No abstract

inflammation ͉ therapy

Abstract-The functional interaction, or "cross-talk," between estrogen receptor (ER) and the proinflammatory transcription factor nuclear factor (NF)-B demonstrated in vitro has been suggested to play a role in estrogen prevention of cardiovascular disease. Here, we demonstrate that this reciprocal cross-talk occurs in vivo. Ovariectomized C57BL/6 mice fed an atherogenic diet had increased hepatic levels of active NF-B and numerous inflammatory genes, including MHC invariant chain (Ii), vascular cell adhesion molecule-1, tumor necrosis factor-␣, and RANTES. Treatment with 17␣-ethinylestradiol (EE) strongly blocked induction of these genes but had no effect on their basal expression levels. ER was required for this activity, because the antagonist ICI 182,780 completely blocked the inhibitory activity of EE. Gene activation by EE was not required for inhibition of inflammatory gene expression, because both the phytoestrogen genistein and low doses of EE were effective in blocking inflammatory gene induction without inducing marker genes such as intestinal trefoil factor (ITF) or myo-inositol-1-phosphate synthase (IPS). The in vivo transcriptional interference was reciprocal, with EE induction of ITF and IPS greatly reduced in animals fed the atherogenic diet versus chow-fed controls. This interference was specific to the liver, because diet had no effect on uterine weight increases produced by EE. Transfection experiments confirmed that the extent of inhibition of ER-mediated transcription by inflammatory stimuli correlated with the extent of NF-B activation. These results suggest that the cross-talk between ER and NF-B does occur in vivo and may indeed contribute significantly to the cardioprotective effects of estrogen.

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.

Apolipoprotein AI (apoAI) gene expression in liver depends on synergistic interactions between transcription factors bound to three distinct sites (A, B, and C) within a hepatocyte-specific enhancer in the 5-flanking region of the gene. In this study, we showed that a segment spanning sites A and B retains substantial levels of enhancer activity in hepatoblastoma HepG2 cells and that sites A and B are occupied by the liver-enriched hepatocyte nuclear factors (HNFs) 4 and 3, respectively, in these cells. In non-hepatic CV-1 cells, HNF-4 and HNF-3␤ activated this minimal enhancer synergistically. This synergy was dependent upon simultaneous binding of these factors to their cognate sites, but it was not due to cooperativity in DNA binding. Separation of these sites by varying helical turns of DNA did not affect simultaneous binding of HNF-3␤ and HNF-4 nor did it influence their functional synergy. The synergy was, however, dependent upon the cell type used for functional analysis. In addition, this synergy was further potentiated by estrogen treatment of cells cotransfected with the estrogen receptor. These data indicate that a cell type-restricted intermediary factor jointly recruited by HNF-4 and HNF-3 participates in activation of the apoAI enhancer in liver cells and suggest that the activity of this factor is regulated by estrogen.An emerging hallmark of transcriptional regulation in eukaryotes is the assembly of multiprotein complexes at the enhancer and promoter regions of target genes (1). These complexes are formed and stabilized through multiple protein-DNA and protein-protein interactions. Tissue specificity of many liver-specific genes, the expression of which is restricted to the liver, is imparted by combinatorial interactions between liver-enriched and ubiquitous transcription factors (2, 3). The unique expression program of a hepatic gene is thus primarily determined by the configuration of cis-elements containing binding sites for these factors and may be fine tuned by secondary interactions among the proteins.The liver-specific enhancer of the gene encoding apolipoprotein AI (apoAI), 1 the major protein component of high density lipoprotein (reviewed in Ref. 4), contains three cis-acting elements (site A, Ϫ214 to Ϫ192; site B, Ϫ169 to Ϫ146; and site C, Ϫ134 to Ϫ119; Ref. 5). Sites A and C serve as sites of action for many nuclear receptors including retinoid X receptor ␣ (6), ARP-1/COUP-TFII (7, 8), and HNF-4 (9, 10). Site B binds the hepatocyte-enriched factor HNF-3␤ (11). Maximal activity of the apoAI enhancer depends upon the integrity of each of the sites A, B, and C, suggesting that synergistic interactions resulting from a factor binding to them dominate transcription activation from the enhancer (5).In previous studies, we showed that the apoAI enhancer can be activated in non-hepatic cell types if hepatocyte-enriched factors HNF-4 and HNF-3␤ are also provided (11). In the current report, we first define the minimum requirements for efficient enhancer function and then experimentally addre...