Interleukin-6 (IL-6) is involved in physiological and pathological processes. Different pharmacological agents have been developed to block IL-6 deleterious effects and to recover homeostatic IL-6 signaling. One of the proposed nanostructures in pre-clinical investigations which reduced IL-6 concentrations is polyglycerol dendrimer, a nano-structure with multiple sulfate groups. The aim of the present study was to uncover the type of binding between critical positions in the human IL-6 structure available for binding dPGS and compare it with heparin sulfate binding. We studied these interactions by performing docking simulations of dPGS and heparins with human IL-6 using AutoDock Vina. These molecular docking analyses indicate that the two ligands have comparable affinities for the positively charged positions on the surface of IL-6. All-atom molecular dynamics simulations (MD) employing Gromacs were used to explore the binding sites and binding strengths. Results suggest two major binding sites and show that the strengths of binding are similar for heparin and dPGS (-5.5-6.4 kcal/ mol). dPGS or its analogs could be used in the therapeutic intervention in sepsis and inflammatory disorders to reduce unbound IL-6 in the plasma or tissues and its binding to the receptors. We propose that analogs of dPGS could specifically block IL-6 binding in the desired signaling mode and would be valuable new probes to establish optimized therapeutic intervention in inflammation.

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.

The transcription factor nuclear factor-kappa B (NF-κB) is critically involved in inflammation and cancer development. Activation of NF-κB induces the expression and release of several pro-inflammatory proteins, which include the cytokine interleukin-6 (IL-6). Perturbation of the actin cytoskeleton has been previously shown to activate NF-κB signaling. In this study, we analyze the influence of different compounds that modulate the actin cytoskeleton on NF-κB activation, IL-6 signaling and the proteolytic generation of the soluble IL-6 receptor (sIL-6R) in human hepatoma cells. We show that perturbation of the actin cytoskeleton is not sufficient to induce NF-κB activation and IL-6 secretion. However, perturbation of the actin cytoskeleton reduces IL-6-induced activation of the transcription factor STAT3 in Hep3B cells. In contrast, IL-6R proteolysis by the metalloprotease ADAM10 did not depend upon the integrity of the actin cytoskeleton. In summary, we uncover a previously unknown function of the actin cytoskeleton in IL-6-mediated signal transduction in Hep3B cells.

Kaempferia parviflora (KP) has been reported to have anti-cancer activities. We previously reported its effects against cervical cancer cells and continued to elucidate the effects of KP on inhibiting the production and secretion of interleukin (IL)-6, as well as its relevant signaling pathways involved in cervical tumorigenesis. We discovered that KP suppressed epidermal growth factor (EGF)-induced IL-6 secretion in HeLa cells, and it was associated with a reduced level of Glycoprotein 130 (GP130), phosphorylated signal transducers and activators of transcription 3 (STAT3), and Mcl-1. Our data clearly showed that KP has no effect on nuclear factor kappa B (NF-κB) localization status. However, we found that KP inhibited EGF-stimulated phosphorylation of tyrosine 1045 and tyrosine 1068 of EGF receptor (EGFR) without affecting its expression level. The inhibition of EGFR activation was verified by the observation that KP significantly suppressed a major downstream MAP kinase, ERK1/2. Consistently, KP reduced the expression of Ki-67 protein, which is a cellular marker for proliferation. Moreover, KP potently inhibited phosphorylation of STAT3, Akt, and the expression of Mcl-1 in response to exogenous IL-6 stimulation. These data suggest that KP suppresses EGF-induced production of IL-6 and inhibits its autocrine IL-6/STAT3 signaling critical for maintaining cancer cell progression. We believe that KP may be a potential alternative anti-cancer agent for suppressing cervical tumorigenesis.

Neutrophils and their extracellular traps have been shown to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the detailed mechanisms in joints are still unclear, and their regulation remains to be solved. Here, we explored neutrophil extracellular trap (NET)osis in experimental models of arthritis and further investigated the effects of interleukin-6 (IL-6) inhibition in neutrophils and NETosis. In skins of peptide GPI-induced arthritis (pGIA), citrullinated protein was detected as well as citrullinated histone expression in immunized skin but this was not specific to pGIA. Citrullinated histone expression in pGIA joints was specific to pGIA and was merged with neutrophil elastase, suggesting NETosis. Neutrophils in joints tend to upregulate IL-6 receptors when compared with bone marrow neutrophils. Administration of mouse anti-IL-6 receptor antibodies in pGIA suppressed arthritis in association with a decrease in neutrophil infiltration and NETosis in joints. In the plasma of RA patients, citrullinated protein was significantly reduced after tocilizumab treatment. Our results suggest that IL-6 enhances neutrophil chemotaxis and NETosis in inflammatory joints and could be the source of citrullinated proteins.

