Phosphorylation of IRF8 in a pre-associated complex with Spi-1/PU.1 and non-phosphorylated Stat1 is critical for LPS induction of the IL1B gene.

Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction.

Glucocorticoid inhibits the human pro-interleukin lbeta gene (ILIB) by decreasing DNA binding of transactivators to the signal-responsive enhancer.

Elucidating the role of glucocorticoid in regulating gene expression is crucial to developing effective strategies against inflammatory diseases such as arthritis. In this report we demonstrate that glucocorticoid inhibits transcription directed by the IL-lbeta gene (IL1B) upstream induction sequence (UIS) enhancer, and to a much lesser extent by the tissue-specific basal promoter. Within the enhancer, three transcription factor binding sites, previously demonstrated by us to be important for the induction of IL1B by lipopolysaccharide, are now shown to be directly inhibited by the synthetic glucocorticoid, dexamethasone.

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1.

Potent induction of the gene coding for human prointerleukin 1beta (il1b) normally requires a far-upstream inducible enhancer in addition to a minimal promoter located between positions -131 and +12. The transcription factor Spi-1 (also called PU.1) is necessary for expression and binds to the minimal promoter, thus providing an essential transcription activation domain (TAD).

Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils.

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2).

Transcriptional repression of the prointerleukin 1beta gene by heat shock factor 1.

Heat shock factor 1 activates the promoters of heat shock genes at elevated temperatures through its interaction with heat shock elements. We have examined a new role for heat shock factor 1 in the repression of the prointerleukin 1beta gene in human monocytes responding to stimulation with lipopolysaccharide. Both exposure to elevated temperatures and heat-independent heat shock factor 1 expression repressed the transcription of the prointerleukin 1beta gene, and repression was strictly dependent on an intact consensus heat shock element in the prointerleukin 1beta promoter to which heat shock factor 1 bound.

Tumour necrosis factor-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils.

The role of the newly identified p38 mitogen-activated protein kinase (MAP kinase) in terminally differentiated cells, such as human neutrophils, is totally unknown. In order to examine the possible role of this MAP kinase in the phosphorylation and activation of cytoplasmic phospholipase A2 (cPLA2), we tested the effect of the recently synthesized inhibitor of p38 MAP kinase, SB 203580, on the phosphorylation and activation of both p38 MAP kinase and cPLA2. We found that while tumour necrosis factor-alpha (TNF-alpha)-stimulated tyrosine phosphorylation of p38 MAP kinase is affected only slightly by SB 203580, its stimulated kinase activity is greatly reduced in human neutrophils in suspension treated with this inhibitor.

A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene.

Binding of many cytokines to their cognate receptors immediately activates Jak tyrosine kinases and their substrates, STAT (signal transducers and activators of transcription) DNA-binding proteins. The DNA binding targets of STATs are sequence elements related to the archetypal gamma interferon activation site, GAS. However, association of interleukin 1 (IL-1) with Jak-STAT signaling has remained unresolved.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils.

Incubation of human neutrophils with 500 pM granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a rapid and time-dependent increase in the phosphorylation of cytosolic phospholipase A2 (cPLA2), which was reflected in a slower electrophoretic mobility of the enzyme. The GM-CSF-induced phosphorylation of cPLA2 was accompanied by a parallel and time-dependent increase in the enzyme activity. Preincubation of neutrophils with the tyrosine kinase inhibitor genistein caused inhibition of the GM-CSF-stimulated phosphorylation and activity of cPLA2.

A mitogen-activated protein kinase independent pathway involved in the phosphorylation and activation of cytosolic phospholipase A2 in human neutrophils stimulated with tumor necrosis factor-alpha.

Although tumor necrosis factor (TNF)-alpha stimulation of human neutrophils does not result in a significant release of arachidonic acid, it primes the cell for arachidonic acid release when cells are further stimulated by agents that induce an intracellular calcium increase. We demonstrate that TNF-alpha stimulation of neutrophils induces the phosphorylation of cytosolic phospholipase A2 (cPLA2) and also increases its activity. These results indicate that although TNF-alpha, by itself, does not cause the release of arachidonic acid in intact cells, it increases the phosphorylation and activation of the enzyme cPLA2.

Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-alpha on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils.

The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein.

Monocyte expression of the human prointerleukin 1 beta gene (IL1B) is dependent on promoter sequences which bind the hematopoietic transcription factor Spi-1/PU.1.

Interleukin-1 beta (IL-1 beta) is produced primarily by stimulated monocytes, suggesting that the IL1B gene, which codes for this protein, depends upon at least one cell-type-specific factor. Our previous characterization of the IL1B promoter indicated that the region between -131 and +12 is sufficient to direct cell-type-specific expression of a reporter gene (F. Shirakawa, K.

Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.

A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.

Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubule-associated protein kinase (MAPK, p42MAPK) antibody show only one band.