Differential regulation of interleukin-6 receptor and gp130 gene expression in rat hepatocytes.

Interleukin-6 (IL-6) relays an important signal to hepatocytes during the early stages of an acute inflammatory response, causing an alteration in the expression of several major defense proteins. Additional regulation of this signal could occur either by altering the number of IL-6 receptors (IL-6-R) or of the signal transducing protein, gp130. We employed ribonuclease protection assays to measure the expression of IL-6-R and gp130 mRNA in primary rat hepatocytes in response to IL-6, interleukin-1, dexamethasone, and combinations thereof. Dexamethasone increases receptor mRNA levels 2.7-fold above controls but has no detectable effect on that of gp130. Such treatment increased surface expression of IL-6-R from 600 receptors per cell to greater than 6000, without a change in Kd (2.5-4.6 x 10(-10) M). In contrast to the stimulatory effect of the steroid signal, the inflammatory cytokines, individually and together, down-modulated both the mRNA and the cell surface expression of IL-6-R. These findings demonstrate for the first time that a sensitive control system exists between inflammatory mediators and IL-6-R.     

Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.     

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1-D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1-D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein - consisting of two extracellular gp130 regions (D1-D6) dimerised by an IgG1-Fc part - to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1-D3 with respect to the membrane-proximal domains D4-D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.     

Expression of ORIGIN-like sequence in mammalian cells]

There are some genes whose expression increases in non-proliferating hepatocytes after whole-body X-irradiation. We suppose that some of them participate in cell division regulation. To verify our postulate we have studied expression of autonomously replicating sequence pDARC1 in normal, irradiated and dividing rat hepatocytes. We have succeeded earlier in cloning the autonomously replicating sequence pDARC1 and have shown that it could be transcribed in X-irradiated hepatocytes. Here we report on the results of analysis of this sequence expression in dividing mammalian cells. Hybridization with the pDARC1 sequence reveals 4.0 kb mRNA in non-proliferating rat hepatocytes after X-irradiation: this mRNA is absent in normal hepatocytes. Induction of 4.0 kb mRNA is a function of radiation dose; 4.0 kb mRNA content is maximum 6-24 h following whole-body 6 Gy X-irradiation. The 4.0 kb mRNA is also found in dividing rat hepatocytes during the pre-replicative period.     

Biochemical analysis of a 130,000 molecular weight glycoprotein on human melanoma cells

A monomeric sialoglycoprotein of 130,000 molecular weight (gp 130) is a membrane protein detected by mouse monoclonal antibodies on human melanoma cells and in lesser amounts on a wide range of normal and malignant cell types. Eight monoclonal antibodies reacting with gp 130 detect at least four spatially distinct epitopes on the exposed surface of the gp 130 molecule. Biosynthetic studies have shown that gp 130 is synthesized through two precursor forms: a 100 kD glycosylated species and an 80 kD unglycosylated species, presumably the primary translational product of the encoding mRNA.     

Regulation of IL-6 receptor and gp130 expression on human cell lines of lymphoid and myeloid origin.

The expression of 80 kDa interleukin-6 receptor (IL-6R) and the associated molecule gp130 has been studied on human cell lines by FACS- and Northern blot analysis. The effects of dexamethasone, dibutyric-(DB)-cAMP and phorbol-12-myristate-13-acetate (TPA) have been studied on plasmacytoma cell line U266, B cell line BMNH and monocytoid cell line U937. Our data show a definite downregulation of IL-6R and gp130 expression by TPA in U266 and BMNH at both mRNA and cell surface protein levels. In U937 TPA inhibits only the IL-6R expression, without affecting that of gp130. DB-cAMP decreases the expression of both proteins in U937, slightly inhibits the IL-6R expression in U266, but is uneffective in BMNH. Dexamethasone induces considerable upregulation of gp130 only in U266. Our findings suggest separate regulation of IL-6R and gp130 on U266, BMNH and U937 cell lines.     

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.     

c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130

Interleukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding α subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cbl, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.     

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.     

Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor.

BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown. MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.     

