Steroid-activated nuclear receptors and uses therefor

Details Steroid-activated nuclear receptors and uses therefor Steroid-activated nuclear receptors and uses therefor

1999-01-08

2004-10-26

Zeman, Mary K. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S004000, C435S007800, C436S501000, C436S086000, C436S087000, C930S010000

Reexamination Certificate

active

06809178

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to intracellular receptors, nucleic acids encoding same, and uses therefor. In a particular aspect, the present invention relates to methods for the modulation of physiological response to elevated levels of steroid and/or xenobiotic compounds.
BACKGROUND OF THE INVENTION
Nuclear receptors constitute a large superfamily of ligand-dependent and sequence-specific transcription factors. Members of this family influence transcription either directly, through specific binding to the promoters of target genes (see Evans, in
Science
240:889-895 (1988)), or indirectly, via protein—protein interactions with other transcription factors (see, for example, Jonat et al., in
Cell
62:1189-1204 (1990), Schuele et al., in
Cell
62:1217-1226 (1990), and Yang-Yen et al., in
Cell
62:1205-1215 (1990)). The nuclear receptor superfamily (also known in the art as the “steroid/thyroid hormone receptor superfamily”) includes receptors for a variety of hydrophobic ligands, including cortisol, aldosterone, estrogen, progesterone, testosterone, vitamin D
3
, thyroid hormone and retinoic acid, as well as a number of receptor-like molecules, termed “orphan receptors” for which the ligands remain unknown (see Evans, 1988, supra). These receptors all share a common structure indicative of divergence from an ancestral archetype.
Lipophilic hormones such as steroids, retinoic acid, thyroid hormone, and vitamin D3 control broad aspects of animal growth, development, and adult organ physiology. The effects of these hormones are mediated by a large superfamily of intracellular receptors that function as ligand-dependent and sequence-specific transcription factors. The non-steroidal nuclear receptors for thyroid hormone (TR), vitamin D3 (VDR), all-trans retinoic acid (RAR), and fatty acids and eicosanoids (PPAR) form heterodimers with the 9-cis retinoic acid receptor (RXR) that bind bipartite hormone-response elements (HREs) composed of directly repeated half sites related to the sequence AGGTCA (Mangelsdorf and Evans,
Cell
83: 841-850, 1995). In contrast, the steroid receptors function as homodimers and bind to palindromic target sequences spaced by three nucleotides (Beato et al.,
Cell
83: 851-857, 1995). In addition to the known receptors, a large group of structurally-related “orphan” nuclear receptors has been described which possess obvious DNA and ligand binding domains, but lack identified ligands (Mangelsdorf et al.,
Cell
83:835-839, 1995; Enmark and Gustafsson,
Mol. Endocrinol.
10:1293 (1996); and O'Malley and Conneely,
Mol. Endocrinol.
6:1359 (1992)). Each has the potential to regulate a distinct endocrine signaling pathway.
It is widely viewed that the hormone response is a consequence of the release, from an endocrine gland, of a ligand that circulates through the blood, and coordinately regulates responses in target tissues by acting through specific nuclear receptors. Hormone responsiveness is dependent on the ability to rapidly clear ligand from the blood and the body so that, in absence of a stimulus, target tissues return to a ground state. Hormonal homeostasis is thus achieved by the coordinated release and degradation of bioactive hormones. Steroid hormones and their many metabolites are primarily inactivated by reduction and oxidation in the liver. Since hundreds of adrenal steroids have been identified (e.g., dozens of each of the sex steroids (androgens, estrogens and progestins), 25-35 vitamin D metabolites, and likely hundreds of fatty acids, eicosanoids, hydroxyfats and related bioactive lipids), the problem of efficient ligand elimination is critical to physiologic homeostasis. In addition to the existence of a myriad of endogenous hormones, a similar diversity of ingested plant and animal steroids and bioactive xenobiotic compounds must also be degraded.
