Recently we found that interleukin-6 (IL-6) knockout mice develop mature-onset obesity and that a single intracerebroventricular (ICV) injection of IL-6 increases energy expenditure. In the present study we investigated if chronic ICV treatment with IL-6 can suppress body fat mass. IL-6 was injected ICV daily for two weeks to rats fed a high-fat diet. IL-6 treatment but not saline treatment decreased body weight by 8.4% and decreased the relative weights of mesenteric and retroperitoneal fat pads. Consistent with this, circulating leptin levels were decreased by 40% after IL-6 treatment but not after saline treatment. Average food intake per day was decreased in the IL-6 treated group compared to the saline treated rats. IL-6 treatment did not change hepatic expression of the acute-phase protein haptoglobin, serum levels of insulin or insulin-like growth factor-I, or the weights of the heart, liver, kidneys, adrenals, and spleen. We conclude that centrally administered IL-6 can decrease body fat in rats without causing acute-phase reaction.

IL-6 has emerged as an important cytokine upregulated in states of insulin resistance such as type 2 diabetes. We evaluated the chronic effect of IL-6 on insulin signaling in 3T3-F442A and 3T3-L1 adipocytes. First, cells responded to a chronic treatment with IL-6 by initiating an autoactivation process that increased IL-6 secretion. Second, IL-6-treated adipocytes showed a decreased protein expression of IR-beta subunit and IRS-1 but also an inhibition of the insulin-induced activation of IR-beta, Akt/PKB, and ERK1/2. Moreover, IL-6 suppressed the insulin-induced lipogenesis and glucose transport consistent with a diminished expression of GLUT4. IL-6-treated adipocytes failed to maintain their adipocyte phenotype as shown by the downregulation of the adipogenic markers FAS, GAPDH, aP2, PPAR-gamma, and C/EBP-alpha. IL-6 also induced the expression of SOCS-3, a potential inhibitor of insulin signaling. Finally, the effects of IL-6 could be prevented by rosiglitazone, an insulin-sensitizing agent. Thus, IL-6 may play an important role in the set-up of insulin resistance in adipose cell.

IL-6 exerts its pleiotropic activities on its target cells via the IL-6 alpha-receptor (IL-6R), which is expressed on a limited number of cell types. IL-6 can further signal via soluble forms of its receptor (sIL-6R), a process that has been termed trans-signaling. Recently, CD5 was described as an alternative alpha-receptor for IL-6 on B cells leading to the phosphorylation of the transcription factor STAT3 via the signal-transducing β-receptor gp130 in a Jak2-dependent manner. In this study, we sought to investigate whether IL-6 was also able to signal via soluble CD5 (sCD5) analogous to IL-6 trans-signaling. We show that IL-6 indeed binds to sCD5, but that this does not lead to the activation of signal transduction or cell proliferation. Furthermore, sCD5 did also not interfere with IL-6 classic signaling, suggesting that the affinity between the two proteins was too weak to provoke a biological effect. Thus, trans-signaling of IL-6 can only occur via sIL-6R, but not sCD5.

Interleukin-6 (IL-6) has become a target of interest for drug development aiming to treat diabetic retinopathy. Since IL-6 signaling can promote beneficial as well as detrimental effects via two different signaling pathways, the objective of the present study was to investigate the effects of classical IL-6 and IL-6 trans-signaling on human Müller cells (HMC), which are important for the development of diabetic retinopathy. HMCs were cultured in normal (5 mmol/L) and high (25 mmol/L) glucose plus or minus IL-6 or IL-6/sIL-6R. IL-6 receptor expression using immunohistochemistry and flow cytometry and cytokine release using magnetic bead assays were determined. HMCs express the membrane bound form of the IL-6 receptor (mIL-6R), gp130, and can release the soluble forms sIL-6R and sgp130 demonstrating that HMCs are capable of responding to classical IL-6 and IL-6 trans-signaling. IL-6 protected HMCs from glucose toxicity via VEGF-A signaling. IL-6/sIL-6R caused only modest protection, which was not mediated by VEGF-A. Our data show for the first time that classical IL-6 signaling exerts its beneficial effects through VEGF-A action contrary to IL-6 trans-signaling, which was VEGF-A independent. These results have clinical implications for drug development targeting IL-6 since strict anti-IL-6 therapies might further decrease neuroretinal functions in the diabetic retina.