Fibroblasts are an important resident cell population in the heart involved in maintaining homeostasis and structure during normal conditions. They are also crucial in disease states for sensing signals and initiating the appropriate repair responses to maintain the structural integrity of the heart. This sentinel role of cardiac fibroblasts occurs, in part, through their ability to secrete cytokines. β-adrenergic receptors (βAR) are also critical regulators of cardiac function in the normal and diseased state and a major therapeutic target clinically. βAR are known to influence cytokine secretion in various cell types and they have been shown to be involved in cytokine production in the heart, but their role in regulating cytokine production in cardiac fibroblasts is not well understood. Thus, we hypothesized that βAR activation on cardiac fibroblasts modulates cytokine production to influence fibroblast function. Using primary fibroblast cultures from neonatal rats and adult mice, increased interleukin (IL)-6 expression and secretion occurred following β2AR activation. The use of pharmacological inhibitors and genetic manipulations showed that IL-6 elevations occurred through the Gαs-mediated activation of ERK1/2 and resulted in increased fibroblast proliferation. In vivo, a lack of β2AR resulted in increased infarct size following myocardial infarction and impaired wound closure in a murine dermal wound healing assay. These findings identify an important role for β2AR in regulating fibroblast proliferation through Gαs/ERK1/2-dependent alterations in IL-6 and may lead to the development of improved heart failure therapies through targeting fibrotic function of β2AR.

Several pretransplant factors, including CRP (C-reactive protein) levels, reflect the risk of complications after allogeneic stem cell transplantation. IL-6 induces CRP increase, and we therefore investigated the effects of pretransplant IL-6, soluble IL-6 receptors, IL-6 family cytokines and CRP serum levels on outcome for 100 consecutive allotransplant recipients. All patients had related donors, none had active infections and 99 patients were in complete remission before conditioning. The incidence of acute graft versus host disease (aGVHD) requiring treatment was 40%, survival at Day +100 82%, and overall survival 48%. Despite a significant correlation between pretransplant CRP and IL-6 levels, only CRP levels significantly influenced transplant-related mortality (TRM). However, CRP did not influence overall survival (OS). Pretransplant IL-31 influenced late TRM. Finally, there was a significant association between pretransplant IL-6 and early postconditioning weight gain (i.e., fluid retention), and this fluid retention was a risk factor for aGVHD, TRM and OS. To conclude, pretransplant CRP, IL-31 and early posttransplant fluid retention were independent risk factors for TRM and survival after allotransplantation.

The tumor microenvironment, which consists of fibroblasts, smooth muscle cells, endothelial cells, immune cells, epithelial cells, and extracellular matrices, plays a crucial role in tumor progression. Hepatic stellate cells (HSCs), a class of unique liver stromal cells, participate in immunomodulatory activities by inducing the apoptosis of effector T-cells, generation of regulatory T-cells, and development of myeloid-derived suppressor cells (MDSCs) to achieve long-term survival of islet allografts. This study provides in vitro and in vivo evidences that HSCs induce the generation of MDSCs to promote hepatocellular carcinoma (HCC) progression through interleukin (IL)-6 secretion. HSC-induced MDSCs highly expressed inducible nitric oxide synthase (iNOS) and arginase 1 mRNA and presented potent inhibitory T-cell immune responses in the tumor environment. Wild-type HSC-induced MDSCs expressed lower levels of CD40, CD86, and MHC II, and a higher level of B7-H1 surface molecules, as well as increased the production of iNOS and arginase I compared with MDSCs induced by IL-6-deficient HSCs in vitro. A murine-transplanted model of the liver tumor showed that HCCs cotransplanted with HSCs could significantly enhance the tumor area and detect more MDSCs compared with HCCs alone or HCCs cotransplanted with HSCs lacking IL-6. In conclusion, the results indicated that MDSCs are induced mainly by HSCs through IL-6 signaling and produce inhibitory enzymes to reduce T-cell immunity and then promote HCC progression within the tumor microenvironment. Therapies targeting the pathway involved in MDSC production or its immune-modulating pathways can serve as an alternative immunotherapy for HCC.

