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Biomedical subjects

L Graeve

Publications and source records attributed to L Graeve.

At least 19 recordsLinked to original sources
PubMed2001

The vesicular stomatitis virus matrix protein inhibits glycoprotein 130-dependent STAT activation.

Infection of cells by vesicular stomatitis virus (VSV) results in the inhibition of host transcription. We show in this study that infection of HeLa cells with VSV leads to a strongly diminished activation of STAT3 and STAT1 by the inflammatory cytokine IL-6. This effect was mimicked by forced expression of a single viral protein, the matrix (M)-protein of VSV, which blocked STAT activation via chimeric receptors containing the cytoplasmic domain of the IL-6 signal transducer gp130. Western blot analysis revealed that VSV M-protein did not inhibit the nuclear translocation of activated STAT3 but did inhibit its tyrosine phosphorylation. Inhibition of STAT activation was not dependent on tyrosine 759 of the IL-6 signal transducer gp130, suggesting that the inhibitory action of VSV M-protein is not mediated by the induction of the suppressor of cytokine signaling 3. VSV M-protein inhibited gene transcription from cotransfected alpha(2)-macroglobulin or antichymotrypsin promoter/luciferase reporter constructs which contain STAT3-binding sites. However, transcription from a STAT5-dependent construct was not negatively affected. In conclusion, our data suggest that infection by VSV and specifically overexpression of the viral M-protein interferes with an important signaling pathway necessary for triggering antiviral and inflammatory responses.

Antigens, CD↗
PubMed2001

The inhibitory effect of IL-1 beta on IL-6-induced alpha 2-macroglobulin expression is due to activation of NF-kappa B.

The cross-talk between the signal transduction of simultaneous acting cytokines largely determines the final impact of cytokines on their target genes. Both NF-kappaB and STAT3 are transcription factors well known to be activated by many stimuli and to mediate transcriptional activation by binding to specific enhancer sequences. In this study, it is analyzed how IL-1beta inhibits IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. It is shown that IL-1beta prevents STAT3 binding to the two STAT3-responsive sites within the alpha(2)-macroglobulin promoter by association of IL-1beta-activated NF-kappaB to this region. The observation that inhibition of IL-6-induced transcriptional activation of this promoter by IL-1beta is reversed by cotransfection with I-kappaBalpha provides evidence that NF-kappaB activation by IL-1beta is responsible for inhibition of IL-6-mediated trans activation of the alpha(2)-macroglobulin gene. Accordingly, cotransfection of the NF-kappaB subunits p50 or p65 themselves inhibited activation of the alpha(2)-macroglobulin promoter by IL-6. Introduction of point mutations in each of the two NF-kappaB sites overlapping the two STAT3 binding sites within the alpha(2)-macroglobulin promoter provides evidence that each of these two sites counteracts transcriptional activation via STAT3. Most interestingly, at least one functional NF-kappaB consensus site is essential for the IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. Additional data are provided indicating that the activation of NF-kappaB by IL-1beta is also responsible for the inhibition of other IL-6-inducible genes, such as the alpha(1)-antichymotrypsin gene as well as the suppressor of cytokine signaling 3 gene, suggesting a more general relevance of this mechanism for transcriptional regulation.

Animals↗
PubMed2000

Differential inhibition of IL-6-type cytokine-induced STAT activation by PMA.

Prior activation of mitogen-activated protein kinases by phorbol 13-myristate 12-acetate (PMA) results in an inhibition of interleukin (IL)-6-induced activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway which is most likely mediated by the induction of suppressor of cytokine signaling-3 and requires the specific SHP2 binding site Y759 of the IL-6 signal transducer gp130. In this study, we demonstrate that PMA inhibits STAT activation by IL-6 and the related cytokine leukemia inhibitory factor (LIF) but not by oncostatin M (OSM). Since the LIF receptor also contains an SHP2 recruitment site whereas the OSM receptor lacks such a module, we propose that two SHP2 binding modules within a homo- or heterodimeric receptor are necessary to mediate the PMA inhibitory effect.

Amino Acid Motifs↗
PubMed2000

The inhibition of interleukin-6-dependent STAT activation by mitogen-activated protein kinases depends on tyrosine 759 in the cytoplasmic tail of glycoprotein 130.

