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...

Cardiac tissue that undergoes an ischemic episode exhibits irreversible alterations that become more extensive upon reperfusion. Estrogen treatment has been reported to protect against reperfusion injury, but the mechanism remains unknown. The cardioprotective effects of 17␤-estradiol, a biologically active form of the hormone, and 17␣-estradiol were assessed in an in vivo occlusion-reperfusion model. Anesthetized, ovariectomized rabbits were administered 17␤-estradiol (20 g), 17␣-estradiol (1 mg), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending (LAD) coronary artery followed by 4 h of reperfusion. Infarct size as a percentage of area at risk decreased in the 17␤-estradiol-treated group (18.8 Ϯ 1.7) compared with 17␣-estradiol (41.9 Ϯ 4.8; P Ͻ 0.01) or vehicle groups (48 Ϯ 5.5; P Ͻ 0.001). Similar results were obtained when infarct size was expressed as a percentage of total left ventricle. The second objective of the study was to assess fulvestrant (Faslodex, ICI 182,780), an estrogen receptor antagonist, for its effects on infarct size in ovariectomized female rabbits treated with 17␤-estradiol. ICI 182,780 was administered intravenously 1 h before the administration of 17␤-estradiol (20 g) or vehicle. The hearts were subjected to 30-min LAD coronary artery occlusion and 4 h of reperfusion. Pretreatment with ICI 182,780 significantly limited the infarct size sparing effect of 17␤-estradiol when expressed as a percentage of the risk region (53.0 Ϯ 5.0). The results indicate that 17␤-estradiol protects the heart against ischemia-reperfusion injury and that the observed cardioprotection is mediated by the estrogen receptor.

No abstract

Liver-specific expression of the apolipoprotein AI (apoAI) gene is mediated by transcription factors bound to three sites (A, B, and C) in the apoAI enhancer. Sites A and C bind various members of the nuclear receptor superfamily, including the orphan nuclear receptor apolipoprotein regulatory protein-1 (ARP-1); site B binds the liver-enriched factor hepatic nuclear factor-3. The immediate early growth response factor (Egr-1), which is transiently expressed in various pathophysiologic states of the liver, activates the apoAI enhancer and overcomes ARP-1-mediated repression of the enhancer in hepatoblastoma HepG2 cells. Deletion mapping analysis revealed two Egr-1 binding sites, E1 and E2, flanking site A. Erg-1 bound efficiently to both E1 and E2. Sp1 in HepG2 nuclear extracts bound to E2 but not E1. In HepG2 cells, E1 functioned as an Egr-1 response element, whereas E2 had high basal activity and was not further induced by Egr-1. Mutations that prevent Egr-1 binding to the apoAI enhancer abolished its responsiveness to Erg-1, while they had only minor effects on its constitutive activity. These mutations also diminished the ability of Egr-1 to overcome ARP-1-mediated repression. Elimination of transcription factor binding to sites A, B, or C reduced enhancer activity without affecting Egr-1-dependent activation. We argue that Egr-1 is recruited to the apoAI enhancer complex under unusual circumstances, such as those prevailing during liver regeneration, to maintain apoAI transcription levels by overriding prior transcriptional controls.

Androgenetic alopecia (AGA), commonly known as male pattern baldness, is a form of hair loss that occurs in both males and females. Although the exact cause of AGA is not known, it is associated with genetic predisposition through traits related to androgen synthesis/metabolism and androgen signaling mediated by the androgen receptor (AR). Current therapies for AGA show limited efficacy and are often associated with undesirable side effects. A major hurdle to developing new therapies for AGA is the lack of small animal models to support drug discovery research. Here, we report the first rodent model of AGA. Previous work demonstrating that the interaction between androgen-bound AR and beta-catenin can inhibit Wnt signaling led us to test the hypothesis that expression of AR in hair follicle cells could interfere with hair growth in an androgen-dependent manner. Transgenic mice overexpressing human AR in the skin under control of the keratin 5 promoter were generated. Keratin 5-human AR transgenic mice exposed to high levels of 5alpha-dihydrotestosterone showed delayed hair regeneration, mimicking the AGA scalp. This effect is AR mediated, because treatment with the AR antagonist hydroxyflutamide inhibited the effect of dihydrotestosterone on hair growth. These results support the hypothesis that androgen-mediated hair loss is AR dependent and suggest that AR and beta-catenin mediate this effect. These mice can now be used to test new therapeutic agents for the treatment of AGA, accelerating the drug discovery process.