Inflammatory disorders are associated with bone destruction; that is, deterioration in bone cell activities are under the control of the innate immune system. Macrophages play a central role in innate immunity by switching their polarized phenotype. A disturbed immune system causes aberrance in the ordered bone matrix microarrangement, which is a dominant determinant of bone tissue functionalization. However, the precise relationship between the immune system and bone tissue organization is unknown. In this study, the controlled in vitro co-culture assay results showed that M1-polarized macrophages disrupted the osteoblast alignment, which directly modulate the oriented bone matrix organization, by secreting pro-inflammatory cytokines. Notably, interleukin-6 was found to be a key regulator of unidirectional osteoblast alignment. Our results demonstrated that inflammatory diseases triggered bone dysfunction by regulating the molecular interaction between the immune system and bone tissue organization. These findings may contribute to the development of therapeutic targets for inflammatory disorders, including rheumatoid arthritis.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.

The purpose of this study was to determine whether pravastatin's prevention of aortic atherosclerosis via attenuation of IL-6 action depends on modulation of STAT3 activity. Male apoE knockout (apoE-/-) mice fed on a diet containing 1.25% cholesterol (wt/wt) were divided into pravastatin group provided with pravastatin (80 mg kg(-1) per day) and atherosclerosis group. After eight weeks, pravastatin significantly prevented atherosclerotic lesion and reduced levels of IL-6 in serum and lesion, and significantly decreased expressions of phosphorylated STAT3 (pSTAT3) and increased suppressor of cytokine signaling 3 (SOCS3) expressions in lesions. Our results suggested that pravastatin's aortic atherosclerosis preventing action via attenuation of IL-6 action may partially depend on modulation of STAT3 activity.

Postnatal muscle growth and exercise- or injury-induced regeneration are facilitated by myoblasts. Myoblasts respond to a variety of proteins such as cytokines that activate various signaling cascades. Cytokines belonging to the interleukin 6 superfamily (IL-6) influence myoblasts' proliferation but their effect on differentiation is still being researched. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is one of the key signaling pathways identified to be activated by IL-6. The aim of this study was to investigate myoblast fate as well as activation of JAK-STAT pathway at different physiologically relevant IL-6 concentrations (10 pg/mL; 100 pg/mL; 10 ng/mL) in the C2C12 mouse myoblast cell line and primary human myoblasts, isolated from eight young healthy male volunteers. Myoblasts' cell cycle progression, proliferation and differentiation in vitro were assessed. Low IL-6 concentrations facilitated cell cycle transition from the quiescence/Gap1 (G0/G1) to the synthesis (S-) phases. Low and medium IL-6 concentrations decreased the expression of myoblast determination protein 1 (MyoD) and myogenin and increased proliferating cell nuclear antigen (PCNA) expression. In contrast, high IL-6 concentration shifted a larger proportion of cells to the pro-differentiation G0/G1 phase of the cell cycle, substantiated by significant increases of both MyoD and myogenin expression and decreased PCNA expression. Low IL-6 concentration was responsible for prolonged JAK1 activation and increased suppressor of cytokine signaling 1 (SOCS1) protein expression. JAK-STAT inhibition abrogated IL-6-mediated C2C12 cell proliferation. In contrast, high IL-6 initially increased JAK1 activation but resulted in prolonged JAK2 activation and elevated SOCS3 protein expression. High IL-6 concentration decreased interleukin-6 receptor (IL-6R) expression 24 h after treatment whilst low IL-6 concentration increased IL-6 receptor (IL-6R) expression at the same time point. In conclusion, this study demonstrated that IL-6 has concentration- and time-dependent effects on both C2C12 mouse myoblasts and primary human myoblasts. Low IL-6 concentration induces proliferation whilst high IL-6 concentration induces differentiation. These effects are mediated by specific components of the JAK/STAT/SOCS pathway.