Interleukin 6 (IL-6) is a major proinflammatory cytokine involved in several aspects of the immune response. Excessive IL-6 production and dysregulated IL-6 receptor signaling lead to multiple inflammatory and autoimmune diseases, such as asthma, even cancer. Thus, its precise regulatory mechanisms need to be fully addressed. Here we found that knockdown of protein C-ets-2 (Ets2) resulted in higher IL-6 production after TLRs activation in macrophages. Mechanistically, Ets2 associated with an epigenetic modifier histone deacetylase 1 (HDAC1) and promoted its recruitment to the Il6 promoter after TLRs activation. Subsequentially, it enhanced histone deacetylation and inhibited Il6 mRNA transcription. Thus, Ets2 epigenetically suppresses TLRs-induced IL-6 production in both human and murine macrophages via promoting histone deacetylation of the Il6 promoter, serving as a new potential therapeutic target in inflammatory diseases therapy.

A clear understanding of the mechanisms that regulate the alveolar epithelium's barrier is critical to develop new therapeutic strategies to mitigate lung injury. The HER2/HER3 receptor tyrosine kinase complex plays a central role in maintaining the alveolar-capillary barrier. This receptor complex is activated by its ligand, neuregulin-1 (NRG-1). Interleukin-6 (IL-6) is also known to induce HER2 signaling through HER2 transphosphorylation by the IL-6 receptor (IL-6R) complex (1). Due to this interaction, we hypothesized that NRG-1 and IL-6 cooperatively interacted to activate the HER2/HER3 complex. Studies were performed in cultured pulmonary epithelial cells measuring the HER2/IL-6/IL-6R/GP130 interaction and receptor activation by western blotting and confocal microscopy, IL-6 production by ELISA, and IL-6 inhibition using specific antibodies, small molecule inhibitors and shRNA. We found that IL-6 was required for NRG-1 induced activation of HER2 in pulmonary epithelial cells. IL-6 inhibition led to a decrease in NRG-1 induced HER2 activation. The IL-6R and GP130, a subunit of the IL-6R complex, were physically associated with HER2 and were required for NRG-1 induced HER2 activation. Inhibition of GP130, the β-subunit of the IL-6 receptor decreased NRG-1 induced HER2 activation lower than control by 38% Finally, HER2 activation increased IL-6 secretion more than two-fold over resting cells (526 ± 131 vs 231 ± 39.7 pg/ml), and inhibition of HER2 gene expression decreased basal IL-6 secretion over 80% (89 + 4.6 vs 1.3 + 0.8 pg/ml). These findings identify a requirement for IL-6 and the IL-6R complex to allow NRG-1 mediated HER2 activation, and a HER2 driven IL-6 production feedback loop.

The accumulation of senescent cells in aged tissues has been implicated in a variety of age-related diseases, including cancer and neurodegenerative disorders. Recent studies have demonstrated a link between age-associated increase of senescent glial cells in the brain and the pathogenesis of Alzheimer's disease (AD). However, there is a lack of in vitro cellular models of senescent human microglia, which significantly limits our approaches to study AD pathogenesis. Here, we show for the first time that ionizing radiation (IR) dose-dependently induces premature senescence in HMC3 human microglial cells. Senescence-associated β-galactosidase activity, a well-characterized marker of cellular senescence, was substantially increased in irradiated HMC3 cells compared with control cells. Furthermore, we found that phosphorylated p53 levels and p21 expression levels were markedly higher in IR-induced senescent microglia than in control cells. Senescent human microglia exhibited the senescence-associated secretory phenotype (SASP), as evidenced by the increased secretion of pro-inflammatory cytokine interleukin-6 (IL-6). Treatment with an NF-κB inhibitor, BAY 11-7082, inhibits the secretion of IL-6 by senescent HMC3 cells. Collectively, our studies have established an in vitro cellular model of human microglial senescence and suggest that the NF-κB pathway may play a critical role in regulating the SASP of senescent HMC3 cells.

