ESTROGEN REGULATION OF HUMAN INTERLEUKIN 6 GENE PROMOTER

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Nearly one half of women over the age of 50 (post-menopausal) will have bone loss (osteoporosis) so severe that spontaneous fractures of the lumbar vertebrae, hip, or wrist will occur. Decreased estrogen production by post-menopausal women is associated with loss of bone mass and estrogen replacement therapy is a primary treatment modality to preserve bone mass. Enhanced production of the cytokine interleukin-6 (IL-6) by osteoblasts causes bone resorption via osteoclast recruitment and differentiation. In vitro, IL-6 production by human osteoblast-like cells (OLCs) is stimulated by the pro-inflammatory cytokine interleukin- 1beta (IL-1beta) and estrogen modulates this effect of IL-1beta on IL-6 mRNA levels and protein secretion. The mechanism(s) of estrogen modulation of cytokine-induced IL-6 gene expression in human OLCs is poorly understood. Estrogen/estrogen receptor (ER) is suggested to affect the IL-6 gene promoter indirectly through interaction with various transcription factors involved in regulation of IL-6 gene expression. However, our analysis of the IL-6 gene promoter nucleotide sequence indicates an additional potential mechanism, via a putative estrogen receptor/activator protein-1 binding site in the distal segment of the promoter. The hypothesis of this proposal is that estrogen/ERs modulate IL-1beta- induced-IL-6 gene expression in human osteoblasts via interaction with transcription factors and/or binding to an ER element within the IL-6 gene promoter. The specific aims to test this hypothesis are to: 1) Determine the promoter elements of the IL-6 gene that are required for IL-1beta activation in human OLCs. This will be accomplished by using the wild type IL-6 gene promoter (phIL-6/luc+) and a series of shortened promoter fragments (deletion constructs). 2) Determine if these or different regions correspond to those responsive to estrogen regulation. This will be accomplished by treating IL-1beta-stimulated OLCs transfected with phIL-6/luc+ or deletion constructs with 17beta- estradiol. 3) Identify transcription factors involved in estrogen regulation of IL-1beta-induced-IL-6 gene promoter activity. This will be accomplished using nuclear protein extracts of IL-1beta-and 17beta- estradiol-stimulated human OLCs in electromobility shift assays. Biomedical significance. Approximately 1.3 million bone fractures occur every year in the United States that are attributable to osteoporosis, at an estimated annual cost of 10 billion dollars. Identification of the mechanisms by which estrogen regulates IL-6 gene expression will enhance our understanding of the pathobiology of osteoporosis and suggest future strategies aimed at treating and preventing this disease.