Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.

Modular organization of Interleukin-6 and Interleukin-11 α-receptors.

Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.

Interleukin-6 (IL-6) mediated the increased contraction of distal colon in streptozotocin-induced diabetes in rats via IL-6 receptor pathway.

Colonic dysmotility occurs in diabetes and blood plasma interleukin (IL)-6 levels are significantly elevated in type 1 diabetes mellitus. The aim of this study was to investigate whether IL-6 and the IL-6 receptor pathway mediates colonic dysfunction in type 1 diabetes mellitus. Male SD rats were treated with a single intraperitoneally injected dose of streptozotocin (STZ), and those displaying sustained high blood glucose were selected as diabetes mellitus models. Longitudinal muscle strips of colon were prepared to monitor colonic contraction in vitro. Contractile responses of strips of colon were recorded following treatment with IL-6 in control animals, and following anti IL-6 antibody treatment in STZ-induced diabetes in rats. Concentration of IL-6 in plasma and colon were determined by ELISA. Expressions of IL-6 α-receptor and IL-6 ß-receptor in colon tissues were determined by immunohistochemistry or Western blot analysis. The non-diabetes rats treated with IL-6 and the untreated diabetes rats showed increased contraction of distal colon, whereas the diabetes rats treated with anti-IL-6 antibody showed decreased contraction of distal colon compared with the untreated diabetes rats. The IL-6 levels of plasma but not colon increased in diabetes rats. The expression of IL-6 α-receptor increased in diabetes rats. These results indicate that diabetes rats show an increase in the contractions of distal colon partly via the IL-6-IL-6 receptor pathway.

gp130 activation is regulated by D2-D3 interdomain connectivity.

Activation of the IL-6 (interleukin 6) receptor subunit gp130 (glycoprotein 130) has been linked to the formation of complexes with IL-6 and the IL-6 receptor, as well as to gp130 dimerization. However, it has been shown that gp130 is present as a pre-formed dimer, indicating that its activation is not solely dependent on dimerization. Therefore the detailed mechanism of gp130 activation still remains to be deciphered. Recently, deletion mutations of gp130 have been found in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope of gp130 and resulted in a ligand-independent constitutively active gp130. We therefore hypothesized that conformational changes of this particular IL-6-binding epitope precedes gp130 activation. Using a rational structure-based approach we identified for the first time amino acids critical for gp130 activation. We can show that gp130 D2-D3 interdomain connectivity by hydrophobic residues stabilizes inactive gp130 conformation. Conformational destabilization of the EF loop present in domain D2 and disruption of D2-D3 hydrophobic interactions resulted in ligand-independent gp130 activation. Furthermore we show that the N-terminal amino acid residues of domain D1 participate in the activation of the gp130 deletion mutants. Taken together we present novel insights into the molecular basis of the activation of a cytokine receptor signalling subunit.

N-glycans modulate the activation of gp130 in mouse embryonic neural precursor cells.

gp130 is a ubiquitously expressed glycoprotein and signal transducer of interleukin 6 family of cytokines. It has been reported that gp130 has 11 potential N-glycosylation sites in the extracellular domain, and nine of them are actually N-glycosylated. However, the structure and functional role of the carbohydrate chains carried by gp130 are totally unknown. In this study, we examined the functional role of N-glycans of gp130 in mouse neuroepithelial cells. In neuroepithelial cells treated with tunicamycin, an N-glycosylation inhibitor, unglycosylated form of gp130 was detected. The unglycosylated gp130 was not phosphorylated in response to leukemia inhibitory factor stimulation. Although the unglycosylated gp130 was found to be expressed on the cell surface, it could not form a heterodimer with leukemia inhibitory factor receptor. These results suggest that N-glycans are required for the activation, but not for the localization, of gp130 in neuroepithelial cells.

Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.

Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G>C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P<0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P<0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P<0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.

Forced dimerization of gp130 leads to constitutive STAT3 activation, cytokine-independent growth, and blockade of differentiation of embryonic stem cells.

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.