver cancer cells. Cell viability was evaluated by MTT assay. Apoptosis of cells was determined using the Annexin V-FITC detection kit. Mouse xenograft tumor models were utilized to evaluate the effect of BAZ in vivo. Our data showed that BAZ inhibited STAT3 phosphorylation (P-STAT3) and expression of STAT3 downstream genes, inducing apoptosis in liver cancer cells. BAZ inhibited P-STAT3 induced by IL-6, but not by leukemia inhibitory factor. BAZ inhibited P-STAT1 and P-STAT6 less significantly as elicited by interferon-α, interferon-γ and IL-4. In addition, pretreatment of BAZ impeded the translocation of STAT3 to nuclei induced by IL-6. BAZ inhibited cell viability, wound healing and colony formation in vitro. Furthermore, tumor growth in HEPG2 mouse xenografts were significantly inhibited by daily intragastric gavage of BAZ. Our results suggest that BAZ inhibited the growth of hepatocellular carcinoma in vitro and in vivo, indicating another potential strategy for HCC prevention and therapy.

Soluble glycoprotein 130 is inversely related to severity of coronary atherosclerosis.

PURPOSE: Recent studies indicate that the effects of interleukin 6 (IL-6) realized via soluble IL-6 receptor (sIL-6R) facilitate the development of various pathological processes. Soluble gp130 (sgp130) is a naturally occurring inhibitor of signal transduction via this pathway. In this study, we assessed the relationship between circulating levels of IL-6, sIL-6R and sgp130 and severity of coronary atherosclerosis in patients with stable coronary artery disease (CAD). METHODS: Plasma levels of IL-6, sIL-6R and sgp130 were measured in patients with atherosclerotic coronary lesions (n = 128, group 1) and with intact coronary arteries (n = 48, group 2). The severity of coronary atherosclerosis was evaluated by the number of affected arteries and by Gensini Score index. RESULTS: Circulating IL-6 levels in group 1 were significantly higher than those in group 2. The levels of sIL-6R did not differ considerably in both the groups. The levels of sgp130 in group 1 were significantly lower than in group 2. A negative correlation has been revealed between sgp130 levels and the number of affected coronary arteries and Gensini Score index. CONCLUSIONS: Serum concentration of sgp130 in patients with stable CAD is inversely related to severity of coronary damage. Low sgp130 level may serve as an additional indicator of coronary atherosclerosis severity.

Blocking gp130 signaling suppresses autotaxin expression in adipocytes and improves insulin sensitivity in diet-induced obesity.

Autotaxin (ATX), which is highly expressed and secreted by adipocytes, functions as the key enzyme to generate lysophosphatidic acid (LPA) from lysophosphatidylcholine. Adipose tissue is the main source of circulating ATX that modulates plasma LPA levels. Upregulation of ATX expression in obese patients and mice is closely related with insulin resistance and impaired glucose tolerance. However, the mechanism of ATX expression in adipocytes remains largely unknown. In this study, we found that glycoprotein 130 (gp130)-mediated Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) activation was required for abundant ATX expression in adipocytes. Through gp130, the interleukin 6 (IL-6) family cytokines, such as IL-6, leukemia inhibitory factor, cardiotrophin-1, and ciliary neurotrophic factor, upregulated ATX expression in adipocytes. ATX contributes to the induction of insulin resistance and lipolysis in IL-6-stimulated adipocytes. Oral administration of gp130 inhibitor SC144 suppressed ATX expression in adipose tissue, decreased plasma ATX, LPA, and FFA levels, and significantly improved insulin sensitivity and glucose tolerance in high-fat diet-fed obese mice. In summary, our results indicate that the activation of gp130-JAK-STAT3 pathway by IL-6 family cytokines has an important role in regulating ATX expression in adipocytes and that gp130 is a promising target in the management of obesity-associated glucose metabolic diseases.

Murine Oncostatin M Acts via Leukemia Inhibitory Factor Receptor to Phosphorylate Signal Transducer and Activator of Transcription 3 (STAT3) but Not STAT1, an Effect That Protects Bone Mass.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are IL-6 family members with a wide range of biological functions. Human OSM (hOSM) and murine LIF (mLIF) act in mouse cells via a LIF receptor (LIFR)-glycoprotein 130 (gp130) heterodimer. In contrast, murine OSM (mOSM) signals mainly via an OSM receptor (OSMR)-gp130 heterodimer and binds with only very low affinity to mLIFR. hOSM and mLIF stimulate bone remodeling by both reducing osteocytic sclerostin and up-regulating the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) in osteoblasts. In the absence of OSMR, mOSM still strongly suppressed sclerostin and stimulated bone formation but did not induce RANKL, suggesting that intracellular signaling activated by the low affinity interaction of mOSM with mLIFR is different from the downstream effects when mLIF or hOSM interacts with the same receptor. Both STAT1 and STAT3 were activated by mOSM in wild type cells or by mLIF/hOSM in wild type and Osmr-/- cells. In contrast, in Osmr-/- primary osteocyte-like cells stimulated with mOSM (therefore acting through mLIFR), microarray expression profiling and Western blotting analysis identified preferential phosphorylation of STAT3 and induction of its target genes but not of STAT1 and its target genes; this correlated with reduced phosphorylation of both gp130 and LIFR. In a mouse model of spontaneous osteopenia caused by hyperactivation of STAT1/3 signaling downstream of gp130 (gp130Y757F/Y757F), STAT1 deletion rescued the osteopenic phenotype, indicating a beneficial effect of promoting STAT3 signaling over STAT1 downstream of gp130 in this low bone mass condition, and this may have therapeutic value.