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 μg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation.

Cardiac myxoma (CM) is a prevalent primary cardiac tumor. miR-217 plays a vital role in tumorigenesis of various cancers, however, its role and underlying molecular mechanism in human CM remain poorly understood. Here, we reported that the expression of miR-217 was downregulated in CM tissues and inversely correlated with the expression of Interleukin-6 (IL-6) mRNA. Gain-of-function analysis indicated that overexpression of miR-217 inhibited the proliferation and promoted the apoptosis of the primary CM cells. Bioinformatics analysis showed that IL-6 was a direct target gene of miR-217, which is confirmed by the dual luciferase assays. Moreover, downregulation of IL-6 by small interference RNA (siRNA) mimicked the tumor-suppressive effects of miR-217 in CM. Furthermore, rescue experiments pointed out that restoration of IL-6 expression abrogated the anti-proliferative and pro-apoptotic effect induced by miR-217 overexpression in CM cells. Taken together, we validated that miR-217 could act as a tumor suppressor in CM by directly targeting 3'UTR of IL-6 gene, indicating that manipulation of miR-217 may be a potential therapeutic strategy for CM patients.

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.

The cornea is the most sensitive tissue in the human body, with the dense nerve endings of the cornea being derived from the first division of the ophthalmic nerve. The existence of such organized nerve fibers reflects the role of neural regulation in corneal homeostasis, with the proper distribution and function of these nerve fibers thus being required for maintenance of a healthy cornea. We recently established an in vitro model, based on the coculture of human corneal epithelial cells and fibroblasts on opposite sides of a collagen vitrigel membrane. We have now examined the role of neural cells in corneal homeostasis with the use of a similar coculture system. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that the presence of neural cells (differentiated PC12 cells) increased the expression of matrix metalloproteinase-1 (MMP-1) in human corneal fibroblasts at both the mRNA and protein levels. The expression of MMP-2 and MMP-9 in corneal fibroblasts was not affected by PC12 cells. Furthermore, a multiplex assay showed that, among various cytokines assayed, only the release of interleukin-6 in cocultures of the two cell types was markedly greater than that in cultures of corneal fibroblasts alone. These results thus suggest that factors released from neural cells may play an important role in regulation of the function of corneal fibroblasts and thereby contribute to the maintenance of corneal structure and function.

Stearoyl-CoA desaturase 1 (SCD1), a lipogenic enzyme that adds a double bond at the delta 9 position of stearate (C18: 0) and palmitate (C16: 0), has been proven to be important in the development of obesity. Mice with skin-specific deficiency of SCD1 (SKO) display increased whole-body energy expenditure, which is protective against adiposity from a high-fat diet because it improves glucose clearance, insulin sensitivity, and hepatic steatosis. Of note, these mice also display elevated levels of the "pro-inflammatory" plasma interleukin-6 (IL-6). In whole skin of SKO mice, IL-6 mRNA levels are increased, and protein expression is evident in hair follicle cells and in keratinocytes. Recently, the well-known role of IL-6 in causing white adipose tissue lipolysis has been linked to indirectly activating the gluconeogenic enzyme pyruvate carboxylase 1 in the liver, thereby increasing hepatic glucose production. In this study, we suggest that skin-derived IL-6 leads to white adipose tissue lipolysis, which contributes to the lean phenotype of SKO mice without the incidence of meta-inflammation that is associated with IL-6 signaling.

