Interleukin-6 is a key mediator of the host response to tissue injury (e.g. the """"""""acute phase"""""""" plasma protein response) and has been recently identified as one of the key cytokines involved in the pathophysiology of HIV reactivation. IL-6 is induced by inflammation-associated cytokines and by endotoxin, cAMP and phorbol ester. Glucocorticoids strongly inhibit IL-6 gene expression in fibroblasts and monocytes; estradiol-17beta downregulates IL-6 gene expression in endometrial stromal cells. We have made the novel observation that glucocorticoid receptor (GR) binds not only to the entire IL-6 multiple cytokine-inducible enhancers (MRE I and MRE II), but also to the IL-6 core promoter (TATA box and RNA start sites) and strongly represses the activity of these IL-6 functional elements in cotransfection experiments. We propose to investigate the detailed molecular and cellular mechanisms that underlie human IL-6 gene repression by steroid hormones using the following lines of approach: I. MOLECULAR STUDIES. a. Enhancer occlusion. The interactions of wild-type, mutant and chimeric """"""""finger-swapped"""""""" GR and estrogen receptor (ER) with wild-type and mutant MRE I, MRE II and IL-6 NF-kappaB regions will be investigated in protein- DNA and protein-hormone receptor interactions using a variety of different techniques. b. Core-promoter occlusion. An immediate objective is to evaluate whether the IL-6 TATA box and the RNA start site region separately or together confer GR repression to heterologous enhancer/promoter constructs. We propose to investigate in detail the biochemical mechanism of core-promoter occlusion. II. CELLULAR STUDIES. a. Tissue-specificity. We propose to extend our studies of estradiol- 17beta-mediated and dexamethasone-mediated repression of IL-6 gene expression to include cell types such as fibroblasts, primary cultures of human monocytes, EBV transformed B cell lines, human breast carcinoma derived cell lines and human hepatocyte cell lines in an attempt to address (i) the generality of both enhancer and core promoter occlusion in each case and (ii) the wider physiological significance of this repression. b. Antagonistic hormonal interactions. We propose to study the interactions between wild-type, mutant and chimeric GR and ER and their target sites in the IL-6 promoter in the presence of the antiglucocorticoid RU486 and the antiestrogen tamoxifen.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Medical Biochemistry Study Section (MEDB)
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Yale University
Schools of Medicine
New Haven
United States
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Zhang, D H; Yang, L; Cohn, L et al. (1999) Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11:473-82
Zhang, D H; Yang, L; Ray, A (1998) Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3. J Immunol 161:3817-21
Ray, P; Ghosh, S K; Zhang, D H et al. (1997) Repression of interleukin-6 gene expression by 17 beta-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-kappa B by the estrogen receptor. FEBS Lett 409:79-85
Ray, P; Yang, L; Zhang, D H et al. (1997) Selective up-regulation of cytokine-induced RANTES gene expression in lung epithelial cells by overexpression of IkappaBR. J Biol Chem 272:20191-7
Zhang, D H; Cohn, L; Ray, P et al. (1997) Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J Biol Chem 272:21597-603
Siegel, M D; Zhang, D H; Ray, P et al. (1995) Activation of the interleukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements. J Biol Chem 270:24548-55
Ray, A; Zhang, D H; Ray, P (1995) Antagonism of nuclear factor-kappa B functions by steroid hormone receptors. Biochem Soc Trans 23:952-8
Ray, A; Zhang, D H; Siegel, M D et al. (1995) Regulation of interleukin-6 gene expression by steroids. Ann N Y Acad Sci 762:79-87;discussion 87-8
Ray, A; Prefontaine, K E; Ray, P (1994) Down-modulation of interleukin-6 gene expression by 17 beta-estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem 269:12940-6
Ray, A; Prefontaine, K E (1994) Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci U S A 91:752-6

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