Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.     

Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr⁷⁰⁵) and of tyrosine phosphatase SHP2 (Tyr⁵⁸⁰). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18 kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.     

Shedding of the interleukin-6 (IL-6) receptor (gp80) determines the ability of IL-6 to induce gp130 phosphorylation in human osteoblasts

Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R.     

The hepatic interleukin-6 receptor. Down-regulation of the interleukin-6 binding subunit (gp80) by its ligand.

Interleukin-6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6-binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp130). We studied the role of gp80 in binding, internalization and down-regulation of the hepatic IL6-receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp80 with parental cells indicate that gp80 is responsible for low affinity binding (Kd = 500 pM) of IL6. Furthermore, gp80 is rate-limiting in internalization and degradation of IL6. Internalization resulted in a rapid down-regulation (t1/2 approximately 15-30 min) of IL6-binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6-binding sites at the cell surface required greater than 8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down-regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.     

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.     

Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.     

Protein kinase C delta (PKC delta): activation mechanisms and functions

Protein kinase C (PKC)delta was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKC delta is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumor-promoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain. It is also cleaved by caspase to generate a catalytically active fragment, and it is converted to an active form without proteolysis through the tyrosine phosphorylation reaction. Various lines of evidence indicate that PKC delta activated in distinct ways plays critical roles in cellular functions such as the control of growth, differentiation, and apoptosis. This article briefly summarizes the regulatory mechanisms of PKC delta activity and its functions in cell signaling.     

Soluble glycoprotein 130 (gp130) attenuates OSM- and LIF-induced cartilage proteoglycan catabolism

Oncostatin M (OSM) and leukaemia inhibitory factor (LIF) exhibit pleiotropic biological activities and share many structural and genetic features. The two cytokines bind with high affinity to the same receptor (LIF/OSM receptor), which consists of the LIF receptor alpha chain (LIFRalpha) and the signal transduction unit gp130. A soluble form of the beta chain of the receptor complex called soluble gp130 (sgp130) has been cloned. In this study, we sought to determine whether recombinant sgp130 or anti-gp130 Ab could attenuate the resorption of proteoglycans induced by OSM and LIF in articular cartilage explants. The results show that at high concentrations sgp130 is capable of attenuating both LIF and OSM mediated resorption. In contrast, anti-gp130 Ab selectively inhibited the stimulation of proteoglycan (PG) release by OSM, albeit minimally. The failure of anti-gp130 to attenuate LIF stimulated PG resorption may be due to the normal interaction of LIF with LIFRalpha and unfettered heterodimerization of LIFRalpha with gp130 in the presence of the antibody. The results indicate that sgp130 and anti-gp130 can modulate cartilage PG metabolism in vitro. Whether sgp130 may have therapeutic activity in models of arthritis or indeed in arthritic diseases remains to be determined.     

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.     

Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses.

Signal transduction in response to interleukin-6 (IL-6) requires binding of the cytokine to its receptor (IL-6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL-6 and soluble IL-6R (sIL-6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL-6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL-6 and sIL-6R. We created recombinant sgp130 proteins that showed binding to IL-6 in complex with sIL-6R and inhibited IL-6/sIL-6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL-6 stimulated proliferation of BAF/3 cells expressing gp130 and membrane bound IL-6R, indicating that sgp130 did not interfere with IL-6 bound to IL-6R on the cell surface. Additionally, sgp130 partially inhibited proliferation induced by leukemia inhibitory factor (LIF) and oncostatin M (OSM) albeit at higher concentrations. Recombinant sgp130 protein could be used to block the anti-apoptotic effect of sIL-6R on lamina propria cells from Crohn disease patients. We conclude that sgp130 is the natural inhibitor of IL-6 responses dependent on sIL-6R. Furthermore, recombinant sgp130 is expected to be a valuable therapeutic tool to specifically block disease states in which sIL-6R transsignaling responses exist, e.g. in morbus Crohn disease.     

Up-regulation of the interleukin-6-signal transducing protein (gp130) by interleukin-6 and dexamethasone in HepG2 cells.

