IFN-gamma induces a p91/Stat1 alpha-related transcription factor with distinct activation and binding properties.

The 5' flanking region of the mig gene, a member of the chemokine family of small m.w. chemoattractant and growth regulatory factors, contains an IFN-gamma-responsive enhancer, gamma RE-1, consisting of an extended imperfect palindrome. In this report we show that a novel factor, gamma RF-1, which binds to the gamma RE-1 element, is rapidly activated in a variety of primary cell types and tumor cell lines treated with IFN-gamma. Our data indicate that gamma RF-1 is present in a latent form in unstimulated cells and its DNA-binding activity is dependent upon tyrosine phosphorylation. UV cross-linking studies revealed that gamma RF-1 consists of at least two proteins of approximately 95 and 130 kDa, which interact with the gamma RE-1 element. A comparison of gamma RF-1 and GAF, an IFN-gamma-activated transcription factor containing the p91/Stat1 alpha protein (Stat, signal transducer and activator of transcription), showed that these two factors exhibited differences in electrophoretic mobility, responsiveness to IFN-alpha, and kinetics of activation. Using anti-Stat Ab, however, we found that one or more subunits of gamma RF-1 are antigenically related to p91/Stat1 alpha. Our results indicate, therefore, that gamma RF-1 and GAF are distinct IFN-gamma-responsive transcription factors and probably contain closely related members of the Stat protein family.

A unique palindromic element mediates gamma interferon induction of mig gene expression.

To define the molecular mechanisms involved in the action of gamma interferon (IFN-gamma), we have analyzed the transcriptional regulation of the mig (monokine induced by gamma interferon) gene, a member of the platelet factor 4-interleukin-8 cytokine family that is expressed in murine macrophages specifically in response to IFN-gamma. Analysis of mig/CAT chimeric constructs transiently transfected into the RAW 264.7 mouse monocytic cell line revealed a unique IFN-gamma-responsive element (gamma RE-1). The sequence of this cis regulatory element defined by deletion analysis contains an imperfect inverted repeat extending 27 bp. Examination of mig/CAT constructs with mutations in gamma RE-1 revealed that the palindromic positions in the element were essential for activity. Consistent with its function as an enhancer, a single copy of gamma RE-1 conferred IFN-gamma inducibility to a heterologous (herpes simplex virus thymidine kinase) promoter. Exonuclease III protection assays demonstrated symmetrical protection of a mig promoter fragment centered about the gamma RE-1 palindromic sequence. Using the gel electrophoretic mobility shift assay, we identified a factor (gamma RF-1) present in nuclear extracts prepared from IFN-gamma-stimulated RAW 264.7 cells which binds to gamma RE-1. The activation of gamma RF-1 occurred rapidly (within 1 min) in response to IFN-gamma and was independent of protein synthesis. Similar to the expression of mig mRNA, the formation of gamma RF-1 was selectively induced by IFN-gamma and not IFN-alpha. The regulation of gene expression through gamma RF-1 and gamma RE-1 may explain the preferential activation of a subset of interferon-inducible genes by IFN-gamma.

Glucocorticoid induced up-regulation of a pituitary K+ channel mRNA in vitro and in vivo.

Hormones might produce long-term changes in cell excitability by regulating K+ channel gene expression. Recently, we found that dexamethasone increases expression of Kv1.5 K+ channel mRNA in GH3 rat pituitary tumor cells. We wished to test if this effect is specific for the Kv1.5 gene, if it is mediated by activation of glucocorticoid receptors, and whether it occurs in normal pituitary cells. Here we report that dexamethasone treatment of GH3 cells for 3 hours increases Kv1.5 mRNA without affecting Kv1.4 or Kv2.1 K+ channel mRNAs or D Ca2+ channel mRNA. Treatment with sex steroids fails to alter Kv1.5 mRNA levels, while natural glucocorticoids increase expression of the channel mRNA. RU38486, a competitive inhibitor of glucocorticoid receptors, inhibits the response to dexamethasone. We then tested whether Kv1.5 mRNA is induced by dexamethasone in normal rat pituitary cells. To study in vivo effects, channel mRNA levels in pituitaries from adrenalectomized rats were measured with RNAse protection assays. One day following dexamethasone injection Kv1.5 mRNA was increased 8-fold. Dexamethasone induction of Kv1.5 mRNA was also found in primary cultured anterior pituitary cells. We conclude that activated glucocorticoid receptors specifically induce Kv1.5 K+ channel mRNA expression in normal and clonal anterior pituitary cells.

Effect of temperature on production of hypochlorous acid by stimulated human neutrophils.

Under natural conditions or because of therapy with heat or cold, neutrophils may function at times in the human body at temperatures other than 37 degrees C. Therefore, we evaluated the effects of temperature on several functions of these cells. Phagocytosis and superoxide production by stimulated neutrophils were optimal at 37 degrees C and attained at least 70% of this peak value at 42 degrees C. In contrast, production of hypochlorous acid (as measured by an assay using the chlorination of taurine) by stimulated neutrophils was optimal at temperatures less than 37 degrees C and attained only 13% to 15% of this peak value at 42 degrees C. During a 2-hour incubation, the major suppressive effects of the higher temperature occurred during the second hour. This result was not explainable by factors related to the hypochlorous acid assay system or by loss of cell viability or myeloperoxidase activity in the cell supernatants, but rather appeared to be caused by reduced generation of hydrogen peroxide at the higher temperatures. Because the extracellular release of a strong oxidant such as hypochlorous acid might result in significant tissue injury, suppression of the release of this oxidant by elevated temperatures may explain why the application of local heat sometimes benefits certain inflammatory conditions.