The remodeling of the vascular network and collagen in the extracellular matrix is closely associated with the expansion and dysfunction of adipose tissue. In the present study, we investigated the effects of interleukin (IL)-6 and tumor necrosis factor (TNF)-α on the expression of angiogenic factors, collagen, and collagenase and its endogenous inhibitor in premature and mature adipocytes.

Several diseases are related to retinal ganglion cell death, such as glaucoma, diabetes and other retinopathies. Many studies have attempted to identify factors that could increase neuroprotection after axotomy of these cells. Interleukin-6 has been shown to be able to increase the survival and regeneration of retinal ganglion cells (RGC) in mixed culture as well as in vivo. In this work we show that the trophic effect of IL-6 is mediated by adenosine receptor (A2aR) activation and also by the presence of extracellular BDNF. We also show that there is a complex cross-talk between IL-6, BDNF, the Adenosine A1 and A2a receptors that results in neuroprotection of retinal ganglion cells.

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.

We previously reported that cyclic compressive force (CCF) induced interleukin-6 mRNA expression in osteocyte-like MLO-Y4 cells. But little is known about how the stimuli are converted into the biochemical signals in MLO-Y4 cells. The aim of this research was to study the effect of CCF on the IL-6 secretion and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in this process. The cells were exposed to CCF with different magnitudes (1000, 2000 and 4000 μstrain), frequencies (0.5, 1.0 and 2.0 Hz) and durations (10 min, 30 min, 1h, 3h and 6h) by a four-point bending system. The IL-6 secretion and ERK1/2 phosphorylation of the cells were determined by ELISA and Western blotting, respectively. The results showed that IL-6 protein secretion was significantly up-regulated in response to CCF in a magnitude-, frequency- and duration-dependent fashion. The phosphorylation of ERK1/2 also increased in all cases but not depended on the magnitude, frequency or duration of CCF. Furthermore, the inhibition of the ERK1/2 pathway by its specific inhibitor PD098059 decreased but not completely abrogated the IL-6 secretion from stressed MLO-Y4 cells. These findings demonstrate that CCF-induced IL-6 secretion occurs via a mechanism that involves ERK1/2 signaling pathway and suggest that modulation of this event contributes to the pathogenesis of osteoporosis and stress-induced pathological bone resorption as well.

Intraplaque hemorrhage (IPH) promotes the rapid progression of atherosclerotic plaques, resulting in cardiovascular events in a short time. Hepcidin increases iron retention and exerts proinflammatory effects in plaques. However, hepcidin expression levels in hemorrhagic plaques remain unknown. In the present study, we evaluated hepcidin expression in hemorrhagic plaques and the underlying mechanism. To investigate hepcidin expression in hemorrhagic plaques, carotid artery plaques were collected from patients undergoing carotid endarterectomy (CEA) and apolipoprotein E-deficient mice. The hepcidin expression level was increased in the area of IPH and positively correlated with the amount of hemorrhage as shown by immunohistochemistry. Hepcidin expression in macrophages within human plaques was confirmed by immunofluorescence. Furthermore, ferric ammonium citrate (FAC) was found to induce hepcidin and interleukin-6 (IL-6) expression in THP-1 macrophages and mouse peritoneal macrophages. Subsequently, activation of the IL-6/signal transducer and activator of transcription (STAT) 3 pathway was observed in rabbit hemorrhagic plaques. Macrophages were pretreated with antibodies that block IL-6/IL-6R interactions or STAT3 activation and dimerization inhibitor (STATTIC), and the results indicated that FAC induced hepcidin expression through the IL-6/STAT3 pathway. In conclusion, our data indicate that hepcidin levels are increased in hemorrhagic plaques, which correlates with iron-stimulated IL-6/STAT3 pathway activation in macrophages. Therefore, inhibition of the IL-6/STAT3 pathway may be a potential strategy to reduce hepcidin expression and further stabilize hemorrhagic plaques.

