Lutz Graeve

LPS and TNFα 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 TNFα 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 TNFα 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 TNFα.

A Di-leucine Motif and an Upstream Serine in the Interleukin-6 (IL-6) Signal Transducer gp130 Mediate Ligand-induced Endocytosis and Down-regulation of the IL-6 Receptor

The interleukin-6 (IL-6) receptor complex is composed of two different subunits, the IL-6 binding protein (IL-6R, gp80) and the signal transducing component gp130. Our previous studies revealed that the 10-amino acid sequence TQPLLDSEER within the intracellular domain of gp130 is crucial for the efficient internalization of IL-6. Since this sequence contains a putative di-leucine internalization motif, we further analyzed this region by constructing two additional deletions and a series of point mutants. Analyses of these mutants showed that the di-leucine pair (Leu-145 and Leu-146) is essential for ligand internalization, with leucine 145 being less resilient to exchanges. Furthermore, when a chimeric protein (Tac-STQPLL) composed of the Tac antigen fused to the hexapeptide STQPLL of gp130 was studied, we found that this sequence is sufficient to mediate endocytosis and lysosomal targeting of the chimera. Mutational analysis of three serine residues upstream of the di-leucine motif revealed that mutation of serine 139 to an alanine reduces the initial internalization rate by 50%. This finding suggests that a serine phosphorylation may be important for rapid endocytosis.

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 κB. 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.

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 (gp13O). 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 gp8O with parental cells indicate that gp80 is responsible for low affinity binding (K d = 500 pM) of IL6. Furthermore, gp80 is rate‐limiting in internalization and degradation of IL6. Internalization resulted in a rapid down‐regulation (t ≈ 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 >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.

Differential shedding of the two subunits of the interleukin‐6 receptor

cDNAs coding for the two receptor subunits of the interleukin‐6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL‐6 binding protein (IL‐6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL‐6R gp 130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL‐6R and gp 130 released by transfected cells can form a ternary complexe with interleukin‐6 indicating that such complexes also may occur in vivo. gp 130; Interleukin‐6; Interleukin‐6‐receptor; Protein kinase C; Shedding

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-κB. 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 α2-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.

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2) Up-regulation by interleukin-6 and transforming growth factor β1

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of Synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase‐1 (TIMP‐1). We show at mRNA and protein levels that TIMP‐1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up‐regulated by interleukin‐6 (IL‐6), transforming growth factor β1 and phorbol 12‐myristate 13‐acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL‐6‐stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP‐1 mRNA in liver cells.

TIMP-1 protein expression is stimulated by IL-1β and IL-6 in primary rat hepatocytes

Degradation of extracellular matrix proteins is performed by metalloproteinases which are inhibited by tissue inhibitors of metalloproteinases (TIMP). We expressed the murine TIMP‐1 protein in E. coli and prepared a polyclonal antiserum against the recombinant protein. Using this antiserum we studied the biosynthesis and glycosylation of murine TIMP‐1 protein in COS‐7 cells transfected with a TIMP‐1 expression plasmid by metabolic labeling and indirect immunofluorescence studies. In primary rat hepatocytes we show for the first time that TIMP‐1 protein expression is up‐regulated upon stimulation with IL‐1β and IL‐6. Since TIMP‐1 is induced during the acute phase reaction it could possibly be involved in the pathogenesis of liver fibrosis.

Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation

Several cytokines and growth factors activate transcription of their target genes via the JAK/STAT signalling pathway. It has been shown that the interaction between SH2 domains of STAT factors and receptor phosphotyrosine residues plays an essential role in the specific recruitment of STATs. For STAT5, however, the importance of receptor tyrosines is still controversial. Using a chimeric receptor system in COS‐7 cells, we studied the activation of STAT5 through the interleukin‐6 signal transducer gp130. In contrast to previous reports, we did not detect gp130‐mediated STAT5 activation. However, STAT5 activation was achieved when tyrosine motifs of other cytokine receptors were fused to the membrane‐proximal part of gp130. The comparison of the relative potency of different tyrosine motifs revealed that hydrophobic amino acids, preferentially leucine, in positions +1 and +3, and an aspartate residue in position ‐1 or ‐2 with respect to the tyrosine are likely to be required for efficient STAT5 recruitment. In summary, we show here for the first time that phosphotyrosine motifs can confer the ability to activate STAT5 to a heterologous receptor.

The Inhibitory Effect of IL-1β on IL-6-Induced α2-Macroglobulin Expression Is Due to Activation of NF-κB

The cross-talk between the signal transduction of simultaneous acting cytokines largely determines the final impact of cytokines on their target genes. Both NF-κB 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-1β inhibits IL-6-induced transcriptional activation of the α2-macroglobulin promoter. It is shown that IL-1β prevents STAT3 binding to the two STAT3-responsive sites within the α2-macroglobulin promoter by association of IL-1β-activated NF-κB to this region. The observation that inhibition of IL-6-induced transcriptional activation of this promoter by IL-1β is reversed by cotransfection with I-κBα provides evidence that NF-κB activation by IL-1β is responsible for inhibition of IL-6-mediated trans activation of the α2-macroglobulin gene. Accordingly, cotransfection of the NF-κB subunits p50 or p65 themselves inhibited activation of the α2-macroglobulin promoter by IL-6. Introduction of point mutations in each of the two NF-κB sites overlapping the two STAT3 binding sites within the α2-macroglobulin promoter provides evidence that each of these two sites counteracts transcriptional activation via STAT3. Most interestingly, at least one functional NF-κB consensus site is essential for the IL-6-induced transcriptional activation of the α2-macroglobulin promoter. Additional data are provided indicating that the activation of NF-κB by IL-1β is also responsible for the inhibition of other IL-6-inducible genes, such as the α1-antichymotrypsin gene as well as the suppressor of cytokine signaling 3 gene, suggesting a more general relevance of this mechanism for transcriptional regulation.