Oncogenic deletion mutants of gp130 signal from intracellular compartments.

Interleukin 6 (IL-6) and, hence, activation of the IL-6 receptor signalling subunit glycoprotein 130 (gp130; also known as interleukin-6 receptor subunit ß, IL6ST), has been linked to inflammation and tumour formation. Recently, deletion mutations in gp130 have been identified in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope and rendered gp130 constitutively active in a ligand-independent manner. Here, we show that gp130 deletion mutants, but not wild-type gp130, localise predominantly to intracellular compartments, notably the endoplasmic reticulum (ER) and early endosomes. One of the most frequent mutants, gp130 Y186-Y190del (ΔYY) is retained in the ER quality control system because of its association with the chaperone calnexin. Furthermore, we can show that gp130 ΔYY induces downstream signalling from both ER and endosomes, and that both signals contribute to ligand-independent cell proliferation. We also demonstrate that the endosomal localisation of gp130 ΔYY is crucial for fully fledged STAT3 activation. Therefore, aberrant signalling from intracellular compartments might explain the tumorigenic potential of naturally occurring somatic mutations of gp130.

Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

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.

Soluble Glycoprotein 130 and Heat Shock Protein 27 as Novel Candidate Biomarkers of Chronic Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Despite their importance, the current clinical biomarkers of chronic heart failure have limitations. In this study, soluble glycoprotein 130 (sgp130), heat shock protein 27 (hsp27), dipeptidyl peptidase IV (dpp4) and cathepsin S (CTSS) were tested for their potential as novel biomarkers for diagnosing chronic heart failure (CHF) with preserved ejection fraction. METHODS: We compared the circulating levels of sgp130, hsp27, dpp4, and cathepsin S in patients with CHF with preserved ejection fraction (n=50) and in controls (n=50), determined how well these candidate biomarkers distinguish patients with CHF from controls, and assessed whether these candidates are superior to N-terminal pro brain natriuretic peptide (NT-pro-BNP) as diagnostic tools. RESULTS: After adjusting for clinical covariates, patients with CHF showed significantly higher mean concentrations of sgp130 (317.38pg/ml vs. 215.90 pg/ml), hsp27 (2601.02 pg/ml vs. 923.61 pg/ml) and NT-pro-BNP (982.35 pg/ml vs. 331.99 pg/ml), but not dpp4 (6930.9 4pg/ml vs. 7081.37 pg/ml) or CTSS (1050.46 pg/ml vs. 984.96 pg/ml), than did controls. In the receiver operating characteristic curve analysis, hsp27 showed the most notable difference between CHF patients and controls, with the largest area under the curve (AUC) (0.920); the AUC values for sgp130 and NT-pro-BNP were 0.877 and 0.882, respectively. CONCLUSIONS: Soluble glycoprotein 130 and hsp27 are novel candidate biomarkers for diagnosing CHF with preserved ejection fraction and thus warrant further investigation; neither dpp4 nor CTSS showed promise as biomarkers of CHF.

Strawberry notch homolog 2 is a novel inflammatory response factor predominantly but not exclusively expressed by astrocytes in the central nervous system.

Interleukin-6 (IL-6) participates in the host response to injury and infection in the central nervous system (CNS). We identified strawberry notch homolog 2 (Sbno2) as an IL-6-stimulated gene in murine astrocytes. Sbno2 is a mouse homolog of the sno gene in Drosophila but little is known about the regulation or function of the mammalian gene. Here we examined the regulation of the Sbno2 gene in astrocytes in vitro and in the murine CNS following systemic endotoxin administration. In murine and human cultured astrocytes, Sbno2 gene expression was significantly upregulated in a dose- and time-dependent fashion by hyper-IL-6 (IL-6 + soluble IL-6 receptor). The level of Sbno2 mRNA was also upregulated significantly in murine astrocytes by other glycoprotein130 cytokine-family members and the pro-inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha. These changes were reflected by corresponding alterations in the level of the SBNO2 protein. Inhibiting protein synthesis resulted in higher Sbno2 mRNA and did not abolish the upregulation of Sbno2 mRNA mediated by hyper-IL-6. Inhibition of transcription led to a rapid reduction in hyper-IL-6-induced Sbno2 mRNA in astrocytes suggesting that the Sbno2 mRNA is quite unstable. Following intra-peritoneal lipopolysaccharide injection in mice, Sbno2 mRNA levels in the brain were significantly increased. Cellular localization studies revealed that this increase in Sbno2 mRNA occurred predominantly in astrocytes and in the choroid plexus and in some microglia, endothelial cells, and neurons. These findings are consistent with SBNO2 functioning as an acute inflammatory response gene in astrocytes as well as other cells in the CNS.