The mechanism by which a single factor evokes opposite regulatory effects from a specific DNA sequence is not well understood. In this study, a 25-base pair element that resides upstream of the mouse proliferin gene was examined; it conferred on linked promoters either positive or negative glucocorticoid regulation, depending upon physiological context. This sequence, denoted a "composite" glucocorticoid response element (GRE), was bound selectively in vitro both by the glucocorticoid receptor and by c-Jun and c-Fos, components of the phorbol ester-activated AP-1 transcription factor. Indeed, c-Jun and c-Fos served as selectors of hormone responsiveness: the composite GRE was inactive in the absence of c-Jun, whereas it conferred a positive glucocorticoid effect in the presence of c-Jun, and a negative glucocorticoid effect in the presence of c-Jun and relatively high levels of c-Fos. The receptor also interacted selectively with c-Jun in vitro. A general model for composite GRE action is proposed that invokes both DNA binding and protein-protein interactions by receptor and nonreceptor factors.

conformation change ͉ FQNLF ͉ FRET ͉ nuclear receptor ͉ estrogen receptor T he nuclear receptor (NR) superfamily consists of a large group of ligand-regulated transcription factors. Several NRs are implicated in human physiology and disease (1, 2) and activation of the estrogen receptors (ER) and androgen receptors (AR) are predisposing factors for breast (3) and prostate cancer (4). Indeed, pharmacologic antagonists of AR and ER are used as antineoplastic agents in these diseases (4-7). It is commonly believed that understanding NR structure and function will facilitate development of specific drugs that can replace or supplement current therapies (2). Ligand binding alters NR structure, cofactor interactions, and transcriptional activity (8). Transcriptional activation functions are present in the aminoterminal domain (NTD; AF-1) and the ligand binding domain (LBD; AF-2) of many NRs, including AR (9) and ER (10). AF-1 is not conserved at the primary sequence level and is poorly characterized functionally (11). In contrast, AF-2 is highly conserved (12) and consists of amino acids that form a coactivator binding pocket on the surface of most NR LBDs (13-16).In many NRs, both AF-1 and AF-2 activities are suppressed in the absence of ligand and enabled after ligand binding (9, 10), which implies that ligand binding to the LBD somehow unmasks AF-1 activities in the NTD. The molecular͞structural basis for LBD communication with AF-1 in full-length molecules remains uncertain. However, an intermolecular interaction between NTD peptides and the agonist-bound LBD has been extensively characterized in vitro and with intracellular two-hybrid assays for the AR (14, 17-21) and ER (22). In the AR NTD, deletion or mutation of a sequence ( 23 FQNLF 27 ) that can bind the AF-2 coactivator pocket of the LBD (14, 19) diminishes activity of the AR at certain promoter elements (21). This finding suggests that an NTD-LBD interaction is functionally important, but it remains unknown whether the NTD interacts with the LBD within one molecule or whether it participates in an intermolecular interaction with the LBD of a second AR molecule.Of the currently available experimental approaches, FRET (23) uniquely can resolve conformation changes and protein interactions of the intact NR molecule in living cells. FRET allows real-time detection of protein conformation changes based on energy transfer between fluorophores attached to domains of interest. Here, we used FRET to determine the time and subcellular location of ligand-induced conformational changes in AR that underlie its activity as a transcription factor. We contrasted these studies with other members of the NR family, ER␣ and peroxisome proliferator-activated receptor-␥2 (PPAR␥2), and have determined a role for the AR-specific 23 FQNLF 27 motif in coordinating intramolecular AR conformational changes that precede AR self-association, most likely as a dimer. Materials and MethodsPlasmid Construction. Plasmids that express AR, ER␣, or PPAR␥2 as enhanced cyan f luorescent protein ...

Activation of the NLRP3 inflammasome by microbial ligands or tissue damage requires intracellular generation of reactive oxygen species (ROS). We present evidence that macrophage secretion of IL1β upon stimulation with ATP, crystals or LPS is mediated by a rapid increase in the activity of xanthine oxidase (XO), the oxidized form of xanthine dehydrogenase, resulting in the formation of uric acid as well as ROS. We show that XO-derived ROS, but not uric acid, is the trigger for IL1β release and that XO blockade results in impaired IL1β and caspase1 secretion. XO is localized to both cytoplasmic and mitochondrial compartments and acts upstream to the PI3K–AKT signalling pathway that results in mitochondrial ROS generation. This pathway represents a mechanism for regulating NLRP3 inflammasome activation that may have therapeutic implications in inflammatory diseases.

The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular, the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural, pharmacologic, and pharmacokinetic properties of RDEA119/BAY 869766, an allosteric MEK inhibitor, are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo, RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma, colon, and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data, we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice, RDEA119/BAY 869766 has the potential for use as a once-or twice-daily oral treatment for cancer. RDEA119/BAY 869766, an exquisitely selective, orally available MEK inhibitor, has been selected for clinical development because of its potency and favorable pharmacokinetic profile.

Steroidal glucocorticoids are commonly used due to their powerful antiinflammatory activity. However, despite their excellent efficacy, severe side effects frequently limit the use of these drugs. The search for novel glucocorticoids with reduced side effects has been intensified by the discovery of new molecular details regarding the function of the glucocorticoid receptor. These new insights may pave the way for novel, safer therapies that retain the efficacy of currently prescribed steroids.

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

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Elevated serum urate concentration is the primary cause of gout. Understanding the processes that affect serum urate concentration is important for understanding the etiology of gout and thereby understanding treatment. Urate handing in the human body is a complex system including three major processes: production, renal elimination, and intestinal elimination. A change in any one of these can affect both the steady-state serum urate concentration as well as other urate processes. The remarkable complexity underlying urate regulation and its maintenance at high levels in humans suggests that this molecule could potentially play an interesting role other than as a mere waste product to be eliminated as rapidly as possible.