Mitogen-activated protein (MAP) kinases stimulated by phorbol 13-myristate 12-acetate (PMA) have been shown to inhibit interleukin-6-induced activation of STAT3 (Sengupta, T. K., Talbot, E. S., Scherle, P. A., and Ivashkiv, L. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11107-11112). In the present study we demonstrate that in addition to STAT3, also tyrosine phosphorylation of STAT1, signal transducer gp130, and phosphotyrosine-phosphatase SHP2 underlies negative regulation by MAP kinases. Stimulation of Erks by basic fibroblast growth factor or a constitutively active mutant of Raf also led to down-regulation of STAT activity. Using chimeric receptor mutants we show that tyrosine 759 of glycoprotein 130 is crucial for the inhibitory effect of MAP kinases. Inhibition is also dependent on gene transcription and translation indicating that newly synthesized proteins are involved. Both PMA and basic fibroblast growth factor rapidly stimulate mRNA expression of the suppressor of cytokine signaling-3 (SOCS-3) and this induction is strongly reduced by an inhibitor of MAP kinase activation. Together with recent results demonstrating that SOCS-3 can bind in vitro to a phosphorylated tyrosine 759 peptide of glycoprotein 130 these data suggest SOCS-3 to be instrumental in the inhibition of the Janus kinase/STAT pathway by MAP kinases.

Animals↗
PubMed2000

Termination of IL-6-induced STAT activation is independent of receptor internalization but requires de novo protein synthesis.

The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signal transducer gp130. Binding of IL-6 to its receptor results in dimerization of gp130, activation of the Jak/STAT pathway, and in a down-regulation of IL-6 binding sites by endocytosis. The STAT activation after stimulation is transient, being maximal after 15-30 min and disappearing after 60-90 min. The mechanism which leads to the termination of the signal is still unknown. In this paper we have studied whether the down-modulation of the STAT signal requires the endocytosis of the receptor complex. Our results suggest that the desensitization of the IL-6 signal is not due to internalization of the receptor complex but requires de novo protein synthesis.

Animals↗
PubMed2000

In vitro-generated stem cell leukaemia showing altered cell cycle progression with distinct signalling of the tyrosine-phosphorylated rasGAP-associated p62(dok) protein.

In an attempt to gain more insight into the events of leukaemic transformation, a cell line overexpressing MHC class II (DR) was generated by transfecting an early CD34-negative haematopoietic progenitor stem cell line with the appropriate constructs. The stable transfection with genes for DR antigens leads to cellular transformation. The DR(+) transformed cell clones express a tyrosine-phosphorylated DR heterodimer and show a significantly different morphology. DR(+) clones present the morphology of an immature myeloid neoplasia expressing alpha-naphthyl-acetate-esterase (ANAE), but neither myeloperoxidase nor CD34. While D064 cells predominately grow adherent as fibroblast-like cells, the DR(+) clones display a decrease in adherent growth. Although both cell lines express similar amounts of the interleukin-6 (IL-6) signal transducer gp130, the DR-transfected cells still show activation of STAT factors by IL-6, whereas D064 cells do not. Although the transformed clones present acceleration of cell-cycle transition and growth, the G(0)/G(1) progression inhibitor p27(kip-1) is up-regulated, while the expression of proteins involved in the S/G(2) phase transition, such as cyclin B and cdc2 (p34), is suppressed. Instead cyclin D3, one of the G(0)/G(1) progression factors, is up-regulated, as well as tyrosine-phosphorylated p62(dok), suggesting dysregulation of cell cycle-controlling proteins. In addition, DR(+) leukaemia-like cells also overexpress Bcl-2, while bax expression is suppressed, compared with the wild-type (wt) parental haematopoietic stem cell line.

Acute Disease↗
PubMed2000

Quiescence of CD34-negative haematopoietic stem cells is mediated by downregulation of Cyclin B and no stat activation.