IL-6 signal transduction and its physiological roles: the signal orchestration model

Interleukin (IL)-6 is a pleiotropic cytokine that not only affects the immune system, but also acts in other biological systems and many physiological events in various organs. In a target cell, IL-6 can simultaneously generate functionally distinct or sometimes contradictory signals through its receptor complex, IL-6Rα and gp130. One good illustration is derived from the in vitro observations that IL-6 promotes the growth arrest and differentiation of M1 cells through gp130-mediated STAT3 activation, whereas the Y759/SHP-2-mediated cascade by gp130 stimulation has growth-enhancing effects. The final physiological output can be thought of as a consequence of the orchestration of the diverse signaling pathways generated by a given ligand. This concept, the signal orchestration model, may explain how IL-6 can elicit proinflammatory or anti-inflammatory effects, depending on the in vivo environmental circumstances. Elucidation of the molecular mechanisms underlying this issue is a challenging subject for future research. Intriguingly, recent in vivo studies indicated that the SHP-2-binding site- and YXXQ-mediated pathways through gp130 are not mutually exclusive but affect each other: a mutation at the SHP-2-binding site prolongs STAT3 activation, and a loss of STAT activation by gp130 truncation leads to sustained SHP-2/ERK MAPK phosphorylation. Although IL-6/gp130 signaling is a promising target for drug discovery for many human diseases, the interdependence of each signaling pathway may be an obstacle to the development of a nonpeptide orally active small molecule to inhibit one of these IL-6 signaling cascades, because it would disturb the signal orchestration. In mice, a consequence of the imbalanced signals causes unexpected results such as gastrointestinal disorders, autoimmune diseases, and/or chronic inflammatory proliferative diseases. However, lessons learned from IL-6 KO mice indicate that IL-6 is not essential for vital biological processes, but a significant impact on disease progression in many experimental models for human disorders. Thus, IL-6/gp130 signaling will become a more attractive therapeutic target for human inflammatory diseases when a better understanding of IL-6 signaling, including the identification of the conductor for gp130 signal transduction, is achieved.     

gp130-specific antisense oligonucleotides inhibit IL-6 signal inducing junB mRNA transcription in the human hepatoma cell line, HepG2

The biosynthesis of interleukin-6 receptor (IL-6R) and gp130 in vitro was blocked using specific antisense oligonucleotides (ASO) in HepG2 liver cells and the efficacy of various ASOs was tested on the generation of IL-6-induced junB mRNA. We used three ASOs specific for the IL-6 receptor, three specific for gp130 and a control (nonsense) oligonucleotide specific for epsilon-chain of IgE (not expressing in HepG2 cells). Our data indicate that a gp130-specific ASO, g2, was the most effective blocker of IL-6-induced junB mRNA, whilst the IL-6 receptor ASOs alone were ineffective. The mechanism of gene inactivation by ASO treatment was partially elucidated by demonstration of the loss of gp130 mRNA from cells treated with ASOs showing functional efficacy. Our data may help to design antisense oligonucleotides that are effective in therapy (e.g. as anti-inflammatory agents) in the future.     

Deletion of gp130 in myeloid cells modulates IL-6-release and is associated with more severe liver injury of Con A hepatitis

IL-6/gp130 dependent signaling plays an important role in modulating inflammation in acute and chronic diseases. The course of Concanavalin A- (Con A) induced hepatitis can be modulated by different immune-mediated mechanisms. IL-6/gp130-dependent signaling has been shown to be protective in hepatocytes. However, the role of this pathway in myeloid cells has not yet been studied. In our present study we used macrophage/neutrophil-specific gp130 knockout (gp130(ΔLys), KO) animals and analyzed its relevance in modulating Con A-induced hepatitis. Additionally, we performed in vitro studies with gp130(ΔLys)-macrophages. We demonstrate that gp130(ΔLys) animals are more susceptible to Con A-induced hepatitis. This is reflected by higher transaminases, higher lethality and more severe liver injury as shown by histological staining. Using flow cytometry analysis we further could show that increased liver injury of gp130(ΔLys) animals is associated with a stronger infiltration of CD11b/F4/80 double-positive cells compared to wild-type (gp130(flox/flox), WT) controls. To further characterize our observations we studied thioglycolate-elicited peritoneal macrophages from gp130(ΔLys) animals. Interestingly, the LPS-dependent IL-6 release in gp130(ΔLys) macrophages is significantly reduced (p<0.05) compared to WT macrophages. Additionally, IL-6 blood levels in vivo after Con A injection were significantly lower in gp130(ΔLys) animals compared to WT animals (p<0.05). In summary, our results suggest that gp130-deletion in macrophages and granulocytes leads to diminished IL-6 release from these cells, which is associated with more severe Con A-induced hepatitis.     

