Friedemann Horn

The signalling pathways of interleukin-6 and gamma interferon converge by the activation of different transcription factors which bind to common responsive DNA elements.

Interleukin-6 (IL-6) and gamma interferon (IFN-gamma) induce a partially overlapping set of genes, including the genes for interferon regulatory factor 1 (IRF-1), intercellular adhesion molecule 1 (ICAM-1), and the acute-phase protein alpha 2-macroglobulin. We report here that the rat alpha 2-macroglobulin promoter is activated by IFN-gamma in human hepatoma (HepG2) cells and that the IFN-gamma response element maps to the same site previously defined as the acute-phase response element (APRE), which binds the IL-6-activated transcription factor APRF (acute-phase response factor). As was reported for fibroblasts, the IFN-gamma-regulated transcription factor GAF is phosphorylated at tyrosine after IFN-gamma treatment of HepG2 cells. IFN-gamma posttranslationally activates a protein which specifically binds to the alpha 2-macroglobulin APRE. This protein is shown to be identical or closely related to GAF. Although APRF and GAF are shown to represent different proteins, their binding sequence specificities are very similar. APRF and GAF bind equally well to the APRE sequences of various acute-phase protein genes as well as to the IFN-gamma response elements of the IRF-1, ICAM-1, and other IFN-gamma-inducible genes. Transient transfection analysis revealed that the IFN-gamma response elements of the IRF-1 and ICAM-1 promoters are able to confer responsiveness to both IFN-gamma and IL-6 onto a heterologous promoter. Therefore, APRF and GAF are likely to be involved in the transcriptional induction of these immediate-early genes by IL-6 and IFN-gamma, respectively. Taken together, these results demonstrate that two functionally distinct hormones, IL-6 and IFN-gamma, act through common regulatory elements to which different transcription factors sharing almost the same sequence specificity bind.

Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction

The cytokine interleukin‐6 (IL‐6) rapidly activates a latent cytoplasmic transcription factor, acute‐phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate‐early gene induction by IL‐6 and, as we show here, stimulation of the promoters of the genes for α 2‐macroglobulin, Jun‐B, and intercellular adhesion molecule‐1 (ICAM‐1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL‐6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL‐6 signal transduction leading to target gene induction.

Cytokines and Growth factors signal through tyrosine phosphorylation of a family of related transcription factors

The ability of cytokines to activate distinct but overlapping sets of genes defines their characteristic biological response. We now show that IFN gamma, IL-3, IL-4, IL-6, erythropoietin, EGF, and CSF-1 activate differing members of a family of latent cytoplasmic transcription factors. Although these factors have distinct physical and functional properties and exhibit different patterns of expression, they share many important features, including recognition of a related set of enhancer elements, rapid activation, tyrosine phosphorylation, and cross-reactivity to antibodies against p91, a cytoplasmic signaling protein activated by IFN alpha, IFN gamma, and IL-6. These shared features point to either parallel or common patterns of signal transduction. A general model of cytokine signal transduction is presented, in which receptor-associated tyrosine kinases activate ligand-specific members of a family of signal-transducing factors. Once activated, these factors carry their signals to the nucleus, where they bind a family of related enhancer elements.

Protein kinase C interferes with Ni-mediated inhibition of human platelet adenylate cyclase

Addition of phorbol ester‐activated, partially purified protein kinase C to membranes of human platelets had no effect on forskolin stimulation of the adenylate cyclase and increased stimulation by prostaglandin E1 only at high GTP concentrations by preventing inhibition by GTP. Hormonal inhibition of the platelet adenylate cyclase by epinephrine was eliminated or largely impaired. At low GTP concentrations, epinephrine even caused a small increase in cyclase activity. The data suggest that activated protein kinase C interferes with GTP‐ and hormone‐induced adenylate cyclase inhibition probably by phosphorylating the inhibitory guanine nucleotide‐binding regulatory component Ni.

A factor induced by differentiation signals in cells of the macrophage lineage binds to the gamma interferon activation site.

Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, intermediate affinity to the Ly6A/E GAS, and low affinity to the guanylate-binding protein GAS. DIF may belong to a family of cytokine- or growth factor-induced factors binding with variable affinities to GAS-related elements: the interleukin-6-responsive acute-phase response factor associated with GAS from different IFN-inducible promoters but with a different preference of binding compared with DIF. The sis-inducible element of the c-fos promoter bound GAF but not DIF. However, the sis-inducible element could be changed by point mutation to compete for GAF and DIF binding. Our data show DIF to be a novel DNA-binding protein which is activated in response to differentiating signals. Moreover, they suggest that a family of cytokine- or growth factor-regulated proteins integrates and coordinates the responses to cytokines and to growth and differentiation factors by binding to GAS-related elements.

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.

A completely foreign receptor can mediate an interferon-γ-like response

A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK‐binding domains of the gp130 subunit of the interleukin‐6 (IL‐6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)‐γ receptor, efficiently mediates an IFN‐γ‐like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN‐γ‐like response, but less efficiently. The IFNGR1 signal‐transducing subunit of the IFN‐γ receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL‐6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN‐γ‐like response through the chimeric receptors, nor does it mediate an IFN‐γ‐like response to IL‐6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the ‘pathways’ involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK–receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.

Regulation of α2‐Macroglobulin Gene Expression by Interleukin‐6a

Disturbances of the physiological homeostasis of the organism by infection (bacteria, viruses, fungi), trauma, neoplasia, or immunological disorders lead to a local reaction characterized by the activation of monocytes, fibroblasts, and endothelial cells and the release of inflammatory mediators such as interleukin-1 (IL-l), IL-6, IL-8, tumor necrosis factor (Y (TNFa), and interferons. These mediators, via circulation, give rise to a systemic reaction of the organism characterized by fever, pain, activation of the immune and endocrine system, stimulation of hematopoiesis, and a change in the synthesis of the so-called “acute phase proteins” (FIGURE l).’-3 The acute phase proteins are synthesized and secreted by the hepatocytes of liver. The major human acute phase proteins are C-reactive protein, serum amyloid A, fibrinogen, haptoglobin, and a,-antichymotrypsin. Acute phase proteins function as proteinase inhibitors and are involved in blood clotting and fibrinolysis, in the removal of foreign materials from the organism, and in the binding and transport of metals and biologically active compounds.3 We have been interested for several years in the regulation of gene expression of the major acute phase protein in the rat, that is, the high-molecular-weight proteinase inhibitor, al-macroglobulin ((YzM).