The CD34-negative, adherent growing, fibroblast-like canine haematopoietic stem cell line D064 was recently identified as the earliest progenitor population in the bone marrow. D064 cells are predominately quiescent. Quiescence is mediated by the accumulation of the cyclin-dependent kinase inhibitor p27(kip-1)and in parallel, by the downregulation of Cyclin B, leading to an accumulation of quiescent cells in the G(0)/G(1)-phase of the cell cycle. Stem cell factor (SCF), the ligand for the tyrosine kinase receptor c-kit, usually induces differentiation of the CD34-negative stem cells into CD34-positive haematopoietic precursors. SCF also suppresses the expression of c-myc-dependent Cyclin E, which is not transcribed initially, but expression occurs later on. Interleukin 6 (IL-6) instead rather promotes proliferation, but fails to induce proliferation in the majority of CD34-negative stem cells due to no STAT activation in quiescent cells. Nevertheless, the potential of quiescent D064 cells to proliferate eventually, becomes apparent by the low-level expression of IL-6 dependent STAT factors. D064 cells also spontaneously start to express Bax, while Bcl-2 is downregulated in parallel. In summary, CD34-negative haematopoietic stem cells dwell in the marrow or other niches as quiescent cells, until they can respond to autocrine or paracrine growth factor-mediated signals.

Animals↗
PubMed2000

The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized madin-darby canine kidney cells.

The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain.

Amino Acid Motifs↗
PubMed1999

LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages.

Recent findings indicate that cytokine signaling can be modulated by other mediators of simultaneously activated signal transduction pathways. In this study we show that LPS and TNFalpha are potent inhibitors of IL-6-mediated STAT3 activation in human monocyte derived macrophages, rat liver macrophages and RAW 264.7 mouse macrophages but not in human hepatoma cells (HepG2) or in rat hepatocytes. Accordingly, LPS and TNFalpha were found to induce the expression of SOCS3 mRNA in each of the investigated type of macrophages but not in HepG2 cells. Using a specific inhibitor, evidence is presented that the p38 MAP kinase might be involved, especially for the inhibitory effect of TNFalpha.

Animals↗
PubMed1999

The mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity.

Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor kappaB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6.

Animals↗
PubMed1999

Identification of a Leu-lle internalization motif within the cytoplasmic domain of the leukaemia inhibitory factor receptor.

Leukaemia inhibitory factor (LIF) signals via a heterodimeric receptor complex comprised of the LIF receptor (LIFR) and the interleukin (IL)-6 signal transducer gp130. Upon binding to its cognate receptor LIF is internalized. In this study, we show that the LIFR is endocytosed independently of gp130. By using a heterochimaeric receptor system we identified a dileucine-based internalization motif within the cytoplasmic domain of the LIFR. Our findings suggest that a heterodimeric LIFR/gp130 complex and homodimeric gp130/gp130 complex are endocytosed via distinct internalization signals.

Animals↗
PubMed1999

Induction of alpha1-antitrypsin synthesis in human articular chondrocytes by interleukin-6-type cytokines: evidence for a local acute-phase response in the joint.

OBJECTIVE: We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflammatory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein alpha1-antitrypsin in human articular chondrocytes. METHODS: Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of alpha1-antitrypsin protein were determined by enzyme immunoassay. RESULTS: Stimulation of chondrocytes with oncostatin M (OSM) and IL-6 led to a 5-10-fold increase in alpha1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. CONCLUSION: Our data strongly suggest the existence of a local acute-phase response in the joint. Synthesis of the acute-phase protein alpha1-antitrypsin, a major inhibitor of serine proteinases, may be an important protective mechanism of articular chondrocytes to prevent cartilage damage in inflammatory joint diseases.

Acute-Phase Proteins↗
PubMed1999

Regulation of beta2-microglobulin expression in different human cell lines by proinflammatory cytokines.