The hepatic IL-6-receptor is composed of an 80 kDa IL-6-binding protein and a 130 kDa polypeptide (gp130) believed to be involved in signal transduction. Previous experiments have shown that the 80 kDa IL-6-receptor is up-regulated by glucocorticoids, but not by IL-6. Here we demonstrate that IL-6 together with the synthetic glucocorticoid dexamethasone induces the expression of mRNA for gp130 approximately 5-fold in HepG2 cells. The induction was dose- and time-dependent. Dexamethasone alone, interferon-gamma, IL-1 alpha and IL-1 beta had no effect. A possible role for the regulation of the IL-6-signal transducing protein gp130 in various inflammatory states is proposed.     

Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor.

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.     

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.     

Soluble interleukin-6 receptor induces motor stereotypies and co-localizes with gp130 in regions linked to cortico-striato-thalamo-cortical circuits

Soluble cytokine receptors are normal constituents of body fluids that regulate peripheral cytokine and lymphoid activity and whose levels are increased in states of immune activation. Soluble interleukin-6 receptor (sIL-6R) levels positively correlate with disease progression in some autoimmune conditions and psychiatric disorders. Particularly strong links between levels of sIL-6R and the severity of psychotic symptoms occur in schizophrenia, raising the possibility that sIL-6R is involved in this disease. However, there is no evidence that peripheral sIL-6R induces relevant behavioral disturbances. We showed that single subcutaneous injections of sIL-6R (0-1 μg), stimulated novelty stress-induced exploratory motor behaviors in male Balb/c mice within 20-40-min of injection. A progressive increase in vertical stereotypies was observed 40-80 min post injection, persisting for the remainder of the test session. Paralleling these stimulant-like effects, sIL-6R pre-treatment significantly enhanced stereotypy scores following challenge with GBR 12909. We found that peripherally administered sIL-6R crossed the blood-brain barrier, localizing in brain regions associated with cortico-striatal-thalamo-cortical (CSTC) circuits, which are putative neuroanatomical substrates of disorders associated with repetitive stereotypies. Peripherally administered sIL-6R co-localized with gp130, a transmembrane protein involved in IL-6 trans-signaling, in the nucleus accumbens, caudate-putamen, motor and infralimbic cortices, and thalamic nuclei, but not with gp130 in the ventral tegmental area, substantia nigra, or sensorimotor cortex,. The results suggest that peripheral sIL-6R can act as a neuroimmune messenger, crossing the blood brain barrier (BBB) to selectively target CSTC circuits rich in IL-6 trans-signaling protein, and inducing repetitive stereotypies. As such sIL-6R may represent a novel therapeutic agent for relevant psychiatric disorders.     

Protein phosphatase-1 is targeted to DNA polymerase delta via an interaction with the p68 subunit

Protein phosphatase-1 (PP1) is a Ser/Thr protein phosphatase that participates in the phosphorylation/dephosphorylation regulation of a diverse range of cellular processes. The PP1 catalytic subunit (PP1) achieves this by its ability to interact with many targeting subunits such that PP1 activity is thereby specified against phosphoprotein substrates in the microvicinity of its targeting subunit. DNA polymerase delta (Pol delta) is a key enzyme in mammalian chromosomal replication. It consists of four subunits, p125, p50, p68, and p12. We identify p68 as a novel PP1 targeting subunit. PP1 was shown to associate with human DNA polymerase delta by affinity chromatography and coimmunoprecipitation assays from mammalian cell lysates and in vitro by pull-down assays. The binding domain for PP1 was identified as the sequence KRVAL, a variant of the canonical RVxF PP1 binding motif. These studies provide the first evidence for the targeting of PP1 to DNA polymerase delta. We also show that CK2 phosphorylates the Pol delta p125, p68, and p12 subunits and that these phosphorylated subunits are substrates for PP1. These findings identify a new role for p68 as a PP1 targeting subunit that implicates PP1 in the dephosphorylation of Pol delta. Our findings also show that CK2 is a strong candidate for the protein kinase involved in the in vivo phosphorylation of p68.     

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.