Inhibition of GP130/STAT3 and EMT by combined bazedoxifene and paclitaxel treatment in ovarian cancer.

The interleukin 6 (IL­6)/glycoprotein 130 (GP130)/signal transducer and activator of transcription 3 (STAT3) signalling pathway, with GP130 as an intermediate membrane receptor, is involved in the survival, metastasis, and resistance of ovarian cancer. Bazedoxifene, an FDA­approved drug, is an inhibitor of GP130 and a selective estrogen modulator (SERM). We studied the mechanism of the combination therapy of bazedoxifene and paclitaxel in inhibiting the IL­6­mediated GP130/STAT3 signaling pathway in ovarian cancer. Surface plasmon resonance (SPR) was used to assess the binding of bazedoxifene to GP130. Migration, invasion, and apoptosis of ovarian cancer cells were assessed using bazedoxifene and paclitaxel. In addition, we determined the effects of bazedoxifene and paclitaxel alone or in combination on the GP130/STAT3 pathway and epithelial­mesenchymal transition (EMT). The results revealed that the combination of bazedoxifene and paclitaxel suppressed cell viability, migration, and invasion in the ovarian cancer cells. In addition, the combination treatment increased apoptosis. Furthermore, bazedoxifene combined with paclitaxel inhibited the growth of ovarian cancer cells in a xenograft tumour model. This combination reduced STAT3 phosphorylation and suppressed gene expression and EMT. In conclusion, inhibition of GP130/STAT3 signalling and EMT via a combination of bazedoxifene and paclitaxel could be used as a therapeutic strategy by which to overcome ovarian cancer.

Inflammatory Glycoprotein 130 Signaling Links Changes in Microtubules and Junctophilin-2 to Altered Mitochondrial Metabolism and Right Ventricular Contractility.

BACKGROUND: Right ventricular dysfunction (RVD) is the leading cause of death in pulmonary arterial hypertension (PAH), but no RV-specific therapy exists. We showed microtubule-mediated junctophilin-2 dysregulation (MT-JPH2 pathway) causes t-tubule disruption and RVD in rodent PAH, but the druggable regulators of this critical pathway are unknown. GP130 (glycoprotein 130) activation induces cardiomyocyte microtubule remodeling in vitro; however, the effects of GP130 signaling on the MT-JPH2 pathway and RVD resulting from PAH are undefined. METHODS: Immunoblots quantified protein abundance, quantitative proteomics defined RV microtubule-interacting proteins (MT-interactome), metabolomics evaluated the RV metabolic signature, and transmission electron microscopy assessed RV cardiomyocyte mitochondrial morphology in control, monocrotaline, and monocrotaline-SC-144 (GP130 antagonist) rats. Echocardiography and pressure-volume loops defined the effects of SC-144 on RV-pulmonary artery coupling in monocrotaline rats (8-16 rats per group). In 73 patients with PAH, the relationship between interleukin-6, a GP130 ligand, and RVD was evaluated. RESULTS: SC-144 decreased GP130 activation, which normalized MT-JPH2 protein expression and t-tubule structure in the monocrotaline RV. Proteomics analysis revealed SC-144 restored RV MT-interactome regulation. Ingenuity pathway analysis of dysregulated MT-interacting proteins identified a link between microtubules and mitochondrial function. Specifically, SC-144 prevented dysregulation of electron transport chain, Krebs cycle, and the fatty acid oxidation pathway proteins. Metabolomics profiling suggested SC-144 reduced glycolytic dependence, glutaminolysis induction, and enhanced fatty acid metabolism. Transmission electron microscopy and immunoblots indicated increased mitochondrial fission in the monocrotaline RV, which SC-144 mitigated. GP130 antagonism reduced RV hypertrophy and fibrosis and augmented RV-pulmonary artery coupling without altering PAH severity. In patients with PAH, higher interleukin-6 levels were associated with more severe RVD (RV fractional area change 23±12% versus 30±10%, P=0.002). CONCLUSIONS: GP130 antagonism reduces MT-JPH2 dysregulation, corrects metabolic derangements in the RV, and improves RVD in monocrotaline rats.

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

Luhong formula inhibits myocardial fibrosis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Luhong formula (LHF)-a traditional Chinese medicine containing Cervus nippon Temminck, Carthamus tinctorius L., Cinnamomum cassia Presl, Codonopisis pilosula( Franch.) Nannf., Astragalus membranaceus ( Fisch.) Bge. var. mongholicus ( Bge.) Hsiao, Lepidium apetalum Willd-is used in the treatment of heart failure. AIM OF THE STUDY: To investigate the antifibrotic efficacy of LHF in a myocardial infarction-induced rat model of heart failure and to determine its mechanism of action. MATERIAL AND METHODS: Myocardial infarction was induced in rats by coronary artery ligation, and cardiac fibroblasts were isolated. Neonatal rat cardiomyocytes (NRCMs) were isolated from 2 to 3-day-old Sprague-Dawley male rats, and cardiomyocyte hypertrophy was induced by isoprenaline. Histological examination was carried out to estimate the degree of myocardial fibrosis. Expression of gp130/JAK2/STAT3 pathway proteins was measured by western blot. The mRNA levels of downstream genes of gp130/JAK2/STAT3 pathway (i.e., CTGF, TSP-1, and TIMP1) were determined by RT-PCR; while CTGF, TSP-1, and TIMP1 protein levels were measured by ELISA. To investigate paracrine effects, cell proliferation and collagen synthesis was measured after treating cardiac fibroblasts with the conditioned media from isoprenaline-treated NRCMs. RESULTS: Histopathological changes showed that LHF inhibited myocardial fibrosis in heart failure rats. Treatment with LHF up-regulated gp130, JAK2, and STAT3 protein expression in heart tissue, and down-regulated CTGF, TSP-1, and TIMP1 gene expression. Isoprenaline-treated NRCMs displayed lower expression of the gp130, JAK2, and STAT3 pathway proteins and higher secretion of its downstream signaling molecules (CTGF, TSP-1, TIMP1). LHF inhibited cardiac fibroblast proliferation and collagen synthesis after treatment with the conditioned media from isoprenaline-treated NRCMs. CONCLUSION: LHF treatment attenuates myocardial fibrosis in vivo. LHF inhibits cardiac fibroblasts proliferation and collagen synthesis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes, thereby inhibiting the secretion of downstream profibrogenic cytokines.

Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism.

OBJECTIVE: The bacteria Staphylococcus aureus is part of the normal bacterial flora and produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are therefore called superantigens. It was recently discovered that the superantigen SEA binds to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with inflammation of the adipose tissue and gp130 has been shown to play an important role in adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function. MATERIALS/METHODS: Binding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake. RESULTS: We demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis. CONCLUSIONS: SEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes.

gp130: a promising drug target for cancer therapy.

INTRODUCTION: Ubiquitously expressed in the human body, glycoprotein 130 (gp130) is a shared subunit of receptor complexes for at least nine cytokines (IL-6, OSM, LIF, IL-11, CNTF, CLC, IL-27, CT-1, and NP) that mediate highly diverse biological processes. Dysregulation of gp130 expression, activation, or associated signaling pathways are implicated in a variety of human diseases, including cancer. Accumulating evidence indicates that the gp130-mediated signaling networks play important roles in the progression of multiple types of cancer. AREAS COVERED: This review discusses the structural basis of gp130 in signal transduction activity and its role in physiological and pathological conditions, particularly cancer. We analyzed the currently available databases to illustrate the expression of gp130, its coexpression with other molecules involved in the gp130 signaling pathways, and the role of gp130 in cancer progression. Finally, we highlight strategies for blocking gp130 signaling and the currently available antagonists. EXPERT OPINION: As gp130 signaling mediates cancer progression, inhibition of gp130 activity offers a potential and promising approach to cancer therapy. Compared to antibodies blocking individual cytokines, gp130-targeted small-molecule inhibitors present multiple advantages. To achieve successful clinical outcomes for gp130-targeted cancer therapy, dosage determination, duration of therapy, and patient selection are some of the critical factors to be considered.

Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth.

Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.

STAT3 activation in photoreceptors by leukemia inhibitory factor is associated with protection from light damage.

Members of the interleukin-6 cytokine family, including leukemia inhibitory factor (LIF), signal through gp130. The neuroprotective role of gp130 activation has been widely demonstrated in both CNS and PNS, but the mechanism by which this is accomplished is not well established. We investigated temporal and cell-specific activation of signaling pathways induced by LIF in the mature mouse retina. Intravitreal injection of LIF preserved photoreceptor function and prevented photoreceptor cell death from light-induced oxidative damage in a dose-dependent manner (2 days post-injection). A therapeutic dose of LIF induced rapid and sustained activation of signal transducer and activator of transcription (STAT) 3. Activated STAT3 was localized to all the retinal neurons and glial cells, including photoreceptors. Activation of extracellular signal-regulated kinase 1 and 2 was robust but transient in Müller glial cells, and undetectable at the time of light exposure. Akt was not activated by LIF. We also show that at the time of neuroprotection, STAT3 but not extracellular signal-regulated kinase 1 and 2 or the Akt pathways was active in LIF-treated retinas, and activated STAT3 was clearly localized in transcriptionally active areas of photoreceptor nuclei. Our data suggest that photoreceptor protection in response to LIF can be directly mediated by activation of STAT3 in photoreceptors.

Bile acids inhibit interleukin-6 signaling via gp130 receptor-dependent and -independent pathways in rat liver.

Interleukin-6 (IL-6) is a major regulator of the acute phase reaction in the liver and is thought to mediate protective effects in response to hepatotoxins. In this study, the influence of bile acids on IL-6 signal transduction was analyzed. It was shown that hydrophobic bile acids such as glycochenodeoxycholate (GCDC) inhibited IL-6-induced tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 3 in hepatocytes and in perfused rat liver. This inhibition was accompanied by GCDC-mediated downregulation of glycoprotein (gp) 130 expression, whereas gp130 and suppressor of cytokine signaling 3 messenger RNA and gp80 protein levels remained unaffected. The GCDC-induced downregulation of gp130 protein expression was insensitive to inhibition of proteasomal or lysosomal protein degradation but turned out to be sensitive to inhibition of caspase-3 or caspase-8 activity. Accordingly, treatment of cell extracts with active recombinant caspase-3 led to a decay of immunoreactive gp130. Moreover, activation of caspases by CD95 ligand or hyperosmotic stress also resulted in a downregulation of gp130 levels. This indicates that caspase activation antagonizes IL-6 signaling by decay of gp130 levels. However, caspase inhibition did not prevent GCDC-dependent inhibition of IL-6-induced STAT3 activation, which turned out to be at least partially sensitive to suppression of p38(MAPK) activation. In conclusion, hydrophobic bile acids compromise IL-6 signaling through both a caspase-mediated downregulation of gp130 and a p38(MAPK)-dependent inhibition of STAT3 phosphorylation. This may contribute to bile acid-induced hepatotoxicity in cholestasis through counteracting the known hepatoprotective effects of IL-6.

Determinants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130.

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.