BACKGROUND: Proinflammatory monocytic cytokines such as interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and IL-6 have been incriminated in the pathogenesis of elevated beta2-microglobulin (beta2M) serum concentrations in patients undergoing haemodialysis with so-called bioincompatible dialyser membranes. However, neither the source of the elevated serum beta2M nor the precise role of monocytic cytokines in the expression of the beta2M gene have been elucidated conclusively. The aim of the current study was to evaluate whether monocytic cytokines, and in particular IL-6, are regulators of beta2M gene expression in human hepatoma cells, T-lymphocytes and monocytes. METHODS: HepG2 and HuH7 human hepatoma cells, Jurkat T-cells, monocytic MonoMac6 cells, primary human monocytes and synoviocytes were stimulated with IL-1beta, IL-6, interferon-alpha (IFN-alpha), IFN-gamma or conditioned media from lipopolysaccharide (LPS)-treated monocytes. Expression of beta2M mRNA was analysed by Northern blotting, beta2M protein synthesis was determined by metabolic labelling and immunoprecipitation, and beta2M secretion was measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: In all cell types tested, IFN-gamma and, to a lesser extent, IFN-alpha stimulated gene expression of beta2M resulting in an increased synthesis and secretion of beta2M protein. Neither IL-1beta and IL-6 nor supernatants from LPS-treated monocytes were capable of inducing beta2M gene expression, with the exception of a small increase in HuH7 hepatoma cells upon IL-1beta treatment. CONCLUSIONS: The present study provides evidence that interferons are important regulators of beta2M expression. It also shows that proinflammatory monocytic cytokines do not modulate directly the expression of beta2M in cells of hepatic, monocytic and T-lymphocytic origin. Whether they influence beta2M synthesis and secretion indirectly by modulating interferon synthesis needs to be elucidated.

Cell Line↗
PubMed1998

Constitutive internalization and association with adaptor protein-2 of the interleukin-6 signal transducer gp130.

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin-6 (IL-6)-type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal-independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL-6/IL-6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL-6/IL-6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP-2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Soluble IL-6 receptor potentiates the antagonistic activity of soluble gp130 on IL-6 responses.

Soluble receptors for several cytokines have been detected in body fluids and are believed to modulate the cytokine response by binding the ligand and thereby reducing its bioavailability. In the case of IL-6, the situation is more complex. The receptor consists of two components, including a ligand-binding alpha-subunit (IL-6R, gp80, or CD126), which in its soluble (s) form (sIL-6R) acts agonistically by making the ligand accessible to the second subunit, the signal transducer gp130 (CD130). Soluble forms of both receptor subunits are present in human blood. Gel filtration of iodinated IL-6 that had been incubated with human serum revealed that IL-6 is partially trapped in IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was enriched by immunoaffinity chromatography and identified as a 100-kDa protein. Functionally equivalent rsgp130 was produced in baculovirus-infected insect cells to study its antagonistic potential on four different cell types. It was found that in situations in which cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R complexes, sgp130 was a much more potent antagonist than it was on IL-6R-positive cells stimulated with IL-6 alone. In the latter case, the neutralizing activity of sgp130 could be markedly enhanced by addition of sIL-6R. As a consequence of these findings, sIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130. Furthermore, in combination with sIL-6R, sgp130 is a promising candidate for the development of IL-6 antagonists.

Adjuvants, Immunologic↗
PubMed1998

Activation of the Janus kinase/signal transducer and activator of transcription pathway by osmotic shock.

Numerous cytokines, growth, and differentiation factors elicit their intracellular responses via Janus tyrosine kinases (Jaks) and transcription factors of the STAT (signal transducer and activator of transcription) family. Additionally, environmental stress (UV light, heat, aniso-osmolarity, and radicals) has recently been shown to activate intracellular signaling cascades such as the stress-activated protein kinases and nuclear factor-kappaB. In this study, we demonstrate that in different cell lines a particular stress, namely hyperosmolarity, results in tyrosine phosphorylation of the Janus kinases Jak1, Jak2, and Tyk2 and in the activation of STAT1 and/or STAT3. Both transcription factors are phosphorylated at a specific tyrosine residue and translocation to the nucleus was demonstrated by the use of a STAT3/green fluorescent protein fusion protein. A prominent role for Jak1 in the activation of STATs by hypertonicity was demonstrated by the use of Jak-deficient cell lines. Stress-activated STAT1 and STAT3 transactivate a reporter gene containing the acute-phase response element of the rat alpha2-macroglobulin promoter. Experiments using a diffusible solute suggest that not the increase in intracellular osmolarity but the resultant cell shrinkage is the trigger for Jak/STAT activation.

Animals↗