Selye first introduced the concept that exogenous steroids and pharmacologic substances may function to modulate the expression of enzymes that would protect against subsequent exposure to toxic xenobiotic substances (H. Selye,
J. Pharm. Sci.
60:1-28, 1971). These compounds, which Selye called “catatoxic steroids,” are typified by the synthetic glucocorticoid antagonist, pregnenolone-16-carbonitrile (PCN). PCN, and a variety of xenobiotic steroids, induce the proliferation of hepatic endoplasmic reticulum and the expression of cytochrome P450 genes (Burger et al.,
Proc. Natl. Acad. Sci. (USA)
89:2145-2149, 1992; Gonzalez et al.,
Mol. Cell. Biol.
6:2969-2976, 1986; and Schuetz and Guzelian,
J. Biol. Chem.
259:2007-2012, 1984). One consequence of PCN treatment is the induction of nonspecific “protection” against subsequent exposure to such diverse xenobiotics as digitoxin, indomethacin, barbiturates, and steroids (Selye, supra, 1971).
Furthermore, it is known that a variety of such compounds can activate P450 genes responsible for their detoxification or degradation (Fernandez-Salguero and Gonzalez,
Pharmacogenetics
5:S123-128, 1995; Denison and Whitlock,
J. Biol. Chem.
270:18175-18178, 1995; O. Hankinson,
Ann. Rev. Pharmacol. Toxicol.
35:307-340, 1995; and Rendic and Di Carlo,
Drug Metab. Rev.
29:413-580, 1997).
While it appears that such catatoxic compounds regulate the expression of cytochrome P450s and other detoxifying enzymes, two lines of evidence argue that such regulation is independent of the classical steroid receptors. First, many of the most potent compounds (e.g., PCN, spironolactone, and cyproterone acetate) have been shown to be steroid receptor antagonists; whereas others (e.g., dexamethasone) are steroid receptor agonists (Burger, supra, 1992). Second, the nonspecific protective response remains after bilateral adrenalectomy (and presumably in the absence of adrenal steroids), but not after partial hepatectomy (Selye, supra, 1971).
Insight into the mechanism by which PCN exerts its catatoxic effects is provided by the demonstration that PCN induces the expression of CYP3A1 and CYP3A2, two closely related members of the P450 family of monooxygenases (see, for example, Elshourbagy and Guzelian in
J. Biol. Chem.
255:1279 (1980); Heuman et al., in
Mol. Pharmacol.
21:753 (1982); Hardwick et al., in
J. Biol. Chem.
258:10182 (1983); Scheutz and Guzelian in
J. Biol. Chem.
259:2007 (1984); Scheutz et al., in
J. Biol. Chem.
259:1999 (1984); and Gonzalez et al., in
J. Biol. Chem.
260:7435 (1985)). The CYP3A hemoproteins display broad substrate specificity, hydroxylating a variety of xenobiotics (e.g., cyclosporin, warfarin and erythromycin), as well as endogenous steroids (e.g., cortisol, progesterone, testosterone and DHEA-sulfate. See, for example, Nebert and Gonzalez in
Ann. Rev. Biochem.
56:945 (1987) and Juchau in
Life Sci.
47:2385 (1990)). A PCN response element (which is highly conserved in the CYP3A2 gene promoter) has since been identified in subsequent studies with the cloned CYP3A1 gene promoter (see Miyata et al., in
Archives Biochem. Biophysics
318:71 (1995) and Quattrochi et al., in
J. Biol. Chem.
270:28917 (1995)). This response element comprises a direct repeat of two copies of the nuclear receptor half-site consensus sequence AGTTCA.
In addition to inducing CYP3A gene expression, PCN has also been shown to have marked effects on hepatic cholesterol homeostasis. These effects include significant decreases in the levels of HMG-CoA reductase and cholesterol 7a-hydroxylase gene expression, with associated reductions in sterol biosynthesis and bile acid secretion. PCN has also been reported to enhance the formation of cholesterol esters and the hypersecretion of cholesterol into the bile. Thus, PCN affects key aspects of cholesterol metabolism, including its biosynthesis, storage and secretion.
Activation of orphan nuclear receptor(s) by catatoxic steroids provides a possible mechanism for the induction of xenobiotic metabolizing enzymes by compounds that do not activate known steroid receptors. Because such enzymes are activated by high (pharmacological) doses of xenobiotic and natural steroids, such a “sensor

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