A single bout of exercise is known to increase the insulin sensitivity of skeletal muscle; however, the underlying mechanism of this phenomenon is not fully understood. Because a single bout of exercise induces a transient increase in blood interleukin-6 (IL-6) level, we hypothesized that the enhancement of insulin sensitivity after a single bout of exercise in skeletal muscle is mediated at least in part through IL-6-dependent mechanisms. To test this hypothesis, C57BL6J mice were intravenously injected with normal IgG or an IL-6 neutralizing antibody before exercise. Twenty-four hours after a single bout of exercise, the plantaris muscle was harvested to measure insulin sensitivity and glucose transporter (GLUT)-4 expression levels by ex-vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake and Western blotting, respectively. Compared with sedentary mice, mice that performed exercise showed enhanced IL-6 concentration, insulin-stimulated 2-DG uptake, and GLUT-4 expression in the plantaris muscle. The enhanced insulin sensitivity and GLUT4 expression were canceled by injection of the IL-6 neutralizing antibody before exercise. In addition, IL-6 injection increased GLUT4 expression, both in the plantaris muscle and the soleus muscle in C57BL6J mice. Furthermore, a short period of incubation with IL-6 increased GLUT4 expression in differentiated C2C12 myotubes. In summary, these results suggested that IL-6 increased GLUT4 expression in muscle and that this phenomenon may play a role in the post-exercise enhancement of insulin sensitivity in skeletal muscle.

To investigate the mechanism of the inhibitory action of high molecular weight hyaluronic acid (HA) on production of matrix metalloproteinases (MMPs) induced by IL-6 in human chondrocyte. Human chondrocyte were stimulated by interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) with or without HA for 24h and the productions of MMP-1, MMP-3 and MMP-13 were measured. Phosphorylations of extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase kinase (MEK) in IL-6+sIL-6R-treated chondrocytes were detected by western blotting. IL-6+sIL-6R induced MMP-1, MMP-3 and MMP-13 productions from human chondrocyte. Inhibition of the mitogen-activated protein kinase (MAPK) signaling pathway resulted in marked decreases of MMP-1, MMP-3 and MMP-13 induction by IL-6. In contrast, STAT inhibition only slightly attenuated the production of MMPs. HA inhibited MMP-1, MMP-3 and MMP-13 induction by IL-6, which was reversed by the addition of anti-CD44 antibody but not anti-ICAM-1 antibody. Pre-treatment of cells with HA reduced the phosphorylation of ERK, but not MEK. Expression levels of mitogen-activated protein kinase phosphatase-1 (MKP-1) in HA-treated chondrocytes were assessed by western blotting. HA induced the expression of MKP-1, a negative regulator of ERK1/2 in IL-6+sIL-6R-treated or untreated chondrocytes, and the MKP-1 inhibitor and MKP-1 siRNA reversed the HA-induced suppression of MMP induction by IL-6. Our study is the first to demonstrate that HA suppressed MMPs induction by IL-6 in human chondrocyte via MKP-1 induction through CD44 signaling.

Tumor necrosis factor (TNF)-α-inducing protein (Tipα) is a newly identified carcinogenic factor secreted by Helicobacter pylori (H. pylori). Although it has been proved that Tipα is a strong inducer of epithelial-mesenchymal transition (EMT), a crucial process of migration, the exact molecular mechanism is unknown. Current evidence indicates that the oncogenic transcription factor signal transducers and activators of transcription 3 (STAT3) is inappropriately activated in multiple malignancies, including gastric cancer. In this study, we showed that Tipα significantly down-regulated the expression of EMT-related markers E-cadherin as well as up-regulated N-cadherin and vimentin in SGC7901 cells, with typical morphological changes of EMT. Tipα also promoted proliferation and migration of SGC7901 cells. Furthermore, Tipα activated interleukin-6 (IL-6)/STAT3 signaling pathway in SGC7901 cells. The effects of Tipα treatment observed was abolished when we block IL-6/STAT3 signaling pathway. Altogether, our data demonstrated that Tipα may accelerate tumor aggressiveness in gastric cancer by promoting EMT through activation of IL-6/STAT3 pathway.