Douglas C. Harnish

The Role of CBP in Estrogen Receptor Cross-Talk with Nuclear Factor-κB in HepG2 Cells

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-κB (NF-κB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-κB function. In the current study 17β-estradiol-bound ERα interfered with cytokine-induced activation of a NF-κB reporter in HepG2 cells. The estrogen metabolite, 17α-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-κB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-κB interfered with the trans-activation properties of ERα. Ligand-bound ERα did not inhibit NF-κB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-κB transcriptional activity. Coexpression of the ...

Estrogen Regulation of the Apolipoprotein AI Gene Promoter through Transcription Cofactor Sharing

Estrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor α (ERα), 17β-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERα plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERα DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERα binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERα plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3β and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERα coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERα and RIP140 levels. At low ratios of RIP140 to ERα, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERα and E2 but also upon the intracellular balance of ERα and coactivators utilized by ERα and the apoAI enhancer.

CONTROL OF APOLIPOPROTEIN AI GENE EXPRESSION THROUGH SYNERGISTIC INTERACTIONS BETWEEN HEPATOCYTE NUCLEAR FACTORS 3 AND 4

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-3β 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-3β 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.

A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia

The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-alpha agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia.

Estrogen Receptor α Inhibits IL-1β Induction of Gene Expression in the Mouse Liver

Estrogens have been suggested to modulate several inflammatory processes. Here, we show that IL-1β treatment induced the expression of approximately 75 genes in the liver of ovariectomized mice. 17α-Ethinyl estradiol (EE) pretreatment reduced the IL-1β induction of approximately one third of these genes. Estrogen receptor α (ERα) was required for this inhibitory activity, because EE inhibition of IL-1β-stimulated gene expression occurred in ERβ knockout mice, but not in ERα knockout mice. EE treatment induced expression of 40 genes, including the transcriptional repressor short heterodimer partner and prostaglandin D synthase, known modulators of nuclear factor-κB signaling. However, the ER agonists genistein and raloxifene both inhibited IL-1β gene induction without stimulating the expression of prostaglandin D synthase, short heterodimer partner, or other ER-inducible genes, indicating that induction of gene expression was not required for ER inhibition of IL-1β signaling. Finally, the ability of EE to ...

CRP is a novel ligand for the oxidized LDL receptor LOX-1

C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-1, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction.

Synthesis and activity of substituted 4-(indazol-3-yl)phenols as pathway-selective estrogen receptor ligands useful in the treatment of rheumatoid arthritis.

Pathway-selective ligands for the estrogen receptor (ER) inhibit NF-kappaB-mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules, and inflammatory enzymes. SAR development of a series of 4-(indazol-3-yl)phenols has led to the identification of WAY-169916 an orally active nonsteroidal ligand with the potential use in the treatment of rheumatoid arthritis without the classical proliferative effects associated with estrogens.

The utility of pathway selective estrogen receptor ligands that inhibit nuclear factor-κB transcriptional activity in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that produces synovial proliferation and joint erosions. The pathologic lesions of RA are driven through the production of inflammatory mediators in the synovium mediated, in part, by the transcription factor NF-κB. We have identified a non-steroidal estrogen receptor ligand, WAY-169916, that selectively inhibits NF-κB transcriptional activity but is devoid of conventional estrogenic activity. The activity of WAY-169916 was monitored in two models of arthritis, the HLA-B27 transgenic rat and the Lewis rat adjuvant-induced model, after daily oral administration. In both models, a near complete reversal in hindpaw scores was observed as well as marked improvements in the histological scores. In the Lewis rat adjuvant model, WAY-169916 markedly suppresses the adjuvant induction of three serum acute phase proteins: haptoglobin, α1-acid glycoprotein (α1-AGP), and C-reactive protein (CRP). Gene expression experiments also demonstrate a global suppression of adjuvant-induced gene expression in the spleen, liver, and popliteal lymph nodes. Finally, WAY-169916 was effective in suppressing tumor necrosis factor-α-mediated inflammatory gene expression in fibroblast-like synoviocytes isolated from patients with RA. Together, these data suggest the utility of WAY-169916, and other compounds in its class, in treating RA through global suppression of inflammation via selective blockade of NF-κB transcriptional activity.

Improvement of Physiochemical Properties of the Tetrahydroazepinoindole Series of Farnesoid X Receptor (FXR) Agonists: Beneficial Modulation of Lipids in Primates.

In an effort to develop orally active farnesoid X receptor (FXR) agonists, a series of tetrahydroazepinoindoles with appended solubilizing amine functionalities were synthesized. The crystal structure of the previously disclosed FXR agonist, 1 (FXR-450), aided in the design of compounds with tethered solubilizing functionalities designed to reach the solvent cavity around the hFXR receptor. These compounds were soluble in 0.5% methylcellulose/2% Tween-80 in water (MC/T) for oral administration. In vitro and in vivo optimization led to the identification of 14dd and 14cc, which in a dose-dependent fashion regulated low density lipoprotein cholesterol (LDLc) in low density lipoprotein receptor knockout (LDLR(-/-)) mice. Compound 14cc was dosed in female rhesus monkeys for 4 weeks at 60 mg/kg daily in MC/T vehicle. After 7 days, triglyceride (TG) levels and very low density lipoprotein cholesterol (VLDLc) levels were significantly decreased and LDLc was decreased 63%. These data are the first to demonstrate the dramatic lowering of serum LDLc levels by a FXR agonist in primates and supports the potential utility of 14cc in treating dyslipidemia in humans beyond just TG lowering.

Pyrrole[2,3-d]azepino compounds as agonists of the farnesoid X receptor (FXR)

Pyrrole[2,3-d]azepines have been identified as potent agonists of the farnesoid X receptor (FXR). Based on the planar X-ray crystal structure of WAY-362450 1 in the ligand binding domain and molecular modeling studies, non-planar reduced compounds were designed which led to agonists that exhibit high aqueous solubility and retain moderate in vitro potency.