In the heart, the delayed rectifier K current, IK, composed of the rapid (IKr) and slow (IKs) components contributes prominently to normal cardiac repolarization. In lipotoxicity, chronic elevation of pro-inflammatory cytokines may remodel IK, elevating the risk for ventricular arrythmias and sudden cardiac death. We investigated whether and how the pro-inflammatory interleukin-6 altered IK in the heart, using electrophysiology to evaluate changes in IK in adult guinea pig ventricular myocytes. We found that palmitic acid (a potent inducer of lipotoxicity), induced a rapid (~24 h) and significant increase in IL-6 in RAW264.7 cells. PA-diet fed guinea pigs displayed a severely prolonged QT interval when compared to low-fat diet fed controls. Exposure to isoproterenol induced torsade de pointes, and ventricular fibrillation in lipotoxic guinea pigs. Pre-exposure to IL-6 with the soluble IL-6 receptor produced a profound depression of IKr and IKs densities, prolonged action potential duration, and impaired mitochondrial ATP production. Only with the inhibition of IKr did a proarrhythmic phenotype of IKs depression emerge, manifested as a further prolongation of action potential duration and QT interval. Our data offer unique mechanistic insights with implications for pathological QT interval in patients and vulnerability to fatal arrhythmias.

The dorsomedial hypothalamus nucleus (DMH) is an important component of the autonomic nervous system and plays a critical role in regulating the sympathetic outputs of the heart. Stress alters the neuronal activity of the DMH, affecting sympathetic outputs and triggering heart rate variability. However, the specific molecular mechanisms behind stress leading to abnormal DMH neuronal activity have still not been fully elucidated. Therefore, in the present study, we successfully constructed a stressed rat model and used it to investigate the potential molecular mechanisms by which IL-6 regulates GABAA receptors in the DMH through activation of the JAK/STAT pathway and thus affects heart rate variability in rats. By detecting the c-Fos expression of neurons in the DMH and electrocardiogram (ECG) changes in rats, we clarified the relationship between abnormal DMH neuronal activity and heart rate variability in stressed rats. Then, using ELISA, immunohistochemical staining, Western blotting, RT-qPCR, and RNAscope, we further explored the correlation between the IL-6/JAK/STAT signaling pathway and GABAA receptors. The data showed that an increase in IL-6 induced by stress inhibited GABAA receptors in DMH neurons by activating the JAK/STAT signaling pathway, while specific inhibition of the JAK/STAT signaling pathway using AG490 obviously reduced DMH neuronal activity and improved heart rate variability in rats. These findings suggest that IL-6 regulates the expression of GABAA receptors via the activation of the JAK/STAT pathway in the DMH, which may be an important cause of heart rate variability in stressed rats.

Patients with rheumatoid arthritis (RA) are at increased risk of cardiovascular disease. Dyslipidemia is a known adverse effect of tocilizumab (TCZ), an anti-interleukin-6 receptor antibody used in RA treatment. We aimed to assess the effect of TCZ on lipid profile and adipokine levels in RA patients. Height, weight, disease activity scores, lipid profile and atherogenic indices (AI), leptin, adiponectin, resistin, interleukin-6, and high-sensitivity C-reactive protein (CRP) were measured before and four months after initiation of TCZ in 40 RA patients and 40 healthy controls. Following TCZ treatment, total cholesterol, high density lipoprotein (HDL), and triglycerides were significantly elevated, but no significant changes in weight, body mass index (BMI), low density lipoprotein (LDL), and AI were observed. Compared with controls, significantly higher adiponectin levels were measured in the RA group at baseline. Following TCZ treatment, resistin levels and the leptin-to-adiponectin ratio increased, adiponectin levels decreased, and leptin levels remained unchanged. No correlation was found between the change in adipokine serum levels and changes in the disease activity indices, nor the lipid profile. In conclusion, the changes observed suggest a protective role for TCZ on the metabolic and cardiovascular burden associated with RA, but does not provide a mechanistic explanation for this phenomenon.

Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.

Heart failure is a serious and life-threatening disease worldwide. Cadherin-11 (Cad-11) is highly expressed in the heart and closely associated with inflammation. There is currently limited understanding on how Cad-11 contributes to cardiac remodeling and its underline molecular mechanism. We found an increased expression of Cad-11 in biopsy heart samples from heart failure patients, suggesting a link between Cad-11 and heart failure. To determine the role of Cad-11 in cardiac remodeling, Cad-11-deficient mice were used in a well-established mouse transverse aortic constriction (TAC) model. Loss of Cad11 greatly improved pressure overload-induced LV structural and electrical remodeling. IL (interleukin)-6 production was increased following TAC in WT mice and this increase was inhibited in cadherin-11-/- mice. We further tested the effect of IL-6 on myocyte hypertrophy and fibrosis in a primary culture system. The addition of hCad-11-Fc to cultured cardiac fibroblasts increased IL-6 production and fibroblast cell activation, whereas neutralizing IL-6 with an IL-6 antibody resulted in alleviating the fibroblast activation induced by hCad-11-Fc. On the other hand, cardiomyocytes were promoted to cardiomyocyte hypertrophy when cultured in condition media collected from cardiac fibroblasts stimulated by hCad-11-Fc.Similarly, neutralizing IL-6 prevented cardiomyocyte hypertrophy. Finally, we found that MAPKs and CaMKII-STAT3 pathways were activated in both hCad-11-Fc stimulated fibroblasts and cardiomyocytes treated with hCad-11-Fc stimulated fibroblast condition medium. IL-6 neutralization inhibited such MAPK and CaMKII-STAT3 signaling activation. These data demonstrate that Cad-11 functions in pressure overload-induced ventricular remodeling through inducing IL-6 secretion from cardiac fibroblasts to modulate the pathophysiology of neighboring cardiomyocytes.

Kudzu (Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep) is a perennial leguminous vine, and its root and flower have been used for herbal medicine in Asia for a long time. Most dietary flavonoids are reported to be concentrated in its root, not in its aerial parts including leaves. In this study, we investigated whether kudzu leaf and its major constituent, robinin (kaempferol-3-O-robinoside-7-O-rhanmoside) possessed anti-inflammatory activity. To test this hypothesis, we used peritoneal macrophages isolated from BALB/c mice and stimulated the cells with lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. Compared with kudzu root extract, its leaf extract was more potent in inhibiting the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6. Kudzu leaf extract decreased LPS-induced activation of c-Jun N-terminal kinase (JNK) and TANK-binding kinase 1(TBK1) with no effects on nuclear factor-κB and activator protein 1 transcriptional activity. Also, kudzu leaf extract inhibited LPS/IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) activation partly via an altered level of STAT1 expression. Robinin, being present in 0.46% of dry weight of leaf extract, but almost undetected in the root, decreased iNOS protein involving modulation of JNK and STAT1 activation. However, robinin showed no impact on other inflammatory markers. Our data provide evidence that kudzu leaf is an excellent food source of as yet unknown anti-inflammatory constituents.

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.

Bisphosphonates are one of the most widely used synthetic pyrophosphate analogues for the treatment of bone resorbing diseases such as osteoporosis, multiple myeloma, and bone metastases. Although the therapeutic usefulness of bisphosphonates mainly depends on their anti-osteoclastogenic effect, a severe side-effect of bisphosphonates called bisphosphonate-related osteonecrosis of the jaw (BRONJ) could not be explained by the anti-osteoclastogenic effect of bisphosphonates. In the present study, we have evaluated the changes in osteoclastogenesis- or osteoblastogenesis-supporting activities of osteocytes induced by bisphosphonates. Zoledronate, a nitrogen-containing bisphosphonate, markedly increased both the receptor activator of nuclear factor kB ligand (RANKL) as well as sclerostin in osteocyte-like MLO-Y4 cells, which were functionally revalidated by osteoclast/osteoblast generating activities of the conditioned medium obtained from zoledronate-treated MLO-Y4 cells. Of note, the zoledronate treatment-induced upregulation of the RANKL expression was mediated by autocrine interleukin-6 (IL-6) and subsequent activation of the signal transducer and activator of transcription 3 (STAT3) pathway. These results were evidenced by the blunted RANKL expression in the presence of a Janus activated kinase (JAK2)/STAT3 inhibitor, AG490. Also, the osteoclastogenesis-supporting activity was significantly decreased in zoledronate-treated MLO-Y4 cells in the presence of IL-6 neutralizing IgG compared to that of the control IgG. Thus, our results show previously unanticipated effects of anti-bone resorptive bisphosphonate and suggest a potential clinical importance of osteocytes in BRONJ development.