Gp130 signaling promotes development of acute experimental colitis by facilitating early neutrophil/macrophage recruitment and activation

IL-6 is known to play a crucial role in the pathogenesis of chronic intestinal inflammation by modulating T cell functions. In this study, we investigated the role of gp130, the common signal transducer for all IL-6 cytokines, in a murine model of acute T cell independent colitis to better characterize the impact of gp130 on innate immune cells and the early stages of inflammation. Experimental colitis was induced by dextran sulfate sodium treatment of mice with inducible systemic deletion of gp130 (MxCre/gp130(-/-)), macrophage/neutrophil-specific gp130-deficiency (LysCre/gp130(-/-)), or bone marrow chimeric mice and compared with wild-type controls (gp130(f/f)). Systemic deletion of gp130 (MxCre/gp130(-/-)) protected mice from severe colitis and wasting and attenuated the mucosal inflammatory infiltrate as well as local cytokine, chemokine, and adhesion molecule expression. Experiments in newly generated macrophage/neutrophil-specific gp130-deleted animals (LysCre/gp130(-/-)) and gp130 bone marrow chimeric mice, revealed a dual mechanism of proinflammatory effects mediated by gp130. Leukocyte recruitment was impaired in gp130-deleted animals and gp130-deleted recipients of wild-type bone marrow, demonstrating a central role of gp130-dependent signals in nonmyeloid cells for directing leukocytes to sites of inflammation, which was further confirmed in a model of sterile peritonitis. In contrast, macrophage/neutrophil-specific gp130 deficiency delayed and attenuated the disease but only marginally affected the inflammatory infiltrate, indicating a defective activation of mucosal leukocytes. We provide evidence that IL-6 cytokines acting via gp130 are required in the acute stages of intestinal inflammation by modulating the dynamics of innate immune cell recruitment and activation.     

Protein phosphatase type 2A, PP2A, is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp 130 at Ser-782 and degradation of gp 130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp 130. Taken together, present results strongly suggest that degradation of gp 130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp 130 is a potential therapeutic target in cancers.     

IL-6 Trans-Signaling via the Soluble IL-6 Receptor: Importance for the Pro-Inflammatory Activities of IL-6

Interleukin-6 (IL-6) is a cytokine with many activities. It has functions in the regulation of the immune system and the nervous system. Furthermore, IL-6 is involved in liver regeneration and in the metabolic control of the body. On target cells, IL-6 binds to an 80 kDa IL-6 receptor (IL-6R). The complex of IL-6 and IL-6R associates with a second protein, gp130, which thereupon dimerizes and initiates intracellular signaling. Whereas gp130 is expressed on all cells, IL-6R is only present on few cells in the body including hepatocytes and some leukocytes. Cells, which do not express IL-6R cannot respond to the cytokine, since gp130 alone has no measurable affinity for IL-6. Interestingly, a soluble form of IL-6R (sIL-6R) comprising the extracellular portion of the receptor can bind IL-6 with a similar affinity as the membrane bound IL-6R. The complex of IL-6 and sIL-6R can bind to gp130 on cells, which do not express the IL-6R, and which are unresponsive to IL-6. This process has been called trans-signaling. Here I will review published evidence that IL-6 trans-signaling is pro-inflammatory whereas classic IL-6 signaling via the membrane bound IL-6R is needed for regenerative or anti-inflammatory activities of the cytokine. Furthermore, the detailed knowledge of IL-6 biology has important consequences for therapeutic strategies aimed at the blockade of